Recombinant virus expressing foreign dna encoding feline cd80, feline ctla-4 or feline cd86 and its applying

FIELD: biotechnology, veterinary science.

SUBSTANCE: invention relates to therapeutic vector used in therapy of infectious diseases in cats that comprises at least one foreign nucleic acid each of that (a) encodes protein taken among the group consisting of feline protein CD28 represented in SEQ ID NO:8 or its immunogenic moiety; feline protein CD80 represented in SEQ ID NO:2 or 3, or its immunogenic moiety; feline protein CD86 represented in SEQ ID NO:6 or its immunogenic moiety, or feline protein CTLA-4 represented in SEQ ID NO:10 or its immunogenic moiety; and (b) nucleic acid that is able to be expressed in insertion of vector in the corresponding host. Indicated therapeutic vector is used in effective dose as component of vaccine against infectious diseases in cats for their immunization and in methods for enhancement or inhibition of immune response in cats and reducing or eradication of tumor in cats. Invention provides stimulating the activation and proliferation of T cells and to enhance effectiveness of control of infectious diseases in cats.

EFFECT: valuable biological properties of recombinant virus.

41 cl, 13 dwg

 

Interferon-γ and use of

In this application claims the priority of U.S. reg. No. 09/071711, filed may 1, 1998, the contents of which are introduced in the present description by reference. In this application references various publications are given in parentheses. All references to these publications can be found at the end of the description immediately before the list of sequences. For a more complete description of prior art in the field to which the present invention, these publications in their entirety are introduced in the present description by reference.

Prior art

It is assumed that stimulate the activation and proliferation of T cells in response to disease of the host depends on two types of interaction: recognition of T-cell receptor (TCR) immunogenic peptides in the presence of molecules MHC class I, and the secondary interaction of additional ligands, such as CD80 and CD86 with their used CD-28 and/or CTLA-4 on T-cells. The effective interaction of these two ways cascades of reactions leads to activation and proliferation of both CD4+-T-, and CD8+-T-cells and increased production of regulatory immune response cytokines of the Th1 type and Th2. In the absence of adequate co-stimulation of T cells can develop anergicakimi state, resulting in budetuslishatj proliferation of T-cells and secretion of cytokines. Over the last few years it was found that the main regulators of T-cell response are two molecules, CD28 and its ligands, CD80 and CD86. CD28 is a major co-stimulatory T-cell receptor, and after interaction with CD80 and CD86 it enhances the proliferation of T-cells and the synthesis of cytokines, preventing the death of T-cells. CTLA-4 (also known as CD152), a homologue CD-28, also plays an important role in co-stimulation. It is obvious, though it is not exactly known, it inhibits T-cell co-stimulatory responses. The interaction and relationship between CD28, CTLA-4 and their ligands CD80 and CD86 in the processes of co-stimulation play a key role in the overall induction and suppression of immune reactions in response to illness of the owner. (Linsley et al., 1991 a; 1993 a).

Currently there is no effective vaccine to prevent the States of immunodeficiency in cats and infectious peritonitis in cats. Currently available vaccines against the virus feline leukemia, but their effectiveness remains problematic, and in some cases they can even cause the disease. It was shown that the experimental vaccine against infectious peritonitis in cats do not have a protective action or even cause premature death of the animal due to antibody-mediated exacerbation. It is therefore necessary to obtain Lek is stennie funds and composition, which would provide protection against these and other diseases against which there are no vaccines or that would enhance the effectiveness of existing and widely used vaccines. In addition, you need to get vaccines and medicines, which would be induced cell-mediated response in the absence of antibodies, contributing to exacerbation of the disease. Finally, vaccination of kittens presents certain difficulties due to the inability to suppress their production of maternal antibodies. Therefore, it is necessary to obtain a safe and effective means, which would allow to solve these problems.

In the present invention, for producing the desired immune response to specific feline pathogen or pathological condition in cats can regulate T-cell responses by modification of the expression of the feline CD28, feline CTLA-4 and their ligands, and on-stimulatory molecules feline CD80 and feline CD86 by the gain suppression or the diversion of this expression. In particular, these co-stimulatory molecules can be used for vaccination against infectious diseases, treatment of infectious diseases and neoplastic, degenerative, autoimmune and immunodeficiency in cats. The present invention solves problems associated with tattvam the efficiency and effectiveness of modern feline vaccines above.

Brief description of the invention

The present invention relates to a recombinant virus that contains at least one foreign nucleic acid, a built-in non-primary region of the viral genome, where each such alien nucleic acid encodes a protein.

This encoded protein is selected from the group consisting of protein feline CD28 or immunogenic parts of the protein of feline CD80 or immunogenic part, protein feline CD86 or immunogenic portions or protein of feline CTLA-4 or immunogenic parts. This part can be expressed in the introduction of this recombinant virus in an appropriate host.

The present invention also relates to a recombinant virus, which, in addition, contains alien nucleic acid encoding the immunogen derived from the pathogen. The present invention also relates to recombinant viruses, which are able to enhance the immune response in cats.

The present invention also relates to recombinant viruses are able to suppress immune response unosek. Brief description of drawings

Figure 1A: DNA and amino acid sequence of feline CD80 (B7-1) (TAMU)(SEQ ID NO. 1 and 2).

Figure 1B: the Curve of the hydrophobicity of the amino acid sequence of feline CD80 (B7-1) (TAMU).

Figure 2A: DNA and inability sequence of feline CD80 (b7-1) (SYNTRO) (SEQ ID NO. 3 and 4).

Figure 2B: the Curve of the hydrophobicity of the amino acid sequence of feline CD80 (B7-1) (SYNTRO).

Figure 3A: DNA and amino acid sequence of feline CD86 (B7-2)(SEQ ID NO. 5 and 6).

Figure 3B: the Curve of the hydrophobicity of the amino acid sequence of feline CD86 (B7-2).

Figure 4A: DNA and amino acid sequence of feline CD28 (SEQ ID NO. 7 and 8).

Figure 4B: the Curve of the hydrophobicity of the amino acid sequence of feline CD28.

Figure 5A: DNA and amino acid sequence of feline CTLA-4 (CD152)(SEQ ID NO. 9 and 10).

Figure 5B: the Curve of the hydrophobicity of the amino acid sequence of feline CTLA-4 (CD152). Detailed description of the invention

The present invention relates to a recombinant virus that contains at least one foreign nucleic acid, a built-in non-primary region of the viral genome, where each such alien nucleic acid (a) encodes a protein selected from the group consisting of protein feline CD28 or immunogenic parts of the protein of feline CD80 or immunogenic part, protein feline CD86 or immunogenic parts; protein feline CTLA-4 or immunogenic part; and (B) is able to be expressed in the introduction of this recombinant virus in an appropriate host.

In one embodiment, the above-described invention of the decree is hydrated recombinant virus contains, at least two foreign nucleic acids, each of which is embedded in the non-primary region of the viral genome.

In another embodiment of the invention indicated recombinant virus contains at least three alien nucleic acids, each of which is embedded in the non-primary region of the viral genome.

In another embodiment of the invention indicated recombinant virus contains four alien nucleic acids, each of which is embedded in the non-primary region of the viral genome.

In another embodiment of the invention, the specified recombinant virus is, but not limited to, the smallpox virus raccoons, smallpox pigs or herpes virus cats.

In another embodiment, the above-described invention indicated recombinant virus contains more than one foreign nucleic acid, and each of these foreign nucleic acid is integrated into the same non-main area. In another embodiment, the present invention any of the above recombinant virus contains more than one foreign nucleic acid, where all of these foreign nucleic acids are not built in the same non-core region.

In one of the embodiments of the invention any of the above recombinant is Urusov contains alien nucleic acid, encoding the immunogen derived from the pathogen. In yet another embodiment of the invention the recombinant virus that encodes a feline pathogen, rubivirus the pathogen, the pathogen originating from chlamydia, pathogen Toxoplasmosis gondii, pathogen Dirofilaria immitis, the pathogen originating from fleas, or bacterial pathogen. In another embodiment of the invention the recombinant virus contains the coding sequence of human immunodeficiency virus in cats (FIV), leukosis virus cats (FeLV), virus infectious peritonitis in cats (FIP)virus, panleukopenia in cats, feline kalitsivirusa, cat reovirus type 3, feline rotavirus, feline coronavirus, syncytial virus cats sarcoma virus in cats, herpes virus in cats, virus diseases Bourne cats or feline parasites.

In another embodiment of the invention indicated recombinant virus contains at least one foreign nucleic acid which comprises a promoter for expression of foreign nucleic acid. In another embodiment of the invention indicated recombinant virus expresses at least one foreign nucleic acid, which is under the control of a promoter that is endogenous to the virus.

In one of the embodiments of the invention any of the above recombinant Viru is s contains alien nucleic acid, encoding the detected marker. In another embodiment, the invention specified detektivami marker is beta-galactosidase of E. coli.

The present invention also relates to a recombinant virus, codereuse immunogen originating from dad-protease FIV, FIV envelope protein, DBP-protease FeLV or FeLV envelope protein.

The present invention relates to a recombinant virus, which, in addition, contains a nucleic acid comprising a gene of human immunodeficiency virus in cats or part of it. The present invention relates to a recombinant virus, which, in addition, contains a nucleic acid, comprising the genome of the virus leukemia cats or part of it. The present invention relates to a recombinant virus, which, in addition, contains a nucleic acid encoding a feline IL12, GM-CSF, R35 or R40. The present invention also relates to a vaccine comprising an effective immunizing amount of such recombinant virus and the corresponding carrier.

The present invention relates to recombinant herpes virus cats containing non-main area, which is a gene glycoprotein G of herpes virus cats.

The present invention relates to recombinant herpes virus of cats on p.12 of the formula of the invention, designated S-FHV-031 (ATSS, the access number VR-2604). This virus was anirban may 1, 1998 in the American type culture collection (ATSS), 10801 University Boulevard, Manassas, VA 20108-0971, U.S.A. under the Budapest Treaty under the International agreement for the Deposit of microorganisms for the purpose of patent procedure.

The present invention relates to a recombinant virus of smallpox pigs with non-main area in the larger HindIII-BglII-subfragment HindIII-fragment M smallpox pigs. The present invention relates to a recombinant virus of smallpox pigs at 14 claims, designated S-SPV-246 (ATSS, the access number VR-2603). This virus was deposited on 1 may 1998 in the American type culture collection (ATSS), 10801 University Boulevard, Manassas, VA 20108, U.S.A. under the Budapest Treaty under the International agreement for the Deposit of microorganisms for the purpose of patent procedure.

In one embodiment, implementation of the above-described invention relates to the recombinant virus, where a portion of the protein CD28, CD80 or CD86 is soluble part of the protein. In another embodiment, its implementation of the present invention relates to a recombinant virus that contains alien nucleic acid encoding a protein, CTLA-4 cats.

The above invention relates to a vaccine which comprises an effective immunizing amount of the recombinant virus and a suitable carrier. In one of the embodiments in the present invention, the vaccine contains an effective immunizing amount of the recombinant virus, comprising from about 1×105boe/ml to about 1×108boe/ml In another embodiment of the present invention the vaccine, in addition, includes a mixture containing recombinant virus and an effective immunizing amount of the second immunogen.

The present invention relates to a method of enhancing an immune response in cats, introducing these cats an effective immunizing amount of any visionartificial recombinant viruses. The present invention also relates to a method of immunization of cats, introducing these cats an effective immunizing amount of any visionartificial recombinant viruses.

The present invention relates to a method of suppression (suppress) the immune response in cats, introducing these cats effective suppressive amount of a recombinant virus containing soluble CD28, CD80 or CD86. The present invention relates to a method of suppressing the immune response in cats by introducing these cats any effective suppressive amount of a recombinant virus containing protein feline CTLA-4.

The present invention relates to intravenous, subcutaneous, intramuscular, crismachisinau, local, oral or intraperitoneal introduction wicheap the sledge recombinant virus.

In one variation of its implementation of the present invention relates to a method of suppressing the immune response in cats, where this cat is a recipient of transplanted organ or tissue, or subject an immune response.

In another embodiment, its implementation of the present invention relates to a method of suppressing the immune response in cats, introducing these cats antisense nucleic acid capable of gibridizatsiya: (a) mRNA-transcript feline CD28, (b) transcript of feline CD80 or (C) mRNA-transcript feline CD86, or to inhibit their translation where the specified antisense nucleic acid is present in an amount effective for inhibiting translation, and thereby suppresses the immune response in cats.

In one embodiment, implementation of the above-described invention relates to a method of reducing or destroying tumors in cats, introducing into the tumor of this cat recombinant virus containing a nucleic acid which encodes a protein of feline CD80 protein feline CD86 or their combination in a quantity effective to reduce and destroy the tumor.

In one variation of its implementation of the present invention relates to a method of reducing or destroying tumors in cats, where specified, it is recommended pinentry virus in addition, contains and is able to Express the tumor-associated antigen cats, and where the specified process is carried out by the system of introducing this recombinant virus.

The present invention relates to isolated and purified DNA encoding the ligand feline CD80 (B7-1) or ligand feline CD86 (B7-2), or receptor CD28 cats or receptor, CTLA-4 cats (CD152), as well as cloning and expressing vectors containing CD80 or CD86, either CD28 or CTLA-4, or RNA, partially or entirely, and to cells transformed by CD80-coding vectors or CD86-encoding vectors, or CD28-coding vectors or CTLA-4-encoding vectors. Animals of the cat family, from which there is CD80, CD86, CD28 or CTLA-4, which are selected from the group including, but not limited to: domestic cats, lions, Cougars, lynx and cheetahs.

The present invention relates to isolated and purified cDNA feline CD80 (B7-1), containing approximately 941 nucleotide. The present invention also relates to isolated and purified polypeptide of feline CD80, containing approximately 292 amino acids in native membraneassociated or Mature form, and having a molecular weight of about 33485 kDa, isoelectric point of about 9.1, and the total charge at pH 7.0 to 10. Co-expression of CD80, together with co-stimulating molecule CD28 and tumor antigen and the and antigen from a pathogenic organism, contributes to activation or increased activation of T-lymphocytes, which leads to the production of a cytokine that stimulates the immune response and helps regulate the growth of other types of cells. Co-expression of CD80, together with co-stimulatory molecule, CTLA-4, helps regulate the activation of T-lymphocytes.

The present invention relates to isolated and purified cDNA feline CD86 (B7-2), containing approximately 1176 nucleotides. The present invention also relates to isolated and purified polypeptide feline CD86, containing approximately 320 amino acids in native membraneassociated or Mature form, and having a molecular weight of approximately 36394 kDa, isoelectric point of about 9,19, and the total charge at pH 7.0, equal 11,27. Co-expression of CD86, together with co-stimulatory molecule CD28 and tumor antigen or an antigen from a pathogenic microorganism, contributes to activation or increased activation of T-lymphocytes, which leads to production of cytokines that stimulate the immune response and helps regulate the growth of other types of cells. Co-expression of CD86, together with co-stimulating molecule and CTLA-4, helps regulate the activation of T-lymphocytes.

In accordance with the present invention feline CD80 or feline CD86 derived from natural or recombinant sources. In accordance with the crust is ashim the invention of the feline CD80 or CD86 are native or membrane-bound form or secreted form, lacking the transmembrane domain.

The present invention relates to isolated and purified cDNA feline CD28, containing about 689 nucleotides. The present invention also relates to isolated and purified polypeptide feline CD28, containing approximately 221 amino acid in the native membrane-associated or Mature form, and having a molecular weight of about 25319 kDa, isoelectric point of about 9,17 and the total charge at pH 7.0, equal 9,58.

The present invention relates to isolated and purified cDNA feline CTLA-4, containing about 749 nucleotides. The present invention also relates to isolated and purified polypeptides feline CTLA-4, containing approximately 223 amino acids in native membraneassociated or Mature form, and having a molecular weight of about 24381 kDa, isoelectric point of about 6,34, and the total charge at pH 7.0, equal -0,99.

The present invention relates to a recombinant virus of smallpox pigs expressing foreign DNA where a specified foreign DNA encodes the cDNA and polypeptide feline CD80, feline CD86, feline CD28 and feline CTLA-4.

The present invention relates to a recombinant virus of smallpox raccoons expressing foreign DNA where a specified foreign DNA encodes the cDNA and polypeptide feline CD80, feline CD86, feline CD28 and Kosha is LEGO CTLA-4.

The present invention relates to recombinant herpes virus cats expressing foreign DNA where a specified foreign DNA encodes the cDNA and polypeptide feline CD80, feline CD86, feline CD28 and feline CTLA-4.

In another aspect the present invention relates to a method of enhancing an immune response in cats to the immunogen, which is achieved by the introduction of the immunogen before, after, or in General, simultaneously with the introduction of the feline CD80 or feline CD86 with feline CD28 or feline CTLA-4, or without them in the recombinant vector-based smallpox pigs, recombinant vector-based smallpox raccoons, or in recombinant vectors based on herpes virus cats, in an amount effective to enhance the immune response.

In another aspect the present invention relates to a method of suppressing an immune response in cats to the immunogen, which is achieved by the introduction of the immunogen before, after, or in General, simultaneously with the introduction of the feline CD80 or feline CD86 with feline CD28 or feline CTLA-4, or without them, or with antisense RNA or DNA, partially or wholly encoding feline CD80, feline CD86, feline CD28 or feline CTLA-4, recombinant vector-based smallpox pigs, recombinant vector-based smallpox raccoons or recombinant vecto the e on the basis of the herpes virus cats, in amounts effective to suppress the immune response.

In another aspect the present invention relates to a vaccine for inducing an immune response in cats to the immunogen containing the immunogen and an effective amount of feline CD80 in the recombinant vector-based smallpox pigs, recombinant vector-based smallpox raccoons or recombinant vectors based on herpes virus cats, to enhance the immune response. This immunogen is, for example, from feline pathogens such as human immunodeficiency virus in cats, the virus leukemia cats, parvovirus, cats, coronavirus cats, leptodirus cats, etc.

In another aspect the present invention relates to a vaccine for inducing an immune response in cats to the immunogen, which is achieved by introducing a recombinant vector virus-based virus swine recombinant vector-based smallpox raccoons or recombinant vectors based on herpes virus cats expressing the DNA or RNA of the immunogen and the DNA or RNA of additional molecules of feline CD80, CD86, CD28 in any combination, encoding the protein or protein fragment in a quantity effective to modulate an immune response.

Protein of feline CD80 has an amino acid sequence that is at 59% and 46% identical to the protein sequences of human and mouse on the, respectively. Protein feline CD86 has an amino acid sequence that is at 68% and 64% identical to the protein sequences of human and rabbit, respectively. Protein feline CD28 has an amino acid sequence that is at 82% and 74% identical to the protein sequences of human and mouse, respectively. Proteins of feline CTLA-4 have an amino acid sequence that is at 88% and 78% identical to the protein sequences of human and mouse, respectively. Human or murine proteins CD80 or CD86 cannot functionally replace proteins of feline CD80 or CD86. Therefore feline CD80, feline CD86, feline CD28 and feline CTLA-4 are new reagents required for the regulation of immunity in cats.

The present invention relates to additional T-cell regulatory molecules, CD80 (B7-1) or CD86 (B7-2) or CD28 or CTLA-4 (CD152), derived from animals of the cat family. The present invention relates to isolated and purified nucleic acids encoding all or part of feline CD80, feline CD86, feline CD28 or feline CTLA-4, as well as polypeptides CD80, CD86, CD28 or CTLA-4, selected either from native or from recombinant sources. Feline CD80, CD86, CD28 or CTLA-4, produced in accordance with the present invention, can be used to enhance the effectiveness of feline vaccines against the tumors and pathogens and also as a therapeutic agent for the treatment of viral and bacterial diseases of cats. Feline CD80, CD86, CD28 or CTLA-4, produced in accordance with the present invention can also be used to mitigate the disease, due to the high level, an overactive or improperly directed immune response.

Nucleic acids, vectors, transformants

Sequence of cDNA encoding feline CD80 (SEQ ID NO: 1, 3) feline CD86 (SEQ ID NO: 5), feline CD28 (SEQ ID NO: 7) or feline CTLA-4 (SEQ ID NO: 9) is shown in figure 1-5, and the predicted amino acid sequence of feline CD80 (SEQ ID NO: 2, 4) feline CD86 (SEQ ID NO: 6), feline CD28 (SEQ ID NO: 8) or feline CTLA-4 (SEQ ID NO: 10) is shown in figure 1-5. The design of these cat-like polypeptides, such as CD80, CD86, CD28 or CTLA-4 is based on the partial amino acid sequence homology with human, mouse or rabbit homologs of these polypeptides, and the ability of polypeptides CD80 or CD86 contact receptor feline CD28 (see below) or CTLA-4 and activate or stimulate or regulate the activation of T-lymphocytes in some other way. In addition, without pretending to any particular theory, it is possible to assume that the polypeptides of feline CD80 or feline CD86 also possess one or more biological activities: activation of killer cells (n is radnih killers), the stimulation of the maturation of b-cells, activation of the MTL-restrictively cytotoxic T-lymphocytes, proliferation of fat cells, interaction with receptors of cytokines and induced cytokines that regulate the immune response.

Due to the degeneracy of the genetic code (i.e., when multiple codons encode certain amino acids), DNA sequence different from the sequence shown in figure 1-5, can also encode the amino acid sequence of feline CD80, CD86, CD28 or CTLA-4, shown in Fig.1-5. Such other DNA are sequences containing conservative in respect of sequence variants, in which the modification of one or more nucleotides in a given codon does not change the amino acid encoded at that position. In addition, this amino acid residue in the polypeptide can often be replaced without changing the whole conformation and function of the native polypeptide. Such "functionally conservative variants include, but are not limited to, replacement of an amino acid by another amino acid having similar physico-chemical properties, such as, for example, acidic, basic, hydrophobic, hydrophilic, aromatic properties and the like(for example, substitution of lysine for arginine, aspartate to glutamate or glycine to alanine). In addition to the CSO, amino acid sequences can be added or deleterow without disrupting the biological activity of the molecule. So, for example, amino - or carboxy-ends can be added additional amino acid sequences, which serve as labels for cleaning, such as his-tag label (i.e., to provide one-step protein purification, after which they are removed by chemical or enzymatic method). Alternatively, these additional sequences provide additional binding sites on cell surfaces, or in any other way alter the specificity of feline CD80, CD86, CD28 or CTLA-4 relative to the target cell, so that the result is added antigennegative site for antibodies.

cDNA feline CD80, feline CD86, feline CD28 or feline CTLA-4, included in the scope of the present invention, represent the sequence shown in figure 1-5; conservative in respect to the sequence of variant DNA; DNA sequences encoding functionally conservative variant polypeptides; and combinations thereof. The present invention relates to fragments of feline CD80, CD86, CD28 and CTLA-4, which, taken separately or in combination with other sequences or components that have the necessary degree of biological activity. As bodø is explained below, each specialist can implement a predictable manipulation of sequences of CD80, CD86, CD28 or CTLA-4, and to establish whether this variant of feline CD80, CD86, CD28 or CTLA-4 corresponding stability and biological activity for a given application or set, lead changes, which affect the activity of the binding of these molecules to increased efficiency. Each of feline CD80 and CD86 associated with co-CD28 receptor or co-receptor CTLA-4. This can be achieved by expression and purification of variant polypeptides CD80, CD86, CD28 or CTLA-4 in a recombinant system through an analysis of their T-cell stimulatory activity and/or stimulating the growth of activity in cell culture and in animals with subsequent testing for suitability for this application. Option CD80 tested for biological activity by functional binding to receptors CD28 or CTLA-4. Option CD86 are tested for biological activity by functional binding to receptors CD28 or CTLA-4. Similarly option CD28 or CTLA-4 are tested for biological activity.

The present invention also relates to DNA of feline CD80, CD86, CD28 or CTLA-4 (and polypeptides), derived from animals of the family cats, such as, but not ogranichivayas them, domestic cats, lions, tigers, cheetahs, Rys is etc. Homologues of the sequences of feline CD80, CD86, CD28 or CTLA-4, shown in figure 1-5, were easily identifitsirovany by screening cDNA or genomic libraries to identify clones that hybridize with probes containing all or part of the sequence shown in Fig.1-5. Alternatively, a library of expressed sequence sceneroot using antibodies that recognize feline CD80, CD86, CD28 or CTLA-4. Without pretending to a particular theory, it is possible to assume that the genes CD80 or CD86 from other felids have homology with genes of feline CD80, CD86, CD28 or CTLA-4, at least about 70%. In the scope of the present invention also includes DNA that encode a homologue of CD80, CD86, CD28 or CTLA-4, defined as DNA encoding polypeptides having amino acid identity with the feline CD80, CD86, CD28 or CTLA-4, at least about 25%.

Mainly for manipulation of nucleic acids in accordance with the present invention by techniques well known in the art, such as described, for example, in Molecular Cloning: A Laboratory manual, (2nd Ed., Sambrook Fritsch &Maniatis, Cold Spring Harbor Laboratory, Cold Spring Harbor), or Current Protocols in Molecular Biology, (Eds. Ausubel, Brent, Kingston, More, Feidman, Smith & Stuhl, Greene Publ. Assoc. Wiley Interscience, NY, NY, 1992).

The present invention relates to cDNA and RNA sequences, and semantic and antimuslim on what sledovatelnot.

The present invention also relates to the genomic DNA sequences of feline CD80, CD86, CD28 or CTLA-4 and flanking sequences, including regulatory sequences, but not limited to. Nucleic acid sequences encoding the polypeptide(s) of feline CD80, CD86, CD28 or CTLA-4, also associated with heterologous sequences, including promoters, enhancers, elements of the immune response, signal sequences, polyadenylation sequence, introns, 5’- and 3’-noncoding region and other Elements of the regulation of transcription, which is functionally attached to the cDNA sequence(s) of feline CD80, CD86, CD28 or CTLA-4, are, but are not limited to, sequences that can regulate the expression of genes derived from prokaryotic cells, eukaryotic cells, viruses, prokaryotic cells, viruses, eukaryotic cells, and any combinations thereof. Specialists and other known suitable heterologous regulatory sequence.

Nucleic acids of the present invention have been modified by methods known in the art, in order to change their stability, solubility, affinity and binding specificity. For example, these sequences were selectively methylated. Sequence NUS is einwich acids of the present invention were also modified the label, capable of directly or indirectly, to produce the detected signal. Examples of labels include radioisotopes, fluorescent molecules, Biotin, etc.

The present invention also relates to vectors that include nucleic acids, partially or fully, encode polypeptides CD80, CD86, CD28 or CTLA-4. Such vectors are, for example, plasmid vectors for expression in a variety of eukaryotic and prokaryotic hosts. These vectors preferably contain a promoter functionally attached to the part that encodes the polypeptide of feline CD80, CD86, CD28 or CTLA-4. The encoded polypeptides feline CD80, CD86, CD28 or CTLA-4 were expressed using any suitable vectors and host cells described in this application or known to specialists from other sources.

The present invention also relates to vectors which include nucleic acids encoding polypeptide feline CD80, CD86, CD28 or CTLA-4 partially or completely. Such vectors are, for example, vectors based on a live virus for expression in a variety of eukaryotic hosts or for the expression of DNA or RNA vaccines. In one of the embodiments of the invention this vector on the basis of the live virus is attenuated (weakened). In another embodiment of the present invention, this ve the tor on the basis of the live virus was attenuated by deletion of genes. In another embodiment of the present invention, this viral vector was inactivated by chemical treatment or heat treatment. This vector is obtained on the basis of the live virus selected from the group including, but not limited to, the herpes virus, poxvirus, adenovirus, adeno-associated virus, retrovirus, baculovirus, alphavirus, rhabdovirus, picornavirus. This vector is obtained on the basis of the live virus selected from the group including, but not limited to, feline herpes virus, canine herpes virus, avian herpes virus, bovine herpes virus, equine herpes virus, pseudorabies, the smallpox virus of pigs, the smallpox virus of birds, the smallpox virus in poultry, the virus of smallpox raccoons, smallpox Canaries, vaccinia virus, the virus murine leukemia, Malone virus Sindbis and virus Semliki forest.

Vector based on a live virus is a recombinant viral vector expressing foreign DNA, which is part or all cDNA feline CD80, CD86, CD28 or CTLA-4. Foreign DNA is cDNA for an antigen from a pathogenic organism. Recombinant viral vector design methods homologous recombinant or kosmidou reverse engineering, well-known specialists. Preferably, these vectors also include a promoter functionally attached to part of the coding for the soup polypeptide feline CD80, CD86, CD28 or CTLA-4. This promoter is chosen from the group including, but not limited to, the promoter of herpes virus de cats, sinteticheskii early/late promoter poxvirus, pretani the promoter of the human cytomegalovirus, the promoter of pseudoreligious HH. Stimulation of gene expression also provides for the expression of cDNA CD80, CD86, CD28 or CTLA-4, derived from the element internal binding site with the ribosome (IRES)present in the cluster (vector pCITE, Novagen, Madison, WI). Cell lines for culturing viral vectors include, but are not limited to, cells of the cat's kidneys Crandell (CRFK), chicken embryo fibroblasts, kidney cells porcine embryo (ESK-4), cells porcine kidney (RK). Coded(e) the polypeptide(s) of feline CD80, CD86, CD28 or CTLA-4 expressed using suitable vectors and host cells described in this application or known to specialists from other sources.

In a preferred embodiment of the invention, genes encoding feline CD80 and CD28, CD80 and CTLA-4, CD86 and CD28 or CD86 and CTLA-4 in combination with genes for immunogen, derived from a feline pathogen is introduced into one recombinant viral vector, and then on the basis of this vector receive a live vaccine. Genes of feline CD80, CD86, CD28 or CTLA-4, alone or in combination with genes cats, derived from a feline pathogen is injected into recombi is based virus so that the expression of these genes was regulated by the relevant promoter. In another embodiment of the invention, genes encoding feline CD80, CD86, CD28 or CTLA-4, separately or in combination, is inserted into the recombinant viral vector and introduced into a vaccine together with a second recombinant viral vector that encodes the genes for the immunogen(s)occurring(substance) from feline pathogens. These two ways of carrying out the invention allows to produce the desired immune responses in the same cells or in cells that are in close proximity to these cells, which results in enhancement, suppression or reorientation of the desired immune response.

Immunogen selected from the group including, but not limited to, feline pathogens such as human immunodeficiency virus in cats, the virus leukemia cats, the virus infectious peritonitis in cats, the virus feline panleukopenia in cats (parvovirus), feline caliciviruses, cat reovirus type 3, feline rotavirus, feline coronavirus (infectious peritonitis virus), rubivirus, syncytial virus cats sarcoma virus in cats, herpes virus cats (rhinotracheitis virus), a virus disease Bourne cats, chlamydia, Toxoplasmosis gondii, feline parasite, Dirofilaria iimnitis, fleas, bacterial pathogens, etc.

Vectors or vectors based on "live" viruses often include one or bore is only of replication systems for cloning or expression, one or more markers for selection in the host, such as, for example, a token resistance to the antibiotic, or calorimetric markers, such as β-galactosidase (lacZ) or β-glucuronidase (uidA), or fluorescent markers, such as protein, fluorescent in the green range of the spectrum, and one or more gene-expression clusters. Built-coding sequences are synthesized, isolated from natural sources and get back in hybrids or other Ligation of coding sequences with sequences regulating transcription carried out by methods known in the art. Appropriate cell hosts transform/transferout/infect any suitable method including electroporation, the uptake of DNA-mediated l2or liposome, infection by fungi, microinjection, delivery of microparticles by bombing or similar

Suitable vectors designed for use in the present invention include, but are not limited to, YEp352, pcDNAI (Invitrogen, Carlsbad, CA), pRc/CMV (Invitrogen) and pSFVl (GIBCO/BRL, Gaitheersburg, MD). One of the preferred vectors for use in the present invention, is pSFVl. Appropriate cell hosts are E. coli, yeast cells, COS cells RC, cells SNO, GH4C1 cells, cells KSS-21 and the cells of the amphibian melanophores. PlaysForSure in the present invention the preferred cell line host cells are KSS-21. Vectors suitable for the design of the "naked" DNA or gene vaccination vaccines are, but are not limited to, pTarget (Promega, Madison, WI), pSI (Promega, Madison, WI) and pcDNA (Invitrogen, Carlsbad, CA).

Nucleic acid encoding the polypeptide(s) of feline CD80, CD86, CD28 or CTLA-4, were also introduced into the cells using recombinant techniques. For example, such a sequence can be introduced by microinjection into the cell with the implementation of homologous recombination at the site of the endogenous gene encoding the polypeptide, its analogue or pseudogene, or a sequence with substantial identity to the gene, codereuse polypeptide feline CD80, CD86, CD28 or CTLA-4. Can also be used and other methods based on recombination, such as non-homologous recombination and deletion of the endogenous gene by homologous recombination, and in particular, poly potent cells.

The present invention relates to a method of enhancing an immune response in cats to the immunogen, which is achieved by the introduction of the immunogen before introduction after introduction or, basically, simultaneously with the introduction of the feline CD80 or CD86 with feline CD28 or feline CTLA-4, or without them, in a quantity effective to enhance the immune response.

The present invention relates to a method of enhancing an immune response in the cat on the immunogen that d is attained by the introduction of expressing vector, containing the immunogen, derived from a feline pathogen, and additional molecules feline CD80 or CD86 with feline CD28 or feline CTLA-4, or without them, in a quantity effective to enhance the immune response.

The present invention relates to a method of reorientation of the immune response in cats to the immunogen, which is achieved by introducing expressing vector containing the immunogen, derived from a feline pathogen, and additional molecules feline CD80 or CD86 with feline CD28 or feline CTLA-4, or without them, in a quantity effective to enhance the immune response.

The present invention relates to a method of suppressing the immune response in the cat on the immunogen, which is achieved by the introduction of the immunogen before introduction after introduction or, basically, simultaneously with the introduction of the feline CD80 or CD86, together with a feline CD28 or feline CTLA-4, or without them, or with antisense RNA or DNA encoding feline CD80, feline CD86, feline CD28 or feline CTLA-4 in an amount effective for suppressing the immune response.

The present invention relates to a vaccine for inducing an immune response in cats to the immunogen containing the immunogen and an effective amount of feline CD80 or feline CD86 with feline CD28 or feline CTLA-4, or without them, to enhance the immune response or efficiency is th number of feline CD80 or feline CD86 with feline CTLA-4 for suppressing the immune response. In another embodiment, its implementation of the present invention relates to a vaccine comprising expressing a vector containing genes encoding the immunogen(s) for feline pathogens and genes encoding CD80, CD86, together with a feline CD28 or feline CTLA-4, or without them for enhancing or suppressing the immune response.

The polypeptides of feline CD80, CD86, CD28 or CTLA-4

The gene of feline CD80 (DNA and amino acid sequence of which is shown in figure 1 and 2) encodes a polypeptide of approximately 292 amino acids. Gene feline CD86 (DNA and amino acid sequence of which is shown in figure 3) encodes a polypeptide of approximately 320 amino acids. Gene feline CD28 (DNA and amino acid sequence of which is shown in figure 4) encodes a polypeptide of approximately 221 amino acid. Gene feline CTLA-4 (DNA and amino acid sequence of which is shown in figure 3) encodes a polypeptide of approximately 223 amino acids.

Purification of feline CD80, CD86, CD28 or CTLA-4 from natural or recombinant sources provide well-known methods, such as, but not limited to, ion exchange chromatography, reverse-phase chromatography on columns with C4, gel filtration, isoelectric focusing, affinity chromatography, etc. In a preferred embodiment of the invention, large quantities of the biologically active feline CD80, CD86, CD28 or CTLA-4 is produced by constructing a recombinant DNA sequence containing a region encoding feline CD80, CD86, CD28 or CTLA-4, legirovannye with preservation of the reading frame sequence that encodes a 6-terminal his-tag residues in the replicon pSFVl (GIBCO/BRL). mRNA encoded by this plasmid, synthesized using techniques well known in the art, and is introduced into cells KSS-21 by electroporation. These cells synthesize and secrete Mature glycosylated polypeptides feline CD80, CD86, CD28 or CTLA-4 with the 6 C-terminal his-tag residues. Modified polypeptides of feline CD80, CD86, CD28 or CTLA-4 purified from the cell supernatant using affinity chromatography with the use of histidine-binding polymer (His-bind, Novagen, Madison, WI).

Polypeptides feline CD80 or feline CD86, isolated from any source, modify known methods. So, for example, feline CD80, CD86, CD28 or CTLA-4 can be phosphorylated or the dephosphorylated, glycosylated or deglycosylated etc. Particularly effective are those modifications that lead to changes in solubility, stability and specificity and affinity of binding of feline CD80, CD86, CD28 or CTLA-4.

The chimeric molecules of feline CD80, CD86, CD28, CTLA-4

The present invention relates to the production of chem the situations molecules, obtained from fragments of feline CD80, CD86, CD28 and CTLA-4 in any combination. So, for example, to increase affinity binding to CD28, while maintaining enhanced immune response instead of CD28 binding site enter the site of binding of CTLA-4.

In one of the embodiments of the invention binding sites for CD80 or CD86 on CTLA-4 and CD28 replace so that the area of binding to CD28 was replaced with the binding of CTLA-4. The effect of the chimeric molecule CD28 with binding to CTLA-4 should increase the affinity of CD28 in relation to CD80 or CD86 and increase the degree of amplification of the immune response.

In an alternative embodiment of the invention the chimeric molecules CD80 and CD28 or CD86 and CD28 or fragments thereof are membrane-bound and increase the ability of these molecules to strengthen immunity. In an alternative embodiment of the invention the chimeric molecules CD80 and CTLA-4 or CD86 and CTLA-4 or fragments thereof are membrane-bound and increase the ability of these molecules to immune suppression. In an alternative embodiment of the invention the chimeric molecules CD80 and CTLA-4 or CD86 and CTLA-4 or fragments thereof are membrane-bound and contribute to the reorientation of the immune response to achieve the desired effect.

In an alternative embodiment of the invention feline CD80, CD86, CD28 or CTLA-4 is a guy the native protein with another polypeptide. This polypeptide is, but not limited to, immunoglobulin, antigen, tumor antigen, a cell surface receptor or ligand on the cell surface.

Antibodies against feline CD80, CD86, CD28 or CTLA-4

The present invention relates to antibodies that are specific for polypeptides of feline CD80, CD86, CD28 or CTLA-4, identified as described above. These antibodies are polyclonal or monoclonal and recognize feline CD80, CD86, CD28 or CTLA-4 different types, identify functional domains and other Such antibodies are usually produced using the methods and compositions described by Harlow &Lane, Antibodies. A Laboratory Manual, Cold Spring Harbor Laboratory, 1988, and with the help of immunological and hybridoma technology, known in the art. If for inducing an immune response specific for feline CD80, CD86, CD28 or CTLA-4, are peptides derived from natural and synthetic feline CD80, CD86, CD28 or CTLA-4, these peptides usually attached to a suitable carrier, such as KLH and injected into a suitable adjuvant, such as beta-blockers. The selected peptides, preferably, attached to the carrier with the lysine core methods, mainly described Tan (1988) Proc.Natl. Acad. Sci., USA, 85: 5409-5413. The obtained antibodies, and in particular antiidiotypic antibodies carrying "vnutrenniy of the times", can also be obtained by the known methods.

In one of the embodiments of the invention purified feline CD80, CD86, CD28 or CTLA-4 are used for immunization of mice, after which the spleen of these mice was removed and the splenocytes are used to generate cell hybrids with myeloma cells to obtain clones of antibody-secreting cells by methods commonly used by professionals. The obtained monoclonal antibodies secreted by the specified cells, sceneroot using in vitro assays for the following activity: binding of feline CD80, CD86, CD28 or CTLA-4, inhibition receptornegative activity CD80, CD86, CD28 or CTLA-4, and inhibition of T-cell-stimulating activity CD80, CD86, CD28 or CTLA-4.

Antibodies against feline CD80, feline CD86, feline CD28 or feline CTLA-4 is used for identification and quantification of feline CD80, CD86, CD28 or CTLA-4 using immunoassays such as ELISA, RIA, etc. Antibodies against feline CD80, feline CD86, feline CD28 or feline CTLA-4 is also used for immunological depletion extracts feline CD80, feline CD86, feline CD28 or feline CTLA-4. In addition, these antibodies can be used for identification, isolation and purification of feline CD80, CD86, CD28 or CTLA-4 from various sources and to conduct research on their OCCS is emochnoy and histochemical localization.

The application

Ligand feline CD80 (B7-1), the ligand feline CD86 (B7-2), the receptor for feline CD28 or receptor feline CTLA-4 (CD152), produced in accordance with the present invention, can be preferably used as vaccines to prevent infectious disease or to stimulate growth in homologous or heterologous species of cat. So, for example, can be carried out coexpressed CD80 or CD86 co-stimulatory molecules CD28 or CTLA-4 in any combination, and a tumor antigen or an antigen from a pathogenic microorganism. Co-expression of feline CD80 or CD86 with receptor feline CTLA-4 contributes to the inhibition of activation of T-lymphocytes and suppression of the immune response. A concrete example may serve as co-expression of CD80 or CD86 with immunogenum, derived from FIV, FeLV or FIP, viral vector or DNA-expressing vector which, when introduced them as vaccines, should activate, enhance or regulate the proliferation of CD4+- and CD8+-T-lymphocytes and to induce cytokines that regulate the immune response, such as IL-2, IFN-g, IL-12, TNFa, IL-6, etc. Another specific example coexpressed CD80, CD86, CD28 or CTLA-4 in a viral vector or DNA-expressing vector which, when introduced as a therapeutic agent should be adjusted or preoriented the SQL immune response.

Strengthening the immune system through interaction feline CD80 or CD86 with CD28 or CTLA-4 or inhibition of the immune response through interaction feline CD80 or CD86 with CTLA-4 provides the advantage that it uses a natural method of regulation, and not adding foreign substances, which can even have multiple negative effects on the health of the animal throughout its life or for an extended period of time. Molecules CD80, CD86, CD28 or CTLA-4 are administered together with other recombinant molecules, such that encode the antigen required to induce immunity. The gene of feline CD80, CD86, CD28 and/or CTLA-4 built in expressing vector or infect or transferout in the target cell, and the target cell is expressed gene product so that it is anchored in the plasma membrane of this target cells or antigen-presenting cells or is secreted from the target cells or antigen-presenting cells. Expressing the vector such as a plasmid, virus, Semliki forest, poxvirus or herpesvirus, carries this gene in the antigen-presenting cell. The gene of feline CD80, CD86, CD28 and/or CTLA-4 or fragments of genes in any combination embed in DNA - or RNA-expressing vector and inyeccion cats, resulting in these cats is expressed gene is the first product in the form of "naked" DNA/RNA or in the form of genetic vaccines. Such co-expression of the immunogen and CD80, CD86, CD28 and/or CTLA-4 in the target cell or in the body of the cat contributes to the activation, increased activation or regulation of T-lymphocytes, b-lymphocytes and other cells. Alternatively, the expressed protein can be introduced after expression in prokaryotic or eukaryotic system, such as a plasmid, virus, Semliki forest, poxvirus or herpes or other viral or bacterial vector. Proteins of feline CD80, CD86, CD28 or CTLA-4 usually operate in anchored in the cell membrane, as additional molecules of the plasma membrane, but they can be represented in other forms, and in particular, without the membrane anchor.

In one of the embodiments of the invention feline CD80 and feline CD86 are soluble, does not contain a transmembrane domain or a hydrophobic region and interact with co-stimulatory molecules CD28 or CTLA-4 or membrane-associated or soluble form. In an alternative embodiment of the invention feline CD80 and feline CD86 are membrane-bound and co-stimulating molecules CD28 or CTLA-4 are present in soluble form and does not contain a transmembrane domain or a hydrophobic region. Soluble CD28 and CTLA-4, preferably in the dimeric form, can be used to treat diseases, tie is the R with T-cell-mediated immunosuppression in cats. Soluble CD28 or CTLA-4 prevents the rejection of transplanted tissue and can be used to treat autoimmune diseases. In particular, soluble CD28 or CTLA-4 can be used to prevent graft-versus-host with bone marrow transplantation. Soluble CD28 or CTLA-4 prevents the binding of cells containing membraneassociated feline CD80 or CD86.

In another embodiment of the present invention feline CTLA-4 hybridizing with immunoglobulin (Ig). Hybrid CTLA-4-IgG can be used to suppress the immune response or to treat autoimmune diseases. Such autoimmune diseases include, but are not limited to, arthritis, psoriasis, rejection of transplanted organ, graft-versus-host.

In one variant of the invention, the proteins of feline CD80 and/or CD86, ExpressionEngine or associated, or in soluble form, can be used to treat, ie, reduction or destruction of tumors in cats. In particular, proteins of feline CD80 and/or CD86 can be expressively of viral vectors or naked DNA by direct injection into the tumor or systemic injections in combination with (or without) feline tumor-associated antigens that are present in the same vector.

Conservative is in terms of consistency and functionally conservative variants of DNA and polypeptides of feline CD80, CD86, CD28 or CTLA-4 or a biologically active fragment or subfragment feline CD80, CD86, CD28 or CTLA-4 are ligated with preservation of the reading frame with another sequence such as the sequence of the cytokine, interleukin, interferon, colony stimulating factor, an antigen from a pathogen, antibodies, or a sequence for purification, such as his-tag tag or reporter gene, such as lacZ E. coli uidA gene, or protein, fluorescent in the green region of the spectrum.

Vaccines

The present invention relates to methods and compositions for enhancing the efficiency of the immune response in the cat. In this embodiment of the invention feline CD80, CD86, CD28 or CTLA-4 is used in combination with the immunogen against which you want to produce an immune response. So, for example, to regulate the intensity and quality of the immune response, it is desirable that the vaccine containing immunogen from pathogens such as human immunodeficiency virus in cats and leukosis virus cats, and other pathogens such as feline parvovirus, feline leptoporus and feline coronavirus, attended feline CD80, CD86, CD28 or CTLA-4. For this feline CD80, CD86, CD28 or CTLA-4, purified from native or recombinant sources, described above, include in the vaccine formulation at a concentration of that component in the range from about 0.01 to 100.0 mg/VA is the CIN on the cat. Alternatively, a recombinant vector expressing feline CD80, CD86, CD28 and/or CTLA-4, and the immunogen from a feline pathogen is injected into the specified vaccine formulation at a concentration of that component in the range from about 0.01 to 100.0 mg/vaccine at a cat, and preferably at a concentration factor of about 0.25 to about 25 mg/kg/day.

Feline CD80, CD86, CD28 or CTLA-4 is administered in combination with a vaccine based on "live" (i.e. that can replicate) virus or not-can replicate the vaccine. Non-limiting examples can replicate vaccines are vaccines containing native or recombinant viruses or bacteria, such as a modified herpes virus cats or modified smallpox raccoons. Neogranichinymi examples of live viral vaccines, which partially replicated or not replicated in the body of the cat owner, but which Express foreign DNA (such as DNA feline CD80, CD86, CD28 or CTLA-4 or immunogen from a feline pathogen) in the cell-master, are modified smallpox virus in poultry, modified smallpox virus swine virus or Semliki forest. Non-limiting examples rereplacenocase vaccines are vaccines that contain "killed" or inactivated viruses or other microorganisms, either crude or purified antigens derived from native and recombinant and synthetic sources, such as, for example, vaccines based on virus leukemia cats.

Commercial sources feline vaccines known to every expert (Compendium of Veterinary Pharmaceuticals, 1997), and for more effective vaccines they can be used in combination with vaccines of the present invention.

The vaccine for the induction and regulation of immune response in the cat in response to the immunogen consists of an immunogen and an effective amount of feline CD80 and feline CD86 with colacem CD28 or feline CTLA-4, or without them, to enhance the immune response, or feline CD80 and feline CD86 with feline CTLA-4, to suppress the immune response.

This immunogen selected from the group including, but not limited to, feline pathogens such as human immunodeficiency virus in cats, the virus leukemia cats, the virus infectious peritonitis in cats, the virus feline panleukopenia in cats (parvovirus), feline caliciviruses, cat reovirus type 3, feline rotavirus, feline coronavirus (infectious peritonitis virus), rubivirus, syncytial virus cats sarcoma virus in cats, herpes virus cats (rhinotracheitis virus), a virus disease Bourne cats, chlamydia, Toxoplasmosis gondii, feline parasite, Dirofilaria immitis, fleas, bacterial pathogens, etc.

Regulation of growth or regulation of the activation of cells of a particular type, such as T-lymphocytes, suggests that p is untranslated answer or stimulates, or inhibits the growth of cells. Regulation of the immune response in cats indicates that this immune response is either stimulated or suppressed by cure the disease or eliminate the infectious agent in cats.

In a preferred variant of the invention, the genes encoding feline CD80 and CD28, CD80 and CTLA-4, CD86 and CD28, or CD86 and CTLA-4 in combination with genes encoding the immunogen from a feline pathogen is introduced into one recombinant viral vector, and then receive a live vaccine. Genes of feline CD80, CD86, CD28 or CTLA-4, taken separately or in combination with the genes of feline immunogen is injected into the recombinant virus so that the expression of these genes was under the control of the appropriate promoter. The introduction of the vaccine leads to expression of biologically active cat ligands CD80 or CD86, and receptors CD28 or CTLA-4, and to the expression of cat immunogen(new) in the same cell, and thereby contributes to the production of primary and secondary co-stimulating signals necessary to gain the desired immune response. In this embodiment, its implementation of the present invention relates to early localized immune response to cat immunogen and vaccine with high efficacy against diseases in cats.

In another embodiment of the present invention, genes encoding feline CD80,CD86, CD28 or CTLA-4, taken separately or in combination, is introduced into a recombinant viral vector, and introducing the vaccine together with a second recombinant viral vector that includes genes cat immunogen(s), and thus contributes to producing the desired response in the same cells or in cells that are in close proximity to these cells, which results in amplification of the desired immune response to obtaining vaccines with high efficacy against diseases in cats.

Below are examples of recombinant viral vectors used for the expression of feline CD80, CD86, CD28 or CTLA-4 and to receive the vaccine in order to produce enhanced protective immune response in response to infection by a pathogen, namely vectors used for:

1. The expression of partial or complete feline CD80, CD86, CD28 or CTLA-4, taken separately or in any combination, recombinant pox virus swine (built-in to any non-primary site of insertion). For vaccination of cats, but not only cats, nerealizirane vaccines used separately or in any combination with another vaccine or therapeutic agent (recombinant, alive or "dead").

2. The expression of partial or complete feline CD80, CD86, CD28 or CTLA-4, taken separately or in any combination, recomb nananom the herpes virus cats (built-in site de FHV or in any non-primary site of insertion). For vaccination of cats, but not only cats that can replicate the vaccines used alone or in combination with another vaccine or therapeutic agent (recombinant, alive or "dead").

3. The expression of partial or complete feline CD80, CD86, CD28 or CTLA-4, taken separately or in any combination, recombinant smallpox raccoons (built-in to any non-primary site of insertion). For vaccination of cats, but not only cats that can replicate the vaccines used alone or in combination with another vaccine or therapeutic agent (recombinant, alive or "dead").

4. The expression of partial or complete feline CD80, CD86, CD28 or CTLA-4, taken separately or in any combination, recombinant pox virus swine, containing genes for dad-protease and/or shell FIV.

5. The expression of partial or complete feline CD80, CD86, CD28 or CTLA-4, taken separately or in any combination, recombinant herpes virus cats containing genes for dad-protease and/or shell FIV.

6. The expression of partial or complete feline CD80, CD86, CD28 or CTLA-4, taken separately or in any combination, recombinant smallpox raccoons, containing genes for dad-protease and/or shell FIV.

7. The expression of partial or complete feline CD80, CD86, CD28 or CTLA-4, taken separately or in any combination, recombinant pox virus swine, containing the m genes for dad-protease and/or shell FeLV.

8. The expression of partial or complete feline CD80, CD86, CD28 or CTLA-4, taken separately or in any combination, recombinant feline herpes virus containing genes for dad-protease and/or shell FeLV.

9. The expression of partial or complete feline CD80, CD86, CD28 or CTLA-4, taken separately or in any combination, recombinant smallpox raccoons, containing genes for dad-protease and/or shell FeLV.

10. The expression of partial or complete feline CD80, CD86, CD28 or CTLA-4, taken separately or in any combination, recombinant pox virus swine, containing genes for dad-protease and/or shell FeLV genes DBP-protease and/or shell FIV or any combination of them.

11. The expression of partial or complete feline CD80, CD86, CD28 or CTLA-4, taken separately or in any combination, recombinant feline herpes virus containing genes for dad-protease and/or shell FeLV genes DBP-protease and/or shell FIV or any combination of them.

12. The expression of partial or complete, feline CD80, CD86, CD28 or CTLA-4, taken separately or in any combination, recombinant smallpox raccoons, containing genes for dad-protease and/or shell FeLV genes DBP-protease and/or shell FIV or any combination of them.

13. Expression, partial or complete feline CD80, CD86, CD28 or CTLA-4, taken separately or in any combination, smallpox pigs, or smallpox raccoons, or is any other expressing the system, including, but not limited to, the E. coli virus Semliki forest or baculovirus, in order to generate the crude or purified polypeptide. For use in the desired purpose, including but not limited to, the production of polyclonal and monoclonal antibodies, and the production of reagents for the development of functional analysis.

14. The expression of partial or complete feline CD80, CD86, CD28 or CTLA-4, taken separately or in any combination, in the vector based on attenuated virus FIV or FeLV. In one of the embodiments of the invention, the vector-based viruses, FIV or FeLV attenuated by deletion of the gene.

15. Expression, partial or complete, feline CD80, CD86, CD28 or CTLA-4, taken separately or in any combination, expressing the vector containing the gene(s) cat immunogens for its introduction as genetic vaccines or vaccine on the basis of "naked" DNA. Such vectors include, but are not limited to, pTarget (Promega Corp., Madison, WI), pcDNA (Invitrogen, Carlsbad, CA). (J.J. Donnelly, et al., 1997; Hassett & Whitton, 1996).

16. The genes or gene fragments CD80, CD86, CD28 or CTLA-4, taken separately or in any combination, partially or completely, can be introduced or transfected into chromosomes cats or other mammal. This integration of these genes or fragments of these genes can be achieved using retroviral what the sector and can be used in gene therapy.

The present invention relates to methods and compositions designed to improve the cat's resistance to diseases and used in medical and/or commercial purposes. In this embodiment, the present invention feline CD80, CD86, CD28 or CTLA-4, expressed separately or in any combination, partially or completely, and in combination with genes encoding feline immunogen, or without them, introducing cats in whatever way is appropriate introduction. To stimulate growth or to generate resistance to disease, feline CD80, CD86, CD28 or CTLA-4, expressed separately or in any combination, enter into the composition at a concentration factor of about 0.01 to 100.0 mg per vaccine for one cat, and preferably in a concentration factor of about 0.25 to about 25 mg/kg/day. To stimulate growth or to generate resistance to diseases recombinant viral vector expressing feline CD80, CD86, CD28 or CTLA-4, alone or in any combination, enter into the composition at a concentration factor of about 0.01 to 100.0 mg per vaccine for one cat, and preferably in a concentration factor of about 0.25 to about 25 mg/kg/day. It should be noted that the required number of feline CD80, CD86, CD28 or CTLA-4 can be determined by the method routine experimentation well investigateislam, for example, by establishing a matrix of dosages and frequencies of administration and a comparison group of experimental units or subjects of each point in the matrix.

According to the present invention, native or recombinant feline CD80, CD86, CD28 and CTLA-4 is administered in a composition together with a physiologically acceptable carrier, such as, for example, phosphate buffered saline or deionized water. This composition may also contain fillers, including sizing(s), plasticizer(s), usilitel(I) absorption, bactericide(s) and the like, well known to specialists. The polypeptide of feline CD80, CD86, CD28 and CTLA-4 of the present invention is administered in any effective manner, including, but not limited to, intravenous, subcutaneous, intramuscular, trasmissione, local or oral administration. For example, for subcutaneous injection dosage form consists of feline CD80, CD86, CD28 or CTLA-4 in sterile saline. For oral administration or administration by inhalation, feline CD80, CD86, CD28 or CTLA-4 with filler or without sign in micro - or microcapsule, for example, liposomes, and microspheres. Can also be used skin patch (or other dosage form extended release).

Materials and methods

Obtain initial samples of the smallpox virus raccoons

To receive the of the original samples of the smallpox virus raccoons and genomic DNA smallpox raccoons used isolate smallpox raccoons (RPV) ATS VR-838. Other RPV isolate is V71-I-85A, available from the Centre for disease control (CDC; Atlanta, GA). Samples of the smallpox virus raccoons (RPV) was obtained by infection of VERO cells, CRFK cells or MDCK cells at a multiplicity of infection of 0.01 boe/cell modified according to the method Dulbecco environment Needle containing 2 mm glutamine, 100 units/ml penicillin, 100 units/ml streptomycin (these components were obtained from Sigma or similar vendor) and called forth deficient DMEM. Before infection of cell monolayers once washed with deficient DMEM to remove trace amounts of fetal bovine serum. Then these monolayers were left for two hours, redistributing them every half hour for absorption in them RPV contained in the original inoculum (0.5 ml per 10 cm plate, 10 ml of 225 cm2-T-flask). After this period of time the original inoculum brought up to the recommended amount by adding complete medium DMEM (deficient DMEM + 5% fetal bovine serum). These plates were incubated at 37°With 5% CO2until then, until it was complete cytopathic effect. The medium and cells were collected and frozen in 50 ml conical-screw-capped tubes at -70°C. After thawing at 37°With aliquots of the original viral material was divided into 1.0 ml-vessels and again Zamora is supported at -70° C. Titers were mostly about 106boe/ml. Obtain initial samples of the smallpox virus swine

Samples of the smallpox virus of swine (SPV) was obtained by infection of kidney cells porcine embryo (EMSK), cells, ESK-4 cells, RK-15 or Vero cells at a multiplicity of infection of 0.01 boe/cell in a 1:1 mixture of the modified according to the method Dulbecco environment Claims (IMDM and RPMI medium 1640 containing 2 mm glutamine, 100 units/ml penicillin, 100 units/ml streptomycin (these components were obtained from Sigma or equivalent supplier, and hereafter they will be referred to as a scarce environment EMSK). Before infection, cell monolayers once washed scarce environment EMSK to remove trace amounts of fetal bovine serum. Then these monolayers were left for two hours, redistributing them every half hour for absorption in them SPV contained in the original inoculum (0.5 ml per 10 cm plate, 10 ml per 175 cm flask T). After this period of time the original inoculum brought up to the recommended amount by adding complete medium EMSK (scarce environment EMSK + 5% fetal bovine serum). These plates were incubated at 37°With 5% CO2until then, until it was complete cytopathic effect. The medium and cells were collected and frozen at -70°With conical 50 ml tubes with screw cap. After thawing at 37°aliqu is you original viral culture was divided into 1.0 ml of the vessels and was again frozen at -70° C. Titers were mostly about 106boe/ml

Obtaining DNA RPV or SPV

For DNA extraction smallpox raccoons or smallpox pigs, confluent monolayer of VERO cells (RPV) or cells EMSK (SPV) 225 cm2-flask T infected with the virus of smallpox raccoons (ATSS VR-838) at multiple infection of 0.1 and incubated for 3-5 days before until cells were not detected 100% cytopathic effect. Then the infected cells were collected by scraping the cells into the medium and centrifuged for 5 minutes at 3000 rpm in a clinical laboratory centrifuge. Then Wednesday decantation, and the cell precipitate gently resuspendable in 1.0 ml phosphate buffered saline (PBS: 1.5 g Na2HPO4, 0.2 g KH2PO4, 0.8 g NaCl, 0.2 g KCl per liter of H2On) (on T) and subjected to two consecutive cycles of freezing-thawing (from -70 to 37°). After the last thaw these cells (on ice) twice were treated with ultrasound, each time for 30 seconds at intervals of 45 seconds to cool down. Then cell debris was removed by centrifugation (ultra-speed centrifuge Sorvall RC-5B) at 3000 rpm for 5 minutes on the rotor NW at 4°C. Then virions RPV present in the supernatant, was besieged by centrifugation at 15,000 rpm for 20 minutes at 4°at the mouth of the re SS34 (Sorvall) and resuspendable in 10 mm Tris (pH 7.5). Then, this fraction was applied to a 36% sucrose gradient (mass./about. in 10 mm Tris, pH 7.5) and centrifuged (Beckman ultracentrifuge LB-70M) at 18000 rpm for 60 minutes in a SW41 rotor (Beckman) at 4°C. the Precipitate of virion resuspendable in 1.0 ml of 10 mm Tris, pH 7.5, and were treated by ultrasound for 30 seconds on ice. This fraction was applied to a 20-50% continuous sucrose gradient and centrifuged at 16000 rpm for 60 minutes in a SW41 rotor at 4°C. a Strip of virion RPV, localized approximately three quarters below the gradient was collected, diluted with 20% sucrose and precipitated by centrifugation at 18000 rpm./min for 60 minutes on a SW41 rotor at 4°C. Then the precipitate once were washed in 10 mm Tricom, pH 7.5, to remove trace amounts of sucrose and, finally, resuspendable in 10 mm Tris, pH 7.5. After that, the RPV DNA was extracted from purified virions by lysis (4 hours at 60° (C)induced by adding EDTA, LTOs and proteinase K to obtain the final concentrations of 20 mm, 0.5% and 0.5 mg/ml, respectively. After hydrolysis was performed three extraction with a mixture of phenol-chloroform (1:1), and the sample was besieged by adding two volumes of absolute ethanol and incubated at -20°C for 30 minutes. The sample was then centrifuged in a mini-Eppendorf centrifuge for 5 minutes at full speed. The supernatant decantation, and receiving the hydrated precipitate was air-dried and re-hydrational in 0,01M Tris, pH 7.5, 1 mm EDTA at 4°C.

Receiving samples of the original viral material FHV

S-FHV-000 was received from ATS ATS No. 636), a S-FHV-001 was obtained from NVSL (NVSL - tested virus strain SGE, KS Lot). Samples of the original viral culture FHV was obtained by infection of cells of the cat's kidneys Crandel (CRFK) at multiplicity of infection of 1.0 boe/cell modified according to the method of Dul-becco environment Needle containing 2 mm glutamine, 100 units/ml penicillin, 100 units/ml streptomycin (these components were obtained from Irvine Scientific or equivalent supplier, and hereafter they will be referred to as complete medium DME) + 5% fetal bovine serum. After cytopathic effect this environment and the cells were harvested, stratified and frozen at -70°C. the Title was, basically, about 1×107up to 1×108boe/ml

Obtaining DNA virus herpes

Confluently monolayer of CRFK cells in 25 cm2-flask or a 60 mm Petri dish was infected with 100 ml of the virus sample. After incubation over night or after detection cells 100% cytopathic effect these cells were scraped into the medium. These cells and the medium was centrifuged at 3000 rpm./min for 5 minutes in a clinical laboratory centrifuge. This environment decantation, and the cell sediment is slightly resuspendable in 0.5 ml of a solution containing 0.5% NONIDET P-40 (condensate activerelation is Yes, containing an average of 9 moles of ethylene oxide per molecule) (NP-40 was purchased from Sigma Chemical Co., St. Louis, MO). This sample was incubated at room temperature for 10 minutes. Then added 10 ml of stock solution RNase A (Sigma Chemical Co., St. Louis, MO.)(mother solution has a concentration of 10 mg/ml, and was boiled for 10 minutes to inactivate Gnkazy). This sample was centrifuged to precipitate nuclei. The DNA precipitate was removed Pasteur pipette or a wooden stick and discard. The liquid supernatant decantation in 1.5-ml Eppendorf tube containing 25 ml of 20% sodium dodecyl sulfate (Sigma) and 25 ml of proteinase K (10 mg/ml; Boehringer Mannhiem Biochemicals, Indianopolis, IN). This sample was mixed and incubated at 37°C for 30-60 minutes. Then added an equal volume of water-saturated phenol, and the sample is rapidly stirred. The sample was then centrifuged in a mini-Eppendorf centrifuge at full speed for 5 minutes. The upper aqueous phase was collected and placed in a new Eppendorf tube, then added two volumes of absolute ethanol, and the tube was placed for 30 minutes at -20°for deposition nukleinovoi acid. The sample was then centrifuged in a mini-Eppendorf centrifuge for 5 minutes. The supernatant decantation, and the precipitate was air-dried and re-hydrational in ~16 ml of N2O. To obtain large quantities of DNA this process is ur conducted on an enlarged scale, since the roller bottles or 175 cm2-flasks with CRFK cells. DNA was stored in 0.01 M Tris, pH 7.5, 1 mm EDTA at 4°C.

DNA trasverse to generate recombinant virus:

This method is based on the procedure using calcium phosphate Graham and Van der eb [25] with the following modifications. The virus and/or plasmid DNA was diluted up to 298 ml in 0.01 M Tris, pH 7.5, 1 mm EDTA. Then added 40 ml of 2M CaCl2with the subsequent addition of an equal volume of 2-HEPES buffered saline (10 g N-2-hydroxyethylpiperazine-N’-2-econsultancy acid (HEPES), 16 g Nad, 0,74 KCl, 0.25 g of Na2HPO42N2About 2 g of dextrose in 1 liter of N2O and buffered with NaOH to pH 7.4). After that, the mixture was incubated on ice for 10 minutes and then was added dropwise to 80% of the confluent monolayers of CRFK cells, cultured in 60 mm Petri dish in 5 ml of medium (DME + 5% fetal bovine serum). These cells were incubated for 4 hours at 37°C incubator with high humidity, containing 5% CO2. Environment on tablets was aspirated and cells were treated with 20% glycerol in 1x PBS (1,15 g Na2HPO4, 0,2 KN2RHO4, 0.8 g NaCl, 0.2 g KCl in 1 liter of N2A) within one minute. The cells three times washed with 5 ml 1x PBS, and then added 5 ml of medium (DME + 5% fetal bovine serum). Cells were incubated at 37°Since, as described above, in 3-7 days, until, on the and cytopathic effect of the virus was not 50-100%. The virus was collected, as described above, to obtain the source of viral material. This source of viral material called transfection original viral material, and then skanirovali on recombinant virus as described in the Chapter "Screening for recombinant herpesvirus expressing enzymatic marker genes".

Getting infected cell lysates. To obtain cell lysates used a serum-free environment. Confluent monolayer of cells (VERO, CRFK or MDCK) 25 cm2-flask or a 60 mm Petri dish was infected with 100 μl of virus sample. After ensuring the cytopathic effect of the medium and cells were collected, and these cells were besieged at 3000 rpm for 5 minutes in a clinical laboratory centrifuge. Cellular precipitate resuspendable in 250 ál of buffer for lysis (2% sodium dodecyl sulphate, 2% β-mercaptoethanol). The samples were treated with ultrasound for 30 seconds on ice and kept at -20°C.

The procedure for Western blotting. Samples of standard lysates and proteins were subjected to polyacrylamide gel electrophoresis in accordance with the method Laemmli (Laemnli). After gel electrophoresis, proteins are transferred and was processional, as described by Sambrook and others (1989). "First" antibody diluted 1:100 with 5% skim milk powder in a mixture of Tris-sodium chloride and sodium azide (TSA: 6,61 g Tris-Hcl, 0.97 g Tris-base, 9.0 g aCl and 2.0 g of sodium azide in 1 liter of N 2About). "Second" antibody conjugatively with alkaline phosphatase was diluted 1:1000 TSA.

The technique of molecular biology. The technique of manipulation of bacteria and DNA, including procedures such as hydrolysis restrictively by endonucleases, gel electrophoresis, gel extraction of DNA, ligation, phosphorylation kinase, treatment with phosphatase, the cultivation of bacterial cultures, transformation of bacteria with DNA and other methods used in molecular biology described by Sambrook et al.(1989) & Current Protocols in Molecular Biology (1992). In specially stipulated cases, they can be used with minor essentialisation DNA.

DNA sequencing was carried out by conducting the reactions dideoxy-sequencing by fluorescent labels using pre-set to cycle sequencing reactions, containing a dye for termination circuit ABI PRISM (Dye Terminator Cycle Sequencing Ready Reaction) using DNA Amplitaq polymerase, FS (Perkin-Elmer; in accordance with the manufacturer's instructions) and subjected to electrophoresis on an automated DNA sequencing machine model A Perkin Elmer/Applied Biosystems according to the manufacturer's instructions. Reactions using as mixtures dGTP, and mixtures dITP, was carried out to lighten seal areas. Alternatively, the compacted plots were divided in formamide gel is. Matrix represented the subclones of double-stranded plasmid or subclones single-stranded M13, and the primers were either embedded in the vector directly for sequanorum insert, or embed in the prior sequence. The obtained sequence was assembled and compared using the software DNAStar.

Cloning, polymerase chain reaction. To introduce restriction sites suitable for carrying out manipulations with different DNA used polymerase chain reaction (PCR). Used the procedure described by Innis and others (1990). Basically amplificatoare fragments had less than 500 base pairs, and the main areas of the amplified fragments were confirmed by DNA sequencing. The primers used in each case are described in detail below in the description of the design of homologous vectors.

The homologous recombination procedure for generating recombinant RPV, SPV or FHV. This method is based on homologous recombination between the DNA of the virus of smallpox raccoons and DNA homologous plasmid vector that occurs in the tissue culture cells containing DNA smallpox raccoons, and transfected with a plasmid homology vector. For the implementation of homologous recombination monolayers of cells (CRFK, MDCK or VERO) inficon the Wali S-RPV-000 (ATCC VR-838) or S-SPV-001 or S-FHV-001 at a multiplicity of infection of 0.01 boe/cell for introduction can replicate RPV (i.e. for DNA synthesis in these cells. Then DNA plasmid homology vector was transferrable in these cells in accordance with the procedure of infection-transfection. Constructing homologous vectors used in this procedure are described below.

Procedure-infection-transfection. 6 cm-cups with cells (CRFK, MDCK or VERO) confluently about 80% of infected S-RPV-000 or S-SPV-001 or S-FHV-001 at a multiplicity of infection of 0.01 boe/cell scarce in the DMEM and incubated at 37°C in an atmosphere of 5% CO2with high humidity for 2-3 hours. Mainly used the transfection procedure, which is recommended for reagent lipofectin™ (BRL). Briefly, for each 6 cm-cups, 15 μg of plasmid DNA was diluted with H2O to a volume of 100 μl. Separately, 50 micrograms lipofectin reagent was diluted with H2O to a volume of 100 μl. Then 100 μl of the diluted lipofectin reagent was added dropwise to the diluted plasmid DNA contained in polystyrene 5 ml test tube with snap-on lid, and gently stirred. Then this mixture is incubated for 15-20 minutes at room temperature. During this period of time viral inoculum was removed from the 6-cm plates and the cell monolayers were once washed with deficient DMEM. Then 3 ml of scarce environment DMEM was added to the mixture of plasmid DNA/lipofectin, soderjimoe was applied by pipette to the cell monolayer. These cells were incubated overnight (about 16 hours) at 37°C in an atmosphere of 5% CO2with high humidity. The next day, 3 ml of scarce environment DMEM was removed and replaced with 5 ml of complete DMEM medium. These cells were incubated at 37°C in an atmosphere of 5% CO2high humidity within 3-5 days up until the cytopathic effect of the virus was 80-100%. The virus was collected, as described above, to obtain the original viral culture. This culture is called transfectional original culture, and then skanirovali on recombinant virus as described in the section "Screening method BLUOGAL on recombinant smallpox raccoons or screening method CPRG on recombinant virus smallpox raccoons".

Screening for rekomendatsii virus RPV or FHV expressing β-galactoside (analyses BLUOGAL and CPRG) or β-glucuronidase (analysis of X-GLUC)

When vvedeniye marker gene β-galactosidase (lacZ) gene in the recombinant virus plaques containing recombinants, visualized in one of two simple methods. In the first method, chemical Bluogal™ (Life Sciences Technology, Bethesda, MD) was applied (200 μg/ml) on top of the agarose layer during the analysis belascoaran, and plaques expressing active β-galactosidase was purchased by blue color. Then blue plaques were sown on fresh cells (MDCK, CRFK or VERO) and was purified by additional is adelene blue plaques. In the second method CPRG (Boehringer Mannhiem) was applied (400 µg/ml) on top of the agarose layer during the analysis on the formation of plaques and plaques expressing active β-galactosidase, was acquired by red color. Then red plaques were sown on fresh cells (MDCK, CRFK or VERO) and was purified by additional allocations red plaques. In both cases, the virus is usually cleared, using three or four cycles of clearing of plaques.

With the introduction of the marker gene β-glucuronidase (uidA) gene in the recombinant virus plaques containing the recombinants were visualized using chromogenic substrate, X-beta-D-gluUA CHX (X-GLUC; cyclohexylammonium salt 5-bromo-4-chloro-3-indoxyl-beta-D-glucuronic acid; Biosynth AG; Switzerland) was applied (200 μg/ml) on top of the agarose layer during the analysis on the formation of plaques and plaques expressing active β-glucuronidase bought a blue color. Then blue plaques were sown on fresh cells (MDCK, CRFK or VERO) and was purified by additional selection of blue plaques.

Screening for expression of a foreign gene in recombinant RPV using analyses on the formation of black plaques

To analyze expression of foreign antigens expressed by recombinant poxviruses raccoons, monolayers of cells (MDCK, CRFK or VERO) were infected with recombinant RPV, SPV or FHV, inflicted on them pitate the performance communications agarose medium and incubated for 3-5 days at 37° For plaque formation. The top agarose layer was removed from the Cup, the cells were fixed with 100% methanol for 10 minutes at room temperature, and then dried in the air. Fixation of the cells makes it possible to detect cytoplasmic antigen and surface antigen, whereas the expression of specific surface antigen can be detected using flow cells. Then "first" antibody was diluted to the appropriate concentration of 1x reagent "blotto" (5% non-fat dry milk in Tris-sodium chloride and sodium azide (TSA: 6,61 g Tris-HCl, 0.97 g Tris-base, 9.0 g NaCl, and 2.0 g of sodium azide in 1 liter of N2O), and incubated on the cell monolayer for 2 hours at room temperature. Unbound antibody was removed by three times washing of the cells with TS buffer at room temperature. "Second" antibody conjugated with alkaline phosphatase diluted 1:1000 1x reagent "blotto and incubated with cells for 2 hours at room temperature. Then unbound second antibody was removed by three times washing of the cells with TS buffer (of 6.61 g of Tris-HCl, 0.97 g Tris-base, 9.0 g NaCl in 1 liter of H2O) at room temperature. After that, cells were incubated for 15-30 minutes at room temperature with freshly prepared substrate solution (100 mm Tris-HCl, pH of 9.5, 100 mm NaCl, 5 mm MgCl2

Screening for expression of feline CD80 (B7-1) and CD86 (B7-2) in recombinant SPV, RPV or FHV using analysis on the formation of black plaques

To analyze the expression of co-stimulating molecules CD80 or CD86 expressed by recombinant pox viruses of pigs, the viruses of smallpox raccoons or herpes viruses cats on monolayers of cells (MDCK, CRFK, VERO or ESK-4), these cells were infected with recombinant viruses RPV, SPV or FHV expressing CD80 or CD86, and then they were applied nutrient agarose medium and incubated for 3-5 days at 37°for plaque formation. The top agarose layer was removed from the Cup, the cells either were fixed with 100% methanol for 10 minutes at room temperature, and then the cells were air-dried, or left unfixed or left unfixed and immediately was treated with 1x PBS. Fixation of the cells makes it possible to detect cytoplasmic antigen and surface antigen, whereas the expression of specific surface antigen can be detected using flow cells. Then the Chimera huCTLA-4/Fc (R&D Systems, Minn MN, cat. # 325-CT) was diluted to the appropriate dilution of 1x reagent "blotto" (5% nonfat dry milk in Tris-sodium chloride (TS: of 6.61 g of Tris-HCl, 0.97 g Tris-base, 9.0 g Nad and 2.0 g of sodium azide in 1 liter of H2O), and incubated on the cell monolayer for 2 hours at room temperature. Unbound Chimera was removed by three times washing of the cells with TS buffer at room temperature. Antibody for the detection monoclonal antibody against hulgGI fc conjugated with alkaline phosphatase (Zymed, cat.05-3322), diluted to the appropriate concentration of 1x reagent "blotto and incubated with cells for 2 hours at room temperature. Unbound antibody for detection was removed by three times washing of the cells with TS buffer (of 6.61 g of Tris-HCl, 0.97 g Tris-base, 9.0 g NaCl in 1 l of N2O) at room temperature. The cells are then incubated for 15-30 minutes at room temperature with freshly prepared substrate solution (100 mm Tris-HCl, pH of 9.5, 100 mm NaCl, 5 mm MgCl2, 0.3 mg/ml nitrotetrazolium blue and 0.15 mg/ml 5-bromo-4-chloro-3-incorporates). Plaques expressing CD80 or CD86, were painted in black color. To stop the color development reaction solution was used-latch (20 mm Tris-HCl, pH 2.9 and 1 mm EDTA).

Screening for bioactivity of feline interferon-gamma expressed recombinant SPV, RPV or FHV, with ISOE what Itanium analysis to reduce the number of plaques formed under the action of VSV

CRFKS or a suitable feline cell line in 96-well tablets were treated with supernatant from cells infected with recombinant viruses expressing the feline IFN-gamma, and incubated for 6-12 hours at 37°C. Then into the respective wells were added to the virus VSV (100-1000 particles/well) and incubated for 24 hours or until until the control wells containing only cells that were not completely lysed. These wells three times washed with 1x PBS, and the monolayers were fixed with 100% methanol and dried in the air. To each well was added 0.05% solution of crystal violet for 10 minutes at room temperature, and then dried in the air. These wells were evaluated for the presence of purple color. A healthy monolayer of intact cells will absorb the crystal violet dye. Supernatant with the activity of IFN-gamma will provide protection from CRFK VSV-induced cell lysis and painted purple.

The treatment of viral glikoproteinov to use for diagnostic purposes

Viral glycoproteins were purified using affinity columns with antibodies. For production of monoclonal antibodies 8-10 week female BALB/c mice were vaccinated intraperitoneally seven times at intervals of 2-4 weeks 107boe recombinants in the Rus smallpox raccoons. Three weeks after the last vaccination, the mice were injected intraperitoneally with 40 mg of the corresponding viral glycoprotein. Three days after the last dose of antigen in the spleen of mice was removed.

Splenocytes were merged with cells of the mouse plasmacytoma NSl/Ag4 improved method Oi and Herzenberg. Splenocytes and cells plasmacytoma besieged together by centrifugation at 300X g for 10 minutes. 1 ml of 50% solution of polyethylene glycol (molmasse 1300-1600) was added to the cell precipitate with stirring for one minute. To these cells within three minutes added modified according to the method Dulbecco Wednesday Needle (5 ml). Cells were besieged by centrifugation at 300X g for 10 minutes and resuspendable in a medium containing 10% fetal bovine serum, and also containing 100 mm gipoksantin, 0.4 mm of aminopterin and 16 mm thymidine (HAT). Cells (100 ml) was added to the wells 8-10 96-well plates for culturing tissues containing 100 ml layer of normal cells-feeders spleen and incubated at 37°C. These cells were fed fresh medium HAT every three or four days.

Supernatant hybridoma cultures were tested using ELISA in 96-well microtiter tablets coated with 100 ng of viral glycoprotein. Then supernatant from reactive hybridomas were analyzed using but is she on the formation of black plaques and using Western blotting. Selected hybridoma twice cloned by limiting dilution. Ascitic fluid was produced by intraperitoneal injection to mice BALB/c 5×106hybridoma cells processed by the Wharf.

Cell lysates from recombinant poxviruses raccoons were obtained as described above in the section "Getting infected cell lysates". Glycoproteinoses cell lysates (100 ml) was passed through a 2-ml agarose affinity resin, which was immobilized 20 mg monoclonal antibodies against glycoprotein in accordance with the manufacturer's instructions (AFC Medium, New Brunswick Scientific, Edison, N.J.). The column was washed with 100 ml of 0.1% Nonidet P-40 in phosphate buffered saline (PBS) to remove non-specific bound material. Associated glycoprotein was suirable 100 mm carbonate buffer, pH to 10.6 (40). Monitoring the purity of pre - and postanweisung fractions was performed by analyzing the reactivity with monoclonal antibodies against RPV in the ELISA system.

The ELISA

To determine the immune status of the animal after vaccination and control of infection used Protocol standard enzyme-linked immunosorbent assay (ELISA). The solution glycoprotein antigen (100 ml at ng/ml in PBS) was left for 18 hours at 4°for absorption to the wells of microtiter plates. Covered the holes once were washed in PBS. These wells were blocked by adding 250 ml of PBS containing 1% BSA (Sigma) and incubated for 1 hour at 37°C. the Blocked holes once were washed in PBS containing 0.02% tween-20. In these wells was added 50 ml of test serum (pre-diluted 1:2 in PBS containing 1% BSA) and incubated for 1 hour at 37°C. Anticigarette was removed and the wells three times washed with PBS containing 0.02% tween-20. 50 ml of a solution containing antibodies against bovine IgG conjugated to horseradish peroxidase (diluted 1:500 in PBS containing 1% BSA, Kirkegaard and Perry Laboratories, Inc.), added for visualization of the wells containing antibody against the specific antigen. The solution was incubated for 1 hour at 37°S, and then removed, and the wells three times washed with PBS containing 0.02% tween-20. To each well was added 100 ml of substrate solution (ATBS, Kirkegaard and Perry Laboratories, Inc.) and left for 15 minutes for the display of color. The reaction was stopped by adding 0.1 M oxalic acid. Color was recorded when the optical density of 410 nm on an automatic tablet reader.

The strategy of constructing synthetic poxvirus promoter. For vectors based on recombinant pox viruses of pigs, synthetic poxvirus promoters have some advantages, including their ability to regulate the level and stage of expression of a foreign gene. Were the designed three promoter cluster LP1, ER and LP2 on the basis of promoters that have been described in the cowpox virus. Each cluster was designed to contain a DNA sequence specific for vaccinia virus and flanked by restriction Sagami, which can be used to merge these clusters in any order or combination. In each cluster were also introduced initiating methionine to ligation with the preservation of the reading frame can be performed with either EcoRI-site or in the BamHI site. Downstream from the site of ligation with preservation of the reading frame was also constructed a series of translational stop codons in all three reading frames, and signal early termination of transcription. DNA encoding each cluster was synthesized by standard methods and cloned into the corresponding homologous vectors.

The selection of the original fragment CD80

mRNA was extracted from mononuclear cells of peripheral blood (MCPC), stimulated for 16 hours And Con using a reagent for extraction of RNA RNAzolB (Biotexc, Houston, TX). First from this RNA was obtained cDNA by reaction with reverse transcriptase (RT) using oligo dT as the 3’primer. Briefly, RNA and oligo dT were heated to 75°C for 3 minutes to remove secondary structure. Then add RT, dNTP, buffer and distiller annoy water, and the mixture is incubated for 1 hour at 42°C. After this incubation, the sample was heated to 95°C for 5 minutes to inactivate the RT. Then degenerate primers derived from consensus regions of the published sequences of human CD80 and mouse (GenBank, Gaithersburg, MA)was used for the initial amplification 344-nucleotide model.) fragment encoding the Central region of the constant domain of this gene:

5’-primer V7-2: GGC CCG AGT A(CT)A AGA ACC GGA (SEQ ID NO 56)

the 3’primer V7-3 CAG (AT)TT CAG GAT C(CT)T GGG AAA (CT)TG (SEQ ID NO 57).

The amplification product was carried out according to the Protocol polymerase chain reaction (PCR) with a "hot start" using Taq polymerase. To prevent the formation of primer dimers, the reaction mixture not containing Taq enzyme, were first heated to 95°C for 5 minutes in a stage of "hot start". Before initiating the holding of the temperature cycle was added to the enzyme. Then, the PCR mixture was heated to 95°C for 30 seconds to melt double-stranded DNA. After the reaction mixture was cooled to 42°C for 30 seconds to facilitate annealing of degenerate primers. To facilitate binding of the primers, which are not homologous to 100%, used a low temperature annealing. Then to extend the primer and create a copy of the opposite DNA strand of reactio the ing the mixture was heated for 45 seconds and 72° C, the optimal temperature for Taq polymerase. The temperature cycle was repeated 30 times. After conducting 30 cycles spent the final stage of elongation at 72°C for 7 minutes to facilitate lengthening any incomplete products. After visualization on a 1% agarose gel, this product for sequencing ligated into the vector for TA cloning (InVitrogen, San Diego, CA) overnight at 16°C. 2 ml of this reaction mixture for ligation was used to transform InvaF-competent cells. Transformed bacteria were applied streaks on LB-plates (50 mg/ml ampicillin), covered with 40 ml of 50 mg/ml x-gal. The next day, white colonies were collected and inoculable in 5 ml LB-medium containing 100 mg/ml ampicillin, and cultured overnight at 37°With shaking at 225 rpm

To identify clones containing the plasmid with the correct insert were obtained mini-preparations for the night cultures. The plasmids were extracted from these cultures using standard procedures alkaline lysis, after which the DNA was purified by extraction with a mixture of phenol:chloroform (Maniatis et al., 1982). This DNA was besieged in 2 volumes of ethanol, and then hydrolyzed EcoRI. The hydrolysates were visualized on 1% agarose gel to identify colonies with plasmid containing the correct insert. Then the plasmid was purified from positive clones and sec what was enroule or by dimethoxyisoquinoline using S 35-radioactively labelled dideoxythymidine circuit on the basis of sequenase (USB, Cleveland, OH)or by cyclic sequencing by the method of chain termination using a fluorescent dye (Perkin-Elmer, Norwalk CT). From the cDNA sequence, were designed specific to the 3’and 5’-primers for use in the reaction, rapid amplification of 5’-cDNA ends (RACE) and derivatization of the 3’sequence in combination with degenerate primers from the 3’-untranslated region (UTR).

The allocation of the 5’-region of CD80

For derivatization 5’sequences of the gene used Protocol Marathon for amplification of cDNA (Clonetech, Palo Alto, CA). mRNA was produced from MCPC, Con a stimulated for 12 hours and at the same time stimulated LPS for 4 hours. This mRNA was extracted using a reagent for extraction of ULTRASPEC RNA (Biotexc, Houston, TX). To facilitate binding of the primer with the most extreme part of the 5’-end poly-a tail was produced cDNA using the anchor primer oligo dT with degenerate nucleotides at the 5’-end. Then cDNA was transcribable, as described above. Specific linkers ligated to cDNA using DNA T4 ligase. Then this cDNA was carried out by short PCR using the internal 3’-primer that is specific to the field of amplified earlier:

V7-284: TTA TAC TAG GGA CAG GGA AG (SEQ ID N: 58)

V7-190: AGG CTT TGG AAA ACC TCC AG (SEQ ID NO: 59)

and the anchor primer, complementary legirovannoi linker sequence. Short-time PCR, polymerase mixture of KlenTaq (Clontech, Palo Alto, CA) was performed under the following conditions: 1 cycle at 95°C for 5 min; 5 cycles at 95°C for 30 sec, at 72°C for 30 sec and at 68°C for 45 sec; 5 cycles at 95°C for 30 sec, 65°C for 30 sec and at 68°C for 45 seconds; and 25 cycles at 95°C for 30 sec, at 60°C for 30 sec and at 68°C for 45 sec. 1 ml of this reaction mixture was dissolved in 50 ml of water and 5 ml of this dilution is then used in a "nested" PCR reaction (1 cycle at 95°C for 5 min; 30 cycles at 95°C for 30 sec, 65°C for 30 sec and at 68°C for 45 sec; using polymerase mixture of KlenTaq) and using specific anchor primer and a gene-specific 3’-primer, with localized side 5’-end of the original primer (6).

V7-20: TTG TTA TCG GTG ACG TCA GTG (SEQ ID NO: 60)

V7-135: CAA TAA CAT CAC CGA AGT CAG G(SEQ ID NO: 61)

20 ml of each reaction mixture were visualized on a 1.5% agarose gel and the desired fragment was cut out of the gel. cDNA was extracted and purified from agarose gel by centrifugation of the gel slices using the gel dispenser and 0.22 mm filter to microcode (Amicon, everly, MA). Then the purified DNA is sequenced by the direct method termination circuit using dye (Perkin-Elmer, Norwalk, CN).

The allocation of the 3’-region of CD80

the 3’region of the gene was derivateservlet by selecting 5 gene-specific primers from the 344-BP fragment and the previously sequenced the 5’-region:

B7-S220 GTC ATG TCT GGC AAA HUNDRED CAA G (SEQ ID NO: 62)

V7-50 CAC TGA CGT CAC CGA TAA SAA (SEQ ID NO: 63)

B7-140 CTG ACT TCG GTG ATG TTA TTG G (SEQ ID NO: 64)

B7-550 GCC ATC AAC ACA ACA GTT TCC (SEQ ID NO: 65)

B7-620 TAT GAC AAA CAA CCA TAG CTT (SEQ ID NO: 66)

Degenerate 3’-primers were selected from the consensus regions of the 3’-UTR of human and murine CD80.

V7-1281 G(A/G)A AGA (A/T)TG CCT CAT GA(G/T) CC (SEQ ID NO: 67)

B7-1260 CA(C/T)(A/G)AT CCA ACA TAG GG (SEQ ID NO: 68)

cDNA was produced from RNA extracted using ULTRASPEC (Biotexc, Houston, TX) from MCPC stimulated by Con a and LPS, as described previously.

Anchored oligo-dT was used as the source 3’-primer for RNA transcription with the formation of cDNA. This cDNA was performed by PCR-reaction on the basis of Taq polymerase using specific 5’primers and degenerate 3’-primers (1 cycle at 95°C for 5 minutes; 30 cycles at 95°C for 30 seconds at 42°C for 30 seconds, and 72°C for 45 seconds; and finally, at 72°C for 7 minutes).

Before biogas produced a single fragment of the desired size needed two RA is ndow "nesting" reactions. This product is cut from a 1.5% agarose gel, purified as described previously and subjected to cycle sequencing using dye as a chain terminator(Perkin-Elmer,Norwalk, CN).

Based on the sequence 5’- and 3’-regions were designed primers with which it is necessary to amplify the region encoding the entire open reading frame of the gene of feline CD80:

B7-START: ATG GGT CAC GCA GCA AAG TGG (SEQ ID NO:69)

B7-960: CCT AGT AGA GAA GAG HUNDRED AAG AGG (SEQ ID NO:70)

Was used for cDNA MCPC, which was produced previously and about which it is known that it contains DNA encoding the indicated gene. In order to minimize random errors, often associated with Taq polymerase, in this panorama of reaction (1 cycle at 95°C for 5 minutes; 30 cycles at 95°C for 30 seconds at 42°C for 30 seconds, and 72°C for 45 seconds; and 72°C for 7 minutes) used DNA polymerase KlenTaq, enzyme mixture, which retains a certain 5'-ectonucleoside activity. In this reaction amplified a fragment of 960 base pairs (BP), which was cloned into a vector for TA cloning (InVitrogen, San Diego, CA) and sequenced as described previously. The final sequence of this gene consisted of cDNA from two separate animals. To reduce the likelihood of errors resulting from PCR inducion is the R error, every couple of reasons for this gene has been independently verified, at least in three separate sequences obtained from separate PCR reactions.

The selection of the initial fragment of CD28: mRNA was extracted from peripheral blood lymphocytes NK stimulated for 16 hours And Con using a reagent for extraction of RNA RNAzolB (Biotexc, Houston, TX). First from this RNA was obtained cDNA by reaction with participation of reverse transcriptase (RT) using oligo dT as the 3’primer. For this, RNA and oligo dT were heated to 75°C for 3 minutes to remove secondary structure. Then add RT, dNTP, buffer and distilled water and the mixture is incubated for 1 hour at 42°C.

After this incubation, the sample was heated to 95°C for 5 minutes to inactivate the RT. Degenerate primers derived from consensus regions found in the published sequences of nucleic acids of human, mouse and rabbit CD28 (GenBank, Bethesda, MD), and then used for the initial amplification 673-nucleotide fragment encoding a large part of the open reading frames:

CD28-113: CAA CCT TAG CTG CAA GTA CAC (SEQ ID NO 71):

CD28-768: GGC TTC TGG ATA ATA GGG GG (SEQ ID NO 72).

For amplification of this product used a Protocol polymerase chain reaction (PCR) with a "hot start" (1 cycle at 95°during the course the e 5 minutes; 30 cycles at 95°C for 30 seconds, 48°C for 30 seconds, and 72°C for 45 seconds; and 1 cycle at 72°C for 7 minutes). This fragment was visualized on 1% agarose gel, ligated into the vector for TA cloning (InVitrogen, San Diego, CA) and sequenced as described previously. Based on the cDNA sequence, were derived and synthesized specific 3’primers for use in 5’-RACE reactions.

CD28 190: CGG AGG TAG AAT TGC ACT GTC (SEQ ID NO 73):

CD28 239: ATT TTG CAG AAG TAA ATA TCC (SEQ ID NO 74).

The allocation of the 5’-region of CD28

For the rest of the 5’sequence of the molecule feline CD28 was used a modified Protocol 5’-RACE GIBCO (Gibco BRL, Gaithersburg, MD). RNA was extracted from 16 hour culture Con A-stimulated MCPC. For the synthesis of the first chain cDNA used gene-specific 3’-primer. RNA and the primer was heated to 75°C for 5 minutes, and then added other reagents RT. After denaturation, the mixture was cooled to 4°and was added to the reaction buffer, magnesium chloride, dNTP, DTT and Superscript RT (Gibco BRL, Gaithersburg, MD). RT mixture was incubated at 42°C for 30 minutes and then was heated to 70°C for 15 minutes to denature the RT. Then was added a mixture of RNase, and the reaction mixture incubated at 55°C for 10 minutes to remove residual RNA and prevent the wrong end transferases (TdT) UD is inane. Then cDNA was purified on a rotating column GlassMax (Gibco BRL, Gaithersburg, MD) to remove non-included dNTP and primer. Purified cDNA, elyuirovaniya with column extended using TdT. TdT was used to add 20-30-nucleotide tail dC to cDNA. After cDNA denaturation at 95°C for 3 minutes to a mixture of purified cDNA, magnesium chloride, reaction buffer and dCTP were added to the enzyme. The reaction mixture was incubated at 37°C for 10 minutes, after which the enzyme was subjected to thermoinactivation at 70°for another 10 minutes. Longer cDNA amplified by PCR with the "hot start" on the basis of Taq polymerase (95°C for 5 min; 35 cycles at 95°C for 30 sec, at 55°C for 30 sec and 72°C for 45 seconds; and 72°C for 7 min). As primers for this reaction used a 3’-primer, localized by 5’-end primer for cDNA synthesis, and anchor primer specific for the linker dC and consisting mainly of dG with a few remnants of dI. 1 ml of this reaction mixture was dissolved in 50 ml of water and 5 ml of this dilution was used in nested-PCR (1 cycle at 95°C for 5 min; 30 cycles at 95°C for 30 sec, at 55°C for 30 sec and at 72°C for 45 sec with a mixture of KlenTaq polymerases) using anchor 5’-primer with dG/dI and additional 3’-primer specific to above the positioning the gene. Then 30 ml of a mixture for breeding, the reaction was visualized on a 1.5% agarose gel and the gel was extracted desired Fig). cDNA was purified, as described previously, using gel spray and Amicon filter for microcode (Amicon, Beverly, MA). The sample was then purified cDNA sequenced by cyclic sequencing method termination circuit using dye (Perkin-Elmer, Norwalk, CN). All of these fragments was obtained consensus sequence. Based on this sequence, was synthesized primer pair that spans the entire open reading frame of the gene of feline CD28:

fe CD28 5’: CGC GGA TCC ACC GGT AGC ACA ATG ATC CTC AGG (SEQ ID NO: 75)

fe CD28 3’: CGC GGA TCC TCT GGA TAG GGG TCC ATG TCA G (SEQ ID NO: 76)

Using these primers cDNA molecule comprising the entire coding region, amplified from cDNA derived from Sopa-stimulated MCPC EC and ED3. This cDNA MCPC was produced previously and was shown that it contains RNA encoding gene. In this PCR (1 cycle at 95°C for 5 min; 30 cycles at 95°C for 30 sec, 42°C for 30 sec and at 72°C for 45 sec; and 1 cycle at 72°C for 7 min), conducted using DNA polymerase KlenTaq to minimize the number of random errors, often associated with Taq polymerase was produced 754 BP fragment, which was cloned in cloning in ctor and sequenced, as described previously. As in the case of molecules CD80, each nucleotide site was confirmed using at least three independently derived sequences.

Homologous vector 902-49 .46. Plasmid 902-49 .46 designed in order to integrate the foreign DNA into the RPV. It consisted of a marker gene β-galactosidase (lacZ) gene, flanked DNA RPV. Above this foreign gene was the DNA fragment of the RPV, consisting of approximately 906 base pairs. Following from this foreign gene was the DNA fragment of the RPV, consisting of approximately 895 base pairs. Using plasmids in accordance with the procedure described in the homologous recombination Procedure for generating recombinant RPV," received a virus containing DNA encoding these alien genes. It should be noted that the marker gene β-galactosidase (lacZ) is under the control of a late promoter (LP1), and the second foreign DNA is inserted into the EcoRI or BamHI site, where this second foreign DNA is under the control of the late/early promoter (LP2EP2). This plasmid was constructed using standard techniques of recombinant DNA (Sambrook et al.) by joining restriction fragments from the following sources with the synthetic DNA sequences. This plasmid vector was obtained from a HindIII restriction-fragments is and plasmids pSP64, with approximately 2999 base pairs (Promega). Fragment 1 is a restriction HindIII-XbaI-subfragment HindIII restriction fragment U RPV, with approximately 906 base pairs (Knight et al.). Fragment 2 is a restriction BamHI-PvuII fragment of plasmid pJF751 (Ferrari et al.), with approximately 3010 base pairs. Fragment 3 is a restriction XbaI-HindIII-subfragment HindIII fragment U RPV, with approximately 895 base pairs. XbaI sites of fragments 1 and 3 were transformed into a unique NotI sites using NotI-linkers.

Homologous vector 904-63 W. Homologous vector V used for integration of foreign DNA into SPV. It includes marker gene β-galactosidase (lacZ) gene and genes gag/protease and envelope of the virus of immunodeficiency cats (FIV), flanked by SPV DNA. Using this homology vector in accordance with the procedure described in the homologous recombination Procedure for generating recombinant SPV," received a virus containing DNA encoding these alien genes.

It should be noted that the marker gene β-galactosidase (IacZ) is under the control of a synthetic late poxvirus promoter (LP1), and the genes of the FIV gag/protease and shell are controlled by separate but identical synthetic late/early promoter poxvirus (LP2EP2). Clusters ol the motor/gene FIVgag/protease and shell FIV oriented in opposite directions so that the transcription of genes gag/protease and shell passes in direction to each other in order to exclude the possibility of homologous recombination between identical promoters. Homologous vector was constructed using standard techniques of recombinant DNA (Sambrook et al.) by joining restriction fragments from the following sources to the appropriate synthetic DNA sequences. This plasmid vector was obtained from restriction HindIII-BamHI fragment of plasmid S64 with approximately 2972 base pairs (Promega). Fragment 1 is a BglII restriction-AccI-subfragment HindIII restriction fragment M(23) SPV, with approximately 1484 base pairs. Fragment 2 is an EcoRI-BglII fragment of the gene shell FIV, with approximately 2580 base pairs and synthesized by reverse transcription (RT) and polymerase chain reaction (PCR) (15,42) using cDNA from the strain of FIV PPR. The reverse primer (5’-GCCCGGATCCTATGGCAGAAGGGTTTGCAGC-3’ 10/93 .21) (SEQ ID NO: 77) is synthesized from the 5’end of the gene shell FIV and introduces a BamHI site at the 5’end of this gene. Direct primer (5’-CCGTGGATCCGGCACTCCATCATTCCTCCTC-3’ 10/93 .20) (SEQ ID NO: 78) is synthesized from the 3’-end of the gene shell FIV and introduces a BamHI site at the 3’end of this gene and used for reverse transcription and polymerase chain reaction. The PCR product restriction is whether the enzyme BamHI to obtain fragment length 2580 base pairs, the corresponding gene shell FIV. Fragment 3 is a restriction EcoRI-BglII-fragment of the gag gene/FIV protease, with approximately 1839 base pairs and synthesized by reverse transcription (RT) and polymerase chain reaction (PCR) (15,42) using cDNA from the strain of FIV PPR. The reverse primer (5’-GCGTGAATTCGGGGAATGGACAGGGGCGAGAT-3’ 11/94 .9) (SEQ ID NO: 79) is synthesized from the 5’end of the gene DBP/FIV protease and introduces an EcoRI site at the 5’end of this gene. Direct primer (5’-GAGCCAGATCTGCTCTTTTTACTTTCCC-3’ 11/94. 10) (SEQ ID NO: 80) is synthesized from the 3’-end of the gene DBP/FIV protease, introduces WD site at the 3’end of this gene and used for reverse transcription and polymerase chain reaction. The PCR product was restrictively enzymes EcoRI and glII obtaining fragment length of approximately 1839 base pairs corresponding gene DBP/FIV protease. Fragment 4 is a restriction BamHI-PvuII fragment of plasmid pJF751 with approximately 3010 base pairs(Ferrari et al.). Fragment 5 is a restriction AccI-HindIII-subfragment HindIII restriction fragment M SPV, with approximately 2149 base pairs. ACCI site of homologous vector SPV has turned into a unique NotI site using synthetic linkers.

Homologous vector 917-60 W. Plasmid 917-60 V designed in order to integrate the foreign DNA into SPV. It consisted of a marker gene β-galactosidases is (lacZ) gene and the gene of feline IFN-γ (Onions, et al.,(1996); Argyle et al.,(1995), flanked by SPV DNA. Above these alien genes were DNA fragment of SPV, consisting of approximately 1484 base pairs. Following from these alien genes were DNA fragment of SPV, consisting of approximately 2149 base pairs. Using plasmids in accordance with the procedure described in the homologous recombination Procedure for generating recombinant SPV," received a virus containing DNA encoding these alien genes. It should be noted that the marker gene β-galactosidase (lacZ) is under the control of the promoter of OIL smallpox pigs, and gene feline CD28 is under the control of the synthetic late/early poxvirus promoter (LP2EP2). This plasmid can be constructed using standard techniques of recombinant DNA (Sambrook et al.) by joining restriction fragments from the following sources. This plasmid vector was obtained from restriction HindIII-BamHI fragment of plasmid pSP64, with approximately 2972 base pairs (Promega). Fragment 1 is a BglII restriction-Accl-subfragment HindIII restriction fragment M SPV, with approximately 1484 base pairs. Fragment 2 is a restriction EcoRI-BamHI fragment synthesized by reverse transcription and polymerase chain reaction (PCR) with the use of the cation in the matrix quality RNA from Sopa-stimulated cells of the spleen cats. For the synthesis of feline IFN-γ, primer 5’-TCGAGAATTCGATGAATTACACAAGTTTTATTTTCG-3’; 1/97 .4) (SEQ ID NO:81) was synthesized from 5’-end of the gene of feline IFN-γ and introduced EcoRI site at the 5’end of this gene. Primer (5’-TCGAGGATCCTTATTTCGATGCTCTACGGCCTC-3’; 1/97 .3) (SEQ ID NO:82) was used for reverse transcription and PCR was synthesized from the 3’-end of the gene of feline IFN-γand introduced a BamHI site at the 3’-end of the gene. The PCR product was restrictively enzymes EcoRI and BamHI to obtain a fragment length of approximately 504, the base pairs corresponding to the gene of feline IFN-γ. Fragment 3 is a restriction BamHI-PvuII fragment of plasmid pJFV751 with approximately 3010 base pairs (Ferrari et al.). Fragment 4 is an AccI-HindIII-subfragment HindIII-fragment M SPV, with approximately 2149 base pairs. Accl-sites of fragments 1 and 4 were transformed into a unique Notl sites using NotI-linkers. Homologous vector 926-76 .D7.

Homologous vector 926-76 .D7 designed to dellarovere part of the coding region from de herpes virus cats and for integration of foreign DNA. It includes the gene of feline CD80, flanked DNA FHV. The gene of feline CD80 is under control of the promoter de FHV. It was constructed from these DNA sources using standard techniques of recombinant DNA (Sambrook et al.). The plasmid vector was obtained from restriction endonuclease Asp718I-s7181-f is armenta plasmids pSP18/19, with approximately 2958 base pairs. Fragment 1 is an Asp7181-SmaI-subfragment SalI fragment In FHV, with approximately 1415 base pairs. Fragment 2 is an EcoRI-BamHI fragment of the gene of feline CD80, with approximately 879 base pairs and synthesized according to the procedure described in the section "Cloning by polymerase chain reaction". Template for the PCR reaction consisted of RNA from Sopa-stimulated cells of the spleen cats. Reverse primer(SEQ ID NO:52) was synthesized from 5’-end of the gene of feline CD80 and introduced EcoRI site. Direct primer(SEQ ID NO:53) was synthesized from the 3’-end of the gene of feline CD80, introduced a BamHI site at the 3’-end of the gene and was used for the reaction of reverse transcription and polymerase chain reaction. Fragment 3 is a SalI-s7181-subfragment EcoRI fragment E FHV, with approximately 2205 base pairs.

Homologous vector 930-23 A. Plasmid 930-23 A designed to integrate the foreign DNA into SPV. It includes marker gene β-galactosidase (lacZ) gene and the gene of feline CD80, flanked by SPV DNA. Above these alien gene is a DNA fragment of the SPV, with approximately 1484 base pairs. Following from these alien gene is a DNA fragment of the SPV, with approximately 2149 vapor core is there. Using this plasmid in accordance with the procedure described in the homologous recombination Procedure for generating recombinant SPV," received a virus containing DNA encoding these alien genes. It should be noted that the marker gene β-galactosidase (lacZ) is under the control of a synthetic late poxvirus promoter (LP1), and the gene of feline CD80 is under the control of the synthetic late/early poxvirus promoter (LP2EP2). This vector was constructed using standard techniques of recombinant DNA (Sambrook et al.) by joining restriction fragments from the following sources. This plasmid vector was obtained from restriction HindIII-BamHI fragment of plasmid pSP64, with approximately 2972 base pairs (Promega). Fragment 1 is a BglII restriction-AccI-subfragment HindIII restriction fragment M SPV, with approximately 1484 base pairs.

Fragment 2 is a restriction EcoRI-BamHI fragment synthesized by reverse transcription (RT) and polymerase chain reaction (PCR) using as template RNA from ConA-stimulated cells of the spleen cats. For the synthesis of feline CD80, primer(SEQ ID NO: 52) was synthesized from the 5’end of the gene of feline CD80 and introduced EcoRI site at the 5’-end of the g is on. Primer(SEQ ID NO: 53) was used for reverse transcription and PCR were synthesized with a 3’-end of the gene of feline CD80 and introduced a BamHI site at the 3’-end of the gene. The PCR product was restrictively enzymes EcoRI and BamHI to obtain a fragment length of approximately 879 base pairs, corresponding to the gene of feline CD80. Fragment 3 is a restriction BamHI-PvuII fragment of plasmid pJF751 with approximately 3010 base pairs (Ferrari et al). Fragment 4 is an AccI-HindIII-subfragment HindIII-fragment M SPV, with approximately 2149 base pairs. AccI sites of fragments 1 and 4 were transformed into a unique NotI sites using NotI-linkers.

Homologous vector 930-26. A1. Plasmid 930-26. A1 was designed in order to integrate the foreign DNA into SPV. It consisted of a marker gene β-galactosidase (lacZ) gene and the gene of feline CD28, flanked by SPV DNA. Above these alien genes were DNA fragment of SPV, consisting of approximately 1484 base pairs. Following from these alien genes were DNA fragment of SPV, consisting of approximately 2149 base pairs. Using plasmids in accordance with the procedure described in the homologous recombination Procedure for generating recombinant SPV," received a virus containing DNA encoding these alien genes. It should be noted that the marker gene ; -galactosidase (lacZ) is under the control of a synthetic late poxvirus promoter (LP1), and the gene of feline CD28 is under the control of the synthetic late/early poxvirus promoter (LP2EP2). This vector can be constructed using standard techniques of recombinant DNA (Sambrook et al.) by joining restriction fragments from the following sources. This plasmid vector was obtained from restriction HindIII-BamHI fragment of plasmid S64 with approximately 2972 base pairs (Promega). Fragment 1 is a BglII restriction-AccI-subfragment HindIII restriction fragment M SPV, with approximately 1484 base pairs. Fragment 2 is a restriction EcoRI-BamHI fragment synthesized by reverse transcription and polymerase chain reaction (PCR) using as template RNA from Con A-stimulated cells of the spleen cats. For the synthesis of feline CD28, from 5’-end of the gene of feline CD28 was synthesized primer 5’-GATGAATTCCATG-ATCCTCAGGCTGGGCTTCT-3’; 7/97 .1) (SEQ ID NO 54), which was introduced EcoRI site at the 5’end of this gene. For reverse transcription and PCR used primer (5’-GATCAGATCTCAGGAACGGTATGCCGCAA-3’; 7/97 .2) (SEQ ID NO 55), which was synthesized from the 3’-end of the gene of feline CD28 and introduced a BamHI site at the 3’-end of the gene. The PCR product was restrictively enzymes EcoRI and BamHI to obtain a fragment on the other approximately 666 base pairs, corresponding to the gene of feline CD28. Fragment 3 is a restriction BamHI-PvuII fragment of plasmid pJF751 with approximately 3010 base pairs (Ferrari et al). Fragment 4 is an AccI-HindIII-subfragment HindIII-fragment M SPV, with approximately 2149 base pairs. AccI sites of fragments 1 and 4 were transformed into a unique NotI sites using NotI-linkers.

Homologous vector 931-21 .Al. Homologous vector 931-21 .Al used in order to integrate the foreign DNA into SPV. It includes marker gene β-glucuronidase (uidA) gene and the gene of feline CD80, flanked by SPV DNA. Using this homology vector in accordance with the procedure described in the homologous recombination Procedure for generating recombinant SPV," received a virus containing DNA encoding these alien genes. It should be noted that the marker gene β-glucuronidase (uidA) is under the control of the synthetic early promoter poxvirus (ER), and the gene of feline CD80 is under the control of separate and unique synthetic late/early poxvirus promoter (LP2EP2). Homologous vector was constructed using standard techniques of recombinant DNA (Sambrook et al.) by joining restriction fragments from the following sources to the corresponding synthetic is Kim DNA sequences. This plasmid vector was obtained from DraI restriction fragment of plasmid pNEB193 (New England Biolabs), with approximately 2700 base pairs. Fragment 1 is a restriction Dral-EcoRI-subfragment HindIII fragment To the SPV, with approximately 881 couple of reasons. Fragment 2 is a restriction EcoRI-BamHI fragment of the gene of feline CD80, with approximately 879 base pairs and synthesized according to the procedure described in the section "Cloning by polymerase chain reaction". Template for the PCR reaction consisted of RNA from Sopa-stimulated cells of the spleen cats. Reverse primer1/97 .43) (SEQ ID NO 52) was synthesized from 5’-end of the gene of feline CD80 and introduced EcoRI site. Direct primer(SEQ ID NO:53) was synthesized from the 3’-end of the gene of feline CD80, introduced a BamHI site at the 3’-end of the gene, and was used for the reaction of reverse transcription and polymerase chain reaction. Fragment 3 is a restriction EcoRI-SmaI-fragment of plasmid pRAJ260 (Clonetech)with approximately 1823 base pairs. Fragment 4 is a restriction EcoRI-DraI-subfragment HindIII restriction fragment To the SPV, which has approximately 994 base pairs. EcoRI site in the homology vector SPV has turned into a unique NotI site using synthetic Linke the s.

Homologous vector 931-22 A. Plasmid 931-22. A1 was designed in order to integrate the foreign DNA into the RPV. This plasmid was introduced gene of feline CD80, flanked DNA RPV. Above these cwiertnia genes were DNA fragment RPV, consisting of approximately 906 base pairs. Below these cwiertnia genes were DNA fragment RPV, consisting of approximately 895 base pairs. Using plasmids in accordance with the procedure described in the homologous recombination Procedure for generating recombinant RPV," received a virus containing DNA encoding these alien genes. It should be noted that the gene of feline CD80 is under the control of the late/early promoter (LP2EP2). This vector was constructed using standard techniques of recombinant DNA (Sambrook et al.) by joining restriction fragments from the following sources with the synthetic DNA sequences. This plasmid vector was obtained from HindIII restriction fragment of plasmid pSP64 (Promega), with approximately 2999 base pairs. Fragment 1 is a restriction HindIII-XbaI-subfragment HindIII restriction fragment U RPV (Knight et al.), with approximately 906 base pairs. Fragment 2 is an EcoRI-BamHI fragment of the gene of feline CD80, with approximately 879 base pairs and synthesized in with the accordance with the procedure described in "Cloning by polymerase chain reaction". Template for the PCR reaction consisted of RNA from Sopa-stimulated cells of the spleen cats. Reverse primer(SEQ ID NO 52) was synthesized from 5’-end of the gene of feline CD80 and introduced EcoRI site. Direct primer(SEQ ID NO:53) was synthesized from the 3'-end of the gene of feline CD80, introduced a BamHI site at the 3’-end of the gene and was used for the reaction of reverse transcription and polymerase chain reaction. Fragment 3 is an XbaI-HindIII-subfragment HindIII fragment U RPV, with approximately 895 base pairs. XbaI sites in fragments 1 and 3 were transformed into a unique NotI sites using NotI-linkers. Synthetic DNA, located between parts 2 and 3, contains the promoter LP2EP2, EcoRI-BamHI site and a site for insertion of foreign DNA.

Homologous vector 931-32 A. Plasmid 931-32 A designed in order to integrate the foreign DNA into the RPV. It included the gene of feline CD80 and marker gene β-galactosidase (lacZ) gene, flanked DNA RPV. Above these alien genes were DNA fragment RPV, consisting of approximately 906 base pairs. Following from these alien genes were DNA fragment RPV, consisting of approximately 895 base pairs. Using plasmids in accordance with the procedure described in time is hardly "the homologous recombination Procedure for generating recombinant RPV", got a virus containing DNA encoding these alien genes. The vector was constructed using standard techniques of recombinant DNA (Sambrook et al.) by joining restriction fragments from the following sources with the synthetic DNA sequences. This plasmid vector was obtained from HindIII restriction fragment of plasmid pSP64 (Promega), with approximately 2999 base pairs. Fragment 1 is a restriction HindIII-XbaI-subfragment HindIII restriction fragment U RPV (Knight et al.), with approximately 906 base pairs. Fragment 2 is an EcoRI-BamHI fragment of the gene of feline CD80, with approximately 879 base pairs and synthesized according to the procedure described in the section "Cloning by polymerase chain reaction". Template for the PCR reaction consisted of RNA from Sopa-stimulated cells of the spleen cats. Reverse primer(SEQ ID NO 52) was synthesized from 5’-end of the gene of feline CD80 and introduced EcoRI site. Direct primer(SEQ ID NO:53) was synthesized from the 3’-end of the gene of feline CD80, introduced a BamHI site at the 3’-end of the gene and was used for reverse transcription and polymerase chain reaction. Fragment 3 is a restriction BamHI-PvuII fragment of plasmid pJFV751 (Ferrari et al), with approx the positive 3010 base pairs. Fragment 4 is a XbaI-HindIII-subfragment HindIII fragment U RPV, with approximately 895 base pairs. Xbal sites in fragments 1 and 4 were transformed into a unique NotI sites using NotI-linkers. Homologous vector 931-55 W.

Homologous vector 931-55 V used in order to integrate the foreign DNA into SPV. It includes marker gene β-glucuronidase (uidA) gene and the gene of feline IFN-γ (Onions, et al., (1996); Argyle et al., (1995), flanked by SPV DNA. Using this homology vector in accordance with the procedure described in the homologous recombination Procedure for generating recombinant SPV," received a virus containing DNA encoding these alien genes. It should be noted that the marker gene β-glucuronidase (uidA) is under the control of the synthetic early promoter poxvirus (ER), and the gene of feline IFN-γ is under the control of separate and unique synthetic late/early poxvirus promoter (LP2EP2). Homologous vector was constructed using standard techniques of recombinant DNA (Sambrook et al.) by joining restriction fragments from the following sources to the appropriate synthetic DNA sequences. This plasmid vector was obtained from DraI restriction fragment of plasmid pNEB193 (New England Biolabs), with approximately PR grounds. Fragment 1 is a restriction DraI-EcoRI-subfragment HindIII fragment To the SPV, with approximately 881 couple of reasons. Fragment 2 is a restriction EcoRI-BamHI fragment synthesized by reverse transcription and polymerase chain reaction (PCR) using as template RNA from Sopa-stimulated cells of the spleen cats. For the synthesis of feline IFN-γfrom the 5’end of the gene of feline IFN-γ synthesized primer 5’-TCGAGAATTCGATGAATTACACAAGTTTTATTTTCG-3’; 1/97 .4) (SEQ ID NO:81), which was introduced EcoRI site at the 5’end of this gene. For use in the reaction of reverse transcription and PCR of 3’-end of the gene of feline IFN-γ synthesized primer (5’-TCGAGGATCCTTATTTCGATGCTCTACGGCCTC-3’; 1/97 .3) (SEQ ID NO:82), which was introduced a BamHI site at the 3’-end of the gene. The PCR product was restrictively enzymes EcoRI and BamHI to obtain a fragment length of approximately 504, the base pairs corresponding to the gene of feline IFN-γ. Fragment 3 is a restriction EcoRI-SmaI-fragment of plasmid pRAJ260 (Clonetech)with approximately 1823 BP Fragment 4 is a restriction EcoRI-DraI-subfragment HindIII restriction fragment To the SPV, which has approximately 994 base pairs. EcoRI site in the homology vector SPV has turned into a unique NotI site using synthetic linkers.

Homologous vector 846-88 W. Plasmid 846-88 V intercept what was wireway to dellarovere the entire coding region from de herpes virus cats and for integration of foreign DNA. It consisted of a marker gene β-galactosidase (lacZ) gene, integrated in the website deletional region de FHV, flanked DNA HV. Plasmid 846-88 V contains a deletion in 1638 base pairs of a gene de from SmaI-SalI site in the fragment In FHV to the SalI site in the EcoRI-fragment E FHV. SmaI-SalI site in the fragment In FHV and SalI-EcoRI site in the fragment E FHV defines the end point of the deletion of the gene de. Above these alien genes were Asp718-Smal-subfragment SalI fragment In FHV, consisting of about 1415 base pairs and containing the complete coding sequence of a gene gI (370 amino acids). Following from these alien genes were Sll-s718-subfragment EcoRI fragment E FHV DNA, consisting of approximately 2205 base pairs and contains a unique short terminal repeat sequences. Using plasmids in accordance with the procedure described in the homologous recombination Procedure for generating recombinant RPV, SPV or FHV," received a virus containing DNA encoding this foreign gene. It should be noted that the gene lacZ gene is under the control of constitutive promoter de FHV. This plasmid was constructed using standard techniques of recombinant DNA (Sambrook et al.).

Homologous vector 921-65 W. Homologous vector 921-65 V designed to dellarovere gene 15L SPV (approximately 237 BP) and for integration of foreign DNA the SPV. It includes marker gene β-galactosidase (lacZ) gene and genes DBP/protease and envelope of the virus leukemia cats (FeLV), flanked by SPV DNA. Using this homology vector in accordance with the procedure described in the homologous recombination Procedure for generating recombinant RPV, SPV or FHV," received various viruses containing DNA encoding these alien genes. This vector was constructed using standard techniques of recombinant DNA (Sambrook et al.). It should be noted that the marker gene β-galactosidase (lacZ) is under the control of a constitutive late poxvirus promoter (15L), and genes DBP/protease FeLV and shell FeLV are under separate control of the synthetic early promoter of poxvirus ER and ER, respectively. The sequence SPV flanking interturbine alien genes were derived from the genome of 3.2 KBP HindIII fragment N. Above from the sequence of foreign genes is 903 BP fragment containing part of the gene 14L SPV, and below from the sequence of foreign genes is 966 BP fragment containing part of the gene 16L SPV. Gene lacZ gene and open reading frames shell FeLV and gag/protease FeLV are in the same orientation relative to genes 16L SPV and 14L SPV.

Homologous vector 942-03 S. Plasmid 942-03 S designed to dellarovere part of Cody the respective area de of the herpes virus cats and for embedding three foreign genes in the website deletirovanie de. It includes the gene of feline CD80 (~879 BP), gene DBP/FIV protease (~1800 BP) and gene shell FIV (~2600 BP), flanked DNA FHV. The gene of feline CD80 is under control of the promoter de FHV, gene DBP/FIV protease is under control of the promoter DF of pseudoreligious and gene shell FIV is under control pretannage promoter of the gene of cytomegalovirus. Above these alien genes is Asp718-SmaI-subfragment SalI fragment In FHV, consisting of approximately 1415 base pairs. Following from these alien genes is SalI-Asp718 subfragment EcoRI fragment E FHV, consisting of approximately 2205 base pairs and contains a unique short terminal repeat sequences. Using plasmids in accordance with the procedure described in the homologous recombination Procedure for generating recombinant RPV, SPV or FHV," received a virus containing DNA encoding this foreign gene. This plasmid with homologous sequence, 942-03 S, was constructed using standard techniques of recombinant DNA (Sambrook et al.).

Examples

Example 1A

The cDNA cloning of feline CD80 (B7-1)-TMAU, CD80 (B7-1)-SPAH, CD86 (B7-2), CD28 and CTLA-4:

cDNA feline CD80 (B7-1), CD86 (B7-2), CD28 and CTLA-4 were cloned by the first reaction RT-PCR (polymerase chain reaction with reverse transcriptase), amplificating the area between the last two is dovalidate, which were sufficiently conservative to ensure that it was possible to design degenerate primers that interact with the cat mRNA. The source of mRNA were mononuclear cells of peripheral blood (MCPC) or splenocytes, stimulated And Con, at least for 16 hours. This PCR product sequenced. This sequence was used to design primers for RACE (rapid amplification of cDNA ends)-PCR. 5’-end of the amplified first by obtaining cDNA downstream primer, complementary to the newly sequenced conservative area. Oligonucleotide ligated with the 3’-end of the cDNA (complementary to the 5’-end of the mRNA). This sequence served as a binding site for the reverse primer, which is a PCR-compatible with direct PCR primer corresponding to a different area in the newly sequenced region. Degenerate primers were used in multiple cycles of "nesting" reactions with 3’-end. This reverse primer for PCR were designed in such a way that it reacted with the sequence in the newly sequenced region. The products sequenced either by the direct method, or they were cloned into a cloning vector and sequenced from plasmid. The entire open-reading frames in its entirety Clonie is ovali by amplification by PCR using primers designed from the known sequences. These ORFS were cloned and sequenced three times. ORS B7-1 was subcloned into the plasmid pSI using the SV40 promoter in the plasmid SFV. Plasmid pSI was used to establish the functional interaction of B7-1 with a feline CD28.

For RT/PCR cDNA feline CD80 (B7-1) used DNA primers:

5’-primer: 5’-CGCGGATCCGCACCATGGGTCACGCAGCAAAGTGGAAAAC-3’; (SEQ ID NO. 11)

3’-primer: 5’-CCTAGTAGAGAAGAGCTAAAGAGGC-3’ (SEQ ID NO. 12) (see above for the complete list of primers for cDNA feline CD80).

For RT/PCR cDNA feline CD28 used DNA primers:

5’-primer: 5’-CGCGGATCCACCGGTAGCACAATGATCCTCAGG-3’; (SEQ ID NO. 13)

3’-primer: 5’-CGCGGATCCTCTGGATAGGGGTCCATGTCAG-3’ (SEQ ID NO.14) (see above for the complete list of primers for cDNA feline CD28). For RT/PCR cDNA feline CTLA-4 used DNA primers:

1. Degenerate primers for the first PCR product (672 BP)

War’-primer: 5’-ATGGCTT (C) GCCTTGGATTT (C) CAGC (A) GG-3’; (SEQ ID NO. 15)

War’-primer: 5’-TCAATTG (A) ATG (A) GGAATAAAATAAGGCTG-3’ (SEQ ID NO.16).

2. 5’-end of CTLA-4 (455 BP): Degenerate gene-specific (GSP) and "nesting" gene-specific (NGSP) primers:

The first round PCR:

War’-primer: 5’-TGTTGGGTTTC (T) G (A) CTCTG (A) CTT (C) CCTG-3’; (SEQ ID NO. 17)

3’-S-primer: 5’-GCATAGTAGGGTGGTGGGTACATG-3’ (SEQ ID NO.18). "Nested" PCR using the PCR product from the first round:

War’-primer: 5’-TGTTGGGTTTC (T) G (A) CTCTG (A) CTT (C) CCTG 3'; (SEQ ID NO. 19)

3’-NGS-Prime time is: 5’-ACATGAGCTCCACCTTGCAG-3’ (SEQ ID NO.20).

3. 3’-end of the CTLA-4: adaptery primer 1 (API, Clontech, Lab. Inc., Palo Alto, CA); "nesting" adaptery primer (AR, Clontech, Lab.), gene-specific primer (GSP) and "nested gene-specific primer (NGSP):

3’- RACE-PCR:

AP1: 5’-CCATCCTAATACGACTCACTATAGGGC-3’ (SEQ ID NO 21)

5’-GSP: 5’-GTGAATATGGGTCTTCAGGCAATG-3’ (SEQ ID NO 22)

3’ nested RACE-PCR using the product 3’- RACE-PCR: A: 5’-ACTCACTATAGGGCTCGAGCGGC-3’ (SEQ ID NO 23)

5’-NGSP: 5’-GAAATCCGAGTGACTGTGCTGAG-3’ (SEQ ID NO 24)

4. Primers for a gene CTLA-4

.CTLA-4 5’-primer: 5’-AACCTGAACACTGCTCCCATAAAG-3’ (SEQ ID NO 25)

.CTLA-4 3’-primer: 5’-GCCTCAGCTCTTAGAAATTGGACAG-3’ (SEQ ID NO 26).

For RT/PCR cDNA feline CD86 (B7-2) used DNA primers:

1. Degenerate primers for the first PCR product (423 BP)

Exp. 5’-primer: 5’-TAGTATTTTGGCAGGACCAGG-3’; (SEQ ID NO. 27)

Exp. 3’-primer: 5’-CTGTGACATTATCTTGAGATTTC-3’ (SEQ ID NO.28).

2. Degenerate primers for the second PCR product (574 BP)

Exp. 5’-primer: 5’-GA (G) CA (T) GCACT (A) ATGGGACTGAG-3’; (SEQ ID NO. 29)

Exp. 3’-primer: 5’-CTGTGACATTATCTTGAGATTTC-3’ (SEQ ID NO. 30).

3. the 5’-end CD86: API, AP2 (Clontech, Lab.) degenerate 3’gene-specific (GSP) and 3’-nested gene-specific (NGSP) primers:

5’- RACE-PCR:

API: 5’-CCATCCTAATACGACTCACTATAGGGC-3’ (SEQ ID NO 31)

3’-GSP: 5’-TGGGTAACCTTGTATAGATGAGCAGGTC-3’ (SEQ ID NO 32)

"Jack" 5’-R-PCR using the PCR product 5’-RACE:

AP2: 5’-ACTCACTATAGGGCTCGAGCGGC-3’ (SEQ ID NO 33)

3’-NGSP: 5’-CAGGTTGACTGAAGTTAGCAAGCAC-3’ (SEQ ID NO 34).

4. 3’-end V7-2: API, AP2, 5’-GSP and 5’-NGSP:

3’-RACE-PCR:

API: 5’-CCATCCTAATACGACTCACTATAGGGC-3’ (SEQ ID NO 35)

5’-GSP: 5’-GGACAAGGGCACATATCACTGTTTC-3’ (SEQ ID NO 36)

Nested 3’-RACE-PCR using the PCR product from the 3’-RACE:

AP2: 5’-ACTCACTATAGGGCTCGAGCGGC-3’ (SEQ ID NO 37)

5’-NGSP: 5’-CAGTGCTTGCTAACTTCAGTCAACC-3’ (SEQ ID NO 38).

The entire gene CD86:

Kosh. V7 2(1) 5’-primer: 5’-CGGGAATGTCACTGAGCTTATAG-3’ (SEQ ID NO 39)

cosv(1176) 3’-primer: 5’-GATCTTTTTCAGGTTAGCAGGGG-3’ (SEQ ID NO 40).

Example 1V

Cloning CD80 (B7-1)-Syntro/SPAH; Plasmid 917-19-8/16

Cats were isolated spleen cells and were cultured with con-canavalin And within 5 hours. Cells were besieged, washed with PBS and used for selection of full-sized RNA (Qiagen RNeasy Total RNA System). Whole RNA was treated with Dnazol 1 (Boehringer Mannhiem) to remove DNA contaminants from RNA preparations. Then from these drugs were extracted information RNA using spheres of oligotex Qiagen (Santa Clara, CA) and fast speakers. Of mRNA was produced copy of the DNA in the presence of randomized hexamers, dNTP, Rcasino, reverse transcriptase (Promega) and buffer for reverse transcriptase (Promega) and incubated for 30 minutes at 42°C. Then PCR was performed to generate double-stranded full-size cDNA clone open reading frame (ORF) of feline B7-1 using sense primer 5/97 .50 (5’-ATGGGTCACGCAGCAAAGTG-3’ (SEQ ID NO 41) and antisense primer 5/97 .51 (5’-CTATGTAGACAGGTGAGATC-3’) (SEQ ID NO 42), dNTP, cDNA B7-1 (1st circuit), MgSO4, Vent polymerase (BRL) and puff the RA Vent polymerase (BRL). PCR was performed under the following conditions: 1 cycle at 94°sec, 15 sec, 35 cycles at 94°C, 30 sec at 48°C, 2 min at 72°C, 2 min; 1 cycle at 72°C, 10 min, the PCR mixture was subjected to electrophoresis on 1% low-melting agarose gel, and DNA fragments corresponding to the expected size of the LFS B7-1, was isolated, subjected to gel purification using a set of for gel purification (Qiagen''s Gel Purification kit, Santa Clara, CA) and cloned into the plasmid vector pCR-BLUNT using reagents from the kit for PCR cloning (Invitrogen's Zero Blunt PCR Cloning kit, San Diego, CA). DNA extracted from bacterial colonies resistant to kanamycin, was previously skanirovali on the presence of unique Nhel site (contained in feline CD80 (B7-1)-TAMU). Inserts, which have a size of 800-900 BP and contained a Nhel site, sequenced using protocols and equipment for automated fluorescent sequencing ABI (Perkin-Elmer-Cetus; Applied Biosyntems, Inc.). To generate the DNA sequence of primers pCR-Blunt were used gene-specific primers for plasmid vector and B7-1, derived from the previously cloned gene B7-1, namely: 1/97 .36 (5’-CAGGAAACAGCTATGAC-3’); (SEQ ID NO: 43) and 1/97 .37 (5’-AATACGACTCACTATAGG-3’); (SEQ ID NO: 44). Primers specific to the gene of B7-1, are:

12/96 .22 5’-ASSETTRADEX-3’ (SEQ ID NO: 45);

1/97 .33 5’-ATACAAGTGTATTTGCCATTGTC-3’ (SEQ ID NO: 46);

12/96 .20 5’-AGCTCTGACCAATAACATC-3’ (SEQ ID NO: 47);

12/96 .21 5’-ATTAGAAATCCAGTTCACTGCT-3’ (SEQ ID NO: 48);

1/97 .32 5’-TCATGTCTGGCAAAGTACAAG-3’ (SEQ ID NO: 49);

11/96 .32 5’-ATTCACTGACGTCACCGA-3’ (SEQ ID NO: 50);

11/96 .31 5’-AAGGCTGTGGCTCTGA-3’ (SEQ ID NO: 51).

It was determined that two clones contain the full sequence of CD80, corresponding to the original sequence CD80, except for the 2 point mutations of DNA. One such point mutation did not affect the amino acid sequence. The second mutation has led to the replacement of the amino acid leucine to isoleucine. The resulting clone of feline CD80 meant 917-19.8/16. (CD80-Syntro/SPAH).

Example 2

S-SPV-229

S-SPV-229 Wallpaper is the smallpox virus of pigs, which exprcssion at least two foreign gene. Gene β-galactosidase gene (LacZ) gene and the feline CD80 was built in Ass website SPV in larger BglII-HindIII-subfragment genomic HindIII fragment M SPV (a unique NotI restriction site has been replaced by a unique AccI restriction site). Gene LacZ is under the control of the synthetic late promoter (LP1), and the gene of feline CD80 is under the control of the synthetic late/early promoter (LP2EP2).

S-SPV-229 was obtained from S-SPV-001 (strain Kasza). This procedure was performed using homologous vector 930-23 A (see "Materials and methods) and virus S-SPV-001, as described in the homologous recombination Procedure for generating recombinant SPV". The source material for the respectiv was skanirovali, as described in the section "Screening for recombinant virus SPV expressing β-galactosidase (analyses BLUOGAL and CPRG)". In the treatment of red plaques received the recombinant virus designated S-SPV-229. This virus was analyzed for the expression of β-galactosidase, purity and stability of the insert through multiple subcultures, traceable through the analysis on the formation of blue plaques and analysis on the formation of black plaques, as described in "Materials and methods". After the initial three cycles of cleaning all observed plaques had a blue color, indicating that this virus was clean, stable and expressed β-galactosidase (U.S. patent 5382425, which is introduced into the present description by reference).

S-SPV-229 analyzed for the expression of antigens that are specific for β-galactosidase using the procedure described in the section "Screening of black plaques on the expression of a foreign gene in recombinant SPV". It was shown that monoclonal antibody against β-galactosidase specifically react with plaques S-SPV-229 and does not react with the negative control plaques S-SPV-001. All observed plaques S-SPV-229 reacted with a monoclonal antibody against β-galactosidase, which indicates that the virus was stably expresses the foreign gene β-galactosidase. Description is here by analyses were carried out on cells, ESK-4, that indicates that the cells ESK-4 must be a suitable substrate for the production of recombinant SPV-vaccines.

S-SPV-229 analyzed for the expression of antigens that are specific for feline CD80 using the procedure described in the section "Screening for the expression of feline CD80 (B7-1) and CD86 (B7-2) in recombinant SPV, PRV or FHV using analyses on the formation of black plaques". It was shown that chimeric antibody against CTLA-4/Fc man specifically reacts with plaques S-SPV-229 (expressing the feline CD80) and does not react with the negative control plaques S-SPV-001. It was shown that all observed plaques S-SPV-229 was repairable with chimeric antibody against CTLA-4/Fc man, that indicates that the virus was stably expresses the foreign gene of feline CD80.

For confirming the expression product of the gene of feline CD80 cells were infected with S-SPV-229, and samples of infected cell lysates were subjected to electrophoresis in polyacrylamide gel with LTOs. This gel was blokirovala and analyzed as described in the Procedure section Western blotting".

S-SPV-229 was used as vaccines against diseases in cats.

S-SPV-229, when it is used alone or in combination with FIV-and FeLV-, FIP-vaccines or other feline vaccines, contributes to the effectiveness of the vaccine against FIV, FeLV, FIP, or other diseases of cats. S-SPV-22 can also be used for expression of the polypeptide of feline CD80. Cell lysate S-SPV-229-inficirovannyh cells were injected by injection to mice or rabbits to produce them polyclonal monospecific antibodies against feline CD80.

Example 3

S-SPV-230

S-SPV-230 represents the smallpox virus of pigs, which expresses at least two foreign gene. Gene β-galactosidase gene (LacZ) and the gene of feline CD28 have been built into the AccI site-SPV in larger Bg1II-HindIII-subfragment genomic HindIII fragment M SPV (a unique NotI restriction site has been replaced by a unique AccI restriction site). Gene LacZ is under the control of the synthetic late promoter (LP1), and the gene of feline CD28 is under the control of the synthetic late/early promoter (LP2EP2).

S-SPV-230 received from S-SPV-001 (strain Kasza). This procedure was performed using homologous vector 930-26 A (see "Materials and methods) and virus S-SPV-001, as described in the homologous recombination Procedure for generating recombinant SPV". The source material for transfection was skanirovali as described in the section "Screening for recombinant virus SPV expressing β-galactosidase (analyses BLUOGAL and CPRG)". In the treatment of red plaques received the recombinant virus designated S-SPV-230. This virus was analyzed for the expression of β-galactosidase, purity and stability of the insert by multiple the transfers, traced through the analysis on the formation of blue plaques, as described in "Materials and methods". After the initial three cycles of cleaning all observed plaques had a blue color, indicating that this virus was clean, stable and expressed the foreign gene.

S-SPV-230 analyzed for the expression of antigens that are specific for feline CD28, using the procedure described in the section "Screening of black plaques on the expression of a foreign gene in recombinant SPV". This analysis was performed on cells, ESK-4, indicating that cells ESK-4 must be a suitable substrate for the production of recombinant SPV-vaccines.

To confirm the expression product of the gene of feline CD28 cells were infected with S-SPV-230, and samples of infected cell lysates were subjected to electrophoresis in polyacrylamide gel with LTOs. This gel was blokirovala and analyzed as described in the Procedure section Western blotting".

S-SPV-230 used as vaccines against diseases in cats. S-SPV-230, when it is used alone or in combination with FIV-and FeLV-, FIP-vaccines or other feline vaccines, contributes to the effectiveness of the vaccine against FIV, FeLV, FIP, or other diseases of cats. S-SPV-230 can also be used for expression of the polypeptide feline CD28. Cell lysate S-SPV-229-inficon the bathrooms cells were injected by injection to mice or rabbits to produce them polyclonal, monospecific antibodies against feline CD28.

Example 4

S-SPV-225

S-SPV-225 represents the smallpox virus of pigs, which expresses at least two foreign gene. Gene β-galactosidase gene (LacZ) and the gene of feline interferon-γ (feline IFN-γ) have been built into the AccI site of SPV in larger BglII-HindIII-subfragment genomic HindIII fragment M SPV (a unique NotI restriction site has been replaced by a unique AccI restriction site). Gene LacZ is under the control of the promoter of smallpox pigs (O1L), and the gene of feline IFN-γ is under the control of the synthetic late/early promoter (LP2EP2).

S-SPV-225 received from S-SPV-001 (strain Kasza). This procedure was performed using homologous vector 917-60 V (see "Materials and methods) and virus S-SPV-001, as described in the homologous recombination Procedure for generating recombinant SPV". The source material for transfection was skanirovali as described in the section "Screening for recombinant virus SPV expressing β-galactosidase (analyses BLUOGAL and CPRG)". In the treatment of red plaques received the recombinant virus designated S-SPV-225. This virus was analyzed for the expression of β-galactosidase, purity and stability of the insert through multiple subcultures, traceable through the analysis on the formation of blue plaques, as described in section the Le "Materials and methods". After the initial three cycles of cleaning all observed plaques had a blue color, indicating that this virus was clean, stable and expressed the foreign gene.

S-SPV-225 analyzed for the expression of antigens that are specific for feline IFN-γusing the procedure described in the section "Screening of black plaques on the expression of a foreign gene in recombinant SPV". This analysis was performed on cells, ESK-4, indicating that cells ESK-4 must be a suitable substrate for the production of recombinant SPV-vaccines.

To confirm the expression product of the gene of feline IFN-γ cells were infected with S-SPV-225, and samples of infected cell lysates were subjected to electrophoresis in polyacrylamide gel with LTOs. This gel was blokirovala and analyzed as described in the Procedure section Western blotting".

S-SPV-225 analyzed for the expression of biologically active feline IFN-γ using the procedure described in the section "Screening for biological activity of feline interferon-gamma expressed from recombinant SPV, RPV or FHV using analyses to reduce the number of plaques formed under the action of VSV".

S-SPV-225 was used as vaccines against diseases in cats. S-SPV-225, when it is used alone or in combination with FIV-and FeLV-, FIP vaccines is or with other feline vaccines contributes to the effectiveness of the vaccine against FIV, FeLV, FIP, or other diseases of cats.

Example 5

S-SPV-200

S-SPV-200 represents the smallpox virus of pigs, which expresses three foreign gene. Gene DBP/protease of human immunodeficiency virus in cats (FIV), gene shell FIV (full-size) and gene β-galactosidase gene (lacZ) was built into the unique NotI restriction site (NotI-linkers, built in a unique AccI restriction site SPV in OPC 01L HindIII-fragment M SPV). Genes DBP/protease and shell FIV are controlled by separate but identical synthetic late/early promoter (LP2EP2). Gene LacZ is under the control of the synthetic late promoter (LP1).

S-SPV-200 were obtained from S-SPV-001 (strain Kasza). This procedure was performed using homologous vector 9.04-V and virus S-SPV-001, as described in the homologous recombination Procedure for generating recombinant SPV". The source material for transfection was skanirovali, as described in the section "Screening for recombinant virus SPV expressing β-galactosidase (analyses BLUOGAL and CPRG and screening for recombinant herpesvirus expressing enzymatic marker genes)". In the treatment of red plaques received the recombinant virus designated S-SPV-157. This virus was analyzed for the expression of β-galactosidase using the analysis described in the section "Materials and methods". Analysis of purity and stabiles insert after 5 transfers carried out by the detection of FIV gag and β-galactosidase in the analysis on the formation of black plaques and detection gag and shell FIV in Western blot analysis.

S-SPV-200 is a recombinant pox virus swine expressing proteins gag/protease FIV and shell FIV can be used as a vaccine against FIV infection in cats. S-SPV-200 can also be used for expression of proteins gag/protease and shell FIV.

Example 6

S-SPV-233

S-SPV-233 represents the smallpox virus of pigs, which expresses five foreign genes: FIV gag, env FIV, feline CD80, E.coli lacZ and uidA gene. Full gene of feline CD80 gene β-glucuronidase E. coli (uidA) was built into the unique NotI restriction site (Notl-linkers that are built into the unique EcoRI restriction site approximately 3.2 KBP region (SEQ ID NO) of 6,7-KBP-HindIII fragment To the SPV). Genes gag/protease virus immunodeficita cats (FIV) and shell FIV (full-size) and gene β-galactosidase of E. coli have been built into the unique NotI restriction site (NotI-linkers, built in a unique AccI restriction site SPV in OPC 01L HindIII-fragment M SPV). Gene CD80 is under the control of the synthetic late/early promoter (LP2EP2), and the uidA gene is under the control of a separate, unique synthetic early promoter (ER). Genes gag/protease and Obolo the key FIV are under the control of the individual, but identical synthetic late/early promoter (LP2EP2). Gene lacZ is under the control of the synthetic late promoter (LP1). (International PCT application WO 96/22363, which is introduced in the present description by reference).

S-SPV-233 was obtained from S-SPV-200 (containing FIV gag, shell FIV and E.coli lacZ). This procedure was performed using homologous vector 931-21 A and virus S-SPV-200, as described in the homologous recombination Procedure for generating recombinant SPV". The source material for transfection was skanirovali, as described in the section "Screening for recombinant virus SPV expressing β-glucuronidase (X-gLUC and screening for recombinant herpesvirus expressing enzymatic marker genes)". In the final treatment blue/green plaques were obtained recombinant virus designated S-SPV-233.

S-SPV-233 analyzed for the expression of antigens that are specific for FIV gag, env FIV and feline CD80, using the procedure described in the section "Screening of black plaques on the expression of a foreign gene in recombinant SPV". This analysis was performed on cells, ESK-4, which indicates that cells, ESK-4 must be a suitable substrate for the production of recombinant SPV-vaccines.

S-SPV-233 analyzed for the expression of antigens that are specific for feline CD80 using pried the market, described in the section "Screening for the expression of feline CD80 (B7-1) and feline CD86 (B7-2) in recombinant SPV, RPV or FHV using analyses on the formation of black plaques". It was shown that chimeric antibody against CTLA-4/Fc man specifically reacts with plaques S-SPV-233 (expressing the feline CD80) and does not react with the negative control plaques S-SPV-001. It was shown that all observed plaques S-SPV-233 reacted with chimeric antibody against CTLA-4/Fc man, that indicates that the virus was stably expresses the foreign gene of feline CD80.

To confirm the expression of the gene product FIV gag, env FIV and feline CD80, cells were infected with S-SPV-233, and samples of infected cell lysates were subjected to electrophoresis in polyacrylamide gel with LTOs. This gel was blokirovala and analyzed as described in the Procedure section Western blotting".

S-SPV-233 is a smallpox pigs expressing proteins gag/protease FIV and shell FIV, and can be used as a vaccine against FIV infection in cats. S-SPV-233 can also be used for expression of proteins gag/protease and shell FIV.

Example 7

S-SPV-235

S-SPV-235 represents the smallpox virus of pigs, which expresses five foreign genes: gag FIV, FIV env, cat IFN-γ, E.coli lacZ and uidA gene. Full gene of feline ifn-γ gene β-glucurono the basics of E. coli (uidA) was built into the unique NotI restriction site (NotI-linkers, built in the unique EcoRI restriction site approximately 3.2 KBP region (SEQ ID NO) of 6,7-KBP-HindIII fragment To the SPV). Genes gag/protease virus immunodeficita cats (FIV) and shell FIV (full-size) and gene β-galactosidase gene (lacZ) was built into the unique NotI restriction site (NotI-linkers, built in a unique AccI restriction site SPV in OPC 01L HindIII-fragment M SPV). Gene IFN-γ is under the control of the synthetic late/early promoter (LP2EP2), and the uidA gene is under the control of a separate, unique synthetic early promoter (ER). Genes DBP/protease and shell FIV are controlled by separate but identical synthetic late/early promoter (LP2EP2). Gene lacZ is under the control of the synthetic late promoter (LP1).

S-SPV-235 was obtained from S-SPV-200 (containing genes FIV gag, shell FIV and E.coli lacZ). This procedure was performed using homologous vector 931-55 V and virus S-SPV-200, as described in the homologous recombination Procedure for generating recombinant SPV". The source material for transfection was skanirovali as described in the section "Screening for recombinant virus SPV expressing β-glucuronidase (X-GLUC and screening for recombinant herpesvirus expressing enzymatic marker genes)". In the clean blue/green plaques received recombinantively, designated S-SPV-235.

S-SPV-235 analyzed for the expression of antigens that are specific for FIV gag, env FIV and feline IFN-γ using the procedure described in the section "Screening of black plaques on the expression of a foreign gene in recombinant SPV". This analysis was performed on cells, ESK-4, which indicates that cells, ESK-4 must be a suitable substrate for the production of recombinant SPV-vaccines.

S-SPV-235 analyzed for the expression of biologically active feline IFN-γ using the procedure described in the section "Screening for the expression of biological activity of feline interferon-gamma expressed from recombinant SPV, RPV or FHV using analyses to reduce the number of plaques formed under the action of VSV".

To confirm the expression of the gene product FIV gag, env FIV and feline IFN-γ cells were infected with S-SPV-235, and samples of infected cell lysates were subjected to electrophoresis in polyacrylamide gel with LTOs. This gel was blokirovala and analyzed as described in the Procedure section Western blotting".

S-SPV-235 is a recombinant pox virus swine expressing proteins gag/protease FIV and proteins shell FIV, and can be used as a vaccine against FIV infection in cats. S-SPV-235 can also be used for expression of proteins gag/protease and about the glasses FIV.

Example 8:

S-SPV-224

S-SPV-224 represents the smallpox virus of pigs, which expresses three foreign gene. Gene gag/protease leukosis virus cats (FeLV), gene shell FeLV (full-size) and the lacZ gene of E. coli was built into the website deletirovanie 15L SPV, originating from incomplete genome 1869 BP-HindIII-fragment of the n Gene gag/protease FeLV is under the control of the synthetic early promoter poxvirus (ER). Gene shell FeLV is under the control of the synthetic early promoter poxvirus (ER). Gene lacZ is under the control of a constitutive late promoter 15LP SPV.

S-SPV-224 was obtained from S-SPV-001 (strain Kasza). This procedure was performed using homologous vector 921-65 V and virus S-SPV-001, as described in the homologous recombination Procedure for generating recombinant RPV, SPV or FHV". The source material for transfection was skanirovali, as described in the section "Screening for recombinant virus SPV expressing β-galactosidase (analyses BLUOGAL and CPRG) or β-glucuronidase (analysis of X-GLUC)". In the treatment of red plaques received the recombinant virus designated S-SPV-224. This virus was analyzed for the expression of β-galactosidase by analysis of blue plaques, as described in "Materials and methods".

S-SPV-224 analyzed for the expression of proteins DBP/protease FeLV, FeLV env and β-galactosidase used with the em procedure, described in the section "Screening of black plaques on the expression of a foreign gene in recombinant RPV, SPV or FHV using analysis on the formation of black plaques". This analysis was performed on cells, ESK-4, which indicates that cells, ESK-4 must be a suitable substrate for the production of recombinant SPV-vaccines. August 14, 1998.

To confirm the expression of the product gene DBP/protease FeLV and FeLV env cells were infected with S-SPV-224, and samples of infected cell lysates were subjected to electrophoresis in polyacrylamide gel with LTOs. This gel was blokirovala and analyzed as described in the Procedure section Western blotting".

S-SPV-224 is a recombinant pox virus swine expressing proteins DBP/protease FeLV and shell FeLV, and can be used as vaccines against FeLV infection in cats. S-SPV-224 can also be used for expression of proteins gag/protease and shell FeLV.

Example 9:

S-SPV-246

S-SPV-246 represents the smallpox virus of pigs, which expresses five foreign genes: gag/protease FeLV, env FeLV, feline CD80, E.coli lacZ and uidA gene. Full gene of feline CD80 gene β-glucuronidase E. coli (uidA) was built into the unique NotI restriction site (NotI-linker that is built into a unique EcoRI restriction site approximately 3.2 KBP region of 6,7-KBP-HindIII fragment To the SPV). Gene CD80 n is located under the control of the synthetic late/early promoter (LP2EP2), and uidA gene is under the control of the synthetic early promoter poxvirus, ER. Gene gag/protease FeLV, gene shell FeLV (full-size) and gene β-galactosidase gene (lacZ) was built into the website deletirovanie 15L SPV, originating from incomplete genome 1869 ..HindIII-fragment of the n Gene gag/protease is under the control of the synthetic early promoter poxvirus, ER. Gene shell FeLV is under the control of the synthetic early promoter of poxvirus ER. Gene lacZ is under the control of a constitutive late poxvirus promoter 15L.(International PCT application WO 96/22263, which is introduced in this description by reference).

S-SPV-246 was obtained from S-SPV-224 (containing genes gag/protease FeLV, shell FeLV and E.coli lacZ in the website deletirovanie 15L genomic incomplete 1869 KBP-HindIII fragment (N). This was performed using homologous vector 931-21 A and virus S-SPV-224, as described in the homologous recombination Procedure for generating recombinant RPV, SPV or FHV". The source material for transfection was skanirovali, as described in the section "Screening for recombinant viruses RPV, SPV or FHV expressing β-galactosidase (analyses BLUOGAL and CPRG) or β-glucuronidase (analysis of X-GLUC)". In the clean blue/green plaques were obtained recombinant virus designated S-SPV-246.

S-SPV-246 analyzed for the expression of proteins gag/p is ateasy FeLV and shell FeLV using procedures described in the section "Screening of black plaques on the expression of a foreign gene in recombinant RPV, SPV or FHV using analysis on the formation of black plaques". This analysis was performed on cells, ESK-4, which indicates that cells, ESK-4 must be a suitable substrate for the production of recombinant SPV-vaccines.

S-SPV-246 analyzed for the expression of antigen specific for feline CD80 using the procedure described in the section "Screening for the expression of feline CD80 (B7-1) and CD86 (B7-2) in recombinant SPV, RPV or FHV using analyses on the formation of black plaques". It was shown that chimeric antibody against CTLA-4/Fc man specifically reacts with plaques S-SPV-246 (expressing the feline CD80) and does not react with the negative control plaques S-SPV-001. It was shown that all observed plaques S-SPV-246 react with chimeric antibody against CTLA-4/Fc man, that indicates that the virus was stably expresses the foreign gene of feline CD80.

To confirm the expression product of the genes gag/protease FeLV, shell FeLV and feline CD80, cells were infected with S-SPV-246, and samples of infected cell lysates were subjected to electrophoresis in polyacrylamide gel with LTOs. This gel was blokirovala and analyzed as described in the Procedure section Western blotting".

S-SPV-246 the submitted is a recombinant pox virus swine, expressing the proteins gag/protease FeLV and shell FeLV, and can be used as vaccines against FeLV infection in cats. S-SPV-246 can also be used for expression of proteins gag/protease and shell FeLV.

Example 10

Additional examples of recombinant pox virus swine, which can be used as a vaccine against infection with human immunodeficiency virus in cats (FIV)is a virus leukemia cats (FeLV) or virus feline infectious peritonitis (FIP)are:

Recombinant pox virus swine, which expresses five foreign genes. The env gene FIV is under the control of the synthetic early promoter poxvirus, ER, gene gag/protease FIV is under the control of the synthetic early promoter of poxvirus ER, gene lacZ gene is under the control of the promoter of smallpox pigs 15L, the gene of feline CD80 is under the control of the synthetic late/early promoter LP2EP2, gene uidA gene is under the control of the synthetic early promoter of poxvirus ER. Genes shell FIV, gag/protease FIV and E.coli lacZ localized in another non-core insertion site SPV different from the sites of insertion of the gene of feline CD80 and uidA gene of E. coli.

Recombinant pox virus swine, which expresses five foreign genes. The env gene FIV is under the control of the early promoter of poxvirus, ER, gene gag/protease FI is under the control of the synthetic early promoter poxvirus, ER, gene lacZ gene is under the control of the promoter of smallpox pigs 15L, gene feline CD86 is under the control of the synthetic late/early promoter LP2EP2, gene uidA gene is under the control of the synthetic early promoter of poxvirus ER. Genes shell FIV, gag/protease FIV and E.coli lacZ localized in another not-primarily insertion site SPV different from the sites of insertion of the gene of feline CD86 gene and E. coli uidA.

Recombinant pox virus swine expresses two foreign gene. Gene feline CD86 is under the control of the synthetic late/early poxvirus promoter LP2EP2; gene uidA gene is under the control of the synthetic early promoter of poxvirus ER. This virus is used alone or in combination with other recombinant proteins or vaccine.

Other examples of recombinant swine poxvirus used for the production of vaccines against diseases caused by FeLV can serve as viruses, similar to that described above, except that they FIV gene was replaced by the corresponding FeLV-specific genes.

Other examples of recombinant pox viruses of pigs used to obtain proteins for use as vaccines for the production and purification of polyclonal antibodies, are:

Recombinant pox virus swine, which expresses one cogert the th gene. The gene of feline CD80, which lacks the transmembrane domain, is under the control of the synthetic late/early poxvirus promoter LP2EP2. Alternatively, the gene of feline CD80, which lacks the transmembrane domain has a his-tag hybrid tag at the carboxy-end to ensure purification on a Nickel affinity column.

Recombinant pox virus swine, which expresses one foreign gene. Gene feline CD28, which lacks the transmembrane domain, is under the control of the synthetic late/early poxvirus promoter LP2EP2. Alternatively, the gene of feline CD28, which lacks the transmembrane domain has a his-tag hybrid tag at the carboxy-end to ensure purification on a Nickel affinity column.

Recombinant pox virus swine, which expresses one foreign gene. Gene feline CD86, which outstay transmembrane domain is under the control of the synthetic late/early poxvirus promoter LP2EP2. Alternatively, the gene of feline CD86, which lacks the transmembrane domain has a his-tag hybrid tag at the carboxy-end to ensure purification on a Nickel affinity column.

Other examples of recombinant pox viruses of pigs, which are used as CD80 and CD86, and which can be used for p is obtaining vaccines for the treatment of diseases caused by viruses, FIV and FeLV are:

Recombinant pox virus swine, which expresses five foreign genes. The genes of the feline CD86 and feline CD80 expressed in bitestrenos cluster under the control of the synthetic late/early promoter of poxvirus LP1, initiating the transcription of CD80 and CD86 and including an element EMCV IRES located between the two open reading frames; gene uidA gene is under the control of the synthetic early promoter ER. Gene gag/protease FIV is under control of the promoter of the smallpox virus swine OIL, and the lacZ gene of E. coli is under the control of the synthetic late promoter poxvirus LP1. Genes CD80/CD86 and E. coli uidA are a friend and not primarily insertion site SPV different from the sites of insertion of the gene gag/protease FIV and lacZ gene of E. coli.

Recombinant pox virus swine, which expresses five foreign genes. The genes of the feline CD86 and feline CD80 expressed in bitestrenos cluster under the control of the synthetic late/early promoter of poxvirus LP1, initiating the transcription of CD80 and CD86 and including an element EMCV IRES located between the two open reading frames; and gene lacZ gene is under the control of the synthetic late promoter poxvirus LP1. Gene shell FIV is under the control of the synthetic early promoter of poxvirus ER. Gene uidA gene is under to what stralem synthetic late promoter LP1. Genes CD80/CD86 and E. coli uidA localized in another and not primarily insertion site SPV different from the sites of insertion of the gene gag/protease FIV and lacZ gene of E. coli.

Recombinant pox virus swine, which expresses six alien genes. The genes of the feline CD86 and feline CD80 expressed in bitestrenos cluster under the control of the synthetic late/early promoter of poxvirus LP1, initiating the transcription of CD80 and CD86 and including an element EMCV IRES located between the two open reading frames; gene uidA gene is under the control of the synthetic early promoter ER. Gene gag/protease FIV is under the control of the early promoter of poxvirus ER. Gene shell FIV is under the control of the synthetic early promoter of poxvirus ER and gene lacZ gene is under the control of the constitutive promoter of the poxvirus 15L. Genes CD80/CD86 and E. coli uidA embed in website SPV different from the sites of insertion of the gene of the shell FIV, gag/protease FIV and E.coli lacZ.

Other pox viruses of pigs used as FeLV vaccines for cats, can be constructed as described above, with the replacement of FIV genes to compatible FeLV-gene construct.

Example 11

Additional examples of recombinant smallpox raccoons, which can be used as a vaccine against the disease of cats caused by human immunodeficiency virus in cats (FIV)is a virus Le the goat cats (FeLV) or virus feline infectious peritonitis (FIP), are:

Recombinant smallpox raccoons, which expresses two foreign antigen. Gene feline CD86 is under the control of the synthetic late/early poxvirus promoter (LP2EP2), and the LacZ gene of E. coli is under the control of the promoter of smallpox raccoons L1.

Additional examples of recombinant smallpox raccoons, which can be used in order for proteins to be used as vaccines or for the production and purification of polyclonal antibodies, are:

Recombinant smallpox raccoons, which expresses one foreign gene. The gene of feline CD80, no transmembrane domain, is under the control of the synthetic late/early poxvirus promoter LP2EP2. Alternatively, the gene of feline CD80, no transmembrane domain, has a hybrid-his-tag tag at the carboxy-end for purification on a Nickel affinity column.

Recombinant smallpox raccoons, which expresses one foreign gene. Gene feline CD28, no transmembrane domain, is under the control of the synthetic late/early poxvirus promoter LP2EP2. Alternatively, the gene of feline CD28, no transmembrane domain, has a hybrid-his-tag tag at the carboxy-end for purification on a Nickel affinity column.

Recombinant smallpox raccoons, which is expresses one foreign gene. Gene feline CD86, no transmembrane domain, is under the control of the synthetic late/early poxvirus promoter LP2EP2. Alternatively, the gene of feline CD86, no transmembrane domain, has a hybrid-his-tag tag at the carboxy-end for purification on a Nickel affinity column.

Recombinant smallpox raccoons, which expresses four foreign gene. Gene feline CD86 gene and the feline CD80 expressed in bitestrenos cluster, are under the control of a synthetic late/early poxvirus promoter LP2EP2, initiating the transcription of CD80 and CD86 and including an element EMCV IRES located between the two open reading frames, initiating broadcast of the 2nd, lower CD80 gene; gene FIV gag is under control of the promoter of the smallpox virus swine OIL; gene uidA gene is under the control of the synthetic early promoter of poxvirus HER.

Recombinant smallpox raccoons, which expresses four foreign gene. Gene feline CD86 gene and the feline CD80 expressed in bitestrenos cluster, are under the control of a synthetic late/early poxvirus promoter LP2EP2, initiating the transcription of CD80 and CD86 and including an element EMCV IRES located between the two open reading frames and initiating broadcast of the 2nd downstream CD80 gene; gene about the shell FIV is under the control of the synthetic early promoter of poxvirus E1; gene uidA gene is under the control of the synthetic early promoter of poxvirus E2.

Recombinant smallpox raccoons, which expresses five foreign genes. The genes of the feline CD86 and feline CD80 expressed in bitestrenos cluster under the control of the synthetic late/early poxvirus promoter LP2EP2, initiating the transcription of CD80 and CD86 and including an element EMCV IRES located between the two open reading frames and intiraymi broadcast of the 2nd downstream CD80 gene; gene FIV gag is under control of the promoter of the smallpox virus swine OIL; gene shell FIV is under the control of the synthetic early promoter of poxvirus E1; gene uidA gene is under the control of the synthetic early promoter of poxvirus E2.

Additional examples of recombinant smallpox raccoons, which can be used as a vaccine against the disease of cats caused by human immunodeficiency virus in cats (FIV)is a virus leukemia cats (FeLV) or virus feline infectious peritonitis (FIP)are:

Recombinant smallpox raccoons, which expresses two foreign gene. Gene feline CD86 is under the control of the synthetic late/early promoter LP2EP2, gene lacZ gene is under the control of the synthetic late promoter poxvirus LP1.

Other examples of recombinant viruses the Spa raccoons, using as CD80 and CD86, and which can be used in order to obtain a vaccine for the treatment of diseases caused by viruses, FIV and FeLV are:

Recombinant smallpox raccoons, which expresses four foreign gene. Gene feline CD86 gene and the feline CD80 expressed in bitestrenos cluster, are under the control of the synthetic late promoter poxvirus LP1, initiating the transcription of CD80 and CD86 and including an element EMCV IRES located between the two open reading frames. Gene gag/protease FIV is under the control of the synthetic late/early poxvirus promoter LP2EP2; gene uidA gene is under the control of the synthetic early promoter of poxvirus ER. Genes CD80/CD86, gag/protease FIV and uidA was built in the only non-primary site RPV.

Recombinant smallpox raccoons, 4 expresses a foreign gene. Gene feline CD86 gene and the feline CD80 expressed in bitestrenos cluster, are under the control of a synthetic late/early promoter of poxvirus LP2, initiating the transcription of CD80 and CD86 and including an element EMCV IRES located between the two open reading frames; gene lacZ gene is under the control of the synthetic early promoter of poxvirus E1; gene uidA gene is under the control of the synthetic early promoter of poxvirus E2. Genes CD0/CD86, shell FIV and uidA was built in the only non-RPV main website.

Recombinant smallpox raccoons, which expresses six alien genes. The genes of the feline CD86 genes of feline CD80 expressed in bitestrenos cluster under the control of the synthetic late promoter poxvirus LP1, initiating the transcription of CD80 and CD86 and including an element EMCVIRIS located between the two open reading frames; gene lacZ gene is under the control of a late promoter of poxvirus. Gene gag/protease FIV is under the control of the synthetic early promoter ER; gene shell FIV is under the control of the synthetic early promoter of poxvirus ER; gene shell FIV is under the control of the synthetic early promoter of poxvirus ER; gene uidA gene is under control. Genes lacZ gene was embedded in a site different from the site of insertion of the gene of the shell FIV, gag/protease FIV and E. coli uidA.

Other recombinant poxviruses raccoons for use as FeLV vaccines for cats can be constructed as described above, with the replacement of FIV genes to compatible FeLV genes.

Example 12 S-FHV-020

S-FHV-020 is a recombinant herpes virus of cats, which has a deletion of the whole gene de FHV (1638 BP) and the insertion of a lacZ gene of E. coli in the website deletirovanie de. Gene lacZ gene is under the transcriptional control of the constitutive prom the Torah de FHV.

S-FHV-020 received from the S-FHV-001 (strain NVSL). This was done using homologous vector 486-88 V and virus S-FHV-001, as described in the homologous recombination Procedure for generating recombinant RPV, SPV or FHV". The source material for transfection was skanirovali, as described in the section "Screening for recombinant virus RPV, SPV or FHV expressing β-galactosidase (analyses BLUOGAL and CPRG) or β-glucuronidase (analysis of X-GLUC)". In the clean blue plaques were obtained recombinant virus designated S-FJV-020. After 5 transfers conducted analysis of the purity and stability of the insert through detection β-galactosidase, as described in the section "Screening for expression of a foreign gene in recombinant RPV, SPV or FHV using analyses on the formation of black plaques".

Example 13 S-FHV-031

S-FHV-031 is a recombinant herpes virus of cats, which has a deletion of the whole gene de FHV in 1638 base pairs and insert three foreign genes in the website deletirovanie de. Gene CD80 is under transcriptional control of the constitutive promoter de FHV and oriented in the same direction as deleteriously gene de. Gene gag/protease FIV is under control of the promoter of pseudoreligious dH and gene shell FIV is under control pretannage promoter of cytomegalovirus. Genes gag/protease and Obolo the key is oriented in the same direction, but have opposite orientation relative to the CD80 gene.

S-FHV-031 received from the S-FHV-020 (containing the lacZ gene of E. coli, behind the promotor de). This was done using homologous vector 942-03 S (see "Materials and methods) and virus S-FHV-020, as described in "Homologous recombination RPV, SPV or FHV". The source material for transfection was skanirovali, as described in the section "Screening for recombinant viruses RPV, SPV or FHV expressing β-galactosidase (analyses BLUOGALAND and CPRG) or β-glucuronidase (analysis of X-GLUC)". Recombinant plaques were selected and purified by selection of white plaques. This virus is characterized by mapping restricteduse the endonuclease and conduct procedure southern blotting DNA. This analysis confirmed the insertion of genes of feline CD80, gag/protease FIV and shell FIV and a deletion in 1638 base pairs of a gene de FIV. (International PCT application WO 96/13575, which is introduced in the present description by reference).

In the present example, S-FHV-031 analyzed for the expression of antigens specific for feline CD80, gag/protease FIV and to the shell FIV, using procedures Western blotting. This analysis was performed on CRFK cells, indicating that CRFK cells can serve as a suitable substrate for the production of recombinant FHV-vaccines. The lysate of cells infected with the recombinant is a diversified feline herpes virus, found a band of the expected size of the protein of feline CD80, gag/protease FIV and shell FIV.

In the present example, S-FHV-031 analyzed for the expression of antigens that are specific for feline CD80 using the procedure described in the section "Screening for the expression of feline CD80 (B7-1) and CD86 (B7-2) in recombinant SPV, RPV or FHV using analyses on the formation of black plaques". It was shown that chimeric antibody against CTLA-4/Fc man specifically reacts with plaques of recombinant herpes virus cats (expressing the feline CD80) and does not react with the negative control plaques S-FHV-001. It was shown that all observed plaques of recombinant herpes virus cats reacted with chimeric antibody against CTLA-4/Fc man, that indicates that the virus was stably expresses the foreign gene of feline CD80.

S-FHV-031 is a recombinant herpes virus cats expressing the proteins gag/protease FIV, shell FIV and feline CD80, and can be used as a vaccine against FIV infection in cats.

Example 14

Recombinant herpes virus of cats has a deletion of the gene and the insertion of at least one foreign gene into the website datirovannaja de. The foreign gene is a gene of feline CD86 and is under the transcriptional control of the promoter de FHV.

Recombinant virus ger the ena cats, expressing the feline CD86, can be used as vaccines against diseases in cats. This recombinant herpes virus of cats, when used alone or in combination with FIV-and FeLV-, FIP-vaccines or other feline vaccines, contributes to the effectiveness of vaccines against diseases caused by FIV, FeLV, FIP, or other diseases of cats.

Example 15

Additional examples of recombinant herpes virus of cats, which can be used as a vaccine against human immunodeficiency virus in cats (FIV), leukosis virus cats (FeLV) or infectious peritonitis in cats (FIP)are:

Recombinant herpes virus of cats, which expresses three foreign gene in sites deletirovanie gene de FHV. Gene shell FeLV is under control of the promoter of pseudoreligious DF, gene DBP FIV is under control pretannage of cytomegalovirus promoter; gene feline CD80 is under control of the promoter of herpes virus cats de. Recombinant herpes virus cats expresses three foreign gene into the website deletirovanie gene de FHV. The env gene FeLV is under control of the promoter of pseudoreligious DF, gene DBP FIV nahodilsa under control pretannage of cytomegalovirus promoter; gene feline CD86 is under control of the promoter of herpes virus cats de. Recombinant herpes virus cats expresser the em three foreign gene into the website deletirovanie gene de FHV. The env gene FeLV is under control of the promoter of pseudoreligious DF, gene DBP FeLV is under control pretannage of cytomegalovirus promoter; gene feline CD86 is under control of the promoter of herpes virus cats de.

Recombinant herpes virus of cats, which expresses five foreign genes. The genes of the feline CD86 gene and the feline CD80 expressed in bitestrenos cluster under the control of predrainage promoter of cytomegalovirus, initiating the transcription of CD80 and CD86 and including an element EMCV IRES located between the two open reading frames; gene uidA gene is under the control of the promoter of the virus of infectious laryngotracheitis gl. Genes CD80/CD86 and E. coli uidA built in the unique long region of the FHV genome in a site defined as non-core. Gene gag protease FIV, nahodyashayasa under control pretannage of cytomegalovirus promoter; gene and the lacZ gene under the control of the promoter of pseudoreligious dH built into the website deletirovanie de FHV.

Recombinant herpes virus of cats, which expresses five foreign antigens. The genes of the feline CD86 gene and the feline CD80 expressed in bitestrenos cluster under the control of predrainage promoter of cytomegalovirus, initiating the transcription of CD80 and CD86 and broadcast the 2nd downstream open reading frame CD80-controlled element of the EMCV IRES. the Yong uidA gene is under the control of the promoter of the virus of infectious laryngotracheitis gl. Genes CD80, CD86 and E. coli uidA built in the unique long region of the FHV genome in a site defined as non-core. Gene shell FIV controlled pretannage of cytomegalovirus promoter; gene and the lacZ gene under the control of the promoter of pseudoreligious dH built into the website deletirovanie de FHV.

Recombinant herpes virus of cats, which expresses six alien genes. Gene feline CD86 gene and the feline CD80 expressed in bitestrenos cluster under the control of predrainage promoter of cytomegalovirus, initiating the transcription of CD80 and CD86 and including an element EMCV IRES between the two open reading frames. Gene uidA gene is under the control of the promoter of the virus of infectious laryngotracheitis g1. Genes CD80, CD86 and E. coli uidA built in the unique long region of the FHV genome in a non-primary site. Gene shell FIV, nahodyashayasa under control pretannage promoter of cytomegalovirus gene gag/protease FIV, under the control of the promoter of pseudoreligious HHS gene and the lacZ gene under the control of the promoter de FHV built into the website deletirovanie de FHV.

Recombinant herpes virus of cats, which expresses five foreign genes. The genes of the feline CD86 and feline CD80 expressed in bitestrenos cluster under the control of predrainage promoter of cytomegalovirus, initiating the transcription of CD80 and CD86 and including the surrounding element EMCV IRES between the two open reading frames; gene uidA gene is under the control of the promoter of the virus of infectious laryngotracheitis gl. Genes CD80, CD86 and E. coli uidA built in the unique long region of the FHV genome in a non-primary site. Gene gag/protease FeLV, nahodyashayasa under control pretannage of the cytomegalovirus promoter, the gene and the lacZ gene under the control of the promoter of pseudoreligious dH built into the website deletirovanie de FHV.

Recombinant herpes virus cats, kohary expressing page alien genes. Gene feline CD86 gene and the feline CD80 expressed in bitestrenos cluster under the control of predrainage promoter of cytomegalovirus, initiating the transcription of CD80 and CD86 and broadcast the 2nd downstream open reading frame CD80-controlled element of the EMCV IRES. Gene uidA gene is under the control of the promoter of the virus of infectious laryngotracheitis gl. Genes CD80, CD86 and E. coli uidA built in a unique region of the FHV genome in a non-primary site. Gene shell FeLV, nahodyashayasa under control pretannage of the cytomegalovirus promoter, the gene and the lacZ gene under the control of the promoter of pseudoreligious dH built into the website deletirovanie FHV gE.

Recombinant herpes virus of cats, which expresses six alien genes. The genes of the feline CD86 and feline CD80 expressed in bitestrenos cluster under the control of predrainage of the cytomegalovirus promoter initiating transcription of CD80 and CD86 and including an element EMCV IRES between the two open reading frames. Gene uidA gene is under the control of the promoter of the virus of infectious laryngotracheitis gl. Genes CD80, CD86 and E. coli uidA built in the unique long region of the FHV genome in a non-primary site. Gene shell FeLV controlled pretannage promoter of cytomegalovirus gene gag/protease FeLV, under the control of the promoter of pseudoreligious HHS gene and the lacZ gene under the control of the promoter FHV gE built into the website deletirovanie FHV gE.

Example 17

Recombinant herpes virus of cats has a deletion of the gE gene and the insertion of at least one foreign gene, the site of the deletion of the gE. The foreign gene is a gene of feline CD80 and is under the transcriptional control of the promoter FHV gE.

This recombinant herpes virus of cats received from the S-FHV-001 (strain NVSL). This was done using homologous vector 926-76 .D7 (see "Materials and methods) and virus S-FHV-001, as described in the homologous recombination Procedure for generating recombinant herpesvirus". The source material for transfection was skanirovali, as described in the section "Screening for recombinant herpesvirus expressing enzymatic marker genes". This virus is characterized by mapping restricteduse the endonuclease and conduct procedure southern blotting DNA. This analysis confirmed the insertion of a gene of feline CD80 and d is leziy in 1638 base pairs of a gene de FHV. (International PCT application WO 96/13575, which is introduced in the present description by reference).

In this example, recombinant herpes virus cats analyzed for the expression of antigens specific for feline CD80, as described in the section "Screening of black plaques on the expression of a foreign gene in recombinant FHV". This analysis was performed on CRFK cells, indicating that CRFK cells can serve as a suitable substrate for the production of recombinant RPV-vaccines.

In this example, recombinant herpes virus cats analyzed for the expression of antigens specific for feline CD80, using the procedure described in the section "Screening for the expression of feline CD80 (B7-1) and CD86 (B7-2) in recombinant SPV, RPV or FHV using analyses on the formation of black plaques". It was shown that chimeric antibody against CTLA-4/Fc man specifically reacts with plaques of recombinant herpes virus cats (expressing the feline CD80) and does not react with the negative control plaques S-FHV-001. It was shown that all observed plaques of recombinant herpes virus cats reacted with chimeric antibody against CTLA-4/Fc man, that indicates that the virus was stably expresses the foreign gene of feline CD80.

To confirm the expression product of the gene of feline CD80, to EDI infected recombinant kosack the herpes virus of the present example, and samples of infected cell lysates were subjected to electrophoresis in polyacrylamide gel with LTOs. This gel was blokirovala and analyzed as described in the Procedure section Western blotting". The lysate of cells infected with recombinant feline herpes virus, discovered a band of the expected size of the protein of feline CD80.

Example 18

Recombinant herpes virus of cats has a deletion de gene FHV and inserting at least one foreign gene into the website division de. This foreign gene is a gene of feline CD86 and is under the transcriptional control of the promoter de FHV.

This recombinant herpes virus cats expressing the feline CD86, can be used as vaccines against diseases in cats. This recombinant herpes virus of cats when it is used alone or in combination with FIV-and FeLV-, FIP-vaccines or other feline vaccines contributes to the effectiveness of vaccines against diseases caused by FIV, FeLV, FIP, or other diseases of cats.

Example 19

Additional examples of recombinant herpes virus of cats, which can be used as a vaccine against human immunodeficiency virus in cats (FIV), leukosis virus cats (FeLV) or infectious peritonitis in cats (FIP)are:

Recombinant herpes virus of cats, which expresses three courtn the x gene. The env gene FIV is under control of the promoter of pseudoreligious HHS gene of FIV gag is under control pretannage promoter and cytomegalovirus; and a gene of feline CD80 is under control of the promoter of herpes virus cats gE.

Recombinant herpes virus of cats, which expresses three foreign gene. The env gene FeLV is under control of the promoter of pseudoreligious HHS gene of FeLV gag is under control pretannage of the cytomegalovirus promoter and the gene of feline CD80 is under control of the promoter of herpes virus cats gE.

Recombinant herpes virus of cats, which expresses three foreign gene. The env gene FIV is under control of the promoter of pseudoreligious HHS gene of FIV gag is under control pretannage promoter and cytomegalovirus; and gene feline CD86 is under control of the promoter of herpes virus cats gE.

Recombinant herpes virus of cats, which expresses three foreign gene. The env gene FeLV is under control of the promoter of pseudoreligious HHS gene of FeLV gag is under control pretannage promoter and cytomegalovirus; and gene feline CD86 is under control of the promoter of herpes virus cats gE.

Recombinant herpes virus of cats, which expresses five foreign genes. The genes of the feline CD86 and feline CD80 expressed in bitestrenos cluster under the control of predrainage promo is ora cytomegalovirus, initiating the transcription of CD80 and CD86 and including an element EMCV IRES between the two open reading frames, initiating broadcast of the 2nd downstream CD80 gene; gene uidA gene is under the control of the promoter of the virus of infectious laryngotracheitis gl; gene FIV gag is under control pretannage of cytomegalovirus promoter; lacZ gene of E. coli is under the control of the promoter of pseudoreligious HH. These five foreign genes are present in two different sites of insertion of the herpes virus cats.

Recombinant herpes virus of cats, which expresses five foreign genes. The genes of the feline CD86 and feline CD80 expressed in bitestrenos cluster under the control of predrainage promoter of cytomegalovirus, initiating the transcription of CD80 and CD86 and broadcast the 2nd downstream open reading frame CD80 controlled element EMCV IRES; gene uidA gene is under the control of the promoter of the virus of infectious laryngotracheitis gl; gene shell FIV is under control pretannage of cytomegalovirus promoter; lacZ gene of E. coli is under the control of the promoter of pseudoreligious HH. Recombinant herpes virus cats expresses six alien genes. The genes of the feline CD86 and feline CD80 expressed in bitestrenos cluster under the control of predrainage of the cytomegalovirus promoter that initiates transcription D80 and CD86 and including an element EMCV IRES between the two open reading frames, initiating broadcast of the 2nd downstream CD80 gene; gene uidA gene is under the control of the promoter of the virus of infectious laryngotracheitis gl; gene FIV gag is under control pretannage of cytomegalovirus promoter; gene shell FIV is under control pretannage of cytomegalovirus promoter; lacZ gene of E. coli is under the control of the promoter of pseudoreligious DF.

Recombinant herpes virus of cats, which expresses five foreign genes. The genes of the feline CD86 and feline CD80 expressed in bitestrenos cluster under the control of predrainage promoter of cytomegalovirus, initiating the transcription of CD80 and CD86 and including an element EMCV IRES between the two open reading frames, initiating broadcast of the 2nd downstream CD80 gene; gene uidA gene is under the control of the promoter of the virus of infectious laryngotracheitis gl; gene DBP FeLV is under control pretannage of cytomegalovirus promoter; lacZ gene of E. coli is under the control of the promoter of pseudoreligious HH. These five foreign genes are present in two different sites of insertion of the herpes virus cats.

Recombinant herpes virus of cats, which expresses five foreign genes. Gene feline CD86 gene and the feline CD80 expressed in bitestrenos cluster under the control of predrainage promoter of cytomegalovirus, initiating transcr the option CD80 and CD86 and broadcast the 2nd downstream open reading frame CD80, controlled element EMCV IRES; gene uidA gene is under the control of the promoter of the virus of infectious laryngotracheitis gl; gene shell FeLV is under control pretannage of cytomegalovirus promoter; lacZ gene of E. coli is under the control of the promoter of pseudoreligious DF.

Recombinant herpes virus of cats, which expresses six alien genes. Gene feline CD86 gene and the feline CD80 expressed in bitestrenos cluster under the control of predrainage promoter of cytomegalovirus, initiating the transcription of CD80 and CD86 and including an element EMCV IRES between the two open reading frames, initiating broadcast of the 2nd downstream CD80 gene; gene uidA gene is under the control of the promoter of the virus of infectious laryngotracheitis gl; gene DBP FeLV is under control pretannage of cytomegalovirus promoter; gene shell FeLV is under control pretannage of cytomegalovirus promoter; lacZ gene of E. coli is under the control of the promoter of pseudoreligious DF.

Example 20

Characterization of cDNA and polypeptide feline CD80 (B7-1)-TAMU, CD86 (B7-2), CD28, CTLA-4 and CD80 (B7-1)-Syntro/SPAH:

An isolated and purified cDNA feline CD80 (B7-1), containing approximately 941 nucleotide that encodes an open reading frame of the polypeptide of feline CD80, consisting approximately of 292 amino acids in the native membrane-associated is Oh or Mature form, and having a molecular weight of about 33485 kDa, isoelectric point of about 9.1, and the total charge at pH 7.0, equal 10,24. The transmembrane domain of the protein is an area approximately the provisions of amino acids 241-271.

Feline CD80-TAMU and CD80-Syntro/SPAH represent cDNA and polypeptides independently selected from two different sources, and their DNA and amino acid sequence are slightly different. Source CD80 mRNA-TAMU served cat mononuclear cells peripheral blood stimulated by ConA, a source of RNA CD80-Syntro/SPAH served cells of the spleen cats, ConA stimulated. The difference in cDNA sequences between CD80-TAMU and CD80-Syntro/SPAH is to replace T With the nucleotide 351 and replacement With on And nucleotide 670. In the amino acid sequence substitution in the nucleotide 351 is "silent", and the replacement to the nucleotide 670 leads to a conservative substitution of a neutral amino acid, leucine to isoleucine, amino acid residue 224.

An isolated and purified cDNA feline CD86 (B7-2), containing approximately 1176 nucleotides that encodes an open reading frame of the polypeptide feline CD86, consisting of approximately 320 amino acids in the native membrane-associated or Mature form, and having a molecular weight of about 36394 kDa, isoelectric point of about 9,19, and the total charge at pH 7.0, equal 11,27.

You Elena and purified cDNA feline CD28, containing approximately 689 nucleotides that encodes an open reading frame of the polypeptide feline CD28, consisting of approximately 221 amino acids in the native membrane-associated or Mature form, and having a molecular weight of about 25319 kDa, isoelectric point of about 9,17 and the total charge at pH 7.0, equal 9,58.

An isolated and purified cDNA feline CTLA-4, containing approximately 749 nucleotides that encodes an open reading frame of the polypeptide feline CTLA-4, comprising, approximately, of 223 amino acids in the native membrane-associated or Mature form, and having a molecular weight of about 24381 kDa, isoelectric point of about 6,34 and the total charge at pH 7.0, equal -0,99.

Coexpressed with CD80 co-stimulating molecules CD28 or CTLA-4 and tumor antigen or an antigen from a pathogenic microorganism contributes to activation or increased activation of T-lymphocytes, and more specifically, The lymphocytes-1 stimulates growth of cells of other types. Coexpressed with CD80 co-stimulating molecule, CTLA-4 contributes to the suppression of activated T-lymphocytes, and more specifically, The lymphocytes-1. Coexpressed CD86 with co-stimulating molecules CD28 or CTLA-4 and tumor antigen or an antigen from a pathogenic microorganism contributes to activation or increased activation of T-lymphocytes, and more specifically, The lymphocytes-1, and stimulates ro the t cells of other types. Coexpressed CD86 with co-stimulating molecule, CTLA-4 contributes to the suppression of activated T-lymphocytes, and more specifically, The lymphocytes-1.

Example 21

The use of feline CD80(B7-1), CD86(B7-2), CD28 and CTLA-4 in vaccines

For evaluation of the immunostimulatory activity of feline CD80, CD86, CD28 and CTLA-4 in vaccines against diseases of cats were conducted the following experiments.

Feline CD80, CD86, CD28 or CTLA-4 was built into vectors based on recombinant virus (derived from the herpes virus cats, smallpox pigs or smallpox raccoons)used for expression of recombinant proteins in cats (see International application WO 96/22363 or WO 96/13575). Vectors based on recombinant virus expressing all four immunostimulatory molecules or, alternatively, expressing pairwise combinations of CD80 and CD28 or CD80 and CTLA-4, or CD86 and CD28, or CD86 and CTLA-4, is administered orally or intramuscularly cats aged 8 weeks at a dose of from 0.1 to 10.0 mg per kg of body weight, or dose, component, approximately 104-109plaque-forming units (boe) or, preferably, in a dose comprising about 106boe Abednico vaccine for FIV or FeLV or vaccines based on viral vectors for FIV or FeLV (see above) were introduced in the minimum protective dose simultaneously with immunostimulatory vaccine, with the holding vector-based CD-80, CD86, CD28 or CTLA-4. After 3-4 weeks the cats were given a second dose of this vaccine. These cats were subjected to infection with virulent strain of FIV (PPR or Petaluma or strain Rickard FeLV (injected with methylprednisolone for suppression of immunity in cats) when the level of contamination, the appropriate standard dose USDA, and regularly observed within 12 weeks for razvitiia viremia. The group vaccinated cats were observed up to 12 months in relation to the development of tumors caused by FeLV. The incidence of these cats was compared with a control group of cats, which was not introduced vaccine, or with a group of cats, which was introduced FIV or FeLV vaccine without immunostimulatory molecules. The results of this control of infection showed that more than 60% of the cats, which was not introduced vaccine and who were then infected with FeLV or FIV, was observed persistent viremia; 75% of cats vaccinated soyedineniya FIV or FeLV vaccine, and then subjected to the control of infection, were protected from viremia; and cats vaccinated soyedineniya FIV or FeLV vaccine and a vaccine containing a combination of immunostimulatory vaccines on the basis of CD80-, CD86-, CD28 or CTLA-4-vectors, and then subjected to the control of infection, were 100% protected from viremia. Other preferred aspects of the add vaccine containing the vectors on the basis of feline CD80, CD86, CD28, and TLA-4, gave 100% protection from viremia and/or from the formation of tumors; contributed to a more lasting immunity (>1 year) and early production of immunity; or provided the opportunity to conduct primary vaccination of cats with the use of only one dose instead of two doses currently recommended by all manufacturers. Cats vaccinated with vaccines containing vectors based on viruses, FIV or FeLV, were significantly more protected from infection than females, vaccinated soyedineniya FIV or FeLV vaccine. Cats who have introduced the vaccine containing the vectors on the basis of FIV or FeLV, and a vaccine containing a combination of vectors on the basis of immunostimulatory feline CD80,CD86-, CD28 or CTLA-4, and are then subjected to the control of infection, were 100% protected from viremia. Vaccination of cats with vaccine-based FIV or FeLV-containing vector and vaccine containing a combination of immunostimulatory CD80-, CD86-, CD28 or CTLA-4-vectors, also gave additional primary results described above.

In an alternative procedure, the cats aged 8 weeks intramuscularly were injected with 100 μg plasmid containing cDNA for molecules of feline CD80, CD86, CD28 or CTLA-4 in a mixture with a plasmid containing cDNA for env and FIV gag or env and gag FeLV, or, alternatively, the cat was intramuscularly injected with 100 μg plasmid containing cDNA, the former is reservada pairwise combinations of CD80 and CD28, or CD80 and CTLA-4, or CD86 and CD28, or CD86 and CTLA-4 paired with CD28 or CTLA-4 in a mixture with a plasmid containing cDNA for env and FIV gag or env and gag FeLV. The control group cats were injected with CD80, CD86, CD28 or CTLA-4. Cats were subjected to infection with virulent FeLV or FIV and watched the signs of the diseases described above. The results of this experiment on the control of infection showed that cats that were injected with cDNA-vector containing the feline CD80, CD86, CD28 and CTLA-4, and cDNA-vector containing the genes for FIV or FeLV was found 100% protection from the disease compared with cats, which was introduced only cDNA-vector containing the genes for FIV or FeLV genes, and which found 75% protection from disease.

In an alternative procedure, the cats aged 8 weeks intramuscularly were injected with 0.1 to 100 µg purified protein of feline CD80, CD86, CD28 or CTLA-4, or, alternatively, the cats were injected with pairwise combinations of CD80 and CD86 in tandem with protein CD28 or CTLA-4 from recombinant cDNA vectors described above, and was intramuscularly injected with 0.1 to 100 μg subunit vaccines containing env and FIV gag or env and gag FeLV. The control group cats were injected with CD80, CD86, CD28 or CTLA-4. Cats were subjected to infection with virulent strain of FIV or FeLV strain, and regularly watched the development of the disease. The results of this experiment on the control of infection showed that cats that were injected purified protein molecules, Kosach the x CD80, CD86, CD28 or CTLA-4, and subunit vaccine containing FIV or FeLV, found a significant reduction in morbidity compared with cats, which was introduced only a subunit vaccine containing proteins of FIV or FeLV.

Example 22

The use of cat CD80, CD86, CD28 or CTLA-4 as a prophylactic vaccine to protect against disease.

Feline CD80, CD86, CD28 and CTLA-4 in recombinant vectors on the basis of the smallpox virus swine recombinant vectors on the basis of the smallpox virus raccoons, or recombinant vectors based on herpes virus cats, when administered as described in Example 17, but without the introduction of antigens contained in subunit or viral vectors and derived from pathogenic microorganisms, can be used to stimulate the immune system and The-1-response, whereby is produced a protective immune response during infection viral, parasitic or bacterial pathogens. In an alternative procedure feline CD80 or CD86 in combination with a feline CTLA-4 in viral vectors, when administered as described in Example 3 can be used to suppress the immune response and to protect against autoimmune diseases in cats.

Example 23:

The use of feline CD80, CD86, CD28 and CTLA-4 inhibition and inhibition of growth of tumor cells.

Tumor cells cats were transferrable recombinant etc) the rum-based smallpox pigs, recombinant vector-based smallpox raccoons or recombinant vectors based on herpes virus cats expressing the feline CD80 or CD86 in combination with CD28 or CTLA-4. Then the cat was re-introduced transfected tumor cells, and the presence of CD80, CD86, CD28 and CTLA-4 on the surface of tumor cells caused the production of a significant immune response to transfected and atransferrinemia tumor cells, leading, thus, to the cytolysis of localized and metastatic tumor cells. In an alternative procedure, the vectors expressing the feline CD80 or CD86 in combination with CD28 or CTLA-4, were injected with directly into the tumor cats, leading, thus, to produce a significant immune response to the tumor cells to cytolysis localized and metastatic tumor cells.

Example 24

The use of feline CD80, CD86, CD28 and CTLA-4 as a therapeutic means for the treatment of diseases of cats.

Feline CD80, CD86, CD28 and CTLA-4 in the recombinant vector on the basis of the smallpox virus swine recombinant vector-based smallpox raccoons or recombinant vectors based on herpes virus in cats when administered as described in Example 17, but without the introduction of antigens contained in subunit or viral vectors and derived from pathogenic microorganisms, can the be used to stimulate the immune system for the immune clearance or decrease the pathology of the disease.

Confirmation of the experimental data: SPV 246

The safety and efficacy of vaccines based on recombinant viral vector SPV containing the gag gene and shell FeLV gene and the feline CD80.

Construction of recombinant virus SPV, SPV 246 described above (in the main part of the description of this initial application). SPV 246 contains five foreign genes, including genes encoding proteins gag and shell FeLV and feline CD80, as well as two of the marker gene, β-glucoronidase and β-galactosidase. The expression of the gag gene and shell FeLV and CD80 in cells infected with SPV 246, was confirmed using Western blot analysis. Bands representing specific proteins gag and shell FeLV, were detected using goat polyclonal antibodies against FeLV P27 (Biodesign, ME) and monoclonal antibodies against Dr FeLV (Biodesign, ME), respectively. It is obvious that proteins gag and shell FeLV excision are processed similarly to native viral proteins. Analysis of purity, expression and stability was performed by analyzing the formation of black plaques using antibodies described above. SPV 246 steadily in one case replanted at least 5 times. 100% of plaques generated from cells infected with SPV 246 were positive for gag proteins and membrane FeLV, β-galactosidase and β-glucuronidase.

Expression of feline CD80 was confirmed in a Western blot is the analysis using polyclonal antibodies against CD80 person. It was discovered many bands ranging in size from 30 to 60 kDa specific for feline CD80. These bands represented the picture of the alternating and multiple glycosylation CD80 expressed and modified in the "context" of the SPV in the cells ESK-4.

SPV 246 and the control virus, SPV-003, and other recombinant viruses FHV and SPV FeLV, are candidates for the vaccine were tested for their ability to protect cats from the persistent FeLV infection. For this 8-week kittens, 10 kittens per group, were vaccinated subcutaneously with 1 ml SPV 246, control of virus or other recombinant viruses (in doses, comprising from 7×105boe on the cat until 7×107boe on the cat). Cats were vaccinated three times at intervals of 3 weeks. After vaccination of cats subjected to control nasal infection standard laboratory strain of FeLV Rickard (106,2TCID50/ml/cat), after pre-treatment with methylprednisolone acetate (Depo-Medrol).

Serum taken from these cats, weekly and analyzed for persistent viraemia within 15 weeks after infection. When testing for persistent viraemia was believed that cats have persistent viraemia, if they continuously for 3 weeks showed the presence of FeLV P27.

The results:

The research method of FeLV infection showed that Ko is Ki, vaccinated SPV 246, were partially protected from FeLV viremia. A predicted value of the preventive fraction (PF) for cats treated SPV 246, was 50% (table 1).

Table 1: Number and percentage of cats with persistent viremia after 15 weeks after control of infection. For each group was calculated preventive fraction (PF).

Examples of additional recombinant viruses containing CD80 and CD86

SPV 280

SPV 280 is a recombinant pox virus swine expressing six alien genes. The homology vector designated 992-23 .6 designed as follows: the genes of the feline CD86 and feline CD80 expressed in bitestrenos DNA-cluster under the control of the synthetic late promoter poxvirus LP1, initiating the transcription of CD80 and CD86 and including an element EMCV IRES between the two open reading frames; gene β-glucuronidase E. coli is under the control of the synthetic early promoter ER. SPV 280 received from the SPV 258, solariega genes gag and shell FeLV and β-galactosidase. SPV 258 was pre-designed so that it contained the gene gag/protease FeLV and truncated gene shell FeLV (gp70) under the control of the synthetic early/late promoter poxvirus, LP2EP2; gene β-galactosidase gene is under the control of the constitutive promoter of the poxvirus 15L built in deleteriously incomplete 1869 BP-HindIII-a fragment of the N. Genes CD80/CD86 and β-glucuronidase E. coli cloned in homologous vector 992-23 .6 in the private and not-mostly incomplete HindIII fragment To the SPV.

SPV 280 received from the SPV 258. This was done using homologous vector 992-23 .6 and virus S-SPV-258, as described in the homologous recombination Procedure for generating recombinant RPV, SPV or FHV". The source material for transfection was skanirovali, as described in the section "Screening for recombinant virus SPV, repressively β-galactosidase (analyses BLUOGAL and CPRG) or β-glucuronidase (analysis of X-GLUC)". As a result, multiple cleaning cycles blue/green plaques were obtained recombinant virus SPV 280.

SPV 280 analyzed for gene expression FeLV gag, shell FeLV and marker genes, genes β-galactosidase and β-glucuronidase analysis on the formation of black plaques. Using goat polyclonal antibodies against FeLV gag (Biodesign, ME), and mouse monoclonal antibodies against the envelope protein gp70 FeLV (Biodesign, ME), it was determined that 100% of plaques generated from cells, ESK-4 infected with purified SPV 280, Express a gene gag and shell FeLV.

Expression of feline CD80 and CD86 was confirmed in Western blot analysis using goat polyclonal antibodies against CD80 and CD86 person (R&D Systems,MN), respectively. It was discovered many bands ranging in size from 30 to 60 to the and, specific for feline CD80, and many bands ranging in size from 40 to 70 kDa specific for feline CD86. These bands were given a picture of a striped and multiple glycosylation CD80 and CD86, expressed in "context" SPV in cells, ESK-4.

SPV 281

SPV 281 is a recombinant pox virus swine expressing six alien genes. The homology vector designated 992-23 .6, designed as described above for SPV 280. SPV 281 was obtained from SPV 228, solariega genes gag/protease and shell FIV β-galactosidase of E. coli. Gene gag/protease FIV is under the control of the synthetic early promoter of poxvirus ER; gene shell FIV is under the control of the synthetic early promoter of poxvirus ER, gene β-galactosidase gene is under the control of the constitutive promoter of the poxvirus 15L. Genes gag/protease FIV, shell FIV β-galactosidase of E. coli were built in deleteriously incomplete 1869 n.o.-HindIII-fragment N SPV. Genes CD80/CD86 and β-glucuronidase E.coli were built in separate and non-primary partial HindIII-fragment To the SPV.

SPV 281 received from the SPV 228. This was done using homologous vector 992-23 .6 and virus S-SPV-228, as described in the homologous recombination Procedure for generating recombinant RPV, SPV or FHV". The source material for transfection was skanirovali as described in the section "Screenin is on recombinant virus SPV, expressing β-galactosidase (analyses BLUOGAL and CPRG) or β-glucuronidase (analysis of X-GLUC)". As a result, multiple cleaning cycles blue/green plaques were obtained recombinant virus SPV 281.

SPV 281 analyzed for gene expression FIV gag, shell FIV, marker genes, genes β-galactosidase and β-glucuronidase analysis on the formation of black plaques. Using mouse monoclonal antibodies against FIV gag (P27) and shell FIV (gp100) (Custom Monoclonals, CA; Biodesign International, ME, respectively), mouse monoclonal antibodies against β-galactosidase and rabbit polyclonal antibodies against β-glucuronidase (Biodesign, ME & Molecular Probes, OR, respectively), it was determined that 100% of plaques generated from cells, ESK-4 infected with purified SPV 280, Express a gene of FIV gag, shell FIV, β-galactosidase and β-glucuronidase.

Expression of feline CD80 and CD86 was confirmed by Western blot analysis using polyclonal antibodies against CD80 and CD86 person (R&D Systems, MN). It was discovered many bands ranging in size from 30 to 60 kDa specific for feline CD80, and many bands ranging in size from 40 to 70 kDa specific for feline CD86. These bands represented the picture of the alternating and multiple glycosylation CD80 and CD86, expressed in "context" SPV and cells, ESK-4. The expression of FIV gag and oblock the FIV was also confirmed using Western blot analysis using antibodies described above. Proteins of FIV gag and shell FIV, obviously, are processed in R24 and gp100, respectively.

FHV 043

FHV 043 is a recombinant herpes virus of cats that expresses five foreign genes. The homology vector designated 987-57 A, designed as follows:

the genes of the feline CD86 and feline CD80 cloned in bicistronic cluster under the control of predrainage promoter of cytomegalovirus (CMV IE), initiating the transcription of CD80 and CD86. Translation of the second open reading frame downstream CD80 is under control element EMCV IRES. Gene β-glucuronidase E. coli is under the control of the promoter of the virus of infectious laryngotracheitis gl. Genes CD80, CD86 and β-glucuronidase E.coli was built in the unique long region of the FHV unique EcoRI site, originating from incomplete SalI-fragment N FHV. The insertion was introduced between genome gL and related gene activator of transcription.

FHV 043 was derived from FHV 017 containing genes shell FIV β-galactosidase of E. coli. Gene shell FIV is under control of the promoter of CMV IE, and gene β-galactosidase gene is under the control of the promoter element pseudoreligious HH. Gene shell FIV β-galactosidase of E. coli were built in saddletowne gE US FHV.

FHV 043 received from FHV 017. This was done using homologous vector 987-51 A and FHV 017, as described in the homologous recombination Procedure for generating recombinant RPV, SPV or FHV". The source material for transfection was skanirovali as described in the section "Screening for recombinant virus SPV expressing β-galactosidase (analyses BLUOGAL and CPRG) or β-glucuronidase (analysis of X-GLUC)". As a result, multiple cleaning cycles blue/green plaques were obtained recombinant virus FHV 043.

FHV 043 analyzed for the expression of marker genes, gene β-galactosidase gene and β-glucuronidase analysis on the formation of black plaques. Using mouse monoclonal antibodies against β-galactosidase (Biodesign, ME), and rabbit polyclonal antibodies against β-glucuronidase (Molecular Probes, OR), it was determined that 100% of plaques generated from CRFK cells infected with FHV 043, purified by the method of plaques Express β-galactosidase and β-glucuronidase. It was determined that this virus is stable after at least 5 transfers.

Expression of feline CD80 and CD86 was confirmed by Western blot analysis using polyclonal antibodies against CD80 and CD86 person (R&D Systems, MN). It was discovered many bands ranging in size from 30 to 60 kDa specific for feline CD80, and many bands ranging in size from 40 to 70 kDa specific for feline CD86. Gene expression of what rocki FIV (Dr) was confirmed by Western blot analysis using serum convalescent cats, taken from FIV-infected cats.

Homologous vector 1015-18 A (LP1-CD8 6 / IRES-CD80):

This homologous vector 1015-18 A used to create recombinant viruses RPV expressing CD80 and CD86. For this purpose it was designed plasmid containing poxvirus promoter LP1, element EMCV IRES and the terminator of transcription of Poly (A poxvirus. The gene of feline CD80 were subjected to PCR amplification using primers 1/97 .6 (5’-GCTAGGATCCAATCTATGTAGACAGGTGAGAT-3’) and 3/98 .4 (5’-TCGAGGATCCGGGTCACGCAGCAAAGTGG-3’), each of which contained a BamHI cloning sites. CD80 cloned behind the promoter LP1. Gene feline CD86 were subjected to PCR amplification using primer 1/98 .18 (5’-TCGACAATTGGATGGGCATTTGTGACAG-3’) with MfeI site cloning and using a blunt on the ends of the primer 8/97 .31 (5’-GTGGATCCAGGATCCGGAGCGG-3’). CD86 cloned element EMCV IRES. Then the cluster restrictively enzyme NotI and cloned into HindIII-fragment N RPV vector containing the gene of (3-galactosidase gene under the control of the synthetic late promoter 15L. Finite homological vector 1015-18 A used to create viruses containing the genes for FIV or FeLV genes CD80 and CD86 in accordance with the procedure described in the homologous recombination Procedure for generating recombinant RPV".

S-RPV-045:

S-RPV-045 is a recombinant virus of smallpox raccoons, expressing three foreign gene. S-RPV-045 was obtained the C smallpox raccoons RPV-000 (ATCC VR-838). This was done using homologous vector 1015-18 .8A and parental virus S-RPV-000, as described in the homologous recombination Procedure for generating recombinant RPV". The source material for transfection was skanirovali on recombinant, as described in the section "Screening for recombinant virus SPV expressing β-galactosidase (analyses BLUOGAL and screening for recombinant RPV, expressing enzymatic marker genes)". The virus was purified by the method of plaques and perseval 5 times.

RPV-045 analyzed for the expression of β-galactosidase by analyzing the formation of black plaques. Using rabbit polyclonal antibodies (ICN, IT was determined that 100% of plaques generated from VERO cells infected with purified RPV-045, expressed β-galactosidase.

Expression of feline CD80 and CD86 was confirmed in Western blot analysis, as described in the appropriate section using goat polyclonal antibodies against CD80 and CD86 person (R&D Systems, MN), respectively. It was discovered many bands ranging in size from 30 to 60 kDa specific for feline CD80, and many bands ranging in size from 40 to 70 kDa specific for feline CD86. These bands were given a picture of a striped and multiple glycosylation CD80 and CD86, expressed in the "context" of the RPV and in VERO cells.

S-RPV-046:

RPV-046 represents the t of a smallpox raccoons, expresses five foreign genes. RPV-046 was obtained from smallpox raccoons RPV-036. This procedure was performed using homologous vector 1015-18 A and parental virus RPV-036, as described in the homologous recombination Procedure for generating recombinant RPV". The raw materials for transfection was skanirovali on recombinant virus as described in the section "Screening for recombinant virus RPV expressing β-galactosidase (analyses BLUOGAL and Screening for recombinant RPV, expressing enzymatic marker genes)". The virus was purified by the method of plaques and perseval 5 times. As a result, multiple cleaning cycles blue/green plaques were obtained recombinant virus FHV 046. RPV 046 contains the gene of FIV gag under the control of the synthetic early/late poxvirus promoter LP2EP2; gene β-glucuronidase under the control of the synthetic early promoter of poxvirus ER. These genes are located in a separate and non-largely incomplete HindIII site U RPV. Genes CD80 and CD86 gene and the β-galactosidase are unique and separate not-mostly incomplete HindIII site N RPV.

RPV-046 analyzed for the expression of β-galactosidase and β-glucuronidase by analyzing the formation of black plaques. It was determined that 100% of plaques generated from VERO cells infected with purified RPV-046, expressed β-galactosidase and &x003B2; -glucuronidase.

The expression of CD80 and CD86 was confirmed using Western blot analysis, as described in the appropriate section, using goat polyclonal antibodies against CD80 and CD86 person (R&D Systems, MN), respectively. It was discovered many bands ranging in size from 30 to 60 kDa specific for feline CD80, and many bands ranging in size from 40 to 70 kDa specific for feline CD86. These bands represented the picture of the alternating and multiple glycosylation CD80 and CD86, expressed in "context" RPV in VERO cells. The expression of gag/protease FIV was also confirmed by Western blot analysis using mouse monoclonal antibodies against FIV gag (p27) (Custom Monoclonals, CA).

S-RPV-047:

RPV-047 is a smallpox raccoons that expresses five foreign genes. Was constructed as described above, homologous vector 1015-18 A, which contains a cluster LP1-CD86/IRES-CD80 gene β-galactosidase gene under the control of the synthetic late promoter (15L) in HindIII-fragment N. RPV-047 was obtained from RPV-044 containing genes FIV env and β-glucuronidase E.coli (β- - glucuronidase} in HindIII-fragment U RPV. The env gene FIV is under the control of the synthetic early promoter (ER). Gene β-glucuronidase is under the control of the synthetic late promoter (LP1).

RPV-047 received from smallpox raccoons RPV 044. This was done with the use of the cation homologous vector 1015-18 A and parental virus RPV-044, as described in the homologous recombination Procedure for generating recombinant RPV". The raw materials for transfection was skanirovali on recombinant, as described in the section "Screening for recombinant virus RPV expressing β-galactosidase (analyses BLUOGAL and Screening for recombinant RPV, expressing enzymatic marker genes). The virus was purified by the method of plaques and perseval 5 times. As a result, multiple cleaning cycles blue/green plaques were obtained recombinant virus FHV 047.

RPV-047 analyzed for the expression of β-galactosidase by analyzing the formation of black plaques. Using polyclonal rabbit antibodies (ICN, IT was determined that 100% of plaques generated from VERO cells infected with purified RPV-047, Express β-galactosidase.

Western analysis as described in the appropriate section, using goat polyclonal antibodies against CD80 and CD86 person (R&D Systems, MN), conducted as described in the Procedure section Western blotting", confirmed the expression of CD80 and CD86, respectively. It was discovered many bands ranging in size from 30 to 60 kDa specific for feline CD80, and many bands ranging in size from 40 to 70 kDa specific for feline CD86. These bands represented the picture of the alternating and multiple glycosylation CD80 and CD86, expressed in "is ontext" RPV in VERO cells. The expression of FIV env was also confirmed by Western blot analysis using mouse monoclonal antibodies against FIV env (gp100) (BioDesign International, ME).

S-RPV-048:

RPV-048 is a smallpox raccoons that expresses five foreign genes. Was constructed as described above, homologous vector 1015-18 A, which contains a cluster LP1-CD86/IRES-CD80 gene β-galactosidase gene under the control of the synthetic late promoter (15L) in HindIII-fragment N. RPV-048 was derived from RPV-038 containing genes gag/protease FeLV and β-glucuronidase E.coli in HindIII-fragment U RPV. Gene gag/protease FeLV is under the control of the synthetic late/early promoter (LP2EP2). Gene β-glucuronidase is under the control of the synthetic late promoter (LP1).

RPV 048 received from recombinant smallpox raccoons RPV 038. This was done using homologous vector 1015-18 A and parental virus RPV-038, as described in the homologous recombination Procedure for generating recombinant RPV". The raw materials for transfection was skanirovali on recombinant virus as described in the section "Screening for recombinant virus RPV expressing β-galactosidase (analyses BLUOGAL and Screening for recombinant RPV, expressing enzymatic marker genes)". The virus was purified by the method of plaques and perseval 5 times. In the result of the multiple cleaning cycles blue/green plaques were obtained recombinant virus FHV 048.

RPV-048 analyzed for the expression of β-galactosidase by analyzing the formation of black plaques. Using polyclonal rabbit antibodies (ICN, IT was determined that 100% of plaques generated from VERO cells infected with purified RPV-046, expressed β-galactosidase.

The expression of CD80 and CD86 was confirmed by Western blot analysis, as described in "Procedure for Western blotting, using goat polyclonal antibodies against CD80 and CD86 person (R&D Systems, MN), respectively. It was discovered many bands ranging in size from 30 to 60 kDa specific for feline CD80, and many bands ranging in size from 40 to 70 kDa specific for feline CD86. These bands represented the picture of the alternating and multiple glycosylation CD80 and CD86, expressed in "context" RPV in VERO cells. The expression of gag/protease FeLV was also confirmed by Western blotting using rabbit polyclonal antibodies against FeLV gag (P27)(BioDesign International, ME).

S-RPV-052:

RPV-052 is a smallpox raccoons, expressing the six alien genes. Was constructed as described above, homologous vector 1015-18 A, which contains a cluster LP1-CD86/IRES-CD80 gene β-galactosidase gene under the control of the synthetic late promoter (15L) in HindIII-fragment N. RPV-052 was derived from RPV-030 containing the ENES gag/protease FeLV, FeLV env and β-glucuronidase E.coli in HindIII-fragment U RPV. Gene gag/protease FeLV is under the control of the synthetic early promoter (ER). The env gene FeLV is under the control of the synthetic early promoter (ER). Gene β-glucuronidase is under the control of the synthetic late promoter (LP1).

RPV 052 received from smallpox raccoons RPV 030. This was done using homologous vector 1015-18 A and parental virus RPV-030, as described in the homologous recombination Procedure for generating recombinant RPV". The raw materials for transfection was skanirovali on recombinant virus as described in the section "Screening for recombinant virus RPV expressing β-galactosidase (analyses BLUOGAL and Screening for recombinant RPV, expressing enzymatic marker genes)". The virus was purified by the method of plaques and perseval 5 times. As a result, multiple cleaning cycles blue/green plaques were obtained recombinant virus FHV 052.

RPV-052 analyzed for the expression of β-galactosidase, β-glucuronidase, FeLV gag and shell FeLV by analyzing the formation of black plaques. The expression of CD80 and CD86 was confirmed by Western blot analysis, as described in "Procedure for Western blotting, using goat polyclonal antibodies against CD80 and CD86 person (R&D Systems, MN), with the NGOs. The expression of gag/protease FeLV and shell FeLV was also confirmed by Western blot analysis using rabbit polyclonal antibodies against FeLV gag (p27) (BioDesign International, ME), and mouse monoclonal antibodies against FeLV env (gp100) (BioDesign, ME).

S-RPV-053:

RPV-053 is a smallpox raccoons, expressing the six alien genes. Was constructed as described above, homologous vector 1015-18 A, which contains a cluster LP1-CD86/IRES-CD80 gene β-galactosidase gene under the control of the synthetic late promoter (15L) in HindIII-fragment N. RPV-053 was obtained .RPV-034, which contains the genes gag/protease FIV, FIV env and β-glucuronidase E.coli in HindIII-fragment U RPV. Gene gag/protease FIV is under the control of the synthetic early promoter (ER). The env gene FIV is under the control of the synthetic early promoter (ER). Gene β-glucuronidase is under the control of the synthetic late promoter (LP1).

RPV-053 received from smallpox raccoons RPV-034. This was done using homologous vector 1015-18 A and parental virus RPV-034, as described in the homologous recombination Procedure for generating recombinant RPV". The raw materials for transfection was skanirovali on recombinant virus as described in the section "Screening for recombinant virus RPV expressing β-galactosidase (analyses BLUOGAL Screening for recombinant RPV, expressing enzymatic marker genes)". The virus was purified by the method of plaques and perseval 5 times. As a result, multiple cleaning cycles blue/green plaques were obtained recombinant virus FHV 053.

S-SPV 275

S-SPV-275 is a recombinant pox virus swine that expresses five foreign genes. The homology vector designated 992-23 .6, designed as follows: the genes of the feline CD86 and CD80 were expressed in bitestrenos DNA-cluster under the control of the synthetic late promoter poxvirus LP1, initiating the transcription of CD80 and CD86 and including an element EMCV IRES between the two open reading frames. Gene β-glucuronidase E. coli is under the control of the synthetic early promoter of poxvirus ER. As the parent virus used S-SPV 046, which contains the gene gag/protease FIV-driven synthetic late/early poxvirus promoter LP2EP2, and gene β-galactosidase under the control of the constitutive promoter of the poxvirus OIL. Handed/FIV protease gene β-galactosidase activity was embedded in the partial HindIII-fragment M SPV genes and CD86/CD80 and β-glucuronidase embedded in the partial HindIII-fragment To the SPV.

S-SPV 275 was obtained from S-SPV 046. This was done using homologous vector 992-23 .6 and virus S-SPV-046, as described in the Procedure section of homologous recombination degenerative recombinant SPV". The source material for transfection was skanirovali, as described in the section "Screening for recombinant virus SPV expressing β-glucuronidase (analysis of X-GLUC)". As a result, multiple cleaning cycles green/blue plaques were obtained recombinant virus SPV 275.

S-SPV 275 analyzed for gene expression FIV gag, marker genes and genes β-glucuronidase analysis on the formation of black plaques. Using mouse monoclonal antibodies against FIV gag (Custom Monoclonals, CA), it was determined that 100% of plaques generated in cells, ESK-4 infected with purified S-SPV 275, Express a gene of FIV gag and remain stable after 5 transfers.

The expression of FIV gag, CD86 and CD80 was confirmed by Western blot analysis using mouse monoclonal antibodies against FIV gag, and using goat polyclonal antibodies against CD86 and CD80 person (R&D Systems, MN) for feline CD86 and CD80. Were two separate strips 50 and 27 kDa specific for FIV gag. It was discovered many bands ranging in size from 40 to 70 kDa specific for feline CD86, as well as band size from 30 to 60 kDa specific for feline CD80.

S-FHV 040

S-FHV 040 is a recombinant herpes virus of cats that expresses five foreign genes. The homology vector designated 957-87 A, designed as follows: the genes of the feline is their CD86 and CD80 expressed in bitestrenos DNA-cluster under the control of predrainage promoter of cytomegalovirus (CMV IE), initiating the transcription of CD80 and CD86 and including an element EMCV IRES located between the two open reading frames. Gene β-glucuronidase E. coli is under the control of the promoter of the virus of infectious laryngotracheitis gl. Genes CD80, CD86 and β-glucuronidase E.coli was built in the unique long region of the FHV unique EcoRI site, originating from incomplete Sail-fragment N FHV between genome gL and related gene activator of transcription. As the parent virus used S-FHV-019, which contains the gene of FeLV gag-driven IE CMV, and gene β-galactosidase gene under the control of the promoter element pseudoreligious DF; both of these gene localized in the unique short (US) deletions de FHV.

S-FHV 040 received from the S-FHV 019. This was done using homologous vector 987-57 A and virus S-FHV 019, as described in the homologous recombination Procedure for generating recombinant FHV". The source material for transfection was skanirovali, as described in the section "Screening for recombinant virus FHV expressing β-glucuronidase (analysis of X-GLUC)". As a result, multiple cleaning cycles green/blue plaques were obtained recombinant virus S-FHV 040.

S-FHV 040 analyzed for the expression of FeLV gag, marker genes β-glucuronidase and β-galactosidase analysis on the formation of black b is ASEC. It was determined that 100% of plaques generated in CRFK cells, expressed β-glucuronidase and β-galactosidase. Expression of FeLV gag was also confirmed by analysis on the formation of black plaques using goat polyclonal antibodies against gp27 FeLV (Biodesign, ME). This virus was found stability after five subcultures.

Expression of feline CD80 and CDS 6 and FeLV gag was confirmed using Western blot analysis. For feline CD80 and CD86 used goat polyclonal antibodies against CD80 and CD86 person (R&D Systems, MN). It was discovered many bands ranging in size from 30 to 60 kDa specific for feline CD80, and stripe size from 40 to 70 kDa specific for feline CD86. The expression of FeLV was confirmed using goat polyclonal antibodies against gp27 FeLV (Biodesign, ME).

S-FHV 042

S-FHV 042 is a recombinant herpes virus of cats that expresses five foreign genes. The homology vector designated 957-87 A, designed as follows: the genes of feline CD80 and CD86 expressed in bitestrenos DNA-cluster under the control of predrainage promoter of cytomegalovirus (CMV IE), initiating the transcription of CD80 and CD86 and including an element EMCV IRES located between the two open reading frames. Gene β-glucuronidase E. coli is under the control of the promoter of the virus infectious laryngotracheo is the gl. Genes CD80, CD86 and β-glucuronidase embedded in the unique long region of the FHV unique EcoRI site, originating from incomplete SalI-fragment N FHV, between genes gL and adjacent genes activator of transcription. As the parent virus used S-FHV-018, which contains the gene shell FeLV-driven IE CMV, and gene β-galactosidase of E. coli, nahodiysa under the control of the promoter element pseudoreligious DF; both of these gene localized in the unique short (US) deletions de FHV.

S-FHV 042 was obtained from S-FHV 018. This was done using homologous vector 987-57 A and virus S-FHV 018, as described in the homologous recombination Procedure for generating recombinant FHV". The source material for transfection was skanirovali, as described in the section "Screening for recombinant virus SPV expressing β-glucuronidase (analysis of X-GLUC)". As a result, multiple cleaning cycles green/blue plaques were obtained recombinant virus S-FHV 042. S-FHV 042 analyzed for the expression of FeLV env, marker genes β-glucuronidase and β-galactosidase analysis on the formation of black plaques. It was determined that 100% of plaques generated in CRFK cells, expressed β-glucuronidase and β-galactosidase. Expression of FeLV env was also confirmed by analysis on the formation of black plaques and with the use of mouse monoclonal antibodies against Dr FeLV (Biodesign, ME). This virus was stable after five subcultures.

Expression of feline CD80 and CD86 and FeLV env confirmed using Western blot-analia. For feline CD80 and CD86 used goat polyclonal antibodies against CD80 and CD86 person (R&D Systems, MN). It was discovered many bands ranging in size from 30 to 60 kDa specific for feline CD80, and bands ranging in size from 40 to 70 kDa specific for feline CD86. Using mouse monoclonal antibodies against gr (Biodesign, ME) was discovered band of 100 kDa FeLV env.

S-FHV 044

S-FHV 044 is a recombinant herpes virus of cats that expresses five foreign genes. The homology vector designated 957-87 A, designed as follows: the genes of feline CD80 and feline CD86 expressed in bitestrenos DNA-cluster under the control of predrainage promoter of cytomegalovirus (CMV IE), initiating the transcription of CD80 and CD86 and including an element EMCV IRES located between the two open reading frames. Gene β-glucuronidase E. coli is under the control of the promoter of the virus of infectious laryngotracheitis gl. Genes CD80, CD86 and β-glucuronidase embedded in the unique long region of the FHV unique EcoRI site, originating from incomplete SalI-fragment N FHV between genome gL and related gene activator of transcription. As the parent virus used S-FHV-016, which contains the EN gag/protease FIV, stimulated CMV IE (with a deletion of nine amino acids at the 5’end of the protease gene, and gene β-galactosidase gene, which is under the control of the promoter of pseudoreligious DF; where both gene localized in the unique short (US) deletions de FHV.

S-FHV 044 was obtained from S-FHV 016. This was done using homologous vector 987-57 A and virus S-FHV 016, as described in the homologous recombination Procedure for generating recombinant FHV". The source material for transfection was skanirovali, as described in the section "Screening for recombinant virus FHV expressing β-glucuronidase (analysis of X-GLUC)". As a result, multiple cleaning cycles green/blue plaques were obtained recombinant virus S-FHV 044.

S-FHV 044 analyzed for the expression of FIV gag and marker genes β-glucuronidase and β-galactosidase analysis on the formation of black plaques. Using mouse monoclonal antibodies (Biodesign, ME), it was determined that 100% of plaques generated in CRFK cells that expressed both the marker gene. The expression of FIV gag was also confirmed by analysis on the formation of black plaques using mouse monoclonal antibodies against FIV gag (Custom Monoclonals, CA). This virus was stable after five subcultures.

Expression of feline CD80 and CD86 and FeLV env confirmed using Western Blot analysis. For feline CD80 and CD86 used goat polyclonal antibodies against CD80 and CD86 person (R&D Systems, MN). It was discovered many bands ranging in size from 30 to 60 kDa specific for feline CD80, and bands ranging in size from 40 to 70 kDa specific for feline CD86. Using mouse monoclonal antibodies against FIV gag were two separate strips 50 and 27 kDa specific for FIV gag.

Additional examples of co-vectors, including feline CD80 and CD86, and TPS partial or full genomes FIV or FeLV

Note: Recombinant viral vectors containing CD80, CD86, CTLA-4 or CD28 in recombinant virus with partial or full genomic polypeptide FIV and/or FeLV, and with the genes of feline IL-12 R35 and R40 or without them. These recombinant viruses can be used as vaccines against diseases caused by FIV and FeLV in cats.

1. Expression of feline CD80, CD86, CD28 and CTLA-4, taken separately or in any combination, recombinant pox virus swine, containing full or partial genome of FIV.

2. Expression of feline CD80, CD86, CD28 and CTLA-4, taken separately or in any combination, recombinant feline herpes virus, containing full the th or incomplete genome of FIV.

3. Expression of feline CD80, CD86, CD28 and CTLA-4, taken separately or in any combination, recombinant smallpox raccoons, containing full or partial genome of FIV.

4. Expression of feline CD80, CD86, CD28 and CTLA-4, taken separately or in any combination, recombinant pox virus swine, containing complete or incomplete genome FeLV.

5. Expression of feline CD80, CD86, CD28 and CTLA-4, taken separately or in any combination, recombinant feline herpes virus, containing complete or incomplete genome FeLV.

6. Expression of feline CD80, CD86, CD28 and CTLA-4, taken separately or in any combination, recombinant smallpox raccoons, containing complete or incomplete genome FeLV.

7. Expression of feline CD80, CD86, CD28 and CTLA-4, taken separately or in any combination, recombinant pox virus swine, containing complete or incomplete FIV genome and genes of feline IL-12, GM-CSF, R35 and R40.

8. Expression of feline CD80, CD86, CD28 and CTLA-4, taken separately or in any combination, recombinant feline herpes virus, containing a complete or partial genome of FIV genes and feline IL-12, GM-CSF, R35 and R40.

9. Expression of feline CD80, CD86, CD28 and CTLA-4, taken separately or in any combination, recombinant smallpox raccoons, containing complete or incomplete FIV genome and genes of feline IL-12, GM-CSF, R35 and R40.

10. Expression of feline CD80, CD86, CD28 and CTLA-4, taken separately or in any combination, recom is inetnum the smallpox virus swine containing complete or incomplete FeLV genome and genes of feline IL-12, GM-CSF, p35 and R40.

11. Expression of feline CD80, CD86, CD28 and CTLA-4, taken separately or in any combination, recombinant feline herpes virus, containing complete or incomplete FeLV genome and genes of feline IL-12, GM-CSF, p35 and R40.

12. Expression of feline CD80, CD86, CD28 and CTLA-4, taken separately or in any combination, recombinant smallpox raccoons, containing complete or incomplete FeLV genome and genes of feline IL-12, GM-CSF, p35 and R40.

Examples

Homologous vector 1007-70 .A2 (SPV N/CMV-FIV genome(ΔLTR)/15L-lacZ). Homologous vector E used for integration of foreign DNA insertion in HindIII site N SPV. It includes marker gene β-galactosidase of E. coli and a full-sized genome of FIV (8,5 KBP) without flanking elements with long terminal repeats (LTR). This cluster was flanked by SPV DNA homologous non-primary site in HindIII-fragment N SPV. Using this homology vector in accordance with the procedure described in the homologous recombination Procedure for generating recombinant SPV," received a virus containing DNA encoding these alien genes. It should be noted that the marker gene of (3-galactosidase is under the control of the constitutive promoter of the poxvirus 15L and FIV genome (ΔLTR) is under the control of predrainage is promotora cytomegalovirus (CMV IE). This homologous vector was constructed using standard techniques of recombinant DNA (Sambrook et al.). The genome of FIV (ΔLTR) was synthesized by cloning polymerase chain reaction. As a matrix for PCR was proviral DNA from plasmids containing full-sized virus FIV PPR. The reverse primer (5’-ACGCGTCGACCAGCTAACAAGGTAGGAGAGACTCT-3’ 11/23 .98BW/3) was synthesized from the 5’end of the FIV genome above the Gag-coding region and introduced a unique SalI site. Direct primer (5’-TCGAGTCGACTTGTGACAGTTCTTAGTCCATAAGC-3’ 11/11/98BW.1) was synthesized from the 3’end of the FIV genome below 2nd Rev exon and introduced unique Sall site. Finite homological vector, A, used to create recombinant viruses containing the FIV genome (without LTR) and feline CD80 and CD86, or containing the FIV genome (minus LTR) and genes of feline CD80 and CD86 and feline IL-12, R35 and R40, in accordance with the procedure described in the homologous recombination Procedure for generating recombinant SPV, RPV and FHV".

Homologous vector 1005-95 .1 (RPV U/CMV-FIV genome(ΔLTR)/15L-lacZ). Plasmid 1005-95 .1 designed in order to integrate the foreign DNA into the RPV. This plasmid enters the genome-ALTR FIV gene β-glucuronidase E.coli, flanked DNA RPV. Above these cwiertnia genes were DNA fragment RPV, consisting of approximately 906 base pairs. Below these cwiertnia genes were DNA fragment RPV, the state is the seer of approximately 895 base pairs. Using plasmids in accordance with the procedure described in the homologous recombination Procedure for generating recombinant RPV," received a virus containing DNA encoding these alien genes. It should be noted that genome-ΔFIV LTR is under control pretannage of the cytomegalovirus promoter, and the gene β-glucuronidase is under the control of the synthetic early promoter of poxvirus ER. This homologous vector was constructed using standard techniques of recombinant DNA (Sambrook et al.) by joining restriction fragments from the following sources with the synthetic DNA sequences. This plasmid vector was obtained from HindIII restriction fragment of plasmid pSP64 (Promega), with approximately 2999 base pairs. Fragment 1 is a restriction HindIII-XbaI-sub-fragment HindIII restriction fragment U RPV, with approximately 906 base pairs (Knight et al.). Fragment 2 is 8.5 KBP-SalI fragment of the genome of FIV without LTR elements, and was synthesized by cloning polymerase chain reaction (PCR). As a matrix for PCR was proviral DNA from plasmids containing full-sized virus FIV PPR. The reverse primer (5’-ACGCGTCGACCAGCTAACAAGGTAGGAGAGACTCT-3’ 11/23 .98BW/3) was synthesized from the 5’end of the FIV genome above the Gag-coding areas and and have introduced a unique SalI site. Direct primer (5’-TCGAGTCGACTTGTGACAGTTCTTAGTCCATAAGC-3’ 11/11/ 98BW.1) was synthesized from the 3’end of the FIV genome below 2nd Rev exon and introduced a unique SalI site. Fragment 3 is a fragment size of approximately 2.0 so BP, containing the gene β-glucuronidase E.coli. Fragment 4 is a restriction HindIII-XbaI-subfragment HindIII restriction fragment U RPV, with approximately 895 base pairs. Finite homological vector 1005.95.1 used to create recombinant viruses containing the FIV genome (ΔL) and genes of feline CD80 and CD86, or to create recombinant viruses containing the genome (ΔLTR) FIV genes and feline CD80 and CD86 and feline IL-12, R35 and R40, in accordance with the procedure described in the homologous recombination Procedure for generating recombinant SPV, RPV and FHV".

Homologous vector 1016-74 A (FHVΔgE/CMV-FIV genome-ΔLTR/gX-lacZ). Homologous vector 1016-74 A designed to dellarovere part of the de-coding region of the herpes virus cats and incorporation of foreign DNA. It includes FIV genome (minus LTR) and gene β-galactosidase .li, flanked DNA FHV. Genome-ΔL FIV is under control of the cytomegalovirus promoter IE, a gene β-galactosidase is under the control of the promoter of pseudoreligious HH. It was constructed from these DNA sources using standard techniques recombin ntih DNA (Sambrook et al.). The plasmid vector was obtained from restriction endonuclease s7181-s7181-fragment plasmids pSP18/19, with approximately 2958 base pairs. Fragment 1 is an s7181-SmI-subfragment SalI fragment In FHV, with approximately 1415 base pairs. Fragment 2 is an SalI-fragment (size approximately 8.5 KBP) of the FIV genome without the LTR elements and was synthesized by cloning polymerase chain reaction. As a matrix for PCR was proviral DNA from plasmids containing full-sized virus FIV PPR. The reverse primer (5’-ACGCGTCGACCAGCTAACAAGGTAGGAGAGACTCT-3’ 11/23.98BW.3) was synthesized from the 5’end of the FIV genome above the Gag-coding region and introduced a unique SalI site. Direct primer (5’-TCGAGTCGACTTGTGACAGTTCTTAGTCCATAAGC-3’ 11/11/98BW.1) was synthesized from the 3’end of the FIV genome below 2nd Rev exon and introduced a unique SalI site. Fragment 3 is a fragment of the gene β-galactosidase size approximately 3.5 KBP Fragment 4 is a SalI-Asp718I-subfragment EcoRI fragment E FHV, with approximately 2205 base pairs. Finite homological vector E used to create recombinant viruses containing the genome (ΔLTP) FIV genes and feline CD80 and CD86, or to create recombinant viruses containing gene (ALTR) FIV genes and feline CD80 and CD86 and feline IL-12, R35 and R40 in accordance with the procedure described in time is hardly "the homologous recombination Procedure for generating recombinant SPV, RPV and FHV".

SPV 288

SPV 288 is a recombinant pox virus swine expressing the full complement ORFS contained in the genome of FIV, and four additional foreign gene. SPV 288 was obtained from SPV 282. SPV 282 contains the gene of feline CD86 and feline CD80 expressed in bitestrenos DNA-cluster under the control of the synthetic late promoter poxvirus LP1, initiating the transcription of CD80 and CD86 and including an element EMCV IRES between the two open reading frames; gene β-glucuronidase E. coli under the control of the synthetic early promoter OR in genomic HindIII fragment To the SPV. Homologous vector 992-23 .6 used to construct SPV 282, as described in the homologous recombination Procedure for generating recombinant SPV, RPV and FHV". Genes CD80 and CD86 gene and the β-glucuronidase E.coli was built in a separate and non-core partial HindIII-fragment To the SPV. The genome of CMV-FIV genes β-galactosidase activity was embedded in a separate and non-primary partial HindIII-fragment N SPV.

SPV 288 received from the SPV 282. This was done using homologous vector 1007-70 A (see above) and virus SPV-282, as described in the homologous recombination Procedure for generating recombinant RPV, SPV or FHV". The source material for transfection was skanirovali, as described in the section "Screening for recombinant Viru the SPV, expressing β-galactosidase (analyses BLUOGAL and CPRG) or β-glucuronidase (analysis of X-GLUC)". As a result, multiple cleaning cycles blue/green plaques were obtained recombinant virus SPV 288.

SPV 288 analyzed for the expression of FeLV gag gene, a gene of the shell of FeLV FIV genome and marker genes β-galactosidase and β-glucuronidase analysis on the formation of black plaques. Using goat polyclonal antibodies against FeLV gag (BioDesign, ME), and mouse monoclonal antibodies against membrane FeLV, gp70 (BioDesign, ME), it was determined that 100% of plaques generated in cells, ESK-4 infected with purified SPV 280, expressed the gene of FeLV gag gene and the shell FeLV.

Expression of feline CD80 and CD86 was confirmed by Western blot analysis using goat polyclonal antibodies against CD80 and CD86 person (R&D Systems, MN), respectively. It was discovered many bands ranging in size from 30 to 60 kDa specific for feline CD80, and many bands ranging in size from 40 to 70 kDa specific for feline CD86. These bands represented the picture of the alternating and multiple glycosylation CD80 and CD86, expressed in "context" SPV in cells, ESK-4. The expression of proteins encoded in the genome of FIV, was confirmed by Western blot analysis using cat sera from FIV-infected cats.

FHV 054

FHV 054 represents the t of a recombinant herpes virus cats, expressing the full complement ORFS contained in the genome of FIV, and 2 additional foreign gene. The homology vector designated 1016-75 .B1 designed for embedding genome (ΔLTR) FIV and gene β-galactosidase in the unique long incomplete SalI-fragment N FHV. The box was built between genome gL and related gene activator of transcription. The genome of FIV is under control of the promoter of CMV IE, and gene β-galactosidase gene is under the control of the promoter element pseudoreligious DF.

FHV 054 was derived from FHV 30, containing the gene of feline CD80 in the website deletirovanie de FHV. This was done using homologous vector 1016-75 .B1 and virus FHV 030, as described in the homologous recombination Procedure for generating recombinant RPV, SPV or FHV". The source material for transfection was skanirovali, as described in "Methods of screening for recombinant virus FHV expressing β-galactosidase (analyses BLUOGAL and CPRG) or β-glucuronidase (analysis of X-GLUC)". As a result, multiple cleaning cycles blue/green plaques were obtained recombinant virus FHV 054.

FHV 054 analyzed for gene expression β-galactosidase analysis on the formation of black plaques. Using murine monoclonal antibodies (Biodesign, ME), it was determined that 100% of plaques generated in the cell is CRFK, infected with the virus FHV 054, purified by the method of plaques expressed gene β-galactosidase. It was determined that this virus is stable, at least after 5 transfers.

Expression of feline CD80, FIV gag and shell FIV was confirmed by Western blot analysis using polyclonal antibodies against CD80 person (R&D Systems, MN), mouse monoclonal antibodies against FIV gag (Custom Monoclonals, CA), mouse monoclonal antibodies against membrane FIV (Biodesign). The expression of the full complement FIV genes present in the genome was confirmed by Western blot analysis using sera from convalescent FIV-infected cats.

FHV 055

FHV 055 is a recombinant herpes virus cats expressing the full complement ORFS contained in the genome of FIV, and 3 additional foreign gene. The homology vector designated 1016-75 .B1, designed for embedding genome (ALTR) FIV and gene β-galactosidase in the unique long incomplete SalI-fragment N FIV. The box was built between genome gL and related gene activator of transcription. The genome of FIV is under control of the promoter of CMV IE, and gene β-galactosidase gene is under the control of the promoter element pseudoreligious DF.

FHV 055 was derived from FHV 041, containing the gene of feline CD86 gene and the β-glucuronidase in with the continue deletirovanie de FHV. Gene feline CD86 is under control of the promoter de FHV, and gene β-glucuronidase is under control of the promoter of pseudoreligious HH. This was done using homologous vector 1016-75 .B1 and virus FHV 041, as described in the homologous recombination Procedure for generating recombinant RPV, SPV or FHV". The raw materials for transfection was skanirovali, as described in "Methods of screening for recombinant virus FHV expressing β-galactosidase (analysis X-BLUOGAL and CPRG) or β-glucuronidase (analysis of X-GLUC)". As a result, multiple cleaning cycles blue/green plaques were obtained recombinant virus FHV 055.

FHV 055 analyzed for the expression of β-galactosidase and β-glucuronidase analysis on the formation of black plaques. Using murine monoclonal antibodies (BioDesign, ME), and rabbit polyclonal antibodies (Molecular Probes, OR), it was determined that 100% of plaque from CRFK cells infected with FHV 055, purified by the method of plaques, expressed β-galactosidase and β-glucuronidase, respectively. It was determined that this virus is stable, at least after 5 transfers.

Expression of feline CD80, FIV gag and shell FIV was confirmed by Western blot analysis using polyclonal antibodies against CD86 person (R&D Systems, MN), mouse on the different monoclonal antibodies against FIV gag (Custom Monoclonals, CA) and mouse monoclonal antibodies against membrane FIV (BioDesign). The expression of the full complement FIV genes present in the genome was confirmed by Western blot analysis using sera from convalescent FIV-infected cats.

RPV-055

RPV-055 is a recombinant virus of smallpox raccoons, expressing polnorazmernyi complement ORFS contained in the genome of FIV, and 4 additional foreign gene. RPV 055 was derived from RPV-045, containing the gene of feline CD86 gene and the feline CD80 expressed in bitestrenos DNA-cluster under the control of the synthetic late promoter poxvirus LP1, initiating the transcription of CD80 and CD86 and including an element EMCV IRES between the two open reading frames; gene β-glucuronidase E. coli under the control of the synthetic early promoter OR in incomplete genomic HindIII fragment N RPV. Homologous vector 1005-95 .1 used for constructing RPV 055, as described in the homologous recombination Procedure for generating recombinant SPV, RPV and FHV".

Genes CD80 and CD86 gene and the β-galactosidase of E. coli was built in a separate and non-primary partial HindIII-fragment N RPV. The genome of CMV-FIV gene β-glucuronidase built in a separate and non-core partial HindIII-fragment U RPV.

RPV 055 was derived from RPV 045. This was done using g mologogo vector 1005-95 .1 and virus RPV 045, as described in the homologous recombination Procedure for generating recombinant RPV, SPV or FHV". The source material for transfection was skanirovali, as described in the section "Screening for recombinant virus SPV expressing β-galactosidase (analyses BLUOGAL and CPRG) or β-glucuronidase (analysis of X-GLUC)". As a result, multiple cleaning cycles blue/green plaques were obtained recombinant virus RPV 055.

RPV 055 analyzed for gene expression FeLV gag, shell of FeLV FIV genome and marker genes β-galactosidase and β-glucuronidase analysis on the formation of black plaques. Using goat polyclonal antibodies against FeLV gag (BioDesign, ME), and mouse monoclonal antibodies against membrane FeLV, gp70, (BioDesign, ME), it was determined that 100% of plaques generated from VERO cells infected with purified virus RPV 055, expressed the gene of FeLV gag and shell FeLV (BioDesign, ME).

Expression of feline CD80 and feline CD86 was confirmed by Western blot analysis using goat polyclonal antibodies against CD80 and CD86 person (R&D Systems, MN), respectively. It was discovered many bands ranging in size from 30 to 60 kDa specific for feline CD80, and many bands ranging in size from 40 to 70 kDa specific for feline CD86. These bands represented the picture of the alternating and multiple glycosylation CD80 and D86, expressed in "context" RPV in VERO cells. Expression of proteins encoded in the genome of FIV, was confirmed by Western blot analysis using the cat sera from FIV-infected cats.

BIBLIOGRAPHY

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1. Therapeutic vector used in therapy of infectious diseases of cats, which contains at least one foreign nucleic acid, each of which (a) encodes a protein selected from the group consisting of protein feline CD28, is presented in SEQ ID NO:8, or immunogenic parts; of the protein of feline CD80, presented in SEQ ID NO:2 or 4, or immunogenic parts; of the protein of feline CD86 presented in SEQ ID NO: 6, or immunogenic parts; or of the protein of feline CTLA-4, presented in SEQ ID NO: 10, or immunogenic part, and (b) is able to be expressed when introducing the vector into an appropriate host.

2. Therapeutic vector according to claim 1, characterized in that the vector is a therapeutic recombinant virus with at least one foreign nucleic acid, a built-in non-essential region of the viral genome of the virus.

3. Therapeutic vector according to claim 2, from which causesa fact, that contains at least two foreign nucleic acids, each of which is embedded in a non-essential region of the viral genome.

4. Therapeutic vector according to claim 3, characterized in that it contains at least three alien nucleic acids, each of which is embedded in a non-essential region of the viral genome.

5. Therapeutic vector according to claim 4, characterized in that it contains four alien nucleic acids, each of which is embedded in a non-essential region of the viral genome.

6. Therapeutic vector according to claim 2, wherein said virus is a virus of smallpox raccoons, smallpox pigs or herpes virus cats.

7. Therapeutic vector according to any one of claim 2 to 6, characterized in that it contains more than one foreign nucleic acid, each of which is embedded in the same non-core region.

8. Therapeutic vector according to any one of claim 2 to 6, characterized in that it contains more than one foreign nucleic acid, all of these foreign nucleic acid is not built in the same non-core region.

9. Therapeutic vector according to any one of claim 2 to 6, characterized in that it further comprises a foreign nucleic acid encoding the immunogen derived from the pathogen.

10. Therapeutic vector according to claim 9, wherein the pathogen is a cat PA is oven, rubivirus, chlamydia, Taxoplasma gondii, Dirofilaria immitis, a flea or a bacterial pathogen.

11. Therapeutic vector of claim 10, wherein a feline pathogen is human immunodeficiency virus cats (FIV), the virus leukemia cats (FeLV), the virus infectious peritonitis in cats (FIP)virus, panleukopenia in cats, feline caliciviruses, cat reovirus type 3, feline rotavirus, feline coronavirus, syncytial virus cats sarcoma virus in cats, herpes virus in cats, the virus disease Bourne cats or cat parasite.

12. Therapeutic vector according to any one of claim 2 to 6, characterized in that at least one foreign nucleic acid comprises a promoter for expression of foreign nucleic acids.

13. Therapeutic vector according to any one of claim 2 to 6, characterized in that the expression of at least one foreign nucleic acid is under the control of a promoter that is endogenous to the virus.

14. Therapeutic vector according to any one of claim 2 to 6, characterized in that it further comprises a foreign nucleic acid, encoding the detected token.

15. Therapeutic vector according to 14, characterized in that detektivami marker is beta-galactosidase .li.

16. Therapeutic vector according to claim 11, characterized in that the immunogen feline pathogen is gag-protease FIV, FIV envelope protein, gag-p is Oteiza FeLV or FeLV envelope protein.

17. Therapeutic vector according to any one of claim 2 to 6, characterized in that the virus is a herpes virus cats, and non-core region is the gene for glycoprotein E of the herpes virus cats.

18. Therapeutic vector according to 17, characterized in that the herpes virus cats denoted as S-FHV-031 (ATSS, the access number VR-2604).

19. Therapeutic vector according to any one of claim 2 to 6, characterized in that the virus is the virus of smallpox pigs, and non-core area is larger HindIII-BglII - subfragment HindIII-fragment M smallpox pigs.

20. Therapeutic vector according to claim 19, characterized in that the smallpox virus swine marked as S-SPV-246 (ATSS, the access number VR-2603).

21. Therapeutic vector according to any one of claim 2 to 6, wherein the foreign nucleic acid encodes immunogenic portion of the protein of feline CD28, CD80 or CD86, while the immunogenic portion is soluble.

22. Therapeutic vector according to any one of claim 2 to 6, wherein the foreign nucleic acid encodes a protein of feline LA-4.

23. Therapeutic vector according to claim 2, characterized in that it further comprises nucleic acid encoding the genome of the virus feline immunodeficiency or part.

24. Therapeutic vector according to claim 2, characterized in that it further comprises nucleic acid encoding the genome of the virus leukemia cats or part.

25. Therapeutic vector according to item 23 or 24, characterized in that it further comprises nucleic acid encoding a feline GM-CSF, feline IL-12 R35 or feline IL-12 R40.

26. Therapeutic vector according to claim 2, characterized in that the viral genome is double-stranded nucleic acid.

27. Therapeutic vector according to claim 2, characterized in that the viral genome is single-stranded nucleic acid.

28. Therapeutic vector according to item 27, wherein the single-stranded nucleic acid is antisense.

29. Therapeutic vector according to item 27, wherein the single-stranded nucleic acid is meaningful.

30. Therapeutic vaccine used in therapy of infectious diseases of cats, containing an effective immunizing amount of a therapeutic vector according to any one of claim 2 to 6, and a suitable carrier.

31. Therapeutic vaccine according to item 30, wherein the effective immunizing amount of a therapeutic vector is from about 1×105to about 1×108boe/ml

32. Therapeutic vaccine according to item 30, characterized in that it further includes a mixture containing a therapeutic vector and an effective immunizing amount of the second immunogen.

33. Method of strengthening the immune response in cats, including the introduction of the cat efficiency is effective immunizing amount of a therapeutic vector according to any one of claim 2 to 6.

34. The method of immunization of cats against infectious diseases, including the introduction of the cat effective amount of therapeutic vector according to any one of claim 2 to 6.

35. A method of suppressing an immune response in cats, including the introduction of the cat effective suppressive amount of a therapeutic vector according to item 21 or 22.

36. The method according to p or 34, characterized in that the introduction is carried out intravenously, subcutaneously, intramuscularly, trasmision, topically, orally or intraperitoneally.

37. The method according to p, characterized in that the cat is a recipient of transplanted organ or tissue, or subject an immune response.

38. A method of suppressing an immune response in cats, including the introduction of the cat nucleic acid capable of gibridizatsiya with nucleic acid therapeutic vector item 27 and capable of gibridizatsiya with (a) mRNA-transcript feline CD28, (b) mRNA-transcript feline CD80 or (C) mRNA-transcript feline CD86 and to inhibit their translation, with the specified nucleic acid is present in an amount effective for inhibiting translation, thereby suppressing the immune response in cats.

39. A method of reducing or destroying tumors in cats, including the introduction of the cat therapeutic vector according to claim 2, in this case, nucleic acid encodes a protein of feline D80, protein feline is his CD86 or their combination in a quantity effective to reduce or destroy the tumor.

40. The method according to § 39, wherein therapeutic vector further comprises a feline antigen associated with a tumor, and are able to Express, with the introduction of a system.

41. Therapeutic vaccine used in therapy of infectious diseases of cats, containing an effective immunizing amount of a therapeutic vector according to item 23 or 24, and a suitable carrier.



 

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