Isolated nucleic acid encoding feline ligand cd86, diagnostic oligonucleotide, cloning vector, vaccine, methods for induction or suppression of immunity in cat
FIELD: biotechnology, genetic engineering, immunology.
SUBSTANCE: invention proposes: isolated nucleic acid encoding feline ligand CD86; diagnostic oligonucleotide; cloning vector; vaccine for modulation of the immune response in cat; method for induction, enhancement and suppression of immune response in cats. Proposed group of inventions allows designing effective vaccines used in prophylaxis of immunodeficiency in felines and infectious peritonitis in domestic cats. Invention can be used in veterinary science.
EFFECT: valuable properties of nucleic acid.
27 cl, 13 dwg, 5 tbl, 8 ex
This application has priority on patent application U.S. No. 09/071699, filed may 1, 1998, the full contents of which are incorporated in this application by reference. In the text of this application references to other publications are given in parentheses. The full bibliography of these references can be found at the end of the text immediately before the list of sequences. The content of these publications in full included in the present application as reference for a more complete description of the problem in the art to which the present invention relates.
Background of the invention
There are currently no effective vaccine for the prevention of feline immunodeficiency and feline infectious peritonitis in cats. Now available vaccine virus feline leukemia, but their effectiveness remains questionable, and in some cases they can cause disease. Therefore, in the art there is a need for the agents and compositions that would protect against these and other diseases, against which there is no vaccine or existing and commonly used vaccines against which you want to improve. In addition, hampered vaccination of kittens due to the inability to resist maternal antibodies in them. Hence, there is also a need for safe and effective Agay the tah to overcome such barriers.
Stimulation in the body activation and proliferation of T-lymphocytes in response to disease, as is defined by the two interactions: recognition of T-cell receptor (TCR) immunogenic peptides involving molecules of class I major histocompatibility complex (MHC) and the secondary interaction of auxiliary ligands, such as CD80 and CD86, with their corresponding receptors CD28 and (or) CTLA-4 on the surface of T-lymphocyte. The effective interaction of these two mechanisms leads to activation and proliferation and CD4-positive, and CD-8 positive T-cells and increases the production of immunoregulatory cytokines types, Th1 and Th2. In the absence of adequate costimulate T cells can develop anergicakimi a condition in which T-cells are unable to proliferate and secrete cytokines. For many years the key regulators of T-cell responses were considered two molecule - receptor CD28 and its ligands CD80 and CD86. CD28 is a primary T-cell co-stimulatory receptor, which upon binding to CD80 and CD86 enhances the proliferation of T-cells and the synthesis of cytokines, preventing the death of T-cells. CTLA-4 (also known as CD152), which is a homologue of CD28, also plays an important role in the process of costimulation. Although it is not precisely defined, it is assumed that suppresses co-stimulatory T-cell responses. Interaction is between CD28, CTLA-4 and their ligands CD80 and CD86 in the process of costimulation is the key to the induction and suppression of immune responses to disease in the body as a whole. By manipulating these four co-stimulatory molecules apparently you can regulate T-cell response towards activation, inhibition, or changes direction, it is possible to amplify the desired immune response against a specific pathogen or disease. In particular, they can be used for vaccination against infectious diseases, for the treatment of infectious diseases and neoplastic degenerative, autoimmune and immunodeficiency.
T-lymphocytes of the immune system of mammals to perform both regulatory and effector functions. Precursor T cells arise in the bone marrow from stem cells and migrate to the thymus. In the thymus processes of maturation and breeding with the formation of a population of naive immune cells that can recognize the antigen in its presentation in conjunction with the major histocompatibility complex (MHC), but are not self-reactive. After maturation in the thymus, each T-cell carries clonal T-cell receptor (TCR), which determines its antigenic specificity. In addition, T cells two main subclasses found in most adult mammalian is affected - CD44+and CD8+bear TCR composed αand β-subunits (Allison & Lanier, 1987).
Polypeptide and gene organization of the TCR protein similar to that characteristic of the molecules of the immunoglobulin (Ig), and it has many similar characteristics compared to the membrane-bound Ig b-lymphocytes (Allison & Lanier, 1987). As the molecule Ig, TCR potentially must recognize a huge number of possible antigenic sequences. From this point of view the organization and rurangirwa TCR gene is similar in its complexity to those in b cells (Davis & Bjorkman, 1988). As in the case of immunoglobulins In-cell education idiotypical diversity of T-cells due to the presence of multiple gene copies variable domains (V) in the germline occurring randomly rearrangeable αand β-subunits and variability arising from phenomena compounds and inserts (Davis & Bjorkman, 1988). However, unlike b cells, the formation of the diversity of T cells does not seem to be associated with somatic mutations, although the potential repertoire of TCR molecules is not inferior to that of Ig molecules (Lechler et al., 1990).
Molecule TCR though and is responsible for the recognition of antigen is not capable of signal transmission (Allison & Lanier, 1987). Conformational changes after TCR binding involving antigen MHC, located on antigenpresenting the affected cells (APC), determines the signal transmission through non-covalent complex of surface molecules, including CD3 and ζ-subunit (Clevers et al., 1988). Binding to TCR leads to the phosphorylation D3 complex, which indirectly leads to the introduction into the cell of calcium ions, resulting in the initiation of the production of IL-2 and IL-2R (Weiss & Littman, 1994). This cascade is considered as the initial event in the activation of T cells.
TCR recognizes antigen only when it is presented with the participation of the MNF. Known two classes of MHC proteins that are associated with the process of prezentowania antigen on T-cells. Molecules of class I MHC is found on almost all nucleated cells of the body, and their function is the transfer of endogenous peptides on the surface of cells (Matasumura et al., 1992). The peptide expressed with the participation of MHC class I, recognized by T-cells expressing CD8 in connection with the TCR (Littman, 1987). CD8-positive T cells function in the immune surveillance of destruction of cells infected with the virus and tumor cells. The recognition of T-cell CD8+"foreign" molecules (peptides or modified their peptides that may indicate the development of a tumor) causes the destruction of the cell, involving cytotoxic T-lymphocytes (CTL) (Berke, 1994).
Molecules of class II MHC, representing the second group of factors glavnog the histocompatibility complex, OK found only in specialized antigen-presenting cells, including b cells, macrophages/monocytes and dendritic cells, although the possibility of their induction and in some other cell types in response to specific stimuli (Germain, 1993). The molecule class II MHC is responsible for prezentowanie foreign antigen on CD4-positive T-cell. The antigen, which was fagalicious, endochitinase or associated surface Ig with subsequent absorption by the cell undergoes endogenous processing and binds with a molecule of class II MHC (Unanue, 1987). Then this molecule enters the cell surface and becomes available for its recognition of CD4-positive T-cells αβ-TCR (Littman, 1987). Recognition of antigen by T-cells CD4+causes the release of cytokines and growth factors required for the initiation and propagation of the different elements of an active immune response (Mosmann &Coffman, 1987).
The differentiation of groups of T cells αβ-TCR is determined by the presence of CD4 and CD8, which is associated with the determination of the functions of each of these groups. The simultaneous presence of CD4 and CD8 T-cell excluded (Littman, 1987). I.e. in the process of selection and maturation in the thymus T cells-αβ receive only CD4 or CD8. These molecules provide the stability of the interaction between TCR and MHC-associated antigen and determine for a given T-cells, ka is their class of MHC molecules (I or II) prezentuetsya antigen (Littman, 1987). Binding domain of the molecule CD4 or CD8 recognizes appropriate polimorfny portion of the molecule class I or class II (Clayberger et al., 1994). Binding of CD4 or CD8 with these specific areas ensures the stability of the interaction between TCR and MHC-associated antigen from the point of view of the initiation of T-cell activation (Littman, 1987). Thus, CD4-positive T cells are functionally interact only with cells APCS presenting antigen with class II molecules and thereby initiating T-helper response, whereas CD8-positive T-cells only recognize antigen pretentiously involving class I molecules after binding triggers a cytotoxic response (Germain, 1993). Two distinct phenotype of T - helper and CTL - can be identified by surface-cell expression of either CD4 or CD8.
The majority of CD4-positive T-lymphocytes are generally considered to T-helper population, although there is supposed CD4-positive CTL-subtype (Yasukawa et al., 1989). CD4-positive T-helper cells are the main regulators of the immune response due to the generation of a series stimulatory and suppressor cytokines (Mosmann &Coffman, 1987). Produced by these cells factors are important mediators in the initiation and humoral, i.e. mediated by antibodies, and cell, i.e. a delayed hypersensitivity is of type (DTH), responses (Mosmann &Coffman, 1987). In order T cells CD4+activated on the development of soluble growth factors, there should be a complex cascade of processes. The antigen detected and undergoes endocytosis specific ARS-cells, which normally are macrophages (Unanue, 1984). ARS denatured antigen protein and divide it into smaller pieces, then 15-18-amino acid peptides bound by MHC molecules in the endoplasmic reticulum and then transferred to the cell surface (Rotzschke et al., 1994). Transferred to the surface antigen is the "visible" for T-cells and can be recognized groups of T-cells expressing CD4 and have the appropriate TCR idiotype (Germain, 1993). When accurate recognition of antigen by T-cell and formed the exact auxiliary signals, is the differentiation of naive lymphocytes and starts its clonal proliferation. Still not established reasons is the preferred formation of the answer of the 1st type (cellular) against the response of the 2nd type (humoral) (Mosmann &Coffman, 1989).
Part of T-helper cells is necessary to ensure activity and humoral and cellular responses. Dependent on T-cell In-cell response is required for the production of antibodies to most antigens, T-helpers need to ensure is ecene correct maturation of b-cells (Chesnut et al., 1986). Once expressed. B-cell surface Ig bound antigen occurs internalization, processing and output of antigen on the surface molecules of class II MHC (Germain, 1993). Direct intercellular contact between T-cell CD4+having the exact TCR idiotype, and In-cell contributes to the activation and proliferation of T-cells (Chesnut et al., 1986). Activated T-helper initiates the response of the 2nd type due to the secretion of factors necessary for the growth and differentiation of b-cells (Mosmann &Coffman, 1989). These factors are the interleukins IL-4, IL-5 and IL-13, which is able to induce activation and proliferation of b-cells and is also important in the process izotopicheskogo switching molecules, antibodies, whereas IL-10 prevents the initiation of the response of the 1st type, which in turn is a factor in the negative regulation of the humoral response (Mosmann &Coffman, 1989).
Cellular responses (1st type) induced not as humoral responses (2nd type) (Sher et al., 1992). After activation of T cells and their maturation to answer the 1st of this type of T-cells produce factors that promote cellular immunity. IL-2 is a T-cell growth factor, which activates CTL responses, while γ-interferon activates macrophages, CTL and neutrophils (Wang et A1., 1993).
Thus, T-helpers are able to mediate two principal is but mutually exclusive answer. Sign secretion of cytokines associated with the initiation of the humoral response includes factors that are suppressors of cellular response, and Vice versa (Mosmann &Coffman, 1989). It is still unclear what causes the T cell to show any sign of the 1st type (production of IL-2, γ-interferon and lymphotoxin), or the sign of the 2nd type (IL-4, IL-5, IL-6, IL-10 and IL-13), although it is anticipated that emerging variant cytokine profile can influence the type of APC that presents the antigen or soluble factors produced by this ARS (Mosmann &Coffman, 1989). In addition to the helper cells of the 1-St and 2-nd type, there is the so-called 0-th type helper cells, which is characterized by the parameters of cytokine secretion, intermediate in relation to the 1st and 2nd types (Gajewski et al., 1989). Although the types of helpers were mainly characterized in vitro experiments, i.e. may reflect cultural artifacts, they are important models of the role of T-helper cells play in controlling the development of specific responses in the situation in vivo.
In addition to CD4-positive T-helper lymphocytes second population of T cells-αβ composed of CD8-positive cytotoxic lymphocytes (CTL). It is believed that CTL CD8+play a leading role in immune surveillance, whose main function is associated with destruction of cells infected by viruses or intracellular what bacteria as well as tumor cells (Berke, 1994). These cells are also ways to produce cytokines, but, in General, only those that are associated with the induction of cellular responses (IL-2, γ-interferon and TNF) (Fong & Mosmann, 1990). Receptor TCR of these cells with the participation of CD8 recognizes antigen presented by using molecules of class I MHC (Littman, 1987). In General, all nuclear cells are characterized by surface expression of class I molecules presenting intracellular synthesized peptides (Matasumara, 1992). Specific immune activity areas, including the brain and the testes, are characterized by a low level of protein synthesis, although he is induced in these areas under the influence of interferon (Moffett & Paden, 1994).
Proteins produced in the endoplasmic reticulum during the normal functioning of cells, denature, partially split and with the help of molecules of class I MHC brought to the surface (Engelhard, 1994). These polypeptides proteoliticeski linearized and contact in the form of 9-12-amino acid epitopes by class I molecules, which are then brought to the surface of the cell (Engelhard, 1994). Theoretically thus on the surface can be any synthesized intracellular proteins, so the selection in the thymus due to perfect the elimination of all self-reactive T cells and the immune system of supervision which may detect the presence of virus-infected or transformed cells (Berke, 1993). The recognition of a foreign peptide expressed by class I molecules, antigen-specific T-cell receptors on the surface of the CTL CD8+(Lechler et al., 1990). Activation requires contact between effector cells and target (Berke, 1994). When the antigen is considered as alien, detected by receptor TCR, the interaction of molecules is stabilized by CD8 binding to class I molecule on the surface of infected cells (Littman, 1987). After recognition and activation of T-cells and forms a conjugate between cell-target and effector T-cell, after which the effector cell is destroyed (Taylor & Cohen, 1992). Thus, in this mechanism when changing their own proteins or breach of cellular mechanisms in the attack of pathogen peptides to be recognized by the immune system of supervision, resulting in this element of the immune system will allow for the destruction of diseased cells (Berke, 1994).
Cytotoxicity is believed, is due to one of two basic mechanisms. Or in the cell undergo apoptosis, or it literoitca involving cytotoxic granules secreted by CTL (Berke, 1993). Apoptosis in the target cells is induced by secretion cells CTL factors that induce the expression of genes contributing to the death of cells (Russel, 1983). The advantage of this is mechanism is the absence of lysis of the cells, that reduces the likelihood of potentially dangerous infectious contents of this cell (Nagata & Golstein, 1995). However, cell lysis can be the most common mechanism, according to which it is directed cell killing. The main component produced by cells of CTL "cytotoxic granules" is a protein perforin, which pierced through the membrane of target cells (Liu et al., 1995). Although there are other types of cells involved in this form of immune surveillance, it is believed that CTL are the main component of antiviral and antitumor immunity and are considered as a necessary element in the protection against specific pathogens (Kupfer & Singer, 1989).
The original surface-cellular proteins were used for differentiation of specific cell populations. Later were established functional aspects of many of these molecules, and taking into account their importance for the differentiation of cell populations became more familiar with their important role in the functioning of many cells.
A different accessory molecules and adhesion molecules that are involved in the development of the immune response is expressed by T-cells and antigen-presenting cells (van Seventer et al., 1991). Adhesion molecules are expressed at some level by most cells of the immune system. They are important for maintaining cells in ODA is divided by area and for the initiation and maintenance of intercellular contacts (Mescher, 1992).
Two sets of adhesion molecules CD2/LFA-3 (CD58) and LFA-1/ICAM-1 is involved in the stabilization of the interaction between T cells and cells of ARS and increasing activity (Springer et al., 1987). CD2 is one of the earliest markers expressed by precursors of T-lymphocytes, and exists throughout the life of the cell, whereas LFA-1 is expressed by T-cells and later positively regulated in the memory cells or by type of inducibility (Springer et al., 1987).
Complexes of auxiliary molecules also exhibit adhesive properties, but their main function appears to be a transfer of intercellular signal after binding of the ligand (Anderson et al., 1988). After establishing interaction between a receptor and its ligand is such a change in the conformational structure of the molecule, which determines the transmission signal in the cytoplasm of one or both cells (Hutchcroft & Bierer, 1994). Passed these molecules signals perform a number of functions to facilitate the development of T cells, however, in the absence of signals mediated by these molecules, T cells can become anergicakimi (Leung & Lindsley, 1994).
The interaction of CD28/CD80 is a major component of intensive immune response mediated by T-cells (Linsley et al., 1993a). The interaction of auxiliary molecules CD28 with their respective ligand CD80 necessary for full activation of the and and proliferation of naive T cells (Linsley et al., 1991a). Also this interaction is considered, plays a key role in the proliferation of activated CD4-positive T-cell memory and in preventing apoptotic cell death (Linsley et al., 1991a). The establishment of such interaction and the deciphering of its mechanisms is key to understanding the processes of immunity, mediated by T-cells.
The present invention relates to isolated and purified DNA encoding the ligand CD80 (B7-1) cats, ligand CD86 (B7-2) cats, receptor CD28 cats or receptor CTLA-4 (CD152) cats, as well as vectors comprising nucleic acid encoding CD80 cats, CD86 cats, CD28 cats or CTLA-4 cat. The present invention relates to a cell host transformed CD80-coding vectors, CD86-coding vectors, CD28-coding vectors or CTLA-4-encoding vectors. The present invention relates to polypeptides encoded by nucleic acid CD80 cats, CD86 cats, CD28 cats or CTLA-4 cat.
The present invention relates to a vaccine containing an effective amount of polypeptides encoded by nucleic acid CD80 cats, CD86 cats, CD28 cats or CTLA-4 cat. Also the present invention relates to vaccines which additionally contain immunogen derived from pathogens. The present invention relates to vaccines, can enhance the immune response. Takanassee the invention relates to vaccines, able to suppress the immune response.
Brief description of drawings
Figure 1A. Nucleotide and amino acid sequence of CD80 (B7-1) cat (TAMU) (SEQ ID NO: 1 and 2).
Figure 1C. Range of hydrophobicity of amino acid sequence of CD80 (B7-1) cat (TAMU).
Figure 2A. Nucleotide and amino acid sequence of CD80 (B7-1) cat (SYNTRO) (SEQ ID NO: 3 and 4).
Figure 2B. Range of hydrophobicity of amino acid sequence of CD80 (B7-1) cat (SYNTRO).
Figure 3A. Nucleotide and amino acid sequence of CD86 (B7-2) cat (SEQ ID NO: 5 and 6).
Figure 3C. Range of hydrophobicity of amino acid sequence CD86 (B7-2) cat.
Figure 4A. Nucleotide and amino acid sequences of CD28 cat (SEQ ID NO: 7 and 8).
Figure 4B. Range of hydrophobicity of amino acid sequence of CD28 cats.
Figure 5A. Nucleotide and amino acid sequence of CTLA-4 (CD152) cat (SEQ ID NO: 9 and 10).
Figure 5B. Range of hydrophobicity of amino acid sequence of CTLA-4 (CD152) cat.
Detailed description of the invention
The present invention is selected nucleic acid encoding the ligand CD80 cats or soluble ligand CD80 cats. Also the present invention is selected nucleic acid encoding the ligand CD86 cats or soluble ligand CD86 cats. Also the present invention is vyd is certain nucleic acid, encoding the receptor CD28 cats or soluble receptor CD28 cats. Also the present invention is selected nucleic acid encoding the receptor, CTLA-4 cat or soluble receptor, CTLA-4 cat.
In one implementation of the present invention is a nucleic acid encoding the ligand CD80 cats, which is characterized by the amino acid sequence shown in figa, since the residue of methionine and ending with the residue of threonine (SEQ ID NO: 1). In another implementation of the present invention is a nucleic acid encoding the ligand CD86 cats, which is characterized by the amino acid sequence shown in figa, since the residue of methionine and ending with a glutamine residue (SEQ ID NO: 5). In another implementation of the present invention is a nucleic acid encoding the receptor CD28 cats, which is characterized by the amino acid sequence shown in figa, since the residue of methionine and ending with serine residue (SEQ ID NO: 7). In another implementation of the present invention is a nucleic acid encoding the receptor, CTLA-4 cats, which is characterized by the amino acid sequence shown in figa, since the residue of methionine and ending with residue asparagine (SEQ ID NO: 9).
In the implementation described above and is gaining nucleic acid is DNA or RNA. In another implementation of the DNA is cDNA or genomic DNA.
The present invention provides oligonucleotide consisting of at least 12 nucleotides, characterized by complementarity in relation to a unique sequence present in the composition of the nucleic acid encoding CD28, CD80, or CD86, CTLA-4, described above. In another implementation of the present invention is represented oligonucleotide consisting of at least 15 or 16 nucleotides which is characterized by complementarity in relation to a unique sequence present in the composition of the nucleic acid encoding CD28, CD80, or CD86, CTLA-4, described above.
In another implementation of the invention described above is represented oligonucleotide that is marked-defined label. In one implementation detectable label is a radioactive isotope, a fluorophore or Biotin. In another implementation of such oligonucleotide selectively methylated.
The present invention is a vector comprising nucleic acid encoding the ligand CD80 cats or soluble ligand CD80 cats. In another implementation of the present invention is represented plasmid vector, designated PSI-B7-1/871-35 (Collection of ADS, Depository No. 209817). This plasmid was deposited at the American type culture collection (ATSS) 29 AP is El 1998 (the address of which is 10801 University Boulevard, Manassas, VA 20108-0971, USA) in accordance with the Budapest Treaty on International rules of the Deposit of microorganisms for the purposes of patenting procedures.
The present invention is a vector comprising nucleic acid encoding the ligand CD86 cats or soluble ligand CD86 cats. In another implementation of the present invention is represented plasmid vector, designated B7-2#19-2/011298 ADS, Depository No. 209821). This plasmid was deposited at the American type culture collection (ATSS) April 29, 1998 (the address is 10801 University Boulevard, Manassas, VA 20108-0971, USA) in accordance with the Budapest Treaty on International rules of the Deposit of microorganisms for the purposes of patenting procedures.
The present invention is a vector comprising nucleic acid encoding the receptor CD28 cats or soluble receptor CD28 cats. In another implementation of the present invention is represented plasmid vector, designated PSI-CD28-#7/100296 ADS, Depository No. 209819). This plasmid was deposited at the American type culture collection (ATSS) April 29, 1998 (the address is 10801 University Boulevard, Manassas, VA 20108-0971, USA) in accordance with the Budapest Treaty on International rules of the Deposit of microorganisms for the purposes of the procedure is atestowanie.
The present invention is a vector comprising nucleic acid encoding the receptor, CTLA-4 cat or soluble receptor, CTLA-4 cat. In another implementation of the present invention is represented plasmid vector, denoted by CTLA-4-#1/091997 ADS, Depository No. 209820). This plasmid was deposited at the American type culture collection (ATSS) April 29, 1998 (the address is 10801 University Boulevard, Manassas, VA 20108-0971, USA) in accordance with the Budapest Treaty on International rules of the Deposit of microorganisms for the purposes of patenting procedures.
The present invention is described above vector which further includes a promoter functionally attached to the nucleic acid. In another implementation of the present invention is a cell host, which carries any of the above-mentioned vectors. In one implementation of such cell-host, supporting one of the above vector is a eukaryotic or prokaryotic cell. In another implementation of the host-cell cells are E. coli, yeast cells, COS cells, cell RS, cells SNO or GH4C1 cells.
The present invention is a polypeptide encoded by a nucleic acid ligand CD80 cats or soluble ligand CD80 cats. In the implementation of the present invention is predstavljaetsja polypeptide, encoded by the nucleic acid ligand CD86 cats or soluble ligand CD86 cats. In another implementation of the present invention is the polypeptide encoded by the nucleic acid receptor CD28 cats or soluble receptor CD28 cats. The present invention is a polypeptide encoded by a nucleic acid receptor, CTLA-4 cat or soluble receptor, CTLA-4 cat.
In another implementation of the present invention is the method of production mentioned above polypeptides by culturing a host cell that expresses these polypeptides, and the allocation of these polypeptides thus obtained.
The present invention is a vaccine containing an effective amount of the above-mentioned polypeptide and a suitable carrier. In another implementation of the invention appears to be the vaccine, which consists of an effective amount of the above-mentioned polypeptide and a suitable carrier is an amount from about 0.01 mg to about 100 mg / 1 dose. In another implementation of the invention appears to be the vaccine, in which an effective amount of the above-mentioned polypeptide and a suitable carrier is an amount from about 0.25 mg per 1 kg of body weight cats per day to about 25 mg per 1 kg of body weight cats a day.
Further, the present invention presents yet mentioned above vaccine, which further comprises an immunogen derived from a pathogen. In another implementation of the invention, such an immunogen in the vaccine composition is derived from a feline pathogen, rabies virus, chlamydia, Toxoplasma gondii, Dirofilaria immitis, fleas or bacterial pathogens. In another implementation of the invention appears to be the vaccine, in which the pathogen is a feline immunodeficiency virus (FIV), the virus feline leukemia (FeLV), the virus feline infectious peritonitis (FIP)virus, feline panleukopenia, feline caliciviruses, reovirus cat 3rd type, feline rotavirus, feline coronavirus, respiratory syncytial virus, feline, feline sarcoma virus, feline herpesvirus, a virus disease Bourne cats or cat parasite.
The present invention also provides a method of inducing immunity in cats, which includes the introduction of the cat dose of a vaccine containing any of okazavshihsya above immunogens. The present invention also provides a method of enhancing an immune response in cats, which includes an effective dose of the polypeptide immunogen and a suitable carrier. The present invention provides a method of introducing the above-mentioned vaccine subcutaneously, intramuscularly, systemically, topically or orally.
In another implementation of the present invention is the method of suppressing the immune resp is that of a cat, which includes the introduction of the cat effective in suppressing the immune response quantities of the polypeptide encoded by the nucleic acid CTLA-4 cat. In another implementation of the invention is the method of suppressing an immune response in cats, including the introduction of the cat effective in suppressing the immune response of quantity of a soluble polypeptide encoded CD80 cats, CD86 cats or CD28 cats.
In another implementation of the present invention is the method of suppressing an immune response in cats by introducing a polypeptide encoded by a nucleic acid CTLA-4 cats, in the amount of from about 0.25 mg per 1 kg of body weight per day to 25 mg per 1 kg of body weight per day. In another implementation of the invention is the method of suppressing an immune response in cats by introducing a polypeptide encoded CD80 cats, CD 86 cats or CD28 cats, in the amount of from about 0.25 mg per 1 kg of body weight per day to 25 mg per 1 kg of body weight per day.
The present invention also provides a method of suppressing an immune response in cats diagnosed with an autoimmune disease or a cat-recipient transplant tissue or organ by introducing the cat effective in suppressing the immune response quantities of the polypeptide encoded by the nucleic acid CTLA-4 cat.
The present invention also provides a method noise reduct is of immune response in cats diagnosed with an autoimmune disease or a cat-recipient transplant tissue or organ by introducing the cat effective in suppressing the immune response quantities of the polypeptide, encoded CD80 cats, CD86 cats or CD28 cats.
The present invention is isolated and purified cDNA CD80 (B7-1) length of about 941 nucleotide. The invention is also isolated and purified polypeptide CD80 cats, consisting of approximately 292 amino acids, which is associated with the membrane or the Mature form with a molecular mass of 33,485 kDa, isoelectric point about pI=9.1 and the total charge at pH 7.0 is equal to 10. Coexpressed with CD80 co-stimulatory molecule CD28 and with tumor antigen or an antigen of a pathogenic organism shows the ability to activate or enhance the activation of T-lymphocytes inducyruya thereby the production of immunostimulatory cytokines, and growth regulation of other cell types. Coexpressed with CD80 co-stimulatory molecule, CTLA-4 shows the ability to regulate the activation of T-lymphocytes.
The present invention is isolated and purified cDNA CD86 (B7-2) with a length of about 1176 nucleotides. The invention is isolated and purified polypeptide CD86 cats, consisting of approximately 320 amino acids, which is associated with the membrane or the Mature form with a molecular mass of 36,394 kDa, isoelectric point about pI=9,19 and the total charge at pH 7.0, equal 11,27. Coexpressed CD86 with co-stimulatory molecule CD28 and with tumor antigen or an antigen of a pathogenic organism PR which is the ability to activate or enhance the activation of T-lymphocytes, inducyruya thereby the production of immunostimulatory cytokines, and growth regulation of other cell types. Coexpressed CD86 with co-stimulatory molecule, CTLA-4 shows the ability to regulate the activation of T-lymphocytes.
CD80 or CD86 cats of the present invention were obtained from native or recombinant sources. CD80 or CD86 cats in the present invention include natural and associated with the membrane form or secreterial form, lost transmembrane domain.
The present invention is isolated and purified CD28 cDNA length of about 689 nucleotides. The invention is isolated and purified polypeptide CD28 cats, consisting of approximately 221 amino acids, which is associated with the membrane or the Mature form with a molecular mass of 25,319 kDa, isoelectric point about pI=9,17 and net charge at pH 7.0, equal 9,58.
Also present is isolated and purified cDNA CTLA-4 length of about 749 nucleotides. The invention is isolated and purified polypeptide CTLA-4 cats, consisting of approximately 223 amino acids, which is associated with the membrane or the Mature form with a molecular mass of 24,381 kDa, isoelectric point about pI=6,34 and net charge at pH 7.0, equal to 0,99.
In another aspect the present invention provides a method of enhancing immunogenicity a cat on the immunogen, what is achieved by the introduction of the immunogen before, after or substantially simultaneously with CD80 cats or CD86 cats together with CD28 cats or CTLA-4 cat or without them, in a quantity effective for strengthening the immune response.
In another aspect of the present invention is the method of suppressing an immune response in cats to the immunogen, which is achieved by the introduction of the immunogen before, after or substantially simultaneously with CD80 cats or CD86 cats together with CD28 cats or CTLA-4 cat or without them or with antisense RNA or DNA, partially or fully encoding CD80 cats, or CD86 cats, or CD28 cats, or CTLA-4 cats, in an amount effective to suppress the immune response.
The following aspect of the present invention is the vaccine for the induction of immune response in cats to the immunogen containing the immunogen and the amount of CD80, effective for strengthening the immune response. The immunogen is derived, for example, from feline pathogens such as human immunodeficiency virus cats, the virus leukemia cats, parvovirus, cats, coronavirus cats, leptodirus cats and the like.
In another aspect of the present invention is the vaccine for the induction of immune response in cats to the immunogen, which is achieved by the introduction of DNA or RNA immunogen and DNA or RNA auxiliary molecules CD80, CD86, CD28 cats in any combination that encode the be the key or protein fragments in a quantity effective for modulation of the immune response.
Protein CD80 cats is characterized by the amino acid sequence that is at 59% and 46% identical to the polypeptides of human and mouse, respectively. Protein CD86 cats is characterized by the amino acid sequence, which is 68% and 64% identical to such human proteins and rabbit, respectively. Protein CD28 cats is characterized by the amino acid sequence, which is 82% and 74% identical to the polypeptides of human and mouse, respectively. Protein CTLA-4 cats is characterized by the amino acid sequence that is at 88% and 78% identical to the polypeptides of human and mouse, respectively. Proteins CD80 or CD86 mouse or human cannot functionally replace the cat proteins CD80 or CD86. Therefore, proteins CD80 cats, CD86 cats, CD28 cats and CTLA-4 cats are new reagents necessary for the control of immunity in cats.
The present invention covers the T-cell regulatory auxiliary molecules CD80 (B7-1) or CD86 (B7-2), or CD28, or CTLA-4 (CD152) domestic cats. The present invention is isolated and purified nucleic acid encoding a partially or fully CD80 cats, CD86 cats, CD28 cats and CTLA-4 cats, as well as polypeptides CD80, CD86, CD28 or CTLA-4, purified either from native or from recombinant sources. Developed in accordance with this from what rutenium CD80, CD86, CD28 or CTLA-4 cats use to improve the efficiency of feline vaccines against tumors and pathogens, as well as medicines for the treatment of viral and bacterial diseases of domestic cats. Developed in accordance with the present invention CD80, CD86, CD28 or CTLA-4 cats are also used to mitigate the disease due to overactive, hyperactive or redirected immune responses.
Nucleic acids, vectors, transformants
Sequence of cDNA encoding CD80 cat (SEQ ID NO: 1), CD86 cat (SEQ ID NO: 5), CD28 cat (SEQ ID NO: 7) or CTLA-4 cats (SEQ ID NO: 9) is shown in figure 1-5, and the decoded amino acid sequence of CD80 cat (SEQ ID NO: 2), CD86 cat (SEQ ID NO: 6), CD28 cat (SEQ ID NO: 8) or CTLA-4 cats (SEQ ID NO: 10) is shown in figure 1-5. Identification of these cat-like polypeptides as CD80, CD86, CD28 or CTLA-4 is based on a certain level of homology of amino acid sequences and homologous polypeptides man or mouse, or rabbit, and on the ability of polypeptides CD80 or CD86 contact receptor CD28 cats (see below) or CTLA-4 and activate or stimulate, or any other way to regulate the activation of T-lymphocytes. Moreover, without being limited to any theory, predicts that the polypeptides CD80 cats or CD86 cats also exhibit one or more of sleduyuschischy activities: activation of NK cells (native killer cells), stimulation of the maturation of b-cells, activation of bounded-MTL cytotoxic T-lymphocytes, proliferation of fat cells, interaction with receptors of cytokines and the induction of immunoregulatory cytokines.
Due to the degeneracy of the genetic code (i.e. the presence of more than one codon coding for some amino acids) DNA sequences that do not match are shown in figure 1-5, can also encode the amino acid sequence of CD80, CD86, CD28 or CTLA-4 cats, shown in Fig.1-5. Such "other DNA" include those sequences that include "structurally conservative" changes, where the change of one or more nucleotides in the composition of the codon does not change the amino acid encoded by that codon. Moreover, this amino acid residue in the polypeptide can often be changed without altering the overall conformation and function of the native polypeptide. Such "functionally conservative variants include, thereby not limited to, replacement of amino acids at the amino acid characterized by similar physical-chemical parameters, such as, for example, acidic, basic, hydrophobic, hydrophilic, aromatic and similar properties (for example, substitution of lysine for arginine, aspartic acid, glutamic acid or glycine to alanine). In addition, the amino acid at the sledovatelnot added or removed without disrupting the biological activity of this molecule. For example, additional amino acid sequence added to either the N-or C-end, which serve as labels for the purification of this protein, such as polyhistidine labels (i.e. to provide a one-step purification of this protein, after which they are removed by chemical or catalytic means). On the other hand, additional sequences provide additional site surface binding or in any other way alter the specificity of CD80, CD86, CD28 or CTLA-4 cats against target cells, for example, by adding antigennegative site for antibodies.
Falling in the scope of the present invention CD80 cDNA cats or CD86 cats, or CD28 cats, or CTLA-4 cats correspond to the sequences shown in figure 1-5, structural-conservative variants of DNA, the DNA sequences encoding functionally-conservative variants of the polypeptides, and their combinations. The present invention encompasses fragments of CD80, CD86, CD28 or CTLA-4 cats that are effective degree of biological activity separately, and in combination with other sequences or components. As will be explained hereinafter, the competence of a person skilled in the art to predict the results of the manipulation of sequences of CD80, CD86, CD28 or CTLA-4, and to determine whether this option CD80, CD86, CD2 or CTLA-4 cat to take reasonable stability and biological activity in relation to this application or to determine the parameters, which disrupt the binding activity of these molecules, which will result in increased efficiency. Each of CD80 and CD86 cats associated with the coreceptor CD28 or receptor CTLA-4. This can be achieved by expression and purification of variant polypeptides CD80, CD86, CD28 or CTLA-4 in a recombinant system and assess their T-stimulatory activity and (or) activity by activation of the growth in cell cultures and in animals with subsequent testing for possible use. Option CD80 tested for its biological activity by functional binding to receptors CD28 or CTLA-4. Option CD86 tested for its biological activity by functional binding to receptors CD28 or CTLA-4. Similarly option CD28 or CTLA-4 are tested for their biological activity.
The present invention also encompasses DNA CD80, CD86, CD28 or CTLA-4 cats (and polypeptides)that are derived from other species of big cats, including, but not limited to, domestic cats, lions, tigers, cheetahs, lynxes, etc. Homologues CD80, CD86, CD28 or CTLA-4 cats in relation to shown. figure 1-5 can be easily identified by screening cDNA libraries or genomic clonetech to identify clones that hybridize with probes comprising all or part of the sequence shown in Fig.1-5. On the other hand, expression bib is iteki subjected to screening using antibodies that recognize CD80, CD86, CD28 or CTLA-4 cat. Out of touch with any theory can be considered that the genes CD80 or CD86 cats other species of big cats will be at least 70% homologous genes CD80, CD86, CD28 or CTLA-4 cat. Also in the scope of the present invention are DNA that encode homologues of CD80, CD86, CD28 or CTLA-4, defined as DNA-encoded polypeptides, characterized by at least approximately 25%level of identity with the amino acid sequences of CD80, CD86, CD28 or CTLA-4 cat.
In General, the manipulation of nucleic acids in accordance with the present invention by techniques which are well known in science, such as those described, for example, the guides Sambrook, Fritsch &Maniatis, 1989, "Molecular Cloning: A Laboratory Manual", 2nd ed.. Cold Spring Harbor or "Current Protocols in Molecular Biology", eds. Ausubel, Brent, Kingston, More, Feidman, Smith & Stuhl, Greene Publ. Assoc., Wiley-Interscience, NY, 1992.
The present invention encompasses sequences of cDNA and RNA in the sense and antisense orders. Also the invention comprises the sequence of genomic DNA CD80, CD86, CD28 or CTLA-4 cats and flanking sequences, including, but not limited to, the regulatory sequence. Nucleic acid sequences encoding polypeptides CD80, CD86, CD28 or CTLA-4 cats, also associated with heterologous sequences, including promoters, enhancers, reg is attornye elements, signal sequences, polyadenylation signals, introns, 5'- and 3'-non-coding segments, and the like. Transcriptional regulatory elements that are functionally associated with the sequences of cDNA CD80, CD86, CD28 or CTLA-4 cats, are without any restrictions sequences capable of controlling gene expression, derived from prokaryotic cells, eukaryotic cells, viruses, prokaryotes, viruses, eukaryotes, and any combinations thereof. Also in the art of famous and other heterologous regulatory sequence.
Nucleic acids of the present invention modify using methods known in the art, to change their stability, solubility, affinity and binding specificity. For example, the sequence was identified in a selective manner. Also the nucleotide sequence of the present invention modifies a label capable of providing effective direct or indirect signal. Examples of labels are radioactive isotopes, fluorescent molecules, Biotin and the like.
Also, the present invention represents the vectors that include nucleic acids encoding polypeptides CD80, CD86, CD28 or CTLA-4 partially or completely. Such vectors include plasmid vectors, art is meant for expression in a variety of prokaryotic and eukaryotic organisms, organisms owners. Preferably, the vectors include a promoter functionally attached to the land, encoding CD80, CD86, CD28 or CTLA-4 cat. Of the encoding feline CD80, CD86, CD28 or CTLA-4 expressed using any suitable vectors and host cells as described herein and in accordance with well-known specialists in this field of technology.
Suitable vectors for use in the practice of the present invention are thus not limited to, YEp352, pcDNAI (Invitrogen, Carlsbad, CA), pRc/CMV (Invitrogen) and pSFV1 (Gibco BRL), Gaithersburg, MD). One of the preferred for use in the present invention the vector is pSFV1. Suitable cell host cells are E. coli, yeast cells, COS cells, cell RS, cells SNO, GH4C1 cells, cells KSS-21 and the cells of the amphibian melanophores. Cells KSS-21 are the preferred cells of the host from the point of view of use in the practice of the present invention. Suitable vectors for constructing the "naked" DNA or "genetic immunization" are thus not limited to, pTarget (Promega, Madison, WI), pSI (Promega, Madison, WI) and pcDNA (Invitrogen, Carlsbad, CA).
Nucleic acids encoding polypeptides CD80, CD86, CD28 or CTLA-4 cats, also contribute in cells using recombinant techniques. For example, the following sequence introduced into the cell by microinjection, to ensure achiwa homologous recombination at the site of localization of the endogenous gene, encoding such a polypeptide, its analogue or pseudogene, or sequence, characterized by a significant level of homology with the gene coding for CD80, CD86, CD28 or CTLA-4 cat. Can also be used by other recombinant methods, such as non-homologous recombination and the delegation of the endogenous gene by homologous recombination, particularly in the genome of pluripotent cells.
The present invention provides a method of enhancing the cat's immune response to an immunogen, which is achieved by the introduction of the immunogen before, after or substantially simultaneously with CD80 or CD86 cats along with CD28 cats or CTLA-4 cat or without them, in a quantity effective for strengthening the immune response.
The present invention provides a method of enhancing the cat's immune response to an immunogen, which is achieved by the introduction expresarse vector, which includes are derived from a feline pathogen immunogen, before, after or substantially simultaneously with CD80 or CD86 cats along with CD28 cats or CTLA-4 cat or without them, in a quantity effective for strengthening the immune response.
The present invention provides a method of redirecting the cat's immune response to an immunogen, which is achieved by the introduction expresarse vector, which includes are derived from a feline pathogen immunogen, before, after or essentially at once is with CD80 or CD86 cats along with CD28 cats or CTLA-4 cat or without them, in a quantity effective for strengthening the immune response.
The present invention represents a way to suppress the cat's immune response to an immunogen, which is achieved by the introduction expresarse vector, which includes are derived from a feline pathogen immunogen, before, after or substantially simultaneously with CD80 or CD86 cats along with CD28 cats or CTLA-4 cat or antisense RNA or DNA encoding CD80 cats, CD86 cats, CD28 cats or CTLA-4 cats, in an amount effective to suppress the immune response.
The present invention is a vaccine for the induction of the cat's immune response to the immunogen(s)containing the immunogen and an effective amount of CD80 cats, or CD86 cats together with CD28 cats, or CTLA-4 cats, or without them, to enhance the immune response or CD80 cats or CD86 cat with CTLA-4 cat to suppress the immune response. In another implementation of the present invention is containing vaccines expressing vector comprising genes encoding the immunogen(s) feline pathogens and genes CD80, CD86 with CD28 cats, or CTLA-4 cats, or without them, to enhance or suppress the immune response.
Polypeptide CD80, CD86, CD28 or CTLA-4 cats
Gene CD80 cats (cDNA and corresponding amino acid sequence is shown in figure 1 and 2) encodes a polypeptide consisting of approximately 292 amino acids. Gene CD86 cock the (cDNA and corresponding amino acid sequence is shown in figure 3) encodes a polypeptide, consisting of approximately 320 amino acids. Gene CD28 cats (cDNA and corresponding amino acid sequence is shown in figure 4) encodes a polypeptide consisting of approximately 221 amino acids. Gene CTLA-4 cats (cDNA and corresponding amino acid sequence is shown in figure 5) encodes a polypeptide consisting of approximately 223 amino acids.
Cleaning CD80, CD86, CD28 or CTLA-4 cats from natural or recombinant sources carried out using methods well known in the art, including, but not limited to, ion exchange chromatography, reversed-phase chromatography on columns C4, gel filtration, isoelectric focusing, affinity chromatography and the like. In a preferred implementation of a large number of biologically active protein CD80, CD86, CD28 or CTLA-4 cats is produced by constructing a recombinant DNA sequence comprising coding section CD80, CD86, CD28 or CTLA-4 cats, which in General coding frame combined with a sequence encoding 6 C-terminal residues of histidine in the replicon pSFV1 (Gibco BRL). Encoded by this plasmid mRNA synthesized using methods well known to experts in the art, and contribute in cell lines KSS-21 method of electroporation. The cells produce and secrete versions glycoside is consistent polypeptides CD80, CD86, CD28 or CTLA-4 cats, including the C-terminal of hexastylis. Modified cat polypeptides CD80, CD86, CD28 or CTLA-4 purified from the cell supernatant by the method of affinity chromatography using histidinemia resin (His-bind: Novagen, Madison, WI).
Polypeptides CD80 cats or CD86 cats, isolated from any source, modify, using methods known in the data field of technology. For example, feline CD80, CD86, CD28 or CTLA-4 phosphorylate or dephosphorylate, glycosylases or deglycosylated and the like. The most applicable modifications are those that change the solubility, stability, and specificity and binding affinity of the CD80, CD86, CD28 or CTLA-4 cat.
Chimeric molecules CD80, CD86, CD28 gel CTLA-4 cats
The present invention comprises obtaining chimeric molecules, formed by fragments of feline CD80, CD86, CD28 or CTLA-4, in any combination. For example, the introduction of binding site CTLA-4 instead of CD28 binding site should enhance the binding affinity of CD28 with preservation of enhanced immune response.
In one implementation of the binding sites CD80 or CD86 molecules CTLA-4 and CD28 are replaced in such a way that the binding site in CD28 is replaced with the binding site CTLA-4. The effect of the chimeric molecule CD28, which includes the binding site, CTLA-4, is associated with an increased affinity of CD28 in respect of CD80 or CD86 and increased extent and amplification of the immune response. In an alternative implementation of the chimeric molecules CD80 and CD28, or CD86 and CD28, or their fragments are associated with membranes forms and improve the properties of these molecules to strengthen the immune response. In another implementation of the chimeric molecules CD80 and CTLA-4, or CD86 and CTLA-4, or fragments thereof are associated with membranes forms, which are molecules, more effective suppression of the immune response. In another implementation of the chimeric molecules CD80 and CTLA-4 or CD86 and CTLA-4 or fragments thereof are associated with membranes forms that direct the immune response to achieve the desired effect.
Antibodies that are specific in relation CD80, CD86, CD28 or CTLA-4 cats
The present invention encompasses antibodies that are specific against polypeptides CD80, CD86, CD28 or CTLA-4 cats, identified as described above. Antibodies are polyclonal or monoclonal antibodies, separating CD80, CD86, CD28 or CTLA-4 cats and other proteins, identifying their functional domains and such. Such antibodies will receive the standard methods using the methods and compositions described in Harlow &Lane, 1988, ″Antibodies: A Laboratory Manual″, Cold Spring Harbor Lab., as well as using immunological and hybridoma methods known in the art. In the case of native or synthetic peptides, derivatives of the CD80, CD86, CD28 or CTLA-4 cats, for the induction of the cat CD80-, CD86-, CD28 or CTLA-4-specific immune response of these peptides in the standard manner connected with a suitable carrier, such as hemocyanin slug (KLH)and administered with a suitable adjuvant, such as adjuvants Freund.
Preferably the selected peptides are combined with a carrier having a "lysine core", essentially in accordance with the methods Tan, 1988, Proc. Natl. Acad. Sci. USA, 85, 5409-5413. Such antibodies, particularly the so-called antiidiotypic antibodies "internal correspondence", also prepared using known methods.
In one implementation purified feline CD80, CD86, CD28 or CTLA-4 is used for immunization of mice, after which they remove the spleen and splenocytes used for cell hybrids with myeloma cells to obtain clones secreting antibody of cells in accordance with standard methods of the art. The resulting monoclonal antibodies secreted by such cells, subjected to screening tests in vitro on the following activities: associating with the cat proteins CD80, CD86, CD28 or CTLA-4, suppressing the activity of CD80, CD86, CD28 or CTLA-4 binding receptors and suppression of T-cell stimulatory activity CD80, CD86, CD28 or CTLA-4.
Antibodies to CD80 cats, CD86 cats, CD28 cats and CTLA-4 cats used to Ident the classification and quantitative analysis of feline CD80, CD86, CD28 or CTLA-4, applying such immunological tests, such as TYPHOID, RIA and the like. Antibodies to CD80 cats, CD86 cats, CD28 cats and CTLA-4 cats are also used for immunological depletion extracts CD80 cats, CD86 cats, CD28 cats or CTLA-4 cat. In addition, these antibodies can be used for identification, isolation and purification of feline CD80, CD86, CD28 or CTLA-4, originating from different sources, and for studies on cellular and histochemical mapping.
Ligand CD80 (B7-1) cats, ligand CD86 (B7-2) cats, receptor CD28 cats or receptor CTLA-4 (CD152) cats produced in accordance with the present invention can be effectively used as vaccines for preventive infectious disease or promote growth in homologous or heterologous species of cat. For example, coexpression CD80 or CD86 with co-stimulatory molecules CD28 or CTLA-4 in combination with a tumor antigen or an antigen of a pathogenic organism. Coexpressed CD80 or CD86 with cats with the receptor, CTLA-4 cats provides the ability to suppress activation of T-lymphocytes and suppress the immune response. A concrete example should be coexpressed CD80 or CD86 with immunogenum derived from viruses, FIV, FeLV or FIP, viral vector or DNA-expressing vector, which, if introduced in the form of vaccines, will be the act is to promote, to increase or regulate the proliferation of CD4-positive and CD8-positive T-lymphocytes and to induce the production of cytokines, such as IL-2, IFN-γ, IL-12, TNFα, IL-6, etc. Another specific example should be the expression of CD80, CD86, CD28 or CTLA-4 with a viral vector or DNA expresarse vector, which, if introduced in the form of medicines, to regulate or to redirect the immune response.
Strengthening of the immune system due to the interaction of feline CD80 or CD86 with CD28 or CTLA-4 or suppression of the immune response due to the interaction of feline CD80 or CD86 with CTLA-4 provides advantages in the natural process of regulation to a greater extent than adding foreign substances that would multiple and even a harmful effect on health in General or for a long time. Molecules CD80, CD86, CD28 or CTLA-4 is administered along with other recombinant molecules, such as those that encode antigens that are desirable from the point of view of the induction of immunity. Gene CD80, CD86, CD28 or CTLA-4 cats built in the composition expressing vector to infect them or transferout the target cell with subsequent expression of the gene product in that cell in the target so that it is attached to the plasmalemma of this target cells or antigen-presenting cells or secreted into the outside e the th target cells or antigen-presenting cells environment. Expressing the vector such as a plasmid, virus, Semliki Forest, poxvirus or herpes virus that carries the gene into the antigen-presenting cell. Gene CD80, CD86, CD28 or CTLA-4 cat or fragments of these genes in any combination embed in the composition of DNA - or RNA-expressing vector and injected the cat with the expression of this gene product in cats in the form of "naked" DNA/RNA, or genetic vaccines. Coexpressed immunogen and CD80, CD86, CD28 or CTLA-4 in the target cell or in the body of the cat causes activation, enhanced activation or regulation of T-lymphocytes, b-lymphocytes and other cells. On the other hand, downregulation of protein can be introduced after expression from the plasmid. Cat proteins CD80, CD86, CD28 or CTLA-4 in normal function, being attached to the cell membrane, as an auxiliary molecules of the cell membrane, but can be presented in other forms, in particular in the absence of their composition "membrane anchors".
In one implementation CD80 cats and CD86 cats soluble ("free" in cellular and extracellular fluids) due to the loss of the transmembrane domain or a hydrophobic segment and interact with co-stimulatory molecules CD28 or CTLA-4, which is either associated with the membrane, either in soluble form. In another embodiment, CD80 cats and CD86 cats are associated with the membrane form, and co-stimulatory molecules D28 or CTLA-4 - in soluble form, i.e. does not have a transmembrane domain or a hydrophobic segment. Soluble CD28 or CTLA-4, preferably in the form of dimers, are used for treatment of cats associated with immunosuppression mediated by T-cells. Soluble CD28 or CTLA-4 prevents the rejection of transplanted tissue and can be used to treat autoimmune diseases. Specific soluble CD28 or CTLA-4 are used to prevent graft-versus-host with bone marrow transplant. Soluble CD28 or CTLA-4 prevents the binding of cells bearing membrane form cat ligands CD80 or CD86.
Structurally conservative and functionally conservative variants of DNA and polypeptides CD80, CD86, CD28 or CTLA-4 cat or a biologically active fragment or subfragment CTLA-4 unite in a common reading frame with another sequence, such as a cytokine, interleukin, interferon, colony stimulating factor, a pathogen antigen, antibody or necessary for the cleaning sequence, such as polyhistidine label, or gene-reporter, such as genes lacZ and uidA gene or green fluorescent protein.
The present invention encompasses methods and compositions for increasing the efficiency of the immune response in domestic cats. In this implementation of feline CD80,CD86, CD28 or CTLA-4 used with the immunogen, in respect of which it is desirable to induce an immune response. For example, in the composition of vaccines for cats containing immunogen such pathogens like virus feline immunodeficiency virus feline leukemia, and other pathogens such as feline parvovirus, leptodirus cat and feline coronavirus, it is desirable to include CD80, CD86, CD28 or CTLA-4 cat to regulate the extent and effectiveness of the immune response. For this purpose feline CD80, CD86, CD28 or CTLA-4, purified from native or recombinant sources as described above, are included in the vaccine composition at a concentration in the range from about 0.01 to 100.0 mg per vaccinating one cat.
Specialists in the art known commercial sources of vaccines for cats (Compendium of Veterinary Pharmaceuticals, 1997), which can be used in combination with the present invention for a more effective vaccine.
The vaccine for the induction and regulation of the cat's immune response to the immunogen contains an immunogen and an effective amount of CD80 cats, or CD86 cats together with CD28 cats, or CTLA cats, or without them, to enhance the immune response CD80 or cats, or CD86 with CNLA-4 cat to suppress the immune response.
Immunogen selected from the group which includes, thus not limited to, Kosach the pathogens, such as feline immunodeficiency virus, the virus of feline leukemia virus feline infectious peritonitis virus, feline panleukopenia (parvovirus, feline caliciviruses, reovirus 3rd type of cat, feline rotavirus, feline coronavirus (infectious peritonitis), rabies virus, respiratory syncytial virus, feline, feline sarcoma virus, feline herpesvirus (rhinotracheitis virus), a virus disease Bourne cats, chlamydia, Toxoplasmosis gondii, a parasite of cats, Dirofilaria immitis, fleas, bacterial pathogens and the like.
Regulation of growth or regulation of the activation of cell type, such as T-lymphocytes, determines that a regulatory response either stimulates or inhibits the growth of cells. Regulation of immune response in cats specifies that this immune response is either stimulated or inhibited in connection with the treatment of disease or exposure to an infectious agent in cats.
The expression of CD80, CD86, CD28 or CTLA-4 cats individually or in any combination of parts or whole comprising expressing a vector comprising the gene(s) cat immunogens for the purposes of the administration in the form of genetic vaccine or naked DNA vaccines". Vectors are thus not limited to, pTarget (Promega, Madison, WI), pcDNA (Invitrogen, Carlsbad, CA) (J.J. Donnelly et al., 1997; Hassett & Whitton, 1996).
The genes or gene fragments CD80, CD86, CD28 or CTLA-4 alone or in combination, p is nerazmennye or partial, can be embedded or transliterowany into the genome of a cat or other mammal. This integration of the genes or fragments of these genes, which can be achieved using a retroviral vector can be used for the purposes of gene therapy.
The present invention is methods and compositions for increasing resistance to disease in domestic cats, for use in medical and / or commercial purposes. In this implementation of CD80, CD86, CD28 or CTLA-4 cats, expressed individually or in any combination, full or partial, and in combination with genes encoding feline immunogen, or without introducing the cat using the correct method of administration. To promote growth or resistance to disease feline CD80, CD86, CD28 or CTLA-4, expressed individually or in any combination, administered in the form of a composition in a concentration ranging from about 0.01 to 100.0 mg for 1 dose of vaccine 1 cat, preferably in the composition at a concentration from about 0.25 mg/kg / day to about 25 mg/kg / day. It should be clear that the required amount of CD80, CD86, CD28 or CTLA-4 cats may be determined by routine tests, are well known in the art, so as to set the schema of the dosage and frequency of their administration and to compare groups of experimental units or actors who s at each point of such a scheme.
In accordance with the present invention, native or recombinant feline CD80, CD86, CD28 or CTLA-4 is prepared with a physiologically acceptable carrier, such as, for example, phosphate-saline buffer or deionized water. This composition may also contain fillers, including lubricating components, plasticizers, absorption enhancers, bactericides and the like, which is well known in the art. Polypeptides CD80, CD86, CD28 or CTLA-4 cats of the present invention is administered by any effective route, including, thereby not limited to, intravenous, subcutaneous, intramuscular, chrismassy, local or oral route. For subcutaneous administration, for example, the dose includes feline CD80, CD86, CD28 or CTLA-4 in sterile saline. For oral or inhalation of feline CD80, CD86, CD28 or CTLA-4 pack at the micro or macro levels, for example, in liposomes or microspheres. Can also be used skin plaques (or other forms of slow secretion).
Cloning of cDNA CD80 (B7-1)-TAMU, CD86 (B7-2), CD28 and CTLA-4 cats
The sequence of feline CD80 (B7-1), CD86 (B7-2), CD28 and CTLA-4 was first cloned by amplificatore RT-PCR (polymerase chain reaction with repertorium or reverse transcriptase) site between two sequences, which is quite conservative for to form degenerate primers, interacting with the cat mRNA. The source of mRNA were menagerie cells of peripheral blood (RVMS), stimulated by concanavalin A. immunology at least for 16 hours. The obtained PCR product sequenced. The obtained sequence was used to design primers for RACE (rapid amplification of cDNA ends). 5'-end of the amplified first obtaining cDNA from the reverse primer, complementary to the re-sequenced conservative area. Oligonucleotide ligated to the 3'end of the cDNA (complement of 5'-end of mRNA). This sequence was the binding site for the forward primer, which is compatible for PCR reverse PCR primer corresponding to another part of the new sequenced plot. Degenerate primers used in repeated cycles of "nesting" of reactions to obtain the 3'end of the sequence. This direct primer for PCR were designed to interact with a sequence of new sequenced segment. Products are either sequenced directly or cloned into the cloning vector and sequenced from the obtained plasmid. Full coding frame (ORF) was cloned by amplification along its entire length using primers designed on the parameters of the known sequence is linota. ORF cloned and sequenced three times. ORF B7-1 was subcloned into the plasmid pSI, including the SV40 promoter, and plasmid SFV. Plasmid pSI was used to establish the functional interaction of B7-1 with a feline CD28.
DNA primers used for RT-PCR cDNA CD80 (B7-1) cats were as follows:
Direct primer: 5'-CGCGGATCCGCACCATGGGTCACGCAGCAAAGTGGAAAAC-3' (SEQ ID NO: 11)
Reverse primer: 5'-CCTAGTAGAGAAGAGCTAAAGAGGC-3' (SEQ ID NO: 12)
(see above for the complete list of primers for cDNA CD28).
DNA primers used for RT-PCR cDNA CD28 cats, were as follows:
Direct primer: 5'-CGCGGATCCACCGGTAGCACAATGATCCTCAGG-3' (SEQ ID NO: 13)
Reverse primer: 5'-CGCGGATCCTCTGGATAGGGGTCCATGTCAG-3' (SEQ ID NO: 14)
(see above for the complete list of primers for cDNA CD28).
DNA primers used for RT-PCR cDNA CTLA-4 cats, were as follows:
1. Degenerate primers for the first PCR product (672 nucleotides):
Deg-5'-P: 5'-ATGGCTT(C)GCCTTGGATTT(C)CAGC(A)GG-3' (SEQ ID NO: 15)
Deg-3'-P: 5'-TCAATTG(A)ATG(A)GGAATAAAATAAGGCTG-3' (SEQ ID NO: 16)
2. 5'-end sequence of CTLA-4 (455 nucleotides): degenerate gene-specific (GSP) "Jack" gene-specific (NGSP) primers:
The first round PCR:
Deg-5'-P: 5'-TGTTGGGTTTC(T)G(A)CTCTG(A)CTT(C)CCTG-3' (SEQ ID NO: 17)
3'-GSP: 5'-GCATAGTAGGGTGGTGGGTACATG-3' (SEQ ID NO: 18)
"Nested" PCR with the PCR product obtained in the first round:
Deg-5'-P: 5'-TGTTGGGTTTC(T)G(AND)CTCTG(A)CTT(C)CCTG-3' (SEQ ID NO: 19)
3'-NGSP: 5'-ACATGAGCTCCACCTTGCAG-3' (SEQ ID NO: 20).
3. 3'-end sequence of CTLA-4: adapt the RNA 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):
AP1: 5'-CCATCCTAATACGACTCACTATAGGGC-3' (SEQ ID NO: 21)
5'-GSP: 5'-GTGAATATGGGTCTTCAGGCAATG-3' (SEQ ID NO: 22)
the 3'nested RACE with the product 3'-RACE:
AR: 5'-ACTCACTATAGGGCTCGAGCGGC-3' (SEQ ID NO: 23)
5'-NGSP: 5'-GAAATCCGAGTGACTGTGCTGAG-3' (SEQ ID NO: 24).
4. Primers for full-length gene CTLA-4:
Fel CTLA-4 5'-primer: 5'-AACCTGAACACTGCTCCCATAAAG-3' (SEQ ID NO: 25)
Fel CTLA-4 3'-primer: 5'-GCCTCAGCTCTTAGAAATTGGACAG-3' (SEQ ID NO: 26).
DNA primers used for RT-PCR cDNA CD86 (B7-2) cats, were as follows:
1. Degenerate primers for the first PCR product (423 nucleotides):
Deg-5'-P: 5'-TAGTATTTTGGCAGGACCAGG-3' (SEQ ID NO: 27)
Deg-3'-P: 5'-CTGTGACATTATCTTGAGATTTC-3' (SEQ ID NO: 28).
2. Degenerate primers for the second PCR product (574 nucleotide):
Deg-5'-P: 5'-GA(G)CA(T)GCACT(A)ATGGGACTGAG-3' (SEQ ID NO: 29)
Deg-3'-P: 5'-CTGTGACATTATCTTGAGATTTC-3' (SEQ ID NO: 30).
3. the 5'-End CD86: AP1, AP2 (Clontech Lab.), the degeneracy of the 3'gene-specific (GSP) and 3'-"nesting" gene-specific (NGSP) primers:
AP1: 5'-CCATCCTAATACGACTCACTATAGGGC-3' (SEQ ID NO: 31)
3'-GSP: 5'-TGGGTAACCTTGTATAGATGAGCAGGTC-3' (SEQ ID NO: 32).
"Jack" 5'-RACE PCR product with 5'-RACE:
AP2: 5'-ACTCACTATAGGGCTCGAGCGGC-3' (SEQ ID NO: 33)
3'-NGSP: 5'-CAGGTTGACTGAAGTTAGCAAGCAC-3' (SEQ ID NO: 34).
4. 3'-end sequence of B7-2: primers AP1, AP2, 5'-GSP and 5'-NGSP:
AP1: 5'-CCATCCTAATACGACTCACTATAGGGC-3' (SEQ ID NO: 35)
5'-GSP: 5'-GGACAAGGGCACATATCACTGTTTC-3' (SEQ ID NO: 36).
"Jack" 3'-RACE PCR product with 3'-RACE:
AP2: 5'-ACTCATATAGGGCTCGAGCGGC-3' (SEQ ID NO: 37)
5'-NGSP: 5'-CAGTGCTTGCTAACTTCAGTCAACC-3' (SEQ ID NO: 38).
Direct primer Fel-B72 (1): 5'-CGGGAATGTCACTGAGCTTATAG-3' (SEQ ID NO: 39)
Reverse primer Fel-B72 (1176): 5'-GATCTTTTTCAGGTTAGCAGGGG-3' (SEQ ID NO: 40).
Cloning CD80 (B7-1)-Syntro/SPAH; plasmid 917-19-8/16
Selected cells of the cat spleen and cultured them with concanavalin A. immunology for 5 hours. After that, the cells were centrifuged, washed in the FSB and used to allocate the total pool of RNA to the RNeasy system (Qiagen). The total RNA was treated with Dnazol-1 (Boehringer Mannheim) to remove DNA contaminating the RNA preparation. Then from these drugs mRNA was isolated using Oligotex beads (Qiagen, Santa Clara, CA) and high-speed columns. With matrices mRNA synthesized DNA copies in the presence of random hexamers, dNTP, RNAsin, reverse transcriptase (Promega) and operatorassisted buffer (Promega) with incubation for 30 minutes at 42°C. Then, to obtain double-stranded molecules of the full-size cDNA clone coding frame (ORF) V7-1 cat used PCR with sense primer 5/97 .50 (5'-ATGGGTCACGCAGCAAAGTG-3') (SEQ ID NO: 41) and antimuslim primer 5/97 .51 (5'-CTATGTAGACAGGTGAGATC-3') (SEQ ID NO: 42), dNTP, cDNA B7-1 (first circuit), magnesium sulfate, Vent polymerase (Gibco BRL) and Vent polymerase buffer (Gibco BRL). The PCR conditions: 1 cycle of 15 seconds at 94°C; 35 cycles of 30 seconds at 94°C, 2 min at 48°C, 2 min at 72°C; 1 cycle of extension at 72#x000B0; C for 10 minutes. PCR was carried out in 1%low-melting agarose and were isolated DNA fragments corresponding to the expected size of the ORF B7-1, was purified in the gel (set of reagents for gel purification Qiagen, Santa Clara, CA) and cloned into plasmid pCR-BLUNT using a set of Zero Blunt PCR Cloning Kit (Invitrogen, San Diego, CA). DNA extracted from resistant to kanamycin bacterial colonies were subjected to preliminary screening for the presence of a unique NheI site (available in CD80 [B7-13-TAMU cats). Insert size 800-900 base pairs (BP), which included the NheI site, sequenced by fluorescent automated sequencing on the appropriate equipment company Perkin-Elmer-Cetus (Applied Biosystems Inc.). Plasmid vector and B7-1, a gene-specific primers derived from the previously cloned gene B7-1, was used to obtain sequences of pCR-Blunt: primers - 1/97 .36 [5'-CAGGAAACAGCTATGAC-3') (SEQ ID NO: 43) and 1/97 .37 (5'-AATACGACTCACTATAGG-3') (SEQ ID NO: 44). Specific gene V7-1 primers - 12/96 .22 (5'-ASSETTRADEX-3') (SEQ ID NO: 45), 1/97 .33 (5'-ATACAAGTGTATTTGCCATTGTC-3') (SEQ ID NO: 46), 12/96 .20 (5'-AGG-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). Identified two clones that include full-size sequence CD80, corresponding to the original sequence CD80, except the boom point mutations. One such mutation is the amino acid sequence did not change. Another mutation resulted in the substitution of leucine for isoleucine. The resulting clone CD80 cat was marked 917-19.8/16 (CD80-Syntro/SPAH).
To facilitate cloning of the gene CD80 (B7-1) cats behind any promoter of the smallpox virus (poxvirus), including EcoRI and BamHI sites of the cloning, two new primers were designed to introduce restriction EcoRI and BamHI sites of the cloned by 5'- and 3'-ends of the coding frame CD80, respectively. These two primers were: forward primer 1/97 .43(SEQ ID NO: 52) and reverse primer 1/97 .6(SEQ ID NO: 53). The obtained PCR fragment was digested with restrictase EcoRI and BamHI and cloned in the composition of the vector O1L-SPV (AccI site in the sequence of genomic HindIII fragment M poxvirus pigs - SPV) to obtain the recombinant virus SPV. The result was obtained cassette 930-23 A - vector O1L-SPV, including frame encoding CD80 cats behind synthetic "late-early promoter LP2EP2 genome SPV and adjacent to the cassette of the marker gene lacZ gene placed under the control of the synthetic late promoter gene LP2. Plasmid vector 930-23 I was cotranslationally together with SPV-001 obtaining recombinant virus SPV expressing the protein B7-1 cat β-galactosidase .coli.
Subclavian CD28 in homologous poxviruses the genome vector
Coding segment of the gene CD28 was amplificatoare by PCR using synthetic primers, including standard cloning sites to facilitate the cloning of a CD28 for any promoter genome poxvirus during design-specific vector, homologous to the poxvirus. Synthetic primers were formed to introduce EcoRI and BglII sites of the cloned by 5'- and 3'-ends of the PCR fragment, respectively. These two primers were: forward primer 7/97 .1 (5'-GATGAATTCCATGATCCTCAGGCTGGGCTTCT-3') (SEQ ID NO: 54) and the reverse primer 7/97 .2 (5'-GATCAGATCTCAGGAACGGTATGCCGCAA-3') (SEQ ID NO: 55). The resulting PCR DNA fragment was digested with restrictase EcoRI and BglII and cloned into the composition vector O1L-SPV with the purpose of obtaining a recombinant poxvirus. Was received cassette 930-26 A - vector O1L-SPV, including (AccI site in the sequence of genomic HindIII fragment M poxvirus pigs) encoding frame CD28 cats behind synthetic "late-early promoter LP2EP2 genome SPV and adjacent to the cassette of the marker gene lacZ gene placed under the control of the synthetic late promoter gene LP2. Homologous plasmid vector 930-26 I was cotranslationally together with SPV-001 obtaining recombinant virus SPV expressing CD28 protein and cats β-galactos the oxidase of E. coli.
Characterization of cDNA and polypeptide CD80 (B7-1)-TAMU, CD86 (B7-2), CD28, CTLA-4 and CD80 (B7-1)-Syntro/SPAH Koski
An isolated and purified cDNA CD80 (B7-1) cats length of about 941 nucleotide is open frame encoding feline CD80 polypeptide consisting of approximately 292 amino acids, in the form of a native associated with a membrane or a Mature form with a molecular mass of approximately 33,485 kDa, isoelectric point 9.1 and net charge 10,24 at pH 7.0. The transmembrane domain of this protein accounts for approximately amino acids 241-271.
Feline CD80-TAMU and CD80-Syntro/SPAH is the cDNA and corresponding polypeptides that were selected independently from each other from two different sources, and their nucleotide and amino acid sequences are slightly different. Source CD80 mRNA-TAMU were menagerie cells in the peripheral blood of cats, stimulated by concanavalin A. immunology, and the source of CD80 mRNA-Syntro/SPAH splenocytes were cats, stimulated by concanavalin-A. variation in the cDNA sequence between CD80-TAMU and CD80-Syntro/SPAH associated with nucleotide substitutions T→in the 351-m position and With→And 670-m position. Amino acid sequence replacement 351-th nucleotide is Nesmelova and replacement 670-th nucleotide leads to the substitution of a neutral amino acid to neutral - leucine for isoleucine - 224-th position of polypeptide
An isolated and purified cDNA CD86 (B7-2) cats consists of about 1176 nucleotides, corresponding to an open frame that encodes a polypeptide CD86 cats, consisting of approximately 320 amino acids, in the form of a native associated with a membrane or a Mature form with a molecular mass of approximately 36,394 kDa, isoelectric point 9,19 and net charge 11,27 at pH 7.0.
An isolated and purified cDNA CD28 cats consists of about 689 nucleotides, corresponding to an open frame that encodes a polypeptide CD28 cats, consisting of approximately 221 amino acids, in the form of a native associated with a membrane or a Mature form with a molecular mass of approximately 25,319 kDa, isoelectric point 9,17 and net charge 9,58 at pH 7.0.
An isolated and purified cDNA CTLA-4 cats consists of about 749 nucleotides, corresponding to an open frame that encodes a polypeptide CTLA-4 cats, consisting of approximately 223 amino acids, in the form of a native associated with a membrane or a Mature form with a molecular mass of approximately 24,381 kDa, isoelectric point 6,34 and net charge - 0,99 at pH 7.0.
Coexpressed with CD80 co-stimulatory molecule CD28 or CTLA-4 and tumor antigen or an antigen of a pathogenic organism causes the ability to activate or enhance the activation of T-cells, in particular lymphocytes pool Th-1, and activate the growth of other cell types. Coexpressed CD80 with costimulatory the molecule CTLA-4 leads to the ability to suppress the activation of T-lymphocytes, in particular lymphocyte pool Th-1. Coexpressed CD86 with co-stimulatory molecule CD28 or CTLA-4 and tumor antigen or an antigen of a pathogenic organism causes the ability to activate or enhance the activation of T-cells, in particular lymphocytes Th-1, and activate the growth of other cell types. Coexpressed CD86 with co-stimulatory molecule, CTLA-4 leads to the ability to suppress activation of T-cells, specifically lymphocytes Th-1.
|The level of nucleotide identity and amino acid sequence||CD80 cats||CD86 cats||CD28 cats||CTLA-4 cat|
|The human homologue (%identity to the nucleotide sequence)||77||72||85||88|
|Continuation of the table|
|The human homologue (%identity in amino acid sequence)||59||68||82||88|
|The mouse homologue (%identical to the ti nucleotide sequence)||62||-||77||79|
|The mouse homologue (%identity in amino acid sequence)||46||-||74||78|
|The homologue of rabbit (%identity of nucleotide/amino acid sequence)||-||67/64||84/84||-|
|The chicken homologue (%identity of nucleotide/amino acid sequence)||-||-||59/50||-|
The use of feline CD80 (B7-1), CD86 (B7-2), CD28 and CTLA-4 in vaccines
The following experiments were conducted to assess strengthen the immune system activity of feline CD80, CD86, CD28 and CTLA-4 in the composition of vaccines for cats.
In one of the procedures cats aged 8 weeks intramuscularly were injected with 100 μg plasmid comprising cDNA encoding a feline molecules CD80, CD86, CD28 and CTLA-4, mixed with a plasmid comprising cDNA of viral genes env and gag virus FIV or env and gag virus FeLV, or, on the other hand, was intramuscularly injected with 100 μg plasmid containing cDNA expressing pairwise combinations of CD80/CD28 or CD80/CTLA-4, or CD86/CD28, or CD86/CTLA-4, mixed with plasmid comprising cDNA viral genes env and gag (FIV) or env and gag (FeLV). Control specimens injection CD80, CD86, CD28 and CTLA-4 did not. Cats prewar the positive was infected by virulent strains of FeLV or FIV and analyzed the symptoms described above. In the source of infection was found that cats who have introduced the vector with cDNA feline CD80, CD86, CD28 and CTLA-4 and the vector with cDNA genes FIV or FeLV genes showed 100%protection from disease compared to animals that were injected only vector comprising cDNA genes FIV or FeLV genes in this group resistance to disease was 75%.
In another procedure the cats aged 8 weeks intramuscularly were injected with 0.1 to 100 mg of purified protein cat molecules CD80, CD86, CD28 and CTLA-4, or, in another embodiment, is a pairwise combination of CD80 or CD86 in combination with CD28 or CTLA-4, using recombinant vector comprising cDNA described above, as well as intramuscularly were injected with 0.1 to 100 µg vaccine containing proteins env and gag virus FIV or proteins env and gag virus FeLV. The test specimens injection CD80, CD86, CD28 and CTLA-4 did not. Cats previously infected by virulent strains of FeLV or FIV and regularly review the development of the disease. The results showed significantly reduced the incidence in cats, which were administered purified protein CD80, CD86, CD28 and CTLA-4 with a viral vaccine for FIV or FeLV, compared with cats that received a vaccine containing only proteins of FIV or FeLV.
The use of feline CD80 (B7-1), CD86 (B7-2), CD28 and CTLA-4 to inhibit and prevent the growth of tumor cells
Swollen the left cells of the cat was transfusional vector, expressing CD80 or CD86 cats in combination with either CD28 or CTLA-4. Transfetsirovannyh tumor cells were again introduced the same cat, and the presence of CD80, CD86, CD28 and CTLA-4 on the surface of tumor cells increased nonspecific immune response against and transfected, and nitrostilbene tumor cells, which led to the destruction of localized and metastatic tumor cells. In another procedure, the vectors expressing CD80 or CD86 cats in combination with either CD28 or CTLA-4, was directly injected with the cat in the tumor, resulting in the observed non-specific immune response against tumor cells, leading to the destruction of both localized and metastatic tumor cells.
Cloning and sekvenirovanie cDNA CD 80 cats
In addition to cytokines, some surface-cell molecules, as has been shown, can enhance or suppress specific immune response. CD80 (B7-1) is a helper molecule that binds to the corresponding receptor on the surface of T-lymphocytes (Freeman et al., 1989). This interaction ensures the delivery of secondary stimuli, which in combination with the primary signal, mediated by the recognition of T-cell receptor antigen, presented with the participation of MHC molecules, causes asset is the development and proliferation of T-cells (Allison & Lanier, 1994). Although CD80 protein was first described as an antigen In lymphocytes, it was subsequently established that it is expressed next cell types, mainly having the property of prezentowania antigen (Freeman et al., 1989).
In primates and rodents molecule CD80 - polypeptide with a molecular mass of 60 kDa, composed of approximately 290 amino acids (Freedman et al., 1987; Freeman et al., 1989). Confirmation of the proposed amino acid sequence indicates characteristics, according to which it is a member of immunoglobulin superfamily (IgSF) (Peach et al., 1995). It is composed of two extracellular immunoglobulinemia (IgSF) domains, a hydrophobic transmembrane domain and a short cytoplasmic "tail" (Freeman et al., 1989). The extracellular domain of the Mature protein consists of 124 N-terminal residues of variable (V) IgSF domain and then from 100 amino acids const (C-like) IgSF domain (Freeman et al., 1989). In the structure of the homologue of human CD80 there are 8 potential sites of N-glycosylation and, although the Mature protein is characterized by a significant level of glycosylation, these carbohydrate residues, is not associated with the processes of binding to receptors CD28 or CTLA-4, because they are oriented in the opposite from the intended domain binding side (Bajorath et al., 1994). Moreover, removal of carbohydrate residues, see the mu does not affect the ability to bind, and therefore it is assumed that their functions are associated with the increased solubility of the extracellular part of the molecule (Linsley et al., 1994a).
CD80 binds to two different receptors expressed at different times of the process of T-cell activation. The CD28 receptor is found in a variety of thymocytes, naive and activated T-cells and has been demonstrated to participate in the activation and proliferation of T cells (Aruffo, 1987). A second receptor for CD80 - CTLA-4 is usually detected later on fully activated T-cells (Linsley et al., 1991b). Although the role of CTLA-4 while not precisely defined, it is assumed that this molecule may act as a factor of suppression is active, the existing T-cell response (Hutchcroft & Bierer, 1996).
By itself, the molecule CD80 apparently not capable of signal transmission. Its cytoplasmic segment is relatively small and does not include amino acids, which would be confirmed by the signal and catalytic function (Hathcock et al., 1994). The lack of conservatism in the structure of the cytoplasmic sites of the peptides of human and mice is also consistent with the likely absence of signal functions in protein CD80, which is active only on binding to receptors CD28 or CTLA-4 (Linsley et al., 1994a).
The interaction between CD80 and CD28, as shown, is required to transition avnyh T-cells in the activated condition, that initiate primary T-cell response (Damle et al., 1988). Although CD80 was first found on activated b cells (Freedman et al., 1987), after that it was discovered most of the group-specific antigen-presenting cells, including macrophages and monocytes (Freedman et al., 1987), Langerhans cells (Symington et al., 1993), dendritic cells (Liu et al., 1992), activated T-cells (Razi-Wolf et al., 1992) and various tumor cells (Chen et al., 1992). The presence of molecules CD80 cells APC has been shown to be important for activation and CD4-positive and CD8-positive T cells (Allison & Lanier, 1994; Bellone et al., 1994). Although this molecule in normal in a significant amount is present only on specific ARS, in cell lines of some tumors was found increased production of signal molecules (Chen et al., 1992).
It is assumed that the transformation in some opuholerodnyh lines is increased expression of CD80. In these tumor lines not derived from antigen-presenting cells, as well as in some immortalized cell lines CD80 is a surface protein expressed on this level, which is sufficient for conditioning the full activation of T cells (Chen et al., 1992). Although the kinetics of the expression is unclear, it may be that derived from tumor cells CD80 may be the answer to "oncogenic shock" and to reflect evolucio the ing mechanism, by which the immune system is capable of removing transformed or opuholerodnyh cells (Antonia et al., 1995).
Role of the interaction of CD28-B7 is key during the initial activation of T cells. Recognition of the antigen with the participation of MHC molecules T-cell receptors is not sufficient in itself to initiate optimal proliferation and activation of T cells (Schwartz, 1992). TCR stimulation in the absence of auxiliary signals can lead to anergy or decrease the reactivity of T-cell populations (Jenkins et al., 1987). The binding molecule CD28 on T-cell with a molecule CD80 on the antigen presenting cell is treated as transmission of the second signal required for T-cell activation (Schwartz, 1992). When the receptor TCR is loaded, in the absence of the second signal "naive" cells are not activated and can be anergicakimi (Lanier et al., 1995). This fundamental role of the interaction of CD28-CD80 was clearly identified not only for the activation process of "naive" CD4+but also in clones of CD4+Th1 and Th2 and "naive" T cells CD8+derived from small resting peripheral blood lymphocytes (Linsley et al., 1993a).
As in the case of a family of receptors CTLA-4/CD28, also has at least one additional receptor related to CD80. Studies that have attempted to identify the value of the CD80 for the primary immune response, put a number of questions due to the fact that, although the introduction of CTLA-4Ig suppressed immune responses, in contrast, the addition of monoclonal antibodies (mAb) to CD80, as it seemed, had not resulted in a similar effect (Lenschow et al., 1993). Upon receipt of mice, "off" gene CD80, unexpectedly it was found the presence of a second receptor, CTLA-4/CD28 (Freeman et al., 1993). Suggested that these mice would have a similar phenotype to the previously obtained mice, "off" CD28 who had inadequate T-cell response (Freeman et al., 1993). However, it was found that "turned off" by CD80 mouse form the normal response, and their cells APCS capable of forming a secondary signal that is required for maturation of T cells (Freeman et al., 1993). On the basis of these data suggested the existence of a second receptor, which ultimately was selected. The subsequent discovery of related receptor CD86 (B7-2 or B7-0), it appears that removes the contradiction that emerged in the analysis of "off" CD80 mice, and in combination with the similarity of the structure and parameters of the link it points to the possibility that these molecules are characterized by common features and origin (Hathcock et al., 1994).
CD86 (B7-2) shows a certain level of similarity with CD80, in particular on the structure of the extracellular IgSF domains-IgSF V and-C (Freeman et al., 1993). The total level of homology of these the Molek is, however, less than 25%, despite the fact that conservative residues are concentrated at opposite ends of both extracellular domains (Bajorath et al., 1994). Although linking segment has not been set for any of these molecules, homology of sequences allowed us to identify the plot, promising from the point of view of establishing a potential site of interaction (Linsley et al., 1994a). Despite the lack of conservatism, CD80 and CD86 are characterized by similar parameters binding to both receptors, although CD86 characterized by a more rapid release from the receptor CTLA-4 (Linsley et al., A).
CD86 seems to be characterized by similar parameters expression compared with CD80, being expressed on activated b-cells, T-cells, macrophages and monocytes (Azuma et al., 1993a). However, the dynamics of expression of these two molecules in a small degree varies (Hathcock et al., 1994). In General, CD86 appears earlier during an active immune response compared to CD80 and presumably is expressed by monocytes in constitutive type (Freeman et al., 1991). Although CD80 may appear 24 hours after initial stimulation, CD86 appears at an early stage of response or even expressed myeloid cells constantly at a low level (Hathcock et al., 1994). These two surface protein cause similar intracellular reactions being associated with the corresponding the receptors on the surface of T cells as CD4 +and CD8+(Lanier et al., 1995). Apparently there are no differences in the ability of each of these molecules to initiate the activation and proliferation of T cells or to induce CTL activity (Hathcock et al., 1994). Thus, the available evidence suggests that both molecules initiate a similar signaling cascade after binding receptors CD28 or CTLA-4, respectively (Hathcock et al., 1994). Although, it seems that the kinetics of binding of both of these molecules with their receptors the same and no different manifestations of this, it is unclear evolutionary significance of the existence of such a system "a pair of ligands/pair receptors" (Lanier et al., 1995).
CD80 was first described as a marker of b-cells, and high levels and CD80, and CD86 are found on b cells stimulated by lipopolysaccharide (LPS), anti-Ig, anti-CD40, concanavalin A. immunology (Con-A), camp, IL-2 and IL-4 (Hathcock et al., 1994). As has been shown, in b cells of mice γ-interferon and IL-5 increase the production CD86, while there is no similar data for a person or for CD80 in connection with the above immunoregulatory (Azuma et al., 1993a). Dynamics of gene expression in b cells in these two molecules are slightly different. CD86 is expressed almost immediately after stimulation (6 hours), whereas CD80 is missing for almost 24 hours and up to 48 hours does not reach the peak of their expression (Lenschow et al., 1993). It is considered, the positive regulation of CD80 on b cells is under the control of the signaling mechanism, mediated by molecules of class II MHC (Nabavi et al., 1992). Two other surface-cell receptor also seems to be important in connection with the expression of CD80. Cross-linking CD40 expressed on b cells, Ig or T-cells expressing the appropriate receptor, leads to increased expression of CD80 (Azuma et al., 1993b), while cross-linking Fc receptors leads to a decrease in the expression of both molecules (Barcy et al., 1995).
CD40 and the ligand of this receptor CD40L, as expected, are the elements of the mechanism involved in the regulation of expression of CD80 on cells ARS (Page et al., 1994). CD40 expressed in various cell types, including b-lymphocytes, monocytes, dendritic cells, fibroblasts and endothelial cells, and can be positively regulated in these cells in the presence of γ-interferon (de Boer et al., 1993). The CD40 ligand (CD40L) expressives activated CD4-positive T-cells. The binding of CD40 and CD40L, as shown, increases expression of CD80 cells ARS, although apparently it does not induce the expression of other types of cells expressing this receptor, including endothelial cells (Page et al., 1994).
Molecules CD80 and CD86, though showing only 25%amino acid similarity to each other, have structural similarities and are considered to be "remotely related" (Freeman et al., 1993). D is a logical remains concentrated within immunoglobulin-like domains with a small number of conservative residues in the structure of the transmembrane and cytoplasmic domains (June et al., 1995). It is assumed that the family, including the products of the genes V7, also, in addition to CD80 and CD86, covers butyrophilin (W), glycoprotein myelin/oligodendrocytes (MOG), MHC-similar chicken - B-G (Linsley et al., 1994b). TUE, MOG and G are encoded by genes within genomic MHC complex, which indicates the likely evolutionary relationship between major histocompatibility complex and the necessary co-stimulatory molecules (Linsley et al., 1994b).
Resting T-lymphocytes of the mouse and human low Express CD86, whereas T-cells from mice and humans (and T-cell clones), activated by the action of antibodies to CD3, Express CD80 and CD86 on a significant level (Hathcock et al., 1994). The expression of CD80 and CD86 on activated T-cells may reflect the ability of these T cells to proliferate on the mechanism of autocrine costimulation (Azuma et al., 1993b). Interestingly, as has been shown, CD80 positively regulated by HIV-infected CD4-positive T-cells with simultaneous negative regulation of CD28. It is assumed that this is a likely mechanism of viral transmission when uninfected T-cells CD4+initiate mediated by the interaction of the CD28/CD80 contact with infected lymphocytes (Haffar et al., 1993).
Monocytes in human peripheral blood low Express CD80 and at a high level - CD86, while processing the TKA factor GM-CSF or γ -interferon leads to enhanced surface expression and CD80, and CD86 (Barcy et al., 1995). LPS is a potent inducer of the expression of CD80 in monocytes of peripheral blood (Schmittel et al., 1994). While there is no evidence of peritoneal macrophages person, while it is known that resting macrophages mouse Express CD80 and CD86 at low levels (Freeman et al., 1991; Hathcock et al., 1994). Stimulation of LPS and γ-interferon macrophages of mice increases the level of surface expression, although γ-interferon in combination with interleukin-10 reduces the level of synthesis of both of these receptors (Ding et al., 1993).
Dendritic cells of the spleen low Express both molecules and cells of Langerhans low Express CD86, although under cultivation there is a trend towards increased expression in both types of cells (Larsen et al., 1994). The cultivation of dendritic cells CD86 apparently subject to more powerful and earlier positive regulation, which may play an important role in mediating signaling pathways in these cells (Hathcock et al., 1994). Interestingly, although IL-10 has no effect on the expression of CD80 dendritic cells, he is active in the negative regulation of the expression of CD86 (Buelens et al., 1995). It was reported (O Doherty et al., 1993), although initial levels of CD80 in dendritic cells is very low, after their ripening, the existing level of CD80 increases. The expression of CD80 is leccami of Langerhans suppressed and IL-10, and γ-interferon, although the processing of colony-stimulating factor granulocyte/macrophage revertive suppression caused γ-interferon, but not the suppression induced IL-10 (Ozawa et al., 1996).
As has been shown in human and mouse specific cytokines control the expression and CD80, and CD86. Interleukin-4 is a potent inducer of CD86 and to a lesser extent inducer of CD80 in b cells (Stack et al., 1994), while γ-interferon enhances the expression of CD86 in various cell types, including b cells, monocytes and macrophages (Hathcock et al., 1994). Although, as you can see, γ-interferon causes increased expression of CD80 in monocytes, it can lead to weakening of expression and macrophages. IL-10, even in the presence of γ-interferon, reduces the expression of CD80 and CD86 (Ding et al., 1993). Such interaction may reflect the probable mechanism of the switch Th1 response (DTH) to Th2 dysbalance-response (humoral). However, IL-10 does not affect the expression of CD80 in dendritic cells (Buelens et al., 1995). This also may reflect the role of these molecules in the regulation of groups of T-helper cells, because it is believed that dendritic cells play an important role in initiating the immune response of the 2nd type. Interleukin-7 enhances the expression of CD80 in T-cells, although its effect in other cell types is not yet defined (Yssel et al., 1993), while it was found that the expression of CD80 In cells of oposreduemye account of cross-linking with TNF receptor P75, and the expression can be increased in the presence of IL-4 (Ranheim & Kipps, 1995). Interestingly, TNF belongs to the same family of molecules that CD40 - another potential initiator of expression of CD80. Apparently, GM-CSF increases the surface expression of CD80 dendritic cells and Langerhans cells, while γ-interferon causes increased production only CD86 in these cells (Larson et al., 1994).
For a long time it was believed that the recognition, binding and lysis of transformed and virus-infected target cells CD8-positive cytotoxic T-lymphocyte (CTL) mediated by only by TCR recognition of foreign peptides expressed in connection with the molecules of class I MHC (Berke, 1993). Recently it was found that the number of surface-cellular molecules expressing and CTL and target cells necessary for the purpose of full interaction (Mescher, 1992). A key part of this interaction is CD80 and its corresponding receptor CD28. The auxiliary signal generated by the interaction of B7-CD28 required for small resting CD8-positive lymphocytes were differentiated in the lytic state (Mescher, 1992). Interestingly, after differentiation of CTL this secondary signal ceases to be necessary for the manifestation of lytic properties (Hodge et al., 1994).
For a long time it was known that CTL t is Auda key mediators of antiviral immunity. The person in case of infection by the human immunodeficiency virus (HIV) long-term absence of symptoms associated with high levels of CTL memory CD8+specific for viral proteins Gag, Pol and Env, and a very low number of copies of HIV DNA and RNA in managernew peripheral blood cells (Rinaldo, 1995). In contrast, patients in the later stages of AIDS, the number of CTL memory (mCTL) is sharply reduced (Zanussi et al., 1996). The consistency of these data indicates that CTL memory can be a major factor in the control of infection in organizine the recipient and can play a key role in shaping the immune system uninfected persons (Zanussi et al., 1996). In addition, pathogenic HIV infection reflect the probable role of CD80 and CD28 in the development of AIDS.
It was also suggested that CD80 is involved in the pathogenesis of HIV infection (Haffar et al., 1993). In normal T cells Express CD80, but at a low level and only after activation (Schwartz, 1992). In the model analysis of HIV infection in vitro in allostimulatory lines and primary T cells has been shown that CD28 negatively regulated, and the expression of CD80 obviously increases along with the MNF CII (Haffar et al., 1993). Although the exact mechanisms of these processes have not yet been determined, it is possible to assume the existence of two mechanisms of possible damaging effects. The presence of CD80 on the surface together with molecules of class II which may lead to the intensification of contacts between infected T-cells and uninfected CD4-positive cells (Haffar et al., 1993). Although this interaction may lead to an increase in the intensity of the exchange between T-cells, another function may be increased CTL-mediated recognition and destruction of cells by generating a secondary signal resulting from the interaction of CD80 on the surface of infected cells with CD28 receptor expressed by CD8-positive T-cell (Haffar et al., 1993). This may accelerate the decline in the population of lymphocytes CD4+that correlates with the manifestation of diseases associated with AIDS (Haffar et al., 1993).
And in mice, and in humans the expression of the protein family V7 is seen as an important factor in the immune recognition of transformed cells (Chen et al., 1992). Although the expression was installed in some types of transformed cells, the majority of tumors normally do not Express CD80 or CD86, thereby making incredible that in the case of the expression of potentially immunogenic tumor antigen will be fully recognized by T-cells (Chen et al., 1993). However, transfection experiments using molecules CD80 to enhance cytolysis of tumor cells was successful (Hodge et al., 1994).
Retroviral-based vaccinia virus vectors expressing functional molecule CD80, were used for transfection of malignant cells(Li et al., 1994; Hodge et al., 1994). These cells expressing CD80 in addition to bad (OK) recognizable tumor antigens, then brought back to the donor that, as expected, will lead to the emergence of the cellular immune response against tumor antigens expressed on tumour cells (Townsend & Allison, 1993). The results of these experiments with various forms of tumors was surprisingly effective. In many cases, the recipient organism have formed a strong cellular response against malignant tumors, controlling or eliminating it (Hodge et al., 1994). Subsequent reintroduction into the body donor tumor cells, with a surface molecule CD80 or deprived of it, leads to similar levels of antitumor immunity (Hodge et al., 1994). Thus, it is believed that after the establishment of immune memory molecule CD80 is not necessary for maintaining response or to initiate it if reintroduction (Hodge et al., 1994). These experiments showed that the molecule CD80 is an effective mediator of cellular immunity and that specific types of tumor cell responses can be induced by probable control of malignant neoplasms and preventing recurrences (Hodge et al., 1994).
Increased expression of CD80 can have negative consequences that viewsets the development of some forms of autoimmunity. It is believed that CD80 interaction with IL-12 is important in the early stages of multiple sclerosis and causes stimulation of T cells and the development of DTH (Windhagen et al., 1995). Experimentally induced autoimmune encephalomyelitis (EAE) can be partly suppressed by the introduction of remembrance CTLA-4Ig experimental object. Suppression of demyelination by blocking interaction of CD28/CD80 likely reflects the role of this interaction in the exacerbation of the disease (Arima et al., 1996).
The importance molecules CD80 in the development of an effective immune response is obvious. Although the cDNA encoding this protein was identified in rodents and primates, it has not been previously found in other taxonomic groups of mammals. Cat is a common pet and a potential model of retroviral diseases. Cloning of the immunological factors in cats provides an important tool of veterinary research, in particular in connection with research development and prevention of diseases in other species of organisms.
Materials and methods
The selection of the original fragment
mRNA was extracted from managernew peripheral blood cells (RVMS), stimulated for 16 hours Con-And using reagent for RNA extraction RNAzolB (Biotexc, Houston, TX). The original cDNA was synthesized on the matrix of this RNA is by using reverse transcriptase (FROM), where as the reverse primer used oligoimide. Briefly, RNA and oligo-dT was heated to 75°C for 3 minutes in order to remove secondary structures. Then he added, dNTP, buffer and distilled water and the resulting mixture is incubated for 1 hour at 42°C. After this incubation, the sample was heated to 95°C for 5 minutes to inactivate FROM. Degenerate primers derived from consensus sites in the composition of the published nucleotide sequences of human CD80 and mouse (GeneBank, Gaithersburg, MA)was then used for the initial amplification of this gene 344-nucleotide fragment encoding the Central segment comprising a constant domain:
direct primer V7-2: GGC CCG AGT A(CT)A AGA ACC GGA
Reverse primer V7-3: CAG (AT)TT CAG GAT C(CT)T GGG AAA (CT)TG (SEQ ID NO: 56).
For amplification of this product used a Protocol polymerase chain reaction (PCR) with a "hot start" Taq polymerase. At the beginning of the reaction mix without Taq polymerase was heated to 95°C for 5 minutes (the stage of "hot start") to prevent the formation of dimers between the primers. Polymerase was added before the temperature cycle. Then Poland reaction was heated to 95°With 30 seconds to dissociation of double-stranded DNA. Then the reaction was cooled to 42°With 30 seconds to ensure annealing vereide the different primers. Relatively low temperature annealing facilitates the binding of primers if they are not 100%gomologichnosti with the target. Then the reaction was heated to 72°45 seconds, which is the optimal temperature for activity of Taq polymerase, ensuring completion (extension chain) primer and copies the opposite DNA strand. This temperature cycle was repeated 30 times. After these 30 cycles final stage was carried out at 72°C for 7 minutes in order to facilitate the completion of any incomplete products. After visualization in 1%agarose gel resulting product ligated overnight at 16°With part of the cloning vector (InVitrogen, San Diego, CA) for subsequent sequencing. 2 μl of ligation reaction was used to transform competent cells InvαF'. Transformed bacteria were applied stripes on LB plates (50 µg/ml ampicillin)coated with 40 μl of a solution of 50 μg/ml X-Gal. The next day was selected colonies are white and inoculable them in 5 ml of Kul tural Noi LB medium containing 100 μg/ml ampicillin, and cultured overnight at 37°when the rotation speed of 225 rpm
Minipreparation conducted on the material night cultures in order to identify clones that carry a plasmid with the insert in the correct orientation. Plasmid was extracted from cultures using standard is based procedures alkaline lysis followed by purification of the DNA by extraction with a mixture of phenol and chloroform (Maniatis et al., 1982). DNA was besieged by 2 volumes of ethanol and then were digested with restriction enzyme EcoRI. The results of this cleavage was visualizable in 1%agarose gel to identify colonies that carry a plasmid containing the insert in the correct orientation. Then, this plasmid was purified from positive clones and sequenced using reagents for sequencing by the method of the termination circuit with35S-tagging on Sanjuro (Sequenase®, US Biochemicals, Cleveland, OH) or a round-Robin sequencing end with a fluorescent label (Perkin Elmer, Norwalk, CT). According to the nucleotide sequence of the cDNA-specific reverse and direct the primers were designed for use in the method of "rapid amplification of cDNA ends" (5'-RACE) and to highlight the 3'sequence in combination with degenerate primers from the 3'-noncoding segment (UTR).
The allocation of the 5'-segment
Amplication Protocol Marathon cDNA (Clontech, Palo Alto, CA) was used to obtain 5'-sequence of this gene. mRNA was isolated from RVMS, stimulated for 12 hours Kon-a and at the same time for 4 hours with lipopolysaccharide. mRNA was extracted using a reagent for extraction of ULTRASPEC RNA (Biotexc, Houston, TX). cDNA was obtained using the anchor primer" oligo-dT with degenerate nucleotides at the 5'-end to facilitate the binding of this paragraph is Aymara from the far 5'-end polyadenylation "tail". Then cDNA was transcribable as described above. Specific linkers ligated to this cDNA using a DNA ligase of phage T4. PCR "down from the point of ligation was carried out on the matrix cDNA using the internal reverse primer specific regarding the source of the amplified plot:
V7-284: TTA TAC TAG GGA CAG GGA AG (SEQ ID NO: 58)
V7-190: AGG CTT TGG AAA ACC TCC AG (SEQ ID NO: 59), -
and the anchor primer, complementary legirovannoi linker sequence. The PCR parameters "down from point" with chimeric polymerase KlenTaq (Clontech, Palo Alto, CA) were as follows: 5 minutes at 95°1 cycle; 30 seconds at 95°C, 30 seconds at 72°C and 45 seconds at 68°5 cycles; 30 seconds at 95°C, 30 seconds at 65°C and 45 seconds at 68°C - 5 cycles; 30 seconds at 95°C, 30 seconds at 60°C and 45 seconds at 68°25 cycles. 1 μl of this reaction was diluted in 50 μl water and 5 μl of the resulting dilution is then used for "nested" PCR (5 min at 95°1 cycle; 30 seconds at 95°C, 30 seconds at 65°C and 45 seconds at 68°C - 30 cycles, with KlenTaq polymerase) with the linker-specific anchor primer and a gene-specific reverse primer located 5'side with respect to the original primer.
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 µl of each reaction was visualizable 1.5%agarose GE is e, and the exact size of the fragment was cut out of the gel. cDNA was extracted and purified from the agarose by centrifugation of the gel slice through the spray gel and filter Micropure with pores of 0.22 μm (Amicon, Beverly, MA). Then the purified DNA directly sequenced using sequencing end with a fluorescent label (Perkin Elmer, Norwalk, CN).
The selection of the 3'-segment
the 3'Segment of this gene was isolated by screening five genespecific primers for each sequence 344-nucleotide fragment of the previously sequenced 5'-plot:
B7-s220: GTC ATG TCT GGC AAA GTA CAA G (SEQ ID NO: 62)
V7-50: CAC TGA CGT CAC CGA TAA SAA (SEQ ID NO: 63)
V7-140: 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).
Then degenerate reverse primers were selected by consensus sites 3'-UTR CD80 gene between human and mouse:
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 were obtained on material RNA extracted using ULTRASPEC (Biotexc, Houston, TX), isolated from RVMS, stimulated by Con a and LPS as described above. Anchor primer oligo-dT was used as the source of the reverse primer for transcription of RNA into cDNA. PCR with Taq polymerase was performed on the matrix obtained cDNA using specific direct degenerate primers reverse primers (5 minutes at 95°1 qi is l; 30 seconds at 95°C, 30 seconds at 42°C and 45 seconds at 72°C - 30 cycles; 72°C for 7 minutes). Two rounds of "nested" PCR reactions were necessary in order to obtain only a fragment of the correct size. The resulting product was cut from a 1.5%agarose gel, purified as described above and sequenced by the method of sequencing end with a fluorescent label (Perkin Elmer, Norwalk, CN).
According to the sequencing of the 5'- and 3'segments of the designed primers that would amplify a section that encodes the full-size open frame CD80 gene cat:
B7-START: ATG GGT CAC GCA GCA AAG TGG (SEQ ID NO: 69)
V7-960: CCT AGT AGA GAA GAG HUNDRED AAG AGG (SEQ ID NO: 12).
Was used previously obtained on the material RVMS cDNA, for which we know that it has a DNA that encodes a desired gene. In this PCR reaction (5 minutes at 95°1 cycle; 30 seconds at 95°C, 30 seconds at 42°C and 45 seconds at 72°C - 30 cycles; 72°C for 7 minutes) used a chimeric polymerase KlenTaq - this "collective" enzyme retains some 5'-ectonucleoside activity that reduces the frequency of random errors often occur when performing PCR with Taq polymerase. In reaction amplified 960-nucleotide fragment, which was cloned in the composition of the cloning vector (InVitrogen, San Diego, CA) and sequenced in accordance with the previously described the output. In the final sequence of the desired gene were taken into account cDNA from two different animals. Each nucleotide in the sequence of this gene has been independently verified by at least three different sequences obtained in separate PCR reactions, to reduce the likelihood of inaccuracies originating from errors induced by the PCR.
RNA extracted from the tissues of experimental cats NC used for the initial attempts to amplify the gene CD80, but this cat was then put to death and subsequent products were taken from other animals. In the original RNA amplification cells RVMS cats NK about stimulated Con-And received 344-nucleotide product, showing 70%level of identity with the genome of human CD80. The primers were specific area in the centre of the coding sequence corresponding to a constant immunoglobulinemia domain. Although these initial experiments used an additional degenerate primers to amplify regions that encode a larger segment of the peptide, only the combination of primers B7-2 (direct) and B7-3 (reverse) received the product of the appropriate size. Subsequent experiments in which these additional degenerate primers were used along with gene-specific primers, OK what was unsuccessful. Consequently, the 5'-and 3'-areas should be selected with the use of other methods.
A group of six new gene-specific primers formed on the material data sequencing, performed on the original product (direct: V7-20, V7-135, V7-284, V7-190; reverse: V7-140, V7-50). The original reverse primers used in the PCR method, the 5'-RACE, which to some extent similar to the method used for the successful amplification of the 5'-segment of the molecule CD28. However, when using this method, the products have not been obtained.
In amplication system for cDNA Marathon RACE (Clontech, Palo Alto, CA) successfully amplified the plot, which corresponds to the 5'-coding sequence. RNA derived from cells C, stimulated by Con A, was successfully amplified using this Protocol. The initial amplification was performed with primers V7-284 and B7-3 and the anchor primer AP1. In this reaction has not been received distinct single band, so using this product as a matrix held a "nesting" of the reaction using primers V7-20 V7-135 as "Jack" (i.e. internal to the previous primers) reverse primer and anchor primer AP1 as a forward primer. Product suitable length received in each of these reactions.
Data obtained by direct sequencing reaction products RACE, p is svolli to achieve full identity on plots of length 20 and 135 BP, respectively, which overlap with the original sequenced product length 344 BP Products have extended from the known 5'-section through the start codon ATG of the gene cat towards the 5'-untranslated segment. The identity of the 5'-coding sequences derived from these products, and the 5'portion of a gene CD80 person was less than the identity, defined between the 344-BP segment of the gene of cats and a similar portion of a gene of the person. It was found that a similar loss of homology is found between the sequences of human and mouse on the same segments. The comparison of the sequences of areas outside the coding segment was further shown a distinct decrease in the level of conservatism (data not included).
A new group of degenerate primers encoding the 3'-noncoding site, was synthesized according to the parameters of the consensus sites within the 3'UTR sequences of human and mouse. Using these primers successfully amplified cDNA transcribed from RNA extracted from RVMS, stimulated by Con A, taken from a cat ED3. In contrast to the initial amplification to obtain the final product, it was necessary to conduct a series of "nested" reactions. In the primary PCR reactions using these degenerate primers and anchor primers from within the 344-nucleotide is the first sequence and the 5'-segment, failed source to get a clear identifiable bands. However, the "nesting" reaction when using diluted primary product and more specific 5'-primer was allowed to get a product that encodes the remaining 3'-site.
After sequencing of the 3'product, it was found that beyond a constant Ig-like domain level identity again declined. The distal end of the coding sequence indicated a very low level of identity, which is even more decreased after the stop codon.
On the material 3'sequences designed a reverse primer, went beyond the limits of the coding sequence, starting with 960-th nucleotide. With this design, in combination with the primer, covering the start codon, the amplified product of the expected size. Sequencing of the final product showed that all previously installed parts really overlap, and the resulting fragment is a continuous and complete nucleotide sequence of the gene CD80 cats.
For amplification of the final product samples amplified in the material RNA extracted from RVMS, stimulated by Con A, taken from animals ED3 and EC. At least two products from each animal is fully sequenced, and each nucleotide site was inspected and ottbergen at least three correctly read sequences. The product obtained from the animal EK, then cloned in the composition of the cloning vector for subsequent manipulation and as a reference.
Fragment length 960 nucleotides includes the start codon for the 1st position, a stop codon at position 888 and additionally 72 nucleotide 3'-untranslated segment. Among the products that are obtained and sequenced in establishing a full-sized fragment were sequenced an additional 5'- and 3'-plots (data not shown). Sequencing of the site, which is located above the start-codon, the material products of 5'-RACE revealed that the codon ATG, defined in clauses 1-3, is the first coded website (methionine) in the reading frame and occupies a similar position in the sequences of mouse and human. A stop codon located at position 888, as well as confirmed, is in a similar position in the previously sequenced genes. Comparison of the sequences showed levels of identity 77% and 62% with the published nucleotide sequences of CD80 genes between human and mouse, respectively. The level of homology with the sequences of other primates and rodents is comparable with the levels established for human and mouse, respectively. At each level of identity with the genome of CD86 was less than 25%.
Using computer package program is mm for DNA analysis MacVector (IBI, Rochester, NY) the nucleotide sequence of "broadcast" in the amino acid sequence. As a result of this decryption identified 292-amino acid peptide that is similar in size to proteins and mouse, and humans, though not identical to them. Signal segment, as expected, covers the first 25 amino acids. The extracellular domain of this molecule is composed of 115 amino acids of the domain, similar to immunoglobulin variable domain (up to 139-th residue), and 110 amino acids of the domain, similar to immunoglobulin constant domain (approximately 240-th residue). The transmembrane domain, which is composed of the remnants of 241-271, turns into a short cytoplasmic tail, consisting of 21 amino acids. As the molecule man, the polypeptide cats includes 8 potential sites of N-glycosylation, although the location is not identical. The level of homology between proteins cats, humans and mice is significantly less than the level of the identity of the corresponding nucleotide sequences (table 1).
Comparing levels of homology sequences CD80 cats, mice and humans
|The percentage of homology with the sequence cats|
Comparison of the proposed amino acid sequence of CD80 cats with the intended sequence of a person indicates that most of gomologichnosti these two molecules are concentrated in the constant domain (amino acids 140-240). There is little homology between these peptides in the signal segment and it is not beyond the IgV-like domain. As already mentioned, the significant conservatism in the sequence of the constant domain, however, this identity is almost beyond its limits, and a very low level of homology is detected by the transmembrane domain and cytoplasmic "tail" of the peptide cats and similar sites in the molecule man.
Mapping genes CD80 cat, human and mouse genes CD86 mouse and human rights shows that, although there are a very limited level of homology between the two members of the family V7, amino acids, which are considered diagnostic for gene family V7 are available in the protein cat. These molecules include residues, which presumably are involved in spatial stacking ("folding") and constitute the binding site.
Although the level of homology between the follower is awns human CD80 and cats are not so great, as the level of identity between molecules CD28, comparison of the obtained graphs hydrophobicity shows that, although there are a number of substitutions of amino acids in a specific amino acid sequence, these changes are often homologous and apparently do not alter the surface characteristics of this peptide.
The levels of identity of the nucleotide sequences of cats and humans, as well as cat and mouse CD80 average, although the level of homology is not transferred to the peptide. It is assumed that, although the genetic code is degenerate, in some molecules (e.g., CD28) differences between nucleotide sequences substantially do not change the peptide, in the case of CD80 conservatism amino acid sequence as a whole is not so critical, and thus change the length of this molecule in the course of evolution is "more valid".
Although, in General, the nucleotide sequences are very average level of identity, there are certain difficulties in the allocation of the full sequence. The original product CD80 was derived from the constant domain of this molecule - i.e. the area that exhibits the highest level of conservatism in the cDNA sequence of this species. Primers that mark this site and effectively to amplify p is oduct, got a simple way, resulting in a 344-BP fragment covers the most conservative area IgC-similarity. Unfortunately, due to the lack of homology in the sequences of the signal segment of the cytoplasmic domain and the 3'UTR for the selection of the sequences of these sites need to put more effort. The availability of data on the sequencing of the Central site of the protein, however, allows you to define a starting point from which can be defined in other sections of this molecule. CD80 cats is a great example of how by obtaining a short fragment of the desired molecules and applying methods RACE and degenerate primers in combination with anchor primers that meet a segment can be fairly easily obtained full sequence of this molecule.
Comparison of the proposed amino acid sequence of these cloned molecules CD80 shows no overall homology. The polypeptides of the mouse and human showed less than 50%homology in amino acid sequences. This compares with 59%level of identity when comparing proteins cats and humans and 46%identity to the polypeptides of cat and mouse, which may reflect the evolutionary relationship of these species. Comparison of the predicted profiles hydrophilicity of amino acids is oshki and man, which allow to identify those residues that have been affected or displaced by their relative hydrophilicity, showed that, although the amino acid sequence of specific amino acids could not be saved, these changes apparently wore relatively conservative in nature. This indicates a probable saving sign of hydrophilicity/hydrophobicity of the molecule that may correspond to a similar structure in General the polypeptide. For surface-cell protein, obviously, is characterized by the presence of specific amino acids involved in the binding process, as well as other amino acids, which are needed only in order to maintain the structure needed to ensure interaction with the binding site.
Although there are discrepancies amino acid residues in molecules CD80 the Primate, rodent and cat, however, remain signs of IgSF. Molecule CD80 cats includes end IgC-like domain and IgV-like domain located proximally with respect to the area associated with the membrane. As in the case with the level of conservatism when compared CD80 mouse and cat, the level of identity const areas is higher than in the comparison variable regions (Freeman et al., 1989). In General, the conservatism variable domain exceeds 50%, whileas the rate constant of the segment exceeds 70%, despite the fact that a short stretch of amino acids 164-198 (the same plot that was allocated source 344-nucleotide fragment) is characterized by the highest level of identity. This Central 56-amino acid stretch (residues 165-221) in the constant domain shows 87%level of homology between the sequences of human and cat, with a 28-nucleotide stretch (residues 171-198) there is only one difference. Also, this section of the cat sequence, showing a significant level of homology compared with the corresponding residues of the polypeptide of the mouse. The hydrophilic nature of the amino acids in this area indicates a high degree of probability expression on the cell surface, and given a certain level of conservatism between species is likely to participate in interactions "ligand-receptor". It has been suggested that the IgC part of this molecule directly involved in prezentowanie binding domain to bind to the receptor (Peach et al., 1995). However, experiments have found that for efficient binding necessary and constant and variable domains (Peach et al., 1995). The concentration of the homologous residues in IgC-site extracellular domains, along with the high level of divergence transmembrane and cytoplasmic domains, is considered to complement the aspects of the evidence of the role of CD80 as a ligand to a greater extent than factor, capable of generating a signal.
As a human, and mouse CD80 cats is characterized by a high level of glycosylation. Carbohydrate residues are considered to be not directly involved in binding, however, can enhance the solubility of the extracellular segment of this molecule (Peach et al., 1995). Of the eight potential glycosylation sites found in the sequence of the peptide of man, seven were located in positions identical to those in the protein cat. The site, located in the 39th position of the amino acid sequence cats, not reproduced in the molecule CD80 person, while there are customers in the 232 position missing in the sequence cats (Freedman et al., 1987). In the molecule of the mouse has seven glycosylation sites, only two of which are in identical positions, although in principle they are located in the same regions of the molecules, and that molecules in cats and humans (Freeman et al., 1989). The similarity in the number and position of glycosylation sites, as you can assume, reflects the importance of motives for the functioning of a given molecule.
There is a wide range of possible ways of using molecules CD80 cats. In accordance with the discussion above, this molecule is a key element of ensuring accuracy in the development of the response of Mature T cells. Control of expression on the frame of the gene and the RNA level, and at the protein level should help to establish the ways in which the immune system of the cat interacts with the infection. Figuring out how this system affects specific pathogens, based on data obtained from studies in other model systems, will shed more light on the parameters of the human immune system. In addition, contribution to the study of the possibilities of manipulation of the immune system of cats, which is one of the most common types of Pets, will give veterinary medicine new impulses.
An important potential application envisioned for molecules CD80 in other species, is the induction of tumor specific immunity by making CD80 gene in transformed cells with subsequent reintroduction to the donor for the purpose of induction of antitumor immunity mediated by lymphocytes CTL (Townsend & Allison, 1993). As discussed above, it is considered that the surface expression of CD80 tumor cells is a specific CTL response directed at the malignant tumor (Hodge et al., 1994). In addition, the host body is formed population of memory cells, derived from CD8-positive T cells (Hodge et al., 1994). Although this approach generally focuses on anti-tumor immunity, by analogy it can also be extended to the development of antiviral immunity.
As absurd the elk above, long-term latent state of acquired immunodeficiency syndrome is considered as a consequence of the initial formation of a strong CTL-mediated immune response on the BEACH-infection (Landay et al., 1994). It is assumed that those patients who for a long time does not show symptoms of AIDS after infection they are able to form and maintain strong CTL-cell-mediated immunity directed against HIV.
Although most modern vaccines aimed at the formation of the humoral immune response, however, if the vaccine is able to induce the development of population mCTL aimed at HIV and FIV, this population should provide protection similar to that which is typical for long-term asymptomatic AIDS. The introduction of new individuals "gene" vaccine, which consists of FIV proteins combined with protein CD80, should result in surface expression of costimulatory molecules in combination with the presentation of viral epitopes FIV provided by the MHC molecules of CI. If successful, this should lead to proliferation of the population of FIV-specific lymphocytes mCTL. After subsequent exposure to virulent virus in vaccinated individuals will be induction response in cells that were infected by the virus, destroying them before the virus is able to multiply the camping and will begin to destroy components of the immune system.
Cloning and sequencing of cDNA CD28 cats
CD28 is a surface-cell glycoprotein, which normally expressed in the form of glycosilated, composed of identical subunits with a molecular mass of 44 kDa linked by disulfide bonds. He is part of the immunoglobulin superfamily and is characterized by the presence of only the extracellular variable (V) of the site, a transmembrane domain and a short cytoplasmic tail (Aruffo & Seed, 1987). Although this molecule is glycosylated and carbohydrate components apparently not involved in the binding and presumably increase the solubility of the extracellular domain (Peach et al., 1994). cDNA encoding the peptides of human, rat, mouse and rabbit and similar molecule chicken were previously cloned and sequenced (Linsley et al., 1995a).
CD28 is found in the majority of thymocytes CD4+/CD8+and peripheral T cells CD4+/CD8+and is characterized by increased expression in response to stimulation αντl-XΔ3, PHA, PMA and its suppression as a result of binding with the antibody to CD28 (Linsley et al., 1993b). Soon after the discovery of this protein revealed that CD28 plays an important role in the regulation of activated T-cells CD4+/CD8+(June et al., 1990). In addition to stimulating the activation and proliferation of T cells t is the train was installed, the formation of such secondary signal induces cytolytic activity of CTL (Azuma et al., 1993).
CD28 is expressed at early stages of maturation of T cells. While immature D3 negative negative cells and CD28, intermediate cells CD4+/CD8+Express this protein at a low level, and the Mature CD3-positive thymocytes CD4+/CD8+Express CD28 at a high level (Turka et al., 1991). The person after maturation, this receptor is found in almost all T-cells CD4+and more than half of the T cells CD8+(Turka et al., 1991), and almost all of the T-lymphocytes of mice (June et al., 1990). After activation, T-cell surface expression increased, whereas the binding of this molecule with the corresponding ligand or specific monoclonal antibodies causes in activated cells, negative regulation of this gene. and at the transcriptional and translational levels (Linsley et al., 1993a). Although CD28 is found mainly in lymphocytes of T-cell populations, this protein was reported to be detected in plasmacytoma bone marrow biopsy samples (Kozber et al., 1987) and is expressed in the cultures of the line leukemic cells, similar to natural killer cells (Azuma et al., 1992).
CD28 manifests a certain degree of structural homology with other B7-receptor - CTLA-4: they both Rel is relevant to the subfamily in the group IgSF proteins (Linsley et al., 1995a). These two molecules include extracellular IgV domain, a single transmembrane domain and a short cytoplasmic signaling domain (Aruffo et al., 1987). Although the overall level of homology between the two molecules is only 31%, there are two short plot and specific residues, which is fully invariant in these two molecules: this likely reflects the important role of these motifs in the recognition of ligands B7 and maintaining structural integrity (Leung & Linsley, 1994). Hexapeptides the MYPPPY motif remains the same in all selected members of the family of receptors CD28/CTLA-4 (Peach et al., 1994). He mapped in D3-like vydeleny plot of these molecules; if the mutation engine is reduced avidity binding and CD28, and CTLA-4 (Peach et al., 1994). This plot, as expected, is a potential binding site ligands in the structure of both proteins - CD28 and CTLA-4, but it is unknown whether this plot is a direct binding site of the ligand B7 or he represents a structural motifs indirectly involved in binding (Peach et al., 1994). Despite the conservatism of the residues in the sequences of CD28 and CTLA-4, CTLA-4 binds CD80 and CD86 with higher avidity than CD28 (Ellis et al., 1996). Thus, while in activated T-cells in vitro, CTLA-4 expressed on the level of only 2-3% from that of CD28, he binds ligands with greater 20 R is h avidity (Linsley et al., 1995b).
Although molecules CTLA-4 and CD28 evolutionary related and have a common ligands, however, their functions and signal ability apparently differentiated (Balazano et al., 1992). Comparison of signal sections in each of these molecules does not reflect the significant level of identity: therefore, these molecules initiate different signaling mechanisms (Hutchcroft & Bierer, 1996).
While CD28 expressed in resting T-cells and positively regulated in the early response to the activation, expression of CTLA-4 reaches a peak at 48 hours after activation and is returned to the original level after 96 hours after activation (Linsley et al., 1992a). Expression of CTLA-4, as expected, is consistent with the negative regulation of CD28 (Lindsten et al., 1993). In addition, the signaling mechanisms mediated by ligand binding receptor CD28 appears to play an important role in intensifying the expression of CTLA-4 (Linsley et al., A). T cells that are negative for CD28, does not Express CTLA-4 in response to stimulation of PMA or a source of calcium ions (Lindsten et al., 1993).
The full sequence of events mediated by the CD28 signaling pathway is not yet defined, although there is a hypothesis about the composition of this cascade of processes (Hutchcroft & Bierer, 1996).
It has been suggested that the signaling mechanism of CD28 involves the mobilization of intracellular calcium metabolism phosphatidyl is sitala and the induction of phosphorylation of proteins on tyrosine residues (Hutchcroft & Bierer, 1996).
The cytoplasmic tail of the molecule CD28 includes specific motives for which are expected to participate in intracellular signaling processes after binding of ligands CD80 or CD86 (June et al., 1994). Consisting of 41 amino acids intracytoplasmic plot has no detectable catalytic activity, as it does not include intracellular motifs tyrosinemia activation (as in the sequence TCR) or cysteine residues required for the binding of cytoplasmic tyrosinekinase Src family (June et al., 1994). However, some of the potential sites of glycosylation in the composition of the selected sequences conservative (Hutchcroft & Bierer, 1996). Intracellular catalytic activity and milkove interactions are often governed by way of differential protein phosphorylation, although the enzymes that would allow such activity receptor CD28, is not yet defined (Lu et al., 1992). Consensus YMXM motif found within the cytoplasmic domain, is the alleged site of the Src-homology phosphotyrosine binding (SH2 domain), which, in turn, the binding of phosphatidylinositol-3-kinase (PI3 kinase) (Prasad et al., 1995). Although this is only one of the possible signaling mechanisms for participation CD28 was shown that the activity of PI3 kinase does not correlate with the activity is using IL-2; but because the increased production of IL-2 is the primary consequence of the signaling activity of CD28, it appears that other mechanisms influence the activity arising from intracellular signalling processes (June et al., 1994).
Although the importance of these processes is not yet fully deciphered, costimulate CD28 leads to increased production of cytokines by T-cells. In CD28-positive T-cells activated by the action of antibodies to CD3 or PHA, anti-CD28 leads to the excess of initial RNA levels of several cytokines, including IL-1, IL-2, IL-3, IL-4, tumor necrosis factor (TNFα), lymphotoxin, γ-interferon and colony-stimulating factor granulocyte-monocyte (GM-CSF)and receptor of interleukin-2 (Lenschow et A1., 1996). Increasing the initial content of mRNA determined and increased levels of transcripts, and the intensification of transcription (Hutchcroft & Bierer, 1996).
Although costimulate CD28 was first described in clones of CD4-positive T cells (Martin et al., 1986), it is now known that CD28 is involved in the activation of many cell types. Costimulate this mechanism, as shown, regulates the subpopulations "naive" T cells CD4+production γ-interferon, cytokines Th1 response and production of IL-4, which is a cytokine Th2 dysbalance response (Seder et al., 1994). Co-stimulatory mechanism involving CD28 is also important for the activation of CTL CD8+although apparently the n is not necessary for the effector phase of cell killing involving CTL (Hodge et al., 1994). Interestingly, CD28 also presumably plays a role in the pathogenesis of HIV infection. In cultured lymphocytes, derived from some patients with a positive response to AIDS, the binding of CD28 monoclonal antibody may potentiate the replication of the HIV virus (Asjo et al., 1993).
In primates and rodents secondary signal produced by the binding of CD80 to CD28, clearly indicates its role in the initial activation of T cells (Aruffo & Seed, 1987). Recent data, however, suggest that the main consequence of such interaction can be aimed at ensuring proliferation through suppression of apoptosis (Lenschow et al., 1996). Resting T-cells, located in the G0the growth phase can be activated by formation of the TCR complex, but they are not able to proliferate or secrete IL-2 in the absence of CD28 binding: this effect is called anergy (Linsley et al., 1991a). Mature T cells can be activated exclusively by binding of the TCR with MHC molecules on the cell surface of the APC, but it ultimately leads to activation-induced cell death, i.e. apoptosis (Radvanyi et al., 1996). Although other secondary interactions (e.g., costimulate ICAM-1) can form an auxiliary signals for proliferation, it is assumed that CD28-mediated costimulation is unique, milling the IOM), preventing the subsequent occurrence of clonal anergy and apoptosis (Linsley et al., 1993a). It has been shown that CD28 can participate in the regulation of genes with known involvement in the protection of T-lymphocytes from apoptosis (Boise et al., 1995). Constant gain expressionit was found in T-cells, costimulatory binding to the CD28 receptor (Boise et al., 1995). It is believed that costimulate CD28 can stabilize mRNAwhich translates a protein that prevents apoptosis (Radvanyi et al., 1996).
Binding to CD28 has been shown to contribute to increased production of various cytokines by T-helper responses and the 1-St and 2-nd type (Lenschow et al., 1996). It is also assumed that this interaction is involved in the formation of specific types of T-helper cells. "Naive" CD4-positive T-lymphocytes have normal to form the Th1 phenotype, if they are activated in the absence of signaling pathways mediated by the binding of CD28/CD80 (Lenschow et al., 1996). It may be of indirect importance in the development of IL-4 induced by the addition of exogenous IL-2, while the role of CD28 signals in the development of IL-2 has been previously confirmed (Seder et al., 1994). Studies in mice, "off" gene CD28, further confirmed the role of this receptor in the differentiation of Th2 cells.
The mouse, which is homozygote and a null mutant for CD28, were obtained by Sarinana with co-authors with the aim of establishing how the animal adapts to infection in the absence of a secondary signal generated by CD28 (Shahinian et al., 1993). This gene was destroyed in embryonic stem cells by partial substitution of the second exon gene of resistance to neomycin (Shahinian et al., 1993). It was shown that mice, homozygous for the "off" a gene does not Express CD28 on their T-cells, whereas heterozygous individuals CD28 (-/+), as it was found, characterized by reduced surface expression of this receptor (Shahinian et al., 1993). The mitogen stimulation of T cells derived from homozygous off on CD28 mice, reduces the proliferation of T cells and production of cytokines that can only be partially restored by exogenous IL-2 (Shahinian et al., 1993). It was shown that purified T-cells are not activated by pectins in the absence of cells ARS (Unanue, 1984). On the material named "off" line, it was shown that the interaction of the CD28/CD80 necessary for the manifestation of the mitogenic activity of T-cell lectins (Shahinian et al., 1993). It was also found that this interaction is important for mediating the switch isotypes In cells in response to antigen (Shahinian et al., 1993). In contrast to the "off" CD80 mice, the functional role of CD28 can be installed with PR is the application of genetic engineering technologies "off genes. Of mice, "off" gene CTLA-4, has not yet been announced, but the mouse sverkhekspressiya CTLA-4 1g, have already been explored (Lane et al., 1994). As expected, the phenotypic characteristics of this line of mice with similar characteristics lines deficient for CD28 (Lane et al., 1994). Although isolated T cells produce normal amounts γ-interferon after stimulation revealed significantly fewer IL-4 (Ronchese et al., 1994). This leads to the inability of b cells to initiate or to maintain accurate humoral immune response (Ronschese et al., 1994). Although there are various potential ways of differentiation of Th1 and Th2, the interaction of the CD28/B7 clearly affects the differentiation of subpopulations of T-lymphocytes.
Stabilization of mRNA of interleukin-2 may play a key role in binding to CD28, but some other cytokines have been shown to directly or indirectly affect this interaction (Linsley et al., 1991a). The inflammatory mediators IL-1α, IL-6 and TNFα produced by populations of T-cell memory in response to signal molecules CD28, whereas in populations of naive cells is produced only IL-1α (Cerdan et al., 1991; van Kooten et al., 1991). The expression of IL-4 is also regulated with the participation of the signal mechanism CD28 (Seder et al., 1994). This interaction provides positive regulation of IL-5, IL-10 and IL-13, which are important mediators humoral the nogo response (deWaal Malefyt et al., 1993; Minty et al., 1993). In addition, colony-stimulating factors and growth factors including GM-CSF, CSF-1 and IL-3, and chemotactic factors, including IL-8, positively regulated by part of the signal generated by the CD28 receptor (Harlan et al., 1995).
Taking into account what has been discussed above probable use of CD80 in the induction of antitumor immunity, there are a number of other potential methods of clinical application of CD28 and CD80. Preventing the interaction between CD28 and CD80, as has been shown in a rodent model system, contributes to the prevention or treatment of several autoimmune diseases, the prevention of rejection of organs or manifestation of graft-versus-host and prevent the secretion of cytokines associated with sepsis (Harlan et al., 1995; Nickoloff et al., 1993; Thomas et al., 1994; Zhou et al., 1994). The addition of CTLA-4 Ig to block the interaction of the CD28/CD80 mice can prevent the symptoms of lupus-type mice lines NZB/NZW and partial protection from lethal EAE and fatal nephritis in rats (Harlan et al., 1995). Although this immunotherapy approach to autoimmune diseases to humans has not yet been applied, it was found that in medical practice in biopsies from patients with psoriasis and rheumatoid arthritis revealed the expression of CD80, whereas in normal biopsy samples such expression of no is (Nickoloff et al., 1993; Thomas et al., 1994). In transplants of bone marrow and organs in mice and in model experiments on humans in vitro addition of CTLA-4 Ig and preventing the interaction of CD28/B7 may cause at least partial protection against organ rejection, the response "TNX" or the induction of antigen-specific resistance (Harlan et al., 1995). Finally, secretion of cytokines and expression of sepsis, which can lead to septicemia and septic shock can be prevented in mice by in vivo injection of CTLA-4 Ig (Zhou et al., 1994). Manipulation by the interaction of the CD28/CD80 allow a deeper understanding of the processes of T-cell costimulation and allow to approach to the solution of various problems.
Materials and methods
The selection of the initial fragment of CD28
mRNA was extracted from peripheral blood lymphocytes of the animal NC, stimulated for 16 hours Con-And, using a reagent for RNA extraction RNAzolB (Biotexc, Houston, TX). The original cDNA was synthesized on the matrix selected RNA with reverse transcriptase (FROM), using as 3'-primer oligo-dT. Briefly, RNA and oligo-dT was heated to 75°With 3 minutes to remove secondary structures. Then he added, dNTP, buffer and distilled water and the resulting mixture is incubated for 1 hour at 42°C. After incubation, the sample was heated to 95°With 5 minutes to inactivate FROM Degenerate primers, derived from the consensus of the segments found in the published nucleotide sequences of CD28 human, mouse and rabbit (GenBank, Bethesda, MD), and then used for the initial amplification 673-nucleotide fragment encoding a large part of the open frame:
CD28-113: CAA CCT TAG CTG CAA GTA CAC (SEQ ID NO: 70)
CD28-768: GGC TTC TGG ATA ATA GGG GG (SEQ ID NO: 71).
PCR "hot start" based on the use of Taq-polimery was used for amplification product (5 minutes at 95°1 cycle; 30 seconds at 95°C, 30 seconds at 48°C and 45 seconds at 72°C - 30 cycles; 7 minutes at 72°s - 1 cycle). Then the obtained fragment was visualizable in 1%agarose gel and ligated into the composition of the cloning vector (InVitrogen, San Diego, CA) and sequenced as described above. Based on the cDNA sequence were obtained specific 3'primers are synthesized for use in the method of 5'-RACE:
CD28-190: CGG AGG TAG AAT TGC ACT GTC (SEQ ID NO: 72)
CD28-239: ATT TTG CAG AAG TAA ATA TCC (SEQ ID NO: 73).
The allocation of the 5'-segment
Modified by GIBCO method Protocol 5'-RACE (Gibco BRL, Gaithersburg, MD) was used to obtain the remaining 5'sequence of the molecule CD28 cats. RNA was extracted from RVMS, stimulated for 16 hours Con-A. Reverse gene-specific primer used for the synthesis of the first chain cDNA. RNA and the primer was heated to 75°5 minutes from sleduyushim the addition of other reagents. After denaturation, the mixture was cooled to 4°and was added to the reaction buffer, magnesium chloride, dNTP, dithiothreitol and reverse transcriptase SuperScript (Gibco BRL, Gaithersburg, MD). The mixture FROM incubated at 42°C for 30 minutes and then was heated to 70°C for 15 minutes to denature FROM. Then added Recusou mixture and the reaction was incubated at 55°C for 10 minutes to remove residual RNA and prevent incorrect fitting with the participation of terminal transferase (TdT). Then cDNA was purified using a centrifuge column GlassMax (Gibco BRL, Gaithersburg, MD) to remove not incorporated nucleotides and primers. Then the purified cDNA, elyuirovaniya with this column, was completed using TdT. TdT was used to add 20-30-nucleotide deoxycytidine "tail" to the cDNA. This enzyme was added to a mixture of purified cDNA, magnesium chloride, reaction buffer and dCTP after 3 minutes of cDNA denaturation at 95°C. the Reaction was incubated at 37°C for 10 minutes and the enzyme then iactiveaware by heating to 70°C for another 10 minutes. cDNA with attached "tail" amplified using Taq polymerase in PCR reaction with a "hot start" (5 minutes at 95°C; 30 seconds at 95°C, 30 seconds at 55°C and 45 seconds at 72°C - 35 cycles; 72°C for 7 minutes). The primers for this reaction showed the camping reverse primer, the corresponding 5'-site of primer to synthesize cDNA, and the anchor primer specific for dC-linker and consisting mainly of dG residues and a small number of dI. 1 μl of this reaction was diluted by adding 50 μl water and 5 μl of the resulting mixture is then used for "nested" PCR (5 min at 95°1 cycle; 30 seconds at 95°C, 30 seconds at 55°C and 45 seconds at 72°C - 30 cycles; with chimeric polymerase KlenTaq) with direct anchor primer dG/dI and additional top "nesting" gene-specific reverse primer. 30 μl of "nesting" the reaction was then visualizable 1.5%agarose gel and the exact fragment was cut out of the gel. cDNA was purified as described above using the gel dispenser and Amicon filter Micropure (Amicon, Beverly, MA). The purified cDNA sample sequenced by the method of sequencing end with a fluorescent label (Perkin Elmer, Norwalk, CN). On the material of completed fragments identified a consensus sequence. Based on this sequence was synthesized primer pair corresponding to a full coding frame CD28 gene cat:
Direct feCD28: CGC GGA TCC ACC GGT AGC ACA ATG ATC CTC AGG (SEQ ID NO: 13)
Reverse feCD28: CGC GGA TCC TCT GGA TAG GGG TCC ATG TCA G (SEQ ID NO: 14).
Using these primers the cDNA molecule comprising the full-size encoding section, amplified in the material cDNA synthesized n the RNA from RVMS, taken from animals EK and ED3 and stimulated by Con A. This derived from RVMS cDNA was obtained earlier and it was established that included RNA corresponding to this gene. In this PCR (5 min at 95°1 cycle; 30 seconds at 95°C, 30 seconds at 42°C and 45 seconds at 72°C - 30 cycles; 72°C for 7 minutes) used DNA polymerase KlenTaq, which reduces random errors, usually accompany the use of Taq polymerase, and as a result got a 754-BP fragment, which was cloned in the composition of the cloning vector and sequenced in accordance with previously described. As in the case of molecule CD80, each nucleotide was confirmed by at least three independently obtained the sequences of.
Degenerate primers selected according to the parameters of the consensus sites of the sequences of CD28 cDNA of mouse, human and rabbit were used in PCR with the successful receipt of the product, which covers almost the entire coding sequence of the cat. Due to the higher degree of conservatism inherent in the molecule CD28, in the initial amplification using the data of degenerate primers received virtual full-sized molecule. Unlike molecules CD80 cats, which was initially received only a small Central fragment in the coding frame KD is To CD28 not "enough" only 113 "extreme" 5'-nucleotides. This source fragment sequence showed 86%level of homology with the same sequence of man, 86%level of homology with the cDNA of rabbit and 79%homology with the coding sequence of the mouse.
The start codon ATG and an additional 110 nucleotides, as well as some 5'-flanking sequences were isolated using the method of 5'-RACE (Gibco, Gaithersburg, MA). In response to "attach tail" used cDNA obtained on the material RNA cells RVMS cats EC, stimulated by Con A. this material as a result of amplification with primer CD28-786 and anchor primer dG got a small distinct material.
Although not revealed distinct bands when amplification with a combination of primers dG/CD28-786 diluted cDNA from this reaction amplified using "nested" primers for CD28 - CD28-182 and CD28-239. A distinct band was present in the area of about 600 base pairs. This product was isolated from agarose gel and sequenced to confirm the presence of 5'-fragment, including the start codon and flanking sequence beyond.
Based on the nucleotide sequences of these products was formed direct primer, which included the start codon. This primer in combination with the 3'design was used for amplification of cDNA from extra is tov RNA cells RVMS, taken from animals EK and ED3 and stimulated by Con A, and received 754-nucleotide fragment.
At least two products from each animal is fully sequenced and each nucleotide was checked and confirmed by at least three independent just a few sequences. After this full-sized product sequenced included the cloning of THE vector to confirm the accuracy and reproducibility of the obtained product.
In the final 685-nucleotide fragment comprising the complete open frame, the start codon ATG is in position 1, the stop codon is at position 664-666, and 19 nucleotides comprise 3'-UTR. As in the case of molecule CD80 cats, the position of the start codon ATG confirmed by parameters of the sequencing reaction products of 5'-RACE (data not included).
Gene CD28 cats being sequenced, showed the highest level of total identity with sequences of rabbit and man. Homology with the cDNA was also significant, although the identity with the sequence of the chicken was expressed to a lesser extent and was comparable with levels of similarity to other genes that are compared in chicken and mammals (table 2).
Comparison of sequences of CD28 cats and CD28 mouse, human, chicken and rabbit
|The percentage of homology with the sequence cats|
Amino acid sequence was decoded by the nucleotide sequence as described above. The value of the decrypted identity of amino acid sequences with other published sequences was comparable to the identity at the level of nucleotide sequences. The signal peptide segment extends from start-methionine residue to 19 amino acids. It seems that, as in other cloned CD28 polypeptide, the molecule cats only extracellular variable immunoglobulinemia domain account for the remains of 19-153. Hydrophobic transmembrane domain takes the following 27 residues, and 41 comprise amino acid cytoplasmic tail. As the molecule CD28 human polypeptide cats includes five sites of potential N-glycosylation.
Comparing the decoded amino acid sequence CD28 cats and humans showed the presence of areas of homology with certain differences. Most of the changes occur in the transmembrane domain, a signal segment and the N-terminal domain. The highest level of homology is characteristic of the Central IgV-like domain and cytoplasmic tail.
Comparison of molecules CD28 cats with the decoded amino acid sequences of family members CD28/CTLA-4 human and mouse showed that, although the overall level of homology of the members of this group of proteins is only. 25%remain specific areas and amino acids. The MYPPPY motif remains the same in all members of this group. In the sequence of the cat assumes the presence of additional residues important for ensuring structural integrity, including a number of conservative cysteine residues.
The cytoplasmic domain of the molecule in SW characterized by conservatism moderate compared with other published sequences, especially sequences of mammals. It is assumed that different intracellular signaling mechanisms mediated by cross-linking of the extracellular segment of this receptor (Hutchcroft & Bierer, 1995).
Graphics hydrophilicity decoded amino acid sequence of CD28 cats when compared with the same charts polypeptide person additionally show the probability that each of these proteins character.iswhitespace structure. However, if there are substitutions of amino acids is, apparently, does not lead to significant changes in the hydrophilicity of a given molecule: this reflects mainly the homologous nature of these substitutions of amino acids. It should be noted that a very high level of similarity profiles hydrophilicity characterized transmembrane domain peptides of cats and humans, characterized by only 75%level of homology.
All sequences of the cloned molecules CD28 show the average level of evolutionary conservatism. It can be assumed that the involvement of this molecule in the activation and mediating T-cell immunity in a variety of higher vertebrates - from gallinaceous birds "through" rodents and predatory mammals to higher primates.
Comparison aminokislotnihyj sequences of each of these molecules indicates the average level of homology plots extracellular domain, which presumably involved in the binding of the ligand, as well as intracellular sites, presumably involved in the formation of intracellular signals. In General, the highest level of homology is found in the area surrounding the proposed binding site of the ligand - MYPPPY, is located in the composition of the IgV-like domain of the polypeptide cats, amino acids 118-123.
The estimated signal segment for the em site from the initial methionine until the 19th residue (Aruffo et al., 1987). The monomer CD28 is composed of only the extracellular variable immunoglobulinovogo domain, amino acids occupying 19-153 (Aruffo et al., 1987). Hydrophobic transmembrane domain is necessary for the next 27 residues, after which is a 41-amino acid cytoplasmic domain (Aruffo et al., 1987). Protein cat has 5 potential sites of N-glycosylation in identical positions in relation to what was found in the polypeptide person. Interestingly, the glycosylation site found in the residue 105 protein cat, is the motive NQS, while in the sequence it is NQT. This amino acid divergence further confirms that, despite changes in the sequences have a common structural features are preserved.
As might be expected based on the levels of homology shown these proteins, comparison charts hydrophilicity CD28 cats and humans indicates that these molecules are characterized, in principle, similar conformational parameters. However, it is also clear that in the case of replacement of amino acids resulting changes are homologous. Despite the fact that the transmembrane domain is the region of this molecule, characterized by the lowest level of conservatism in a simple preserving properties of hydrophobicity, cytoplasmic house the molecule CD28 cats compared with other published sequences conservative to moderate. Assumes the existence of a number of intracellular signaling mechanisms mediated by binding to this receptor, and, although the intracellular segment of CD28 polypeptide lacks catalytic activity, however, the binding of ligand leads to activation of intracellular effector molecules (Aruffo et al., 1987). There are four conservative residue tyrosine (position 173, 188, 191 and 200), for which it was assumed their phosphorylation (Lu et al., 1992). On the other hand, the motive MNM starting with 193-th amino acid molecules cats, is regarded as the site of the SH2 domain proteins in human and mouse (Prasad et al., 1995). Potential phosphorylation site with the participation of protein kinase-C remains in the residue serine-185, while threonine 202 may be a target for attack proliferatively activity serine-treoninove protein kinases Erk1 or Erk2 (Hutchcroft & Bierer, 1996). As discussed above, the signal function of the CD28 receptor is diverse, so it is no surprise that its cytoplasmic domain includes several potential sites of attack for signaling mediators.
The future use of molecules CD28 cats should include developing ways to detect surface expression of this receptor and control the expression of CD28 after virus infection, such as FIV. If this method can be merge the n with the existing methods of detecting mRNA, it can be obtained valuable information about the level of expression in the infection process. Subsequent communication parameters in the expression of CD28 during chronic FIV infection will allow us to examine the body of a cat as a representative model of HIV infection of humans, which will give more accurate data about infectious processes in both types of organisms.
Protein expression CD28/CD80
Despite the fact that communication in the immune system is largely mediated by "soluble" (remembrance) factors, the initiation of primary T-cell responses in primates and rodents has been found to depend on direct cell-cell contact (Mescher, 1992). Initially it was believed that this interaction involves only the interaction between the TCR on the surface of T cells and MHC molecule on the surface of antigen-presenting cells, but then it became clear that the link between the accessory molecules are also required for full activation of T cells (Schwartz, 1992). As discussed above, evidence has been obtained for the interaction between proteins CD28 and CD80 as mediators of such auxiliary signal (Linsley et al., 1991a).
Many important receptors and ligands in vertebrates belong to the immunoglobulin superfamily (Springer, 1990). These molecules are characterized by the presence immunoglo uliopilaskogu plot usually in the extracellular part of the molecule (Buck, 1992). Although the levels of conservatism varies, often it is limited to those residues that provide the spatial packing of protein for immunoglobulin type (Beale, 1985). Characteristic Ig-domain is the presence of two closely interacting antiparallel (β-chains connected by a loop, providing a conservative topology (Williams & Barclay, 1988). Although there are common features of the structure of members of this family, there is diversity in the parameters of the binding and signaling properties of some representatives of this family (Anderson et al., 1988).
As members of the IgSF family, and CD28, CD80 and to a certain extent similar to the structure of their extracellular domains. The CD28 has a single V-like domain, although it is expressed in the form of heterodimer associated disulfide "bridges" (Aruffo et al., 1987). The extracellular portion of the molecule CD80, however, includes V-and C-like immunoglobulin domains and is expressed as a monomer (Freedman et al., 1989). Since the members of the IgSF family are characterized by the General structural parameters, at least to a limited degree, you can transfer some of the patterns of spatial structure on related molecules that crystallize cannot (Bajorath et al., 1993). Although the crystallization of either CD28 or CD80 was not to implement the and, methods x-ray crystallography were investigated molecules CD2 (Driscoll et al., 1991) and CD8 (Leathy et al., 1992), characterized by similar extracellular domains, which helped to define some of the principles of the structures of related molecules of the IgSF family (Linsley et al., 1995a).
As discussed above, CD80 and CD86 show similar activity by binding to the receptors CD28 and CTLA-4. However, CD28 is less affinity receptor for these ligands, while CTLA-4 is more affine with respect to both of these molecules (Linsley et al., 1994a). Although the probable mechanism is known, it is unclear how low-affinity receptor, characterized by a high rate of cleavage of the ligand, which is typical for CD28, is able to provide the necessary co-stimulatory signal in the differentiation of T cells (Linsley et al., 1995a). It is assumed that binding of CD80 to CD28 on the surface of T cells can promote oligomerization of this receptor, which ensures the effectiveness of binding and generate a signal (Linsley et al., 1995a). It has been shown that CD28 evenly distributed on the surface of activated T-cells, i.e. presumably these molecules move into the membrane after loading T-cells (Damie et al., 1994). High concentration oligomerization CD28 will contribute to reassociate free CD28 in the field of intercellular contact and thereby provide the ü generating a signal, despite the rapid dissociation of the ligand (Linsley et al., 1995a). This process, called "mutual keperawanan", although it is not directly installed in the interaction of CD28/CD80, has been confirmed for other receptors that require similar initiating intercellular contact (Singer, 1992).
Although, as has been shown, the interaction of the CD28/CD80 is key in the development of T-cell immune response, are still questions to clarify the exact mechanism of this signaling mechanism (Linsley et al., 1993a). The existence of two receptor and two ligands in this interaction raises the question of the role of each in the activation of T cells (Linsley et al., 1992b). Although the receptor, CTLA-4 shows a stronger binding, it is expressed much later activation events, and although the signaling mechanisms associated with CD28, and has not been specified, is generated if the signal after binding of the ligand to the receptor, CTLA-4 (Linsley et al., A).
Materials and methods
The following primers were used for amplification of full coding framework genes CD28 and CD80 cats to embed it in expressing vectors:
Direct feCD80: CGC GGA TCC GCA CCA TGG GTC ACG CAG CAA AGT GGA AAA (SEQ ID NO: 11)
feCD80-960: CCT AGT AGA GAA GAG HUNDRED AAG AGG (SEQ ID NO: 12)
Direct feCD28: CGC GGA TCC ACC GGT AGC ACA ATG ATC CTC AGG (SEQ ID NO: 13)
Reverse feCD28: CGC GGA TCC TCT GGA TAG GGG TCC ATG TCA G (SEQ ID NO: 14).
Direct Prime time is for CD80 and both primer for CD28 were designed incorporating BamHI sites and appropriate linkers to facilitate embedding multiple cloning sites. 3'-Located BamHI site was introduced in the sequence of CD80 by splitting the cloning vector. Direct primers included Boxing Kozak and the start codon ATG of both genes. In each case, the primers used for amplification with the matrix, the full coding sequence of each gene, which were previously included in the composition of the cloning vector THAT described above. Approximately 10 ng of each plasmid was used in PCR with Taq-polymerase (5 minutes at 95°1 cycle; 30 seconds at 95°C, 30 seconds at 60°C and 45 seconds at 68°C - 30 cycles; 7 minutes at 68°s - 1 cycle). Amplificatoare products have visualizable by electrophoresis in agarose gel and then ligated into the composition of the cloning vector (InVitrogen, San Diego, CA) as described above. The ligation reaction was used to transform competent cells InvαF' and positive clones were skanirovali and selected as described above.
Cloning in plasmid pSI
For cloning in the composition of the vector pSI intended for transformation of cells COS-7, this plasmid was digested with restriction enzyme EcoRI and then the enzyme was removed using a centrifuge column Micropure EZ (Amicon, Beverly, MA). After removal of restrictase plasmid was treated with a mixture of phenol and chloroform to remove residual protein is precipitated with alcohol. Insertion was tsalala from 50 µg of purified reagents QIAGEN plasmid DNA (Qiagen, Chatsworth, CA) those clones that included the cloning vector with THE correct inserts, using EcoRI sites in the flanking insertion sequences of the vector. 100 µl of the split-off material was subjected to electrophoresis in 1.5%agarose gel and derived fragment was cut out of the gel. The insert was then purified from the agarose using the gel dispenser and filter Microcon (Amicon, Beverly, MA). Processing EcoRI-cleaved plasmid pSI alkaline phosphatase was used to reduce the likelihood of ligation of this vector on itself. By treatment for 1 hour at 37°0.1 units/μg of alkaline phosphatase, calf intestine (SFCT) was dephosphorylated the split ends of the vector. SFCT removed by heat denaturation at 65°C for 30 minutes, followed by purification on a centrifuge column Micropure EZ (Amicon, Beverly, MA). Insert ligated directly into the cut and dephosphorylated vector pSI over night at 16°using DNA ligase of phage T4. The molar ratio of the ligand and the vector was approximately 3:1 with a ratio of 0.05 µg insert CD28 or CD80 0.1 µg pSI. Then 1 µl of the ligation reaction was used to transform competent cells InvαF'. These cells were sown strips on LB plates containing 50 μg/ml ampicillin. lastine incubated over night at 37° And the next day the obtained colonies were perseval in 5 ml liquid LB medium containing 100 μg/ml ampicillin. After incubation over night at 37°rotating at 220 rpm plasmid DNA was extracted by the method of alkaline lysis, DNA was purified by extraction with phenol-chloroform and precipitated with two volumes of 95%ethanol. DNA was treated with RNase and then were digested with processing 10% restrictase EcoRI. The cleavage products were visualizable in 1%agarose gel to identify positive clones. Then plasmid DNA was extracted from overnight culture positive clone a volume of 5 ml using centrifugal QIAprep columns (Qiagen, Chatsworth, CA). Then, the nucleotide sequence of purified DNA was determined by the method of sequencing end with a fluorescent label using the internal 3'-primer that allows you to define the orientation of the insert in this plasmid. The location of this primer is that sequencing "crosses" the docking site of the vector and insert to ensure correct orientation. Then clone each gene in the plasmid with the correct orientation were grown in 100-ml cultures and the plasmids were extracted on maxipriestley QIAGEN column (Qiagen, Chatsworth, CA).
Cloning in SFV
To embed part of the SFV vector insert and the plasmid was treated basically the same education is. 100 µg of SFV vector was digested 120% restrictase BamHI for 1 hour at 37°C. the restriction enzyme was removed from the cleaved product by centrifugation through a filter Micropure EZ (Amicon, Beverly, MA). Then lamido processed SFCT. SFCT iactiveaware by heating, and then the plasmid was purified again on the filter Micropure EZ. The insert was extracted from the purified DNA cloning vector THAT by treatment with restriction enzyme BamHI. The insert was purified and ligated into the composition of the vector as described above. After transformation of competent cells InvαF' plasmid insert and its orientation was confirmed by sequencing method end with a fluorescent label. Large-scale production of plasmids was carried out on the material of the positive clone each gene.
Expression of protein with pSI
For the transformation of eukaryotic cells with plasmid pSI cells COS-7 were obtained from the American type culture collection (ATSS). Frozen block resuspendable in 15 ml of DMEM medium, supplemented with 10% fetal calf serum (TCP). The culture was then grown by a monolayer in flasks T-75. In the evening the day before the transfection, the cells were removed from flasks by trypsinization (0.25% in EDTA) with flushing of the FSB. The cells were then sown at about 20%level of confluence on 100-mm plates and left to grow to confluence approximately 50% until the next day. For each designation is Noah for transfection Cup 5 ml of DMEM-NuSerum (Collaborative Biomedical Products, Bedford, MA) was mixed with 0.2 ml of DEAE-dextran and chloroquine. Then to this mixture was added 10 μg/ml of purified plasmid pSI. Culture medium was aspirated from the cells and COS to these cells was added a solution of DMEM-NuSerum, DEAE-dextran, chloroquine and DNA. The culture was incubated for 3.5 hours in an incubator under 5% carbon dioxide with subsequent removal of culture medium and replace it with 5 ml of 10% DMSO in the FSB. After 2 minutes the solution was aspirated and the cells were cultured overnight in 5 ml of DMEM medium with 10% PTS. The next day the cells were poured into 100-mm culture Cup. After 3 days the medium was aspirated and the transformed cells were selected using the FSB with 0.5 µm EDTA. The mixture FSB/EDTA was added to the cells, which are then incubated for 15 minutes at 37°s Supernatant were selected and combined with subsequent leaching in the FSB. Supernatant and washing then centrifuged. The resulting clot resuspendable in DMEM/TCP and counted cells COS.
Expression of proteins with SFV
Transfection of the vector SFV carried out in relation to renal cells in newborn hamsters (KSS). 30 μg of the purified plasmids were digested with restriction enzyme SpeI for 1 hour at 37°C. Then, the restriction enzyme was removed on the filter Micropure EZ (Amicon, Beverly, MA) and DNA was besieged by 2.5 volumes of 95%ethanol. Then with 1.5 μg of plasmid was used as matrix S6-mediated transcription in vitro. Briefly, DNA was incubated for 1 hour at 37°C: transcription buffer, 100 mm dithiothreitol, 10 mm G(5')PPP(5')G, NTP mixture, water, RNasin and 60 units of RNA polymerase Sp6. After the transcription reaction was divided into aliquots and the sample was visualizable in 1%agarose gel. 45 µl transcription reaction was used for transfection of cells KSS at the level of the confluence of approximately 80% in flasks T-75. Culture medium GMEM supplemented with 10% PTS, was aspirated from the cells and replaced with medium Opti-MEM. After 2 minutes incubation, the medium was replaced with medium Opti-MEM, 9 μg/ml lipofectin and transcribed RNA. Cultures were incubated for 2 hours at 37°With 5% CO2with frequent hand shaking. After 2 hours, the culture medium was removed and replaced with GMEM + 10% PTS. Cultures were incubated for 7-9 hours and cells were then separated by treatment with trypsin.
Cloning into the vector pQE
The bacterial expression vector pQE was also designed with the inclusion of genes CD80 and CD28 cats. Split and processed SFCT plasmid pQE were prepared and purified as described above and ligated using DNA ligase T4 in a molar ratio of insert:plasmid=4:1 with 50 ng of purified gel CD28 or CD80. Reaction of leading incubated for 16 hours at 16°C. Then, 2 μl of this reaction was used to transform competent who's cells INVα F'. Selected positive colonies and the correct orientation of the insert was confirmed by direct sequencing. Conducted large-scale obtaining purified plasmids and obtained was used to transform cells M15 pREP4, which made competent for transformation by treatment with chloride of rubidium. Transformed cells were cultured on LB plates with a content of 50 μg/ml kanamycin and ampicillin order to ensure that both plasmid - pQE and plasmid-assistant pREP4 - remained in these colonies. Positive colonies were then subjected to screening by minipreparation with alkaline lysis and cleavage by the restriction enzyme BamHI. Colonies with proven inserts froze in the mother solution, 50% glycerol for further use.
Tests for binding against transfected cells expressing CD80 cats and CD28 cats, conducted in accordance with the Protocol described by Linsley et al., 1994a. 1 day after transfection cells COS-7 expressing CD28, were taken from the flasks T-75 by treatment with trypsin in EDTA. These cells were left to attach to the wells of 24-well plates at a concentration of 105cells in 1 ml After 2 days, cells COS-7, transfetsirovannyh feCDSO/pSI, were taken from the flasks T-75 using the FSB with 0.5 µm EDTA. These cells were then fluorescently labeled with ISOE what Itanium solution of 5 μm Calcein AM (Molecular Probes, Eugene, OR) in sterile FSB with 1% bovine serum albumin (BSA) for 30 minutes at 37° (Akeson & Woods, 1993). Transfetsirovannyh "dummy" cells COS-7 marked the same way. Labeled cells are then washed three times with DMEM containing 10% PTS, in order to remove the non-aligned labels, counted them and was added directly to the monolayer. These two cell populations were to interact with each other for 1 hour at 37°C. Adherent cells were collected by neat triple rinsing the monolayer with DMEM with addition of 10% PTS. After washing, the level of fluorescence of each well was quantified using a tablet microfluorimetry. The level of fluorescence of the wells containing transfetsirovannyh cell population, compared with wells in which cells expressing CD80, were added to the cells COS-7, transfitsirovannykh only the plasmid pSI.
Tests on competitive binding using chimeric proteins, CTLA-4/Ig and CD80/Ig (courtesy of P.Linsley, Bristol-Meyers Squibb) for the suppression of intercellular interactions were built in such a way as to demonstrate the specificity of this interaction. After labelling callainos, but before adding to the monolayer of cells expressing CD80, Chimera, CTLA-4/Ig in DMEM/title in a concentration of 1 μg/ml were incubated with labeled is passed transfitsirovannykh cells within 30 minutes. Cells are washed twice in DMEM/title and was added to the monolayer. On the other hand, the cell monolayer expressing CD28, were incubated for 30 minutes with Chimera CD80/Ig at a concentration of 1 μg/ml in DMEM/title and then washed, after which was added fluorescently labeled cells expressing CD80. Inhibition of binding of chimeric proteins was assessed by comparing the levels of fluorescence in these holes with binding parameters in the wells containing no competitive factors.
PCR with repertorium
Also transfetsirovannyh COS cells was assessed by transcription of mRNA by PCR with repertorium. 3 days later, RNA was extracted from cells transfected with a plasmid carrying the insert feCD28, feCD80 or without inserts. RNA was treated with Dnazol free from RNase, in order to remove possible contaminating DNA. Then, 0.5 μg of RNA was subjected to reverse transcription to obtain cDNA using primer oligo-dT and reverse transcriptase of the virus MuMLV. Each sample cDNA is then amplified using sets of primers that are specific for CD28, CD80 and G3PDH in the following cycles: 5 minutes at 95°1 cycle; 30 seconds at 95°C, 30 seconds at 55°C, 30 seconds at 72°C - 30 cycles; 5 minutes at 72°s - 1 cycle. Then 20 µl of each reaction was visualizable in 1%agarose gel.
The genes of the feline CD28 and CD80 were among the successfully built the "Trebilcock" expressing vectors (pSI, SFV and pQE). After ligation into the corresponding vectors of these genes were used to transform competent cells INVαF'.
Tests for linkage was performed in order to show the possibility of the expression of functional protein. Initial tests were carried out to determine the relationship between the binding of cells COS-7, transfected with CD28 and CD80, and cells COS-7, transfitsirovannykh CD28 and "phony". The level of fluorescence in the wells, which were added fluorescently labeled nalivshiesya transfetsirovannyh CD80 cells was higher compared to control wells for two initial dilutions. This interaction was dependent on dose and after two initial dilution levels of fluorescence in the wells in which the fused cells Express a surface protein, remained the same as in the controls transfected with the "phony".
In order to show the suppression of this interaction, the lines transfected cells before mixing incubated with soluble (remembrance) receptors. At concentrations of 5×1051×105cell fluorescence of the wells containing the COS cells expressing CD28 and CD80, is similar to that observed in the previous experiment. In the wells in which the fused cells before mixing was plaincourault with contracept the rum CD80/Ig, save fluorescence was comparable with that detected in the wells with the control (transfitsirovannykh "phony") cells. However, when COS cells, transfetsirovannyh pSI with CD80, incubated with soluble CTLA-4 in front of their interaction with cells, transfitsirovannykh pSI with CD28, fluorescence is not fully suppressed. Although these levels were not as significant as those that were detected in eingeborenen group, however, they were clearly more than in the control and the other experimental group.
PCR with repertorium conducted on the material of RNA derived from cells COS-7, transformed designs pSI-CD28, pSI-CD80 and "phony", in order to detect the presence of mRNA specific for each gene in this cell line. Gene glyceraldehyde-3-phosphate dehydrogenase (G3PDH) amplified in each group to show the integrity of the RNA as a positive control in the "empty" transformation. Cells COS-7, transfetsirovannyh pSI-CDSO, expressed CD80 mRNA and G3PDH mRNA, whereas cells COS-7, transfetsirovannyh pSI-CD28, expressed genes CD28 and G3PDH. Cells transfetsirovannyh "phony", expressed only G3PDH.
The lack of suitable antibodies explains that to detect the expression of the peptide could not be conducted a direct test. Available on the commercial the th basis antibodies specific CD28 and CD80 man, were tested on the material isolated lymphocytes to establish their possible cross-reactivity. Analysis of cells by FACS method with the specified antibodies using PCR to detect the expression of mRNA for both surface proteins, have been unsuccessful. This, combined with evidence that these antibodies do not recognize surface expression in cells transfected with the constructs pSI, allowed us to conclude that these antibodies cross (heterologous) reactivity do not possess. But cross-activity of antibodies to CD80 and not expected. Limited homology CD80 humans and cats should limit the potential for cross-activity of monoclonal antibodies specific for them. However, it was very surprising that antibodies specific against human CD28, were not cross-reactive. Although in this case the level of conservatism between the cloned molecules CD28 more substantial, made commercially available antibodies to CD28 man who would cross-react with the same protein mouse, were not found. As in the case of CD80 antibody to human testing of monoclonal antibodies to CD28 person have failed. Therefore, it was necessary to develop a test that would be phospholipiddependent not only the probability of expression of these proteins, but also showed their functionality and interoperability.
cDNA CD80 and CD28 cats were successfully integrated into the group expressing vectors. While the vector pSI meets all the requirements necessary for the implementation of test linking, additional vectors, could facilitate future expression of these proteins.
After transfection, the expression of mRNA of CD28 and CD80 lines of transformed cells COS-7 was confirmed using PCR with repertorium. Processing of RNA Dnazol before PCR reduced the possibility of contamination of genomic or plasmid DNA. Furthermore, it is not expected that cells COS-7 will be the source to Express any of the ligands, which was then confirmed by the absence of mRNA for any of these surface proteins in control, transfetsirovannyh "empty" vectors. The accurate amplification of mRNA on the material of RNA extracted from transfected cells, apparently reflects the fact that cells of the role of matrix performs vector pSI and that of mRNAs encoded by this plasmid, indeed transcribed.
Tested for binding was modeled in accordance with the tests that were carried out P.Linsley, demonstrated similar activity by binding to CD80 and human CD28 (Linsley et al., A). The modified test format used for th is would show, that surface downregulation of CD28 cats is associated with downregulation of surface CD80 cats and that this interaction can be suppressed by soluble receptor. The level of binding may be determined by the conservation of fluorescently labeled cells in a particular hole. Through the use of automated fluorescent flatbed scanner was not necessary in lizirovania cells before measurement of fluorescence.
In the initial test found that the preservation of fluorescently labeled COS cells, transfected with CD80-pSI, was more intense in the hole with merged cells which have transfusional CD28-pSI, compared with wells containing merged cells subjected to "empty" transformation. Control cells, i.e. cells COS transformed a blank, i.e. the vector pSI with no insert, confirmed that neither the presence of the vector (i.e. the process of transfection per se)or adhesive properties of the cells does not cause adhesion (merge) cells, mediated by the interaction between surface downregulation of CD80 and CD28. At an initial dilution of 1 million cells, the level of fluorescence in the wells in which cells expressed CD28, was approximately 5 times higher in comparison with the holes in the cells, transfitsirovannykh "phony", made fluorescently polcann the e cells, transfetsirovannyh CD80. At a concentration of 5×105cells the level of fluorescence was sharply decreased due to the decrease in the number of cells, but the level was still significantly higher fluorescence in control. At a concentration of 1×105cells the difference between experiment and control are statistically unreliable, and if 1×104cells, these levels are almost identical. This test shows that the interaction occurs between COS cells, transfitsirovannykh CD80, and COS cells, transfitsirovannykh CD28, which leads to preservation in this hole fluorescently labeled cells transfected with CD80. However, when fused cells do not Express surface-cellular proteins, cells, transfetsirovannyh CD80, are removed by thorough washing. This effect may be titrated, and at a concentration of 1×104cells the level of fluorescence in the wells of a virtual identical. To confirm that the interaction took place, soluble receptors have been made to suppress the interaction of CD28/CD80.
In the second test used the inclusion of soluble forms of the receptors for each of the peptides to inhibit the interaction between the bonding partners. Soluble receptor for CD80 - huCTLA-4Ig and CD28 - huCD80-Ig, were incubated with the appropriate transfitsirovannykh COS cells, e is pressurewise receptor, associated with each of these molecules, with subsequent mixing of the two cells types. Although the soluble proteins was not from a cat, and suggested that because of a certain level of conservatism found between the putative binding sites in the molecules of humans and cats, this will be enough to achieve cross-reactivity. Moreover, it is known that ligands person associated with specific receptors in the mouse (P.Linsley, mouth. MSG.). Based on the number of cells needed to conduct this test, it was not possible to test with 1×106cells. Initial concentration was 5×105cells, despite the fact that on their own cells, transfetsirovannyh CD28/CD80, characterized by an average level of fluorescence, similar to the level that was determined in the previous experiment. It is not clear why the merged cells, proinsurance with soluble receptor for CD80, give a fluorescent signal that is close to that of control "empty" transfection, while nalivshiesya fluorescently labeled cells, transfetsirovannyh CD80, proinsurance with soluble CTLA-4, are 2-3 times higher levels of fluorescence. Despite the differences, caused by a type of soluble receptor, there was a distinct decrease in the level of fluorescence in the wells, in to is that the receptors were absent. This interaction identified in the previous test, can be suppressed by making the corresponding soluble receptor before mixing cells.
Although monoclonal antibodies specific against the surface of cellular proteins, in principle, appear preferable for tests of this type, in the absence of the necessary reagents described test is an effective approach, which can be demonstrated expression of functional contraceptor. Data source test linking in combination with data from competitive test confirm that you have been selected cDNA functional CD80 and CD28 cats, as well as what is expressed in the form of mRNA proteins to communicate effectively. Although the applicability of such a test on the binding is limited, it remains an effective system for establishing the likelihood of functional surface expression and interaction.
By definition, Lviv viruses are strictly intracellular and potentially pathogenic elements infectious phase, (1) showing only one type of nucleic acid, (2) breeding in the form of his genetic material, (3) unable to grow and pass the state of division, and (4) deprived the system of Lippman" (Lwoff, 1957). Natural viruses are acellular body what smame, the genome of which is in the form of RNA or DNA controls the subsequent synthesis of viral particles to infect a host cell (Luria & Darnell, 1968). Viral diseases represent an interesting system that can be demonstrated practical application of the signal complex B7/CD28. The infection which is the retroviruses HIV in humans and FIV (feline immunodeficiency virus) in cats causes disruption of the normal functioning of the immune system, which is presumably due to the elimination of T-cells and CD4+(Fauci et al., 1984; Pedersen et al., 1987). It is believed that signaling complex CD28/CD80 plays an important role in this disease and that by manipulating the expression of the receptors can aggravate the infection (Harlan et al., 1995).
FIV is a very important clinical problem for cats, as it causes a number of clinical and subclinical symptoms that are very similar to the HIV-infected person (Pedersen et al., 1987). Accumulating information about FIV accordance called disease animal model of AIDS person is becoming more and more obvious, i.e. it is not associated with Primate model that most closely mimics the development of this disease in humans (Siebelink, 1990). Molecular, biological and pathological similarities also confirms that most of the information is received in HIV research, can contribute to a better understanding of FIV infection in cats.
First, HIV infection is manifested in non-persistent lymphopenia with the development mononucleotides syndrome almost simultaneously with seroconversion (Clark et al., 1991). Is short-term decrease in the number of T cells CD4+and expansion of T cells CD8+that leads to the decrease of the ratio of CD4:CD8, and in the next asymptomatic phase of the disease can occur further reduction of this ratio (Cooper et al., 1984). With the appearance of AIDS-related symptoms population of T cells CD4+dramatically depleted, and as the disease progress and final stage is sharply reduced and the total number of lymphocytes (Fauci et al., 1984). Despite the fact that the original lymphopenia, apparently, due to shifts in populations of cells of the immune system induced by corticosteroids, as observed in other viral infections, further loss of T-cells and CD4+and expansion of T cells CD8+, thought to be related to the replication of the virus and pathogenic processes (Fauci & Dale, 1975; Fauci et al., 1984). The formation of informative model systems is a key step in further understanding the mechanisms of infection caused by viruses and diseases.
FIV, which is a T-lymphotropic retrovirus that was first described in a population of domestic cats in the alifornia, which was repeated frequently occurring chronic infection (Pedersen et al., 1987). Although the disease manifests itself similar to the HIV-infected person way and taxonomically viruses are relatively close, antigenic properties FIV different from the causative agent of AIDS person (Siebelink et al., 1990). Transmission of the pathogen occurs through the exchange of body fluids like HIV, but, unlike HIV, which is the predominant sexual transmission, it is assumed that FIV in most cases are transmitted through the saliva when it bites (Yamamoto et al., 1989). Despite the different routes of transmission, final immunodeficiency syndrome is one of the best models related diseases (Siebelink et al., 1990).
Clinical development of FIV is similar to HIV-infection: in particular, the disease is divided into five clinical phases. The initial phase is characterized by fever, malaise and lymphadenopathy, and then after infection comes a long asymptomatic phase, passing in three of the final phase, in which there is weight loss and there are multiple secondary and opportunistic infections (Deutsch et al., 1994). Although it is unclear whether the mechanism of cell infection the same FIV is trophic for CD4-positive and CD8-positive T cells (Brown et al., 1991). Infected with this virus animal is observed decreased activity of T-cells and CD4 +apparently in the education of their entities and their lysis (Siebelink et al., 1990). The onset of the final phase of infection coincides with a significant loss of T-cells and CD4+and the decrease of the ratio of CD4:CD8 (Novotney et al., 1990). Although diseases caused by viruses, FIV and HIV, can oposredovanie different mechanisms to reduce the number of T cells CD4+final phenotype and the nature of damage to the immune system obviously show up in a similar manner.
Although it is clear that infection of T cells CD4+the HIV virus affects the normal development of the immune response, the exact mechanism leading to immunodeficiency, has not been finally determined. In the later phases of the infection processes, leading to a reduction in the number of T cells CD4+not set (Connor et al., 1993). Although HIV infection has been demonstrated formation of entities, induction of apoptosis and elimination involving CTL as factors suppressing T-cells (Schattner & Laurence, 1994; Fouchier et al., 1996), also assumed the existence of a mechanism associated with CD28 (Haffar et al., 1995). As shown, the lines infected T cells under the influence of stimulation alloantigens reduced expression of CD28 on translational and transcriptional levels (Haffar et al., 1995). As discussed above, cross-linking the receptor CD28 is a key signal for RAZVITIJA-cell response (Linsley et al., 1991a). If HIV infection leads to the suppression of surface expression of CD28, the infected T-cells that recognize present antigen more likely to enter into apoptosis than fully activated (Schattner & Laurence, 1994). Although apoptosis is a normal mechanism of death of HIV-infected cells, this mechanism may provide an additional contribution to what is happening in this case, the elimination of T-cells (Brinchmann et al., 1994).
It was stated on the link CD8-positive CTL with the development of long-term viability for HIV infection, with a high level of lymphocytes CTL is associated with long-term suppression of HIV in infected individuals (Landay et al., 1994). In contrast, humoral immunity not only is generally ineffective in the control caused by lentiviruses diseases, but, as has been shown, the antibody may actually exacerbate the disease (Lombardi et al., 1994; Siebelink et al., 1995). The onset of the final clinical phase of HIV infection and associated immune deficiency in most patients is correlated with the switching of the cellular immune response (1st type) on the humoral response (2nd type) (Schattner & Laurence, 1994). This is consistent with the observations, according to which the transition from a healthy state to AIDS is associated with a decrease in antiviral activity, provide CTL CD8+(Lewis et al., 1994). Expr the Russia CD28 on the surface of the CTL CD8 +also seen as related to their antiviral activity, despite the fact that a strong CTL-mediated antiviral activity associated with the expression of CD28 CD8-positive population of lymphocytes in infected patients (Landay et al., 1993).
Surface expression of CD28, although it was assumed the function of a mediator of resistance to HIV-exposed negative effect in the presence of HIV in infected and uninfected T-cells (Caruso et al., 1994). Since asymptomatic phase of HIV infection, there is a decrease in the share of T-cells and CD4+and CD8+bearing CD28 (Lewis et al., 1994). It is assumed that this may be the cause of impaired secretion of cytokines detected in the early phases of infection (Caruso et al., 1994), as modified responses with the participation of CD8-positive T cells in the late phase (Zanussi et al., 1996). In HIV-infected patients decreased proliferation of T-cells CD8+in the early phase of the disease, as expected, is associated with negative regulation of CD28, because only expressing CD28 T cells CD8+proliferate in response to IL-2 (Brinchmann et al., 1994). Unfortunately, those infected T cells CD28-/CD8+can make up to 75% CD8-positive population, while in healthy individuals they represent only 25% of this population (Saukkonen et al., 1993). Thus, although the population CD8 +may remain normal in infected individuals, the effectiveness of this population on its ability to develop effective antiviral immune response may be compromised even in the initial phase of the disease (Caruso et al., 1994).
Also studies have shown that the transmission of the signal from CD28 may be involved in the activity smogó virus (Asjo et al., 1993; Smithgall et al., 1995). Costimulate HIV-infected T cells CD4+peripheral blood with the participation of anti-CD3 and anti-CD28 leads to intensification of the virus compared with stimulation only anti-D3 (Smithgall et al., 1995). This effect can be overcome by making CTLA-4 Ig, as the soluble form of the receptor that is specific for CD80, and to a lesser extent by the introduction of anti-IL2 (Smithgall et al., 1995). In another study, infected T-cells and CD4+in 40% of patients it was found that the binding only CD28 leads to positive regulation of viral replication in the absence of the need for any additional incentives (Asjo et al., 1993).
Pretreatment populations lymphocyte surface glycoprotein BEACH - gp120 - causes negative regulation of CD80 on the surface of antigen presenting cells (Chirmule, 1995). Although the expression of CD28 on T-cells is reduced in HIV infection, these cells the expression of CD80 intensified (Haffar et al., 1993). This is the item of ideologemes mechanism, by which infection can be transmitted to uninfected T-cells, since the interaction between CD28 on the surface of uninfected T cells with CD80 on infected T-cells may contribute to intercellular contact, providing transfer of the virus (Haffar et al., 1993).
While the role of CD28 in HIV infection was investigated to establish the value of this surface protein for FIV so far failed. If the cats can be obtained the results similar to those that have been identified in humans, it will serve as an additional confirmation of the applicability of cats as a model for retroviral infection.
Materials and methods
Infection in vivo
Three adults free from specific pathogen of cats infected intravenously 1×105TCID50virus FIV strain Maryland. Two similar cat was the control, which were injected with not containing virus serum. Blood samples were taken directly before infection and then weekly for 7 weeks. In the first week after infection of cats were examined twice a day in order to ensure that no original response to infection. After development of the disease, animals were monitored daily. During the acute clinical phase of the disease every 5-10 ml of blood was collected for CBC (complete blood count) and allocation RVMS. SVS included p is dscat cell types fast stained smears of blood (Jorgensen Lab., Loveland, CO).
Cells RWMS was isolated from blood by separation in gradient of Histopaque (Sigma, St. Louis, MO). After the initial flush solution Olivera about 5×105cells were collected and divided between the 5 holes 48 hole of the tablet. Cells resuspendable in 500 μl of complete RPMI medium and then labeled using antibodies specific for either CD4 or CD8. After 1 hour incubation at room temperature when used carefully, swing the cells were twice washed FSB. After washing was added to the second antibody is goat antimachine IgG (H+L)labeled with FITC (KP&L, Gaithersburg, MD) in a concentration of 1:500 and incubated at room temperature for 1 hour with careful swing. The cells are then washed three times FSS and fixed with 3.7%formalin. Fluorescently labeled population was then subjected to quantitative analysis by flow cytophotometry FACSCalibur.
The remaining RVS washed with an additional 10 ml of Alsever. After centrifugation the supernatant was removed and for carrying out extraction of RNA was added to 1 ml of ULTRASPEC (Biotexc, Houston, TX). RNA was purified and precipitiously as described above. Then spectrometrically a concentrations were determined by absorption at 260 nm. Then RNA resuspendable in 50 ál DEPC-treated water and frozen to -70°for later use.
Semiquantitative PCR is reverse
Before carrying out PCR amplification of the sample RNA 60 ml of blood was collected from umarsultanova animal. RVMS were isolated as described above. Cells were counted in hemocytometer and shared between 4 flasks at a concentration of 5×105cells in 1 ml of Cells stimulated by concanavalin A. immunology for 0, 8, 16 and 24 hours, followed by centrifugation and extraction of RNA from the cell clot using ULTRASPEC as described above. PCR with repertorium conducted on the material of 1.5 μg RNA, transcribed in cDNA using reverse transcriptase and MMLV reverse primer oligo-dT. A mixture of RNA, primers, oligo-dT and DEPC-treated distilled water, incubated for 5 minutes at 70°With destruction of the secondary structure of RNA and ensure the annealing of the primer. Then added transcription buffer, MgCl2, dNTP and DTT, and the mixture incubated at 42°C for 2 minutes. Then added 1 μl reverse transcriptase reaction was left for half an hour. Then 4 μl of 25 µl obretenitsa reaction was added to each of 3 tubes for amplification CD80 and three tubes for amplification CD28. Then cDNA was added to a mixture of PCR buffer 10×, dNTP and primers specific for CD28 or CD80. The primers for CD80 were as follows:
direct primer B7-S220: CAT GTC TGG CAA AGT ACA AG (SEQ ID NO: 74),
reverse the law the measures V7-284: TTA TAC TAG GGA CAG GGA AG (SEQ ID NO: 75),
while the primers for CD28 were as follows:
direct CD28 start CGC GGA TCC ACC GGT AGC ACA ATG ATC CTC AGG (SEQ ID NO: 13),
reverse CD28-239: ATT TTG CAG AAG TAA ATA TCC (SEQ ID NO: 73).
Then three tubes for each product were incubated at 95°C for 5 minutes and then each tube was added 0.25 ál Taq polymerase in 10 μl of water. Reactions included in this temperature cycle: 95°C - 30 seconds, 55°C - 30 seconds, 72°C - 30 seconds. One tube was removed after 20, 25 and 30 cycles, respectively. 20 µl of each reaction was visualizable in 1%agarose gel. Gels were photographed and determined the number of cycles in which appeared the desired product. After these preliminary experiments, RNA, previously extracted from the blood of patients and healthy animals, amplified in a similar Protocol.
Infection in vitro
Line T-cells infected with FIV in vitro, stimulated Con-And for 16 hours or not stimulated at all, then the expression of CD28 and CD80 was estimated using semi-quantitative PCR with repertorium. Cell line FETJ was a mixed population of T-lymphocytes and grew in the absence of IL-2 in culture medium. Independent subpopulations of these cells were subjected to infection by the virus FIV strains Maryland and Petaluma. About 2×108normal, Petaluma-infected and Maryland-infected cleto is FETJ stimulated for 16 hours Con-A (8 µg/ml) or not stimulated at all. RNA was extracted from these cells after incubation with ULTRASPEC reagent (Biotexc, Houston, TX) and purified as described previously.
Sit-5.4 - line T-cells derived from cats from the colony, characterized multiple FIV-infection, although this line was not chronically infected. Approximately 2×108cells sit-5.4 was palletizable by centrifugation and resuspendable in 5 ml concentrated on FIV the supernatant. Cells at this concentration were incubated for half an hour in an incubator with 5% CO2at 37°With further increase of concentration up to about 5×105and cultivation within 24 hours. After this normal and infected cells sit-5.4 stimulated with Con-A (8 µg/ml) for 16 hours or not stimulated at all. RNA was extracted using ULTRASPEC reagent (Biotexc, Houston, TX) and purified as described above.
For analysis Northern blotting RNA was determined spectrophotometrically at 260 nm. 15 μg of each sample RNA was concentrated to 3 mg/ml and resuspendable in 3 volumes of the boot buffer. Then the samples were heated to 70°C for 15 minutes to denature the RNA and the destruction of its secondary structure. Samples of 20 μl were loaded in a 1%denaturing agarose gel and subjected to electrotorture voltage of 70 V for 2.5 hours until as edge coloring bromophenol blue reaches 2 cm above the bottom edge of the gel. Then the RNA was transferred from the gel onto Genescreen nylon membrane (Dupont NEN, Boston, MA) by capillary effect (blotting). RNA was fixed to the membrane by treatment with low-intensity UV light for 3 minutes and then the track was visualizable clarity ribosomal bands by means of UV-shielding.
The probe specific for CD28 cats, was constructed using labeling of cDNA with random primers. Full-sized molecule CD28 cut from the cloning vector of THE flanking EcoRI sites. The resulting fragment was purified by gel electrophoresis and extracted from the agarose by gel-spray Amicon (Amicon, Beverly, MA). Then 25 ng of purified product were incubated together with random decanucleotide for 5 minutes at 95°With destruction of secondary structures (Ambion, Austin, TX). Then the reaction was instantly frozen in liquid nitrogen, and to the mixture was added dNTP (without dATP) and labeled nucleotide32P-αdATP. After incubation for 1 minute at 37°added 1 μl of fragment maple and the resulting mixture is incubated for 30 minutes. The reaction was stopped by adding 1 μl of 0.5 M EDTA and then purified on a rotating column of Sephadex G-50 (Sigma, St. Louis, MO) to remove neuklyuzhego radioactively marked well is leotide. 1 µl of the reaction was diluted in 1 ml of scintillation fluid and the activity of the probe was determined using a scintillation counter. The blots were prehybridization for 15 minutes at 65°With 5 ml of reagent Rapid-Hyb (Amersham Life Science, Cleveland, OH). 5 µl of the probe at a concentration of 3-5 million pulse/min 1 ál was added to each blot and incubated with rotation for 1.5 hours at 65°C. Then, the probe was removed and the blots washed twice in 1% SSC + 0.1% of SDS at room temperature for 15 minutes. Then the blots were scanned with a Geiger counter and if necessary, re-washed at 65°C. the Results of labeling was assessed quantitatively using the scanner Betagen. Conducted final wash at 65°C for 15 minutes and the blots were placed on film for 16-24 hours at -70°using intensifying screen. Autoradiogram were quantitatively evaluated densitometric. To confirm the integrity of the RNA and its concentration used is specific for G3PDH probe (kindly provided by Prof. J.Piedrahita, Texas A&M University), which were labeled and hybridized in the same way.
Semi-quantitative PCR with reverse on the material in vitro infected cells
Further analysis of the presence of CD28 was used semi-quantitative PCR. As described above, the concentration of extracted RNA was assessed by spectrophotometric what about the reading at 260 nm. 2 µg (final volume 25 μl) was transcribable into cDNA using primer oligo-dT and reverse transcriptase MMLV as described above. Then a 3.5 ál obretenitsa reaction was transferred into 7 tubes for PCR. Three test tubes amplified using primers specific for CD80, three test tubes using primers specific for CD28, and the remaining test tube with primers specific G3PDH:
direct G3PDH: CCT TCA TTG ACC TCA ACT ASA T (SEQ ID NO: 76),
reverse G3PDH: CCA AAG TTG TCA TGG ATG ACC (SEQ ID NO: 77).
As described above, the tubes were removed from the reaction after 20, 25 and 30 cycles. 20 µl of each sample was visualizable in 1%agarose gel. In addition, similarly to the material of the cell lines FETJ and sit-5.4 testing the presence of some other T-cell cytokines. Specific primers were as follows:
direct IL-2: CAA CCC CAA CTC ACT CAG GAT G (SEQ ID NO: 78),
reverse IL-2: GGT CAG CGT TGA GAA GAT GCT TTG (SEQ ID NO: 79),
direct IL-4: TAT TAA TGG GTC TCA CCT ACC (SEQ ID NO: 80),
reverse IL-4: TTG GCT TCA TTC ACA CAG GAA (SEQ ID NO: 81),
direct INFγ: GGG TCG CTT TTC GTA GAC ATT TTG (SEQ ID NO: 82),
reverse INFγ: CAG GCA GGA CAA CCA TTA TTT (SEQ ID NO: 83),
and they were used to amplify cDNA transcribed on the material of 1.25 µg RNA. For each cytokine amplified in 20% of the transcription reaction. The remaining cDNA amplifier the Wali using primers for G3PDH. cDNA amplified for 30 cycles as follows: 5 minutes at 95°1 cycle; 30 seconds at 95°C, 30 seconds at 55°C, 30 seconds at 72°C - 30 cycles; 5 minutes at 72°s - 1 cycle. Then 20 ál of the reaction was visualizable in 1%agarose gel.
Definition infection by PCR with reverse
Infection of cells FETJ and sit-5.4 was confirmed by amplification in RT-PCR sequences of the viral gag gene. RNA in the amount of 1.25 μg was transcribable in cDNA in accordance with the above parameters using reverse transcriptase and MMLV reverse primer specific for the gag. 10 μl obretenitsa reaction amplified by PCR with a "hot start" as follows: 5 minutes at 95°C; 30 seconds at 95°C, 30 seconds at 55°C, 30 seconds at 72°C - 30 cycles; 5 minutes at 72°C. After amplification, 20 μl of each sample was visualizable in 1%agarose gel.
To determine the effect of acute infection in vivo on the expression of CD28, cats AUO4, AUU3 and OAS were infected by intravenous injection, while cats AWG3 and OAE were injected with an "empty" environment. The FACS analysis showed the presence of some differentiation relationship CD4:CD8 in experimental and control animals. While in control generally respected permanent classified the e-level 2:1, in the experimental group there were some fluctuations in this relationship, one animal reduced to 1:1 (table 3).
The ratio of lymphocytes CD4:CD8 among RVMS have suffered acute infection and uninfected cats
|CD4:CD8||week 1||ned-2||week 3||weeks-4||weeks 5||weeks-6||week 8|
Analysis of expression of RNA CD80 and CD28 in time spent to make the show, that mRNA of each protein is present and can be amplified by PCR after 0, 8, 16 and 24 hours after stimulation Con A. This procedure semiquantitative PCR was designed to identify displayable band with a minimum number of cycles of amplification that could be used as a relative measure of the amount of this mRNA. A distinct band specific for CD80, was absent after 20 and 25 cycles through any time after infection, but after 30 cycles the band CD80 mRNA was detected in the gel in each of the experimental groups. Also CD28 mRNA was visualizable on each of the temporalóm phase after 30 cycles of amplification, although on a segment of the 16 hours he was also visualizable (poorly) after 25 cycles.
A similar approach was applied to RNA extracted from RVMS FIV-infected and uninfected cats. Amplification RT-PCR specific for CD28 and CD80 RNA was used to determine the relative amount of transcribed mRNA of each of these proteins. As it was done above in the experiment on the temporalóth dynamics, the samples were removed after 20, 25 and 30 cycles. After 20 cycles mRNA was not detected, although both mRNA CD28 and CD80 was visualizability after 25 cycles (table 4). There were no differences in expression of any of these mRNA between experimental and checkpoints for important locations is th groups. In both cases, there were fluctuations in the characteristic number of cycles, in which the visualization of the product.
Semi-quantitative determination of the products of RT-PCR for CD80 and CD28, amplified by the material RNA of infected and uninfected RVMS at intervals the acute phase of FIV infection
|The number of cycles with visualization||to-INF||week 1||week 3||weeks-4||weeks-6||week 8|
PCR amplification of mRNA from cell lines FETJ and sit-5.4 using FIV/gag-specific primers showed that the cell line sit-5.4 infected and saves the active infectious agent, while cell lines FETJ not allow to amplify the gag-specific RNA. FIV-specific product easily amplificates from RNA extracted from samples of cells sit-5.4, but not from RNA extracted from uninfected control. Similar reactions carried out on the material of cell lines FETJ-treated virus FIV strains Petaluma and Maryland, were not allowed to receive displayable product under the same conditions (data not included).
Northern-blotting and semi-quantitative PCR was performed to detect mRNA CD28 in normal cell lines FETJ and sit-5.4. Cell line sit-5.4 presents infected (experimental) group and uninfected controls, whereas the cell line FETJ used as nopermission control T cells.
The results of semi-quantitative PCR showed that each cell line capable expressed ativate CD28 mRNA. When amplification uninfected FETJ were identified parameters of expression similar to those identified in the above experiment the temporalóth dynamics of expression. If 0 hours after stimulation (in fact without it) band was absent until carrying out 30 cycles, then for the 16th hour after stimulation band was detected after 25 cycles. Similar parameters found in the uninfected cell line sit-5.4 when that band was absent until the 30th cycle at 0 hour and visualizable after 25 cycles after 16 hours of incubation. Interestingly, in the infected cells of the sit-5.4 these parameters differed from those of the control cells. In the experimental group mRNA was not visualizability up to 30 cycles neither 0, nor in the 16th hour. To confirm the integrity of the extracted RNA and its concentration was used to amplify G3PDH mRNA. Thus, the infection affects the expression of RNA CD28.
The analysis Northern blotting was used to confirm the data, which were obtained using semi-quantitative RT-PCR. The strongest hybridization with probe set for RNA from uninfected cells sit-5.4, stimulated Con-And for 16 hours. Nephrotomography sample of uninfected cells was characterized by more intense hybridization in comparison with any infected (and social interactions and or not) cells sit-5.4.
In addition to autoradiographically the evaluation of the measured level of radioactivity using a counter BetaGen. Estimates of hybridization CD28 were standardized according to the estimates obtained for the same blot with a probe for G3PDH (table 5).
The normalized counting Northern blots on CD28 using BetaGen
|Sit-5.4, without stimulation||14995||14921||14807|
|Sit-5.4, stimulation Con-||13336||13270||15430|
|Sit-5.4, INF. + stimulation Con-||15112||11077||10967|
Amplification by PCR with repertorium RNA cytokines from cell lines sit-5.4 showed amplificatori mRNA only for IL-2. Neither IL-4 nor IL-6 or γ-interferon failed to amplify in 30 cycles, although the mRNA of IL-2 was detected quite easily.
mRNA CD28 were analyzed for infections in vivo and in vitro to determine whether to be assessed the expression of CD28 and changes retroviral infection the expression of mRNA. When can be allocated dostat the offered quantity of RNA, mRNA CD28 was measured using Northern blotting, and if such limited selection of used semi-quantitative PCR with repertorium.
According to the results of in vivo experiments, infected three animal virus FIV strain Maryland in accordance with attributable to higher mRNA, specific for CD28 and CD80, amplified from a pool of RNA extracted from RVMS isolated from blood samples of these infected animals and uninfected control. Although the analysis Northern blotting is preferred, semi-quantitative PCR with repertorium used due to the limited number of cells and the available amount of RNA of them. The cats took blood every week, so for each experiment could be taken a maximum of 10 ml of blood.
The FACS analysis of the relationship of CD4:CD8 in RVMS infected animals showed a decrease in 8 weeks in infected animals compared to uninfected animals, in which this ratio has remained relatively constant. There are differences between the two experimental groups. Although the ratio of CD4:CD8 different in infected and uninfected animals, no difference was found according to the parameters of the SHS or the expression of CD80 and CD28 (data not included).
For optimal detection of expression of CD28 necessary purified pools of T cells. Selected fractions RWMS is actually about 40% are T-cells. Among these cells at different stages of the experiment up to half of the cells were CD8-positive T-cells that are not expressed CD28 in the same concentration as T-cells CD4+. This is consistent with the fact that, as in other species, resting T-cells (which in the bloodstream make up the majority of T cells) CD28 at a high level is not expressed, and this explains the necessity of using PCR instead of Northern blotting. Preliminary experiments aimed at detecting mRNA CD28 on the material 20 µg RNA extracted from RVMS, were unsuccessful. In semi-quantitative PCR with repertorium was established the presence of RNA encoding CD28. This method was also applied for amplification of CD80 mRNA.
On the material of cell lines in vitro got pools of RNA, in which CD28 mRNA was detected using Northern blotting. Cell lines sit-5.4 was chosen because, as a line of T-cells, they were potential expresando CD28 mRNA, and she was descended from the line of pre-acquired chronic viral infection. Finally, the advantage of this cell line was the availability of significant quantities of RNA compared with blood lymphocytes taken from a single animal.
There have also been attempts to identify mRNA using Northern blotting. While on resting b cells and monocytes CD0 present in low concentrations, in stimulated monocytes and macrophages highest levels of its expression. Line antigen-presenting cells of the cat was not available, and T-cells normally Express this protein only at a low level. Experiments on the detection of CD80 mRNA in the pools of RNA extracted from RVMS, were unsuccessful, apparently, for the same reasons mentioned above when discussing CD28. On the other hand, when using semi-quantitative PCR with repertorium against cells derived from infected animals were detected mRNA CD80 and CD28. Although this method and does not provide an accurate quantitative assessment of mRNA, however, it indicates the presence of mRNA and the relative amount.
The success of the assays, Northern blotting mRNA CD28 T-cell line cat. When comparing parameters of CD28 mRNA for infected and uninfected cells dierentiated in the parameters of expression. The most numerous CD28 mRNA was in uninfected cells, stimulated by concanavalin A. immunology for 16 hours. Also mRNA was detected in unstimulated and uninfected cells. Despite the fact that this mRNA is detected in stimulated and unstimulated FIV-infected cells, these levels are significantly lower in comparison with uninfected cells. This data is all right consistent with similar data obtained in the method of PCR repertorium.
Detection of CD28 mRNA is not hampered by the same restrictions that had taken place in the case of molecule CD80. However, if it can be allocated to large populations of cells expressing CD80, you could easily identify mRNA using Northern blotting. When will be formed-specific monoclonal antibodies for the data surface-cellular proteins, it will be interesting to compare the mRNA levels with level of surface expression of each of these proteins.
cDNA cytokines amplified to confirm the absence of differences between infected and uninfected lines. Regardless of the status of infection in each group amplified IL-2. the mRNA of any other cytokines to amplify failed.
Data Northern blotting show that in vitro expression of CD28 on the level of mRNA is negatively regulated by the FIV virus. Although such a pattern is yet to be confirmed by measuring the levels of surface expression, it is clear that FIV infection in vitro can influence the expression of CD28, which was previously demonstrated for T-cells in HIV infection (Brinchmann et al., 1994).
Cloning and sequencing of cDNA that encodes a signaling complex molecules CD28 and CD80 cats, led to the receipt of the products, similar molecules allocated in other species. Although the decoded amino acid sequence of the protein of the cats showed a relatively low level of identity in relation to the polypeptides of human and mouse data comparison with the previously cloned molecules, the preservation of the characteristic amino acid residues and the fact that this surface ligand apparently deprived of the direct signal functions, allowed us to conclude that the isolated product is really the cat's similar CD80. On the contrary, the molecule CD28 cats is characterized by an average level of identity at the level of nucleotide and amino acid sequences, i.e. is similar molecules, cloned in other species.
The nature of these molecules further explored by identifying their interaction in the tests on the binding. Monoclonal antibodies specific for similar proteins in other species may not respond to expressed proteins cat. With this in mind, they developed a series of tests on linking aimed at identifying interaction, and that this interaction is inhibited soluble receptor. In these tests the binding was determined by the conservation of fluorescently labeled cells, which can be suppressed by making the appropriate soluble receptors. These tests showed not only that the proteins CD80 and CD28 cats can is to be expressed, but also that these surface-expressed molecules can interact.
Also, the expression of these molecules was characterized during active infection. The expression of CD28 and CD80 were studied models in vivo and in vitro exposed to infection with FIV virus. The expression of CD28, which varies in HIV-infected human cells (Asjo et al., 1993), has also undergone negative changes following FIV-infected T-cells of the cat. The subsequent information concerning expression of each of these molecules during development of the disease should additionally confirm that the body of a cat is an important model for retroviral infection.
Methods long-term application of these molecules can potentially be wide. The study of the structure of the immune systems, evolutionary isolated from the human immune system, can lead only to greater understanding of how the immune system works in the human body. Moreover, the importance of the domestic cat as a model organism retroviral infection, convincingly confirmed (Siebelink et al., 1990). It is assumed that protein CD80, being part of antiretroviral vaccine is a potential adjuvant in the induction of CTL memory. The cat can be extremely informative model from the point of view of testing the effectiveness of this pattern.
1. The selected nucleic acid encoding a ligand CD86 cats, soluble CD86 ligand cats or fragment and a ligand CD86 cat is encoded by the coding sequence of SEQ ID NO: 5.
2. Nucleic acid according to claim 1, where the ligand CD86 cats contains the amino acid sequence of SEQ ID NO: 6.
3. Nucleic acid according to claim 1 or 2, where the nucleic acid is RNA.
4. Nucleic acid according to claim 1 or 2, where the nucleic acid is DNA.
5. Nucleic acid according to claim 4, where the DNA is cDNA.
6. Nucleic acid according to claim 4, where the DNA is genomic DNA.
7. Diagnostic oligonucleotide consisting of at least 12 nucleotides, which is characterized by the sequence complementary to the sequence that uniquely present in the nucleic acid according to claim 1 or 2 having the nucleotide sequence of SEQ ID NO: 5.
8. Diagnostic oligonucleotide according to claim 7, which consists of at least 15 or 16 nucleotides which century
9. Diagnostic oligonucleotide according to claim 7 or 8, wherein the oligonucleotide is labeled detectable label.
10. Diagnostic oligonucleotide according to claim 9, where the detectable label includes a radioactive isotope, a fluorophore or Biotin.
11. Diagnostic oligonucleotide according to claim 7 or 8, wherein the oligonucleotide selectively methylated.
12. The cloning vector containing the nucleic acid according to claim 1 or 2.
13. The vector according to item 12, marked B7-2#19-2/011298 (Depository No. ATS 209821).
14. The vector of claim 10 or 11, containing the promoter is functionally attached to a specified nucleic acid.
15. Vaccine for modulating immune response in cats, containing an effective amount of the polypeptide encoded by the nucleic acid according to claim 1 or 2, and a suitable carrier.
16. The vaccine according to item 15, where the polypeptide is a ligand CD86, soluble CD86 ligand cats or fragment.
17. The vaccine according to item 15 or 16, where the effective amount is an amount from about 0.01 to 100 mg per dose.
18. The vaccine according to item 15 or 16, where the effective amount is an amount from about 0.25 mg/kg of body weight cats per day to about 25 mg/kg of body weight cats per day.
19. The vaccine according to any one of p-18, which further comprises an immunogen derived from a pathogen.
20. The vaccine according to claim 19, where the pathogen is a pathogen of cats, rabies virus, PI is the mussel, Toxoplasma gondii, Dirofilaria immitis, pathogen fleas or bacterial pathogen.
21. The vaccine according to claim 20, where the pathogen of cats is the feline immunodeficiency virus (FIV), the virus feline leukemia (FeLV), the virus feline infectious peritonitis (FIP)virus, feline panleukopenia, feline caliciviruses, reovirus 3 types of cat, feline rotavirus, feline coronavirus, respiratory syncytial virus, feline, feline sarcoma virus, feline herpesvirus, a virus disease Bourne cats or parasite of cats.
22. Method of induction of immunity in cats, introducing cats vaccine according to any one of p-21.
23. Method of enhancing an immune response in cats, introducing cats vaccine according to any one of p-21.
24. The method according to item 22 or 23, where the vaccine is injected subcutaneously, intramuscularly, systemically, topically or orally.
25. A method of suppressing an immune response in cats, introducing cats effectively suppressing the immune response of the quantity of soluble polypeptide encoded by the nucleic acid according to claim 1 or 2,
26. The method according A.25, where the specified number is from about 0.25 mg/kg of body weight per day to about 25 mg/kg of body weight per day.
27. The method according A.25, where the cat suffers from an autoimmune disease or is a recipient for the transplant tissue or organ.
FIELD: medicine, immunobiology, pharmacy.
SUBSTANCE: humanized monoclonal antibody (monAb) or its fragments comprises heavy and/or light chain with the binding rate constant with AILIM 1.0 x 103 (1/M x s) and above, and the dissociation rate constant between monAb and AILIM 1.0 x 10-3 (1/s) or less. MonAb shows also a nucleotide sequence encoding variable region of light and/or heavy chain and corresponding amino acid sequences. Invention relates to DNA and it part encoding monAb or its fragments, and vectors comprising nucleotide sequences encoding antibody or its fragments. The humanized monAb can be prepared by using a genetically recombinant host. MonAb is comprised as a component of pharmaceutical compositions used for inhibition or induction of AILIM-mediated transfer of signal into cell for induction of antibody-dependent cytotoxicity against AILIM-expressing cell and others. Invention can be effective in treatment of different autoimmune diseases associated with AILIM-mediated transfer of co-stimulating signal. Invention can be used in medicine for treatment of diseases associated with AILIM-mediated transfer of co-stimulating signal.
EFFECT: valuable medicinal properties of antibody.
75 cl, 78 dwg, 14 ex
FIELD: genetic engineering, proteins, medicine, pharmacy.
SUBSTANCE: invention relates to a method for preparing a fused protein representing immunoglobulin Fc-fragment and interferon-alpha and can be used in treatment of hepatitis. Method involves construction of a fused protein comprising immunoglobulin Fc-fragment prepared from Ig G1 or Ig G3 in direction from N-end to C-end and the end protein comprising at least one interferon-alpha. Fc-fragment and the end protein are joined directly or by a polypeptide bridge. The fused protein is used for preparing a pharmaceutical composition used in treatment of liver diseases and in a method for targeting interferon-alpha into liver tissues. Invention provides preparing the fused protein eliciting with biological activity of interferon-alpha providing its concentrating in liver and showing enhanced solubility, prolonged half-time life in serum blood and enhanced binding with specific receptors.
EFFECT: improved targeting method, valuable biological properties of fused protein.
10 cl, 5 dwg, 9 ex
FIELD: medicine, immunology, phthisiology.
SUBSTANCE: invention proposes a vaccine composition used in prophylaxis and treatment of tuberculosis infection. The composition comprises a mixture of fused recombinant protein ESAT-HSP70 from Mycobacterium tuberculosis, protein HSP65 from M. tuberculosis and an inert vehicle. Also, the proposed composition can comprise timogen additionally. Also, invention proposes recombinant plasmid DNA pE HSP70 and pQE HSP65 encoding corresponding proteins. The proposed vaccine composition enhances effectiveness in treatment and prophylaxis of tuberculosis infection. Genetic constructions provide carrying out the stable expression of indicated proteins and to enhance purity the end preparing proteins. Invention can be used in treatment and prophylaxis of tuberculosis.
EFFECT: valuable medicinal properties of composition.
5 cl, 6 dwg, 6 ex
FIELD: biotechnology, in particular method for construction and production of mutant transglutaminases (MTG).
SUBSTANCE: invention relates to method for construction and production of mutant transglutaminases based on space structure of transglutaminase obtained from Streptoverticillium mobaraense, as well as to mutant MTG obtained by said method. Also disclosed are method for MTG modification based on space structure and modified transglutaminase with enhanced reactivity relative to substrate. Method of present invention makes it possible to predict MTG binding site to substrate on the base of space structure that is determined by MNG crystal X-ray analysis, and to design mutant transglutaminases by replacement, insertion or deletion of amino acid residues disposed on transglutaminase substrate-binding site.
EFFECT: new method for production and modification of mutant transglutaminases.
6 cl, 60 dwg, 5 ex
FIELD: genetic engineering, in particular genes for cell cycle controlling point.
SUBSTANCE: polynucleotide encoding rad3 polypeptide ATR homologue is cloned into expression vector, having functionality in eucariotic cells. Polypeptide of rad3 polypeptide ATR homologue is obtained by cultivation of eucariotic cell culture, transformed by vector. Monoclonal antibody to rad3 polypeptide ATR homologue is obtained by hybridoma technologies. Polyclonal antibodies are obtained by inoculation of rad3 polypeptide ATR homologue in host animal. Polynucleotide presence in animal tissue sample is detected by contacting of this sample containing DNA or RNA with polynucleotide encoding rad3 polypeptide ATR homologue under hybridization conditions. Polypeptide in biological sample is detected by sample contact with monoclonal or polyclonal antibodies. Substances having anticancer activity are screened on the base of reduced activity of ATR polypeptide on substrate or reduced chelating of ATR homologue in presence of candidate substance. Present invention makes it possible to produce human or S.pombe rad3 polypeptide ATR homologue and is useful in investigation ATR role as gene for cell cycle controlling point in cell culture in vivo or in vitro.
EFFECT: new anticancer substances.
24 cl, 1 dwg
FIELD: biotechnology, in particular epithelial cell growth factors useful in production of new keratinocyte growth factor (KGF).
SUBSTANCE: KGF protein is obtained by cultivation of recombinant host cell, transformed with vector containing DNA which encodes amini acid sequence of KGF protein. Obtained KGF protein in pharmaceutical composition is used for forcing of epithelial cell proliferation. Method of present invention makes it possible to produce KGF protein with specific mitogenic activity of 3.4 x 104 U/mg of protein in relation to keratinocyte cells.
EFFECT: new keratinocyte growth factor.
52 cl, 14 dwg, 3 tbl
FIELD: genetic and tissue engineering, biotechnology, medicine, agriculture.
SUBSTANCE: invention relates to the development of simple with constructive relation peptide vector (PGE-κ) consisting of polypeptide sequence of epidermal growth factor (EGF) and modified sequence of signal peptide T-antigen SV-40. New vector PGE-κ is able to provide the selective delivery of genetic material in target-cell cytoplasm carrying external receptors to EGF and the following its transport across nuclear membrane. Also, invention proposes a method for preparing peptide vector PGE-κ involving its expression as a fused protein "mutant thioredoxine-linker-vector" and cleavage of product expressed in E. coli in the linker region with specific protease. Invention provides preparing the recombinant strain E. coli B-8389 VKPM as a producer of the fused protein comprising PGE-κ. Proposed vector shows simple structure, absence of toxicity and immunogenicity and these properties provide its usefulness for the directed genetic modification of epithelial, embryonic and tumor cells in vivo.
EFFECT: improved preparing method, valuable medicinal properties of vector, improved genetic modification.
7 cl, 12 dwg, 4 tbl, 16 ex
FIELD: medicine, genetic engineering.
SUBSTANCE: invention relates to applying genetic engineering approaches for treatment of autoimmune diseases, in particular, for treatment of cerebrospinal sclerosis. This is achieved by incorporation of one or some recombinant genes encoding autoantigens that represent a target for autoimmune response. In particular, invention claims a method for designation of gene encoding encephalitogenous epitope of proteolipid protein and expression of gene product in vivo by using the recombinant retroviral vector. Expression and secretion of encephalitogenous epitope improves histopathological and clinical indices in experimental autoimmune encephalomyelitis in mice that is used as a model of cerebrospinal sclerosis. The advantage of invention involves the development of a method for recovery the tolerance in treatment of cerebrospinal sclerosis being without suppression of immune system.
EFFECT: improved and valuable method for treatment.
6 cl, 13 dwg, 3 ex
FIELD: biotechnology, genetic engineering, molecular biology.
SUBSTANCE: invention proposes recombinant DNA pcDNA-TCI that has molecular mass 4.3 x 103 kDa and size 6 657 nucleotide pairs. The proposed recombinant plasmid DNA provides expression of artificial gene TCI comprising cytotoxic T-cellular epitopes of HIV-1 in eucaryotic cells. Also, invention proposes recombinant attenuated strain of bacterium Salmonella enteritidis E-23 pcDNA-TCI. The proposed strain is obtained by genetic transformation of the strain Salmonella enteritidis E-23 with plasmid pcDNA-TCI. The strain provides delivery DNA-vaccine in eucaryotic cells as the recombinant plasmid DNA pcDNA-TCI. Proposed groups of inventions provide inducing the expressed specific humoral and cellular immune response to HIV-1 in body. Proposed group of inventions can be used in medicine and virology for construction of live DNA-vaccine against HIV-1.
EFFECT: valuable biological and medicinal properties of strain and vaccine.
2 cl, 3 dwg, 3 tbl, 5 ex
FIELD: genetic engineering, biotechnology, molecular biology, medical-biological and pharmaceutical industry.
SUBSTANCE: invention relates to isolating gene from cells of the strain P. altcromonas producing enzyme that cleaves polysaccharide comprising sulfated fucose and this gene encodes above indicated enzyme, Invention proved the presence of two opened reading frames (ORF-1 and ORF-2) and their nucleotide sequences are determined. Active recombinant forms of enzyme able to hydrolyze sulfated fucose-comprising polysaccharide are obtained by expression of these sequences in E. coli being this polysaccharide is not cleaved by fucoidanase produced by Flavobacterium sp. SA-0082 (FERM BP-5402). Applying the invention provides the possibility for preparing large amounts of qualitative raw for pharmaceutical preparations.
EFFECT: improved preparing method, valuable properties of polypeptide.
5 cl, 5 dwg, 2 tbl, 7 ex
FIELD: biology, genetic engineering, biotechnology, medicine.
SUBSTANCE: invention relates to preparing glycosylated polypeptide (glycoprotein) as a component of human erythropoietin by using the technology of recombinant DNAs. This polypeptide shows ability to increase production of reticulocytes and erythrocytes, to enhance the level of hemoglobin synthesis and consumption of iron by marrow cells and characterized by the higher molecular mass as compared erythropoietin isolated from human urine. Invention describes variants DNA sequences encoding this polypeptide that comprise vector constructions with these sequences, a method for preparing transformed mammalian cell lines producing the recombinant human erythropoietin, and a method for its preparing and purification. Also, invention proposes pharmaceutical compositions comprising glycosylated polypeptide (glycoprotein) of erythropoietin as an active component. Applying this invention provides scaling the process for preparing active human erythropoietin useful for its using in medicine.
EFFECT: improved preparing method, valuable properties of polypeptide.
10 cl, 4 dwg, 21 tbl, 12 ex
FIELD: biotechnology, genetic engineering, pharmaceutical industry.
SUBSTANCE: plasmid DNA pET23-a(+)/PrxVIhumΔ178 with molecular weight of 19691.61 Da is constructed. DNA contains RNA-polymerase T7 promoter; replication initiation site; genetic marker which determinates resistance of cells transformed by said plasmid to ampicillin; and nucleotide sequence encoding N-terminal fragment of human peroxiredoxine VI containing 177 of amino acid residues. E.coli strain BL21/DE3/pET23-a(+)/PrxVIhumΔ178 being producer of N-terminal fragment of human peroxiredoxine VI is obtained by transformation of E.coli cells with plasmid DNA pET23-a(+)/PrxVIhumΔ178. Method of present invention makes it possible to obtain human peroxiredoxine VI fragment having reduced molecular weight, improved tissue permeability, and antioxidant activity of full-scale peroxiredoxine.
EFFECT: human peroxiredoxine VI fragment with improved tissue permeability.
2 cl, 3 dwg, 4 ex
FIELD: biotechnology, in particular epithelial cell growth factors useful in production of new keratinocyte growth factor (KGF).
SUBSTANCE: KGF protein is obtained by cultivation of recombinant host cell, transformed with vector containing DNA which encodes amini acid sequence of KGF protein. Obtained KGF protein in pharmaceutical composition is used for forcing of epithelial cell proliferation. Method of present invention makes it possible to produce KGF protein with specific mitogenic activity of 3.4 x 104 U/mg of protein in relation to keratinocyte cells.
EFFECT: new keratinocyte growth factor.
52 cl, 14 dwg, 3 tbl
FIELD: biotechnology, molecular biology.
SUBSTANCE: method involves transfection of cells HKB with vector pCIS25DTR comprising a selective marker and a sequence encoding protein eliciting procoagulating activity of factor VIII. Cells are selected using the selecting agent and clones with high level for expressing protein eliciting procoagulating activity of factor VIII are isolated. Invention provides preparing the protein eliciting activity of factor VIII with high yield, and strain of cells HKB with improved production under protein-free conditions also. Invention can be used for preparing the protein eliciting activity of factor VIII in industrial scale.
EFFECT: improved preparing and isolating methods.
8 cl,, 6 dwg, 1 tbl, 5 ex
FIELD: genetic engineering, molecular biology.
SUBSTANCE: invention proposes a method for detecting genes encoding membrane-bound transmembrane proteins. Method involves expression of the nucleic acid chimeric sequence in the cell-host consisting of DNA fragment encoding secreting protein that is able to bind antigen and DNA fragment to be tested; interaction of cells expressing the fused protein with antigen; selection of cells on surface of that indicated antigen is bound; isolation of recombinant vector containing in selected cells of DNA fragment to be tested and, if necessary, determination of its sequence. Also, invention proposes the developed vector constructions and comprising their sets designated for realization of the proposed method. Invention provides significant simplifying the screening process of libraries and cloning genes encoding transmembrane proteins. Invention can be used for detecting and preparing genes encoding any membrane-bound proteins used in different branches of science and practice.
EFFECT: improved isolating method, valuable biological properties of protein.
27 cl, 7 dwg, 1 tbl, 8 ex
FIELD: medicine, diagnostics.
SUBSTANCE: the present innovation deals with genetic trials, with diagnostic field of oncological diseases due to analyzing DNA by altered status of gene methylation that take part in intracellular regulation of division, differentiating, apoptosis and detoxication processes. One should measure the status of methylation in three genes: p16, E-cadherine and GSTP1 in any human biological samples taken out of blood plasma, urine, lymph nodes, tumor tissue, inter-tissue liquid, ascitic liquid, blood cells and buccal epithelium and other; one should analyze DNA in which modified genes of tumor origin or their components are present that contain defective genes, moreover, analysis should be performed due to extracting and purifying DNA out of biological samples followed by bisulfite treatment of this DNA for modifying unprotected cytosine foundations at keeping 5-methyl cytosine being a protected cytosine foundation followed by PCR assay of bisulfite-treated and bisulfite-untreated genes under investigation and at detecting alterations obtained according to electrophoretic result of PCR amplificates, due to detecting the difference in the number and electrophoretic mobility of corresponding fractions at comparing with control methylated and unmethylated samples containing normal and hypermethylated forms of genes one should diagnose oncological diseases. The method provides higher reliability in detecting tumors, detection of remained tumor cells after operation.
EFFECT: higher efficiency of therapy.
1 cl, 3 dwg, 4 ex
FIELD: medicine, molecular biology, polypeptides.
SUBSTANCE: invention describes homogenous polypeptide ligand mpI representing polypeptide fragment of the formula: X-hTPO-Y wherein hTPO has amino acid sequence of human fragments TPO (hML); X means a amino-terminal amino-group or amino acid(s) residue(s); Y means carboxy-terminal carboxy-group or amino acid(s) residue(s), or chimeric polypeptide, or polypeptide fragment comprising N-terminal residues of amino acid sequence hML. Also, invention relates to nucleic acid encoding polypeptide and expressing vector comprising nucleic acid. Invention describes methods for preparing the polypeptide using cell-host transformed with vector, and antibodies raised against to polypeptide. Invention describes methods and agents using active agents of this invention. The polypeptide ligand mpI effects on replication, differentiation or maturation of blood cells being especially on megacaryocytes and progenitor megacaryocyte cells that allows using polypeptides for treatment of thrombocytopenia.
EFFECT: valuable medicinal properties of polypeptide.
21 cl, 92 dwg, 14 tbl, 24 ex
FIELD: biotechnology, medicine, proteins.
SUBSTANCE: invention describes new polypeptide in isolated form relating to subfamily of superfamily human immunoglobulins (Ig-Sf). This polypeptide shows at least 70% of homology level with amino acid sequence of murine molecules CRAM-1 or CRAM-2 regulated by the confluence of adhesive (figures 3, 6 are represented in the claim). Also, invention relates to antibodies showing specificity with respect to the polypeptide. Antibodies and soluble polypeptide can be used for treatment of inflammation and tumors. Invention describes polynucleotide or oligonucleotide encoding the full-size polypeptide or its moiety and represents primer, probe, anti-sense RNA and shows the nucleotide sequence that is identical conceptually with human CRAM-1. Invention provides preparing new adhesive proteins from superfamily Ig-Sf that are regulated at the transcription level in endothelium by effect of tumors. Invention can be used for treatment of different diseases, in particular, inflammatory responses.
EFFECT: valuable medicinal properties of polypeptide.
19 cl, 33 dwg, 1 ex