Canine thymic stromal lymphopoietin protein and use thereof

FIELD: chemistry.

SUBSTANCE: disclosed is an isolated thymic stromal lymphopoietin protein (TSLP) or antigenic fragment thereof for inducing immune response, as well as a nucleic acid molecule coding it.

EFFECT: invention can find further use in therapy of atopic diseases.

7 cl, 8 dwg, 2 tbl, 7 ex


Cross-reference to related applications

This application is not provisional application and claims the priority under 35 U.S..§119(e) of provisional application U.S. serial number 60/875, filed December 14, 2006, the contents of which are incorporated herein by reference in its entirety.

The technical field

The present invention relates to dog timesnow stromal lymphoreticular protein (dog MONDAY), nucleic acid molecules, vectors and host cells encoding canine MONDAY, and methods of producing and applying dog MONDAY.

The level of technology

Animals, including humans, suffering from reagin-mediated diseases, such as atopic diseases, hereditary predisposed to the development of direct allergic reactions, including immunoglobulin E (IgE antibodies). Multiple genetic factors contribute to the manifestation of the resulting phenotype observed in these animals. Direct hypersensitivity observed in atopic diseases, is the result of exposure to specific allergens such as house dust mite (Dermatophagoides pteronyssinus), pollen, mold and animal dander. It is not surprising that individuals with atopic disease, the most likely to suffer from asthma, atopic dermatitis, as well as about the other diseases, due to the release of endogenous IgE.

Atopic diseases such as allergic dermatitis, asthma and the like, also appear among the canine species, including domestic dogs. These dogs usually start to show signs of atopy at the age of 1 to 3 years. Because of the hereditary nature of the disease some breeds, including Golden retrievers, most Terriers, Irish setter, Lhasa, Apso, Dalmatians, bulldogs and old English sheep dogs, bear a greater tendency to atopy, although it is known that other types of dogs, including mixed breeds, also suffer from this. The incidence of at least one particular type of atopy, atopic dermatitis, is significantly increased in a similar way as in humans and in dogs.

Dogs with atopy usually begin to RUB, lick, chew, bite or scratch their paws, snout, ears, armpits, or groin that will lead to hair loss, redness and swelling of the skin. In some cases the condition of the skin combine to cause itching of the animal, while only one Allergy could lead to such an itch. These worsening problems can be caused by allergens that are transmitted through the air (pollen, etc), allergens in food and allergens from parasites (Blo and etc). Bacterial and/or yeast skin infections can also increase the itching.

One simple remedy to relieve the irritating symptoms of atopy is the avoidance of allergens pathogens. Unfortunately, this avoidance is usually impossible. First of veterinarians practice was treated for atopic dermatitis in dogs by the use of oral antihistamines, oral or topical anti-inflammatory agents of the class of corticosteroids, other suppressors of the immune system such as cyclosporine or tacrolimus, derivatives of fatty acids, and allergen-specific immunotherapy (which requires the introduction of a specific antigen). However, none of these treatments are not working in all cases. Moreover, such treatments are expensive and/or cause significant side effects. Thus, there is a long-standing need for a safer, more efficient and more cost-effective approaches to the treatment or suppression of symptoms of atopic dermatitis in dogs.

The immune system of mammals is based on a complex series of cellular interactions, which is called "immune network". Many of the immune responses revolve around interactions like network interactions of lymphocytes, macrophages, granulocytes and other cells with soluble proteins called cytokines and play kriticheskie the role in mediating/control/regulation of these cellular interactions. Thus, cytokines and cells of the immune system are used to initiate specific physiological mechanisms or pathways leading to a variety of inflammatory diseases.

Allergic inflammation is the result of a complex immune cascade that leads T cells to the production of unregulated TN-cytokines, such as IL-4, IL-5 and IL-13. These cytokines, in turn, cause bronchial hyperactivity and the production of IgE, eosinophilia, and production of mucus (see, for example, Busse and Lemanske, Jr (2001) N. Engl. J. Med. 344:350-62; Holgate (2000) Br. Med. J. 320:231-234, and Renauld (2001) J Clin Pathol. 54:577-589).

Timony stromal lymphopoiesis protein (MONDAY) is IL-7 in a similar cytokine that was originally opened in mice as a factor that supports: (i) production of in vitro immunoglobulin IgM on the surface of b-cells and (ii) the proliferation of b and T cells (Friend et al., 1994 Exp Hematology 22:321-328, see also Levin et al., 199, J. Immunol. 162:677-683). As you know, MONDAY binds to the cellular receptor, containing the IL-7R-alpha subunit and a unique subunit of the receptor, called MONDAY-R. This interaction triggers the transmission of a signal through the activation of STAT or expression timusewo and regulated by the activation of chemokine (R) in hematopoietic cells, such as myeloid cells, such as monocytes, or dendritic cells (see, for example, U.S. patent No. 6890734 included here in it is the number of links).

MONDAY also can play a significant role in mice in the pathogenesis of allergic diseases such as atopic dermatitis and asthma. For example, a transgenic mouse whose skin was specifically induced the expression of a gene MONDAY, possess immunological and clinical signs of atopic dermatitis such as eczema damage containing inflammatory dermal cellular infiltrates, a sharp increase in the number of TH2-CD4*T cells expressing cutaneous receptors for guidance, and increased levels of IgE in serum. Moreover, the lungs of mice expressing lung specific transgene MONDAY have immunological and clinical features of asthma, including large infiltration of white blood cells, hyperplasia of goblet cells, subepithelial fibrosis, increased cytokines in T-helper type 2 and increased levels of IgE.

Sims et al. got the cDNA sequence of murine MONDAY, used in the expression cloning, but was not able to clone a human homologue of the sample for hybridization based on murine MONDAY (Sims et al. 200. J Exp Med 192:671-680). Subsequently, the human homologue was identified through a detailed EST analysis. Human nucleotide corresponding to MONDAY, as it was discovered, has only 43% homology with the appropriate nucleotide the mice. Thus, there remains a need to provide new and more practical ways of treatment of atopic diseases, including atopic dermatitis and related clinical manifestations in dogs. Furthermore, it is necessary to identify the factors that are involved in the immune cascade that leads to atopic diseases in dogs that could lead to the development of such treatments.

Citation of any reference here should not be construed as acceptance of this reference as prior art for this application.

Brief description of the invention

The present invention provides a new and more practical methods of treatment of atopic diseases in dogs, including atopic dermatitis and related clinical manifestations. Accordingly, the present invention provides a new isolated and/or recombinant timoshii stromal lymphopoiesis proteins (MONDAY), which are involved in the immune cascade that leads to atopic diseases. The present invention further provides an antigenic fragments of such proteins MONDAY. In the private aspect of the present invention protein MONDAY is a dog MONDAY.

Thus, the present invention provides a protein MONDAY, contains an amino acid sequence that is at 0% or more identical to the amino acid sequence of SEQ ID NO: 2, except for the amino acid residue 28 of the signal sequence, which, when assigning protein dog, serves as vaccines, antibodies that bind canine MONDAY protein containing the amino acid sequence of SEQ ID NO: 2, are recognized in the ultimate canine serum obtained from vaccinated dogs. In a similar embodiment, the protein is MONDAY contains the amino acid sequence that is 80% or more identical to the amino acid sequence of SEQ ID NO: 2, except for the 28 amino acid residue signal sequence; and it is cross-reactive antibody to canine MONDAY protein containing the amino acid of SEQ ID NO: 2.

The present invention further provides a protein MONDAY, containing the amino acid sequence that is 80% or more identical to the amino acid sequence of SEQ ID NO: 2 (except for the 28 amino acid residue signal sequence), which binds to a monoclonal antibody specific to the epitope protein dog MONDAY.

In a more private embodiment, the protein is MONDAY contains an amino acid sequence that is 90% or more identical aminosilanes sequence SEQ ID NO: 2, except for the 28 amino acid residue signal sequence. In another embodiment, the imp is in protein MONDAY contains the amino acid sequence, which is 95% or more identical aminosilanes sequence SEQ ID NO: 2, except for the 28 amino acid residue signal sequence.

In the private embodiment of the present invention protein MONDAY is a dog protein MONDAY, which contains the amino acid sequence of SEQ ID NO: 2. In another embodiment, the protein MONDAY is a Mature dog protein MONDAY, which contains amino acid residues 29-155 of SEQ ID NO: 2.

Also in the present invention are antigenic protein fragments MONDAY. Such antigenic fragments include those that contain one or more epitopes defined individually by amino acid sequence SEQ ID NO: 8-101. In the private embodiment, the antigenic fragment of the present invention contains one or more epitopes that contain the amino acid sequence of SEQ ID NO: 30, 31, 32 and/or 34. In another embodiment, the antigenic fragments can have the amino acid sequence within the overlapping amino acid sequences SEQ ID NO: 30, 31, 32 and/or 34, i.e. NPPDCLARIERLTLHRIRGCAS (SEQ ID NO: 118). In the private embodiment, the antigenic fragment of the protein dog MONDAY capable of binding the monoclonal antibody to human MONDAY. The size of the antigenic fragments linakis is now sequence NPPDCLARIERLTLHRIRGCAS (SEQ ID NO: 118) may lie in the approximate range from 5 to 21 amino acid residues.

Also available vaccines, which may include an effective amount of any protein MONDAY the present invention, one or more antigenic fragments of these proteins, or a combination of such full-sized proteins and one or more of these fragments. In one embodiment, the protein MONDAY is the dog's MONDAY, which contains the amino acid sequence of SEQ ID NO: 2. In the private embodiment, the vaccine contains one or more antigenic fragments dog MONDAY protein, which contains from 5 to 22 contiguous amino acids from amino acid residues 71-92 of SEQ ID NO: 2 (defined here as SEQ ID NO: 118). Examples of such antigenic fragments include epitopes shown here, which contain the amino acid sequence of SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 or SEQ ID NO: 34. All vaccines of the present invention may further contain pharmaceutically acceptable adjuvant.

The vaccine of the present invention can be used in the method of producing antibodies to canine MONDAY. One such method includes the immunization of a mammal an effective amount of the vaccine. This method optionally includes a method of regulating the activity of MONDAY on its afferent pathways in dogs and/or method of treatment or prevention of allergic symptoms in soba is with atopy, which includes immunization dog an effective amount of the vaccine. Improved allergic symptoms may include allergic dermatitis, asthma and the like.

The vaccine of the present invention may be administered by intramuscular injection, subcutaneous injection, intravenous injection, intradermal injection, oral administration, intranasal use, scarification and their combinations.

The present invention further provides a nucleic acid molecule encoding a protein MONDAY the present invention or antigenic fragment. In one such embodiment, the nucleic acid molecule encodes the amino acid sequence of SEQ ID NO: 2. In the private embodiment of this type the nucleic acid molecule includes the nucleotide sequence of SEQ ID NO: 1. Fragments of the nucleotide sequence of SEQ ID NO: 1 of about 18 contiguous nucleotides, about 24 contiguous nucleotides, about 36 contiguous nucleotides, about 45 contiguous nucleotides, about 66 adjacent nucleotides or more are also part of the present invention. Nucleic acids of approximately 18 nucleotides, about 24 nucleotides, about 36 nucleotides, about 45 nucleotides, approximately 66 nucleotide is at or greater, including a nucleic acid encoding a full-sized proteins MONDAY, which hybridize with SEQ ID NO: 1 under conditions of exact hybridization, also provided by the present invention. All the molecules of nucleic acids and fragments thereof of the present invention may further comprise a heterologous nucleotide sequence.

The present invention also provides a vector for expression, which includes the previously noted molecules of nucleic acids and/or their fragments. In addition, the present invention provides host cells which contain such vectors for expression. The host cell is not necessarily either prokaryotic or eukaryotic. In one embodiment, the prokaryotic host cell is Esherichia coli. In the private embodiment of this type the host cell is .li BL21(DE3)/pLysS, which contains the gene for T7 RNA polymerase under control of the isopropyl-β-thiogalactopyranoside (IPTG)-inducible lacUV5 promoter.

The present invention further provides a recombinant viral vector and/or empty DNA vector containing one of the aforementioned nucleic acid molecules encoding canine MONDAY, for example SEQ ID NO: 1 and/or fragments of them. Such vectors can be used, for example, in vaccines suitable for appointment to the dogs ate the practical dermatitis. The present invention also provides methods of obtaining protein MONDAY the present invention. One such method includes the cultivation of a host cell of the present invention in a suitable culture medium. This method may further include the extraction and/or purification of the protein from MONDAY cultured host cell or culture medium. The final isolated and/or purified protein MONDAY is also part of the present invention.

Antibodies to MONDAY, derived from a hybridoma of the system under the action of the vaccine of the present invention, are also part of the present invention. In one embodiment of this type is used hybridoma system of mammals. In the private embodiment, the antibody is isolated and/or purified. Antibodies may be either polyclonal or monoclonal. According to the invention a monoclonal antibody derived from the dog does not need to be constructed "isobutene" in order to show the minimal antigenic effect with the introduction of the dog. In some preferred versions of the binding domains of any of the antibodies according to the invention is not necessarily converted into the binding fragments of smaller size than the original antibody, for example, by splitting and/or recombinantly the Fv, Fab and F(ab')2 binding proteins. Derived from antibodies therapeutic proteins that have unique structural and functional properties of naturally occurring antibody heavy chains (for example, NANOBODIES®) are also included in the invention. In addition, substitutes for antibodies that have high affinity to MONDAY and low immunogenicity (e.g., avimanyu proteins constructed from the binding sites of the receptor to MONDAY) is also included in the present invention. Invented avemaria antibodies to canine MONDAY can actively be used in the treatment of allergic symptoms in dogs with atopy by applying an effective amount of antibodies to canine MONDAY.

The present invention also provides a vaccine containing an effective amount of the immunogen is not MONDAY of nature in combination with an effective amount of protein MONDAY the present invention, one or more antigenic fragments of these proteins, or a combination of full-sized protein with one or more such fragment. In the private embodiment of this type, protein MONDAY is a dog protein MONDAY. In a more private embodiment, protein dog MONDAY contains the amino acid sequence of SEQ ID NO: 2. The present invention additionally provides diagnostic methods using soba is LEGO protein MONDAY, its fragments and/or antibody obtained by dog MONDAY and its fragments. In one embodiment, the present invention provides a method for diagnosing atopic dermatitis in dogs, which contains the sample obtained epidermis from dogs and detecting the presence of protein dog MONDAY in the sample epidermis. These and other aspects of the present invention will be better understood by reference to the following figures and detailed description.

Brief description of drawings

Figure 1 illustrates the analysis of a protein from a eukaryotic cell systems for the synthesis of the free protein, expressing the protein dog MONDAY - method of SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Lane 1: Standard protein; Lane 2: Total protein; Lane 3: Soluble protein; Lane 4: Insoluble protein. The protein bands MONDAY marked by arrows.

Figa illustrates the analysis of a protein from a eukaryotic cell systems for the synthesis of the free protein, expressing the protein dog MONDAY - way Western blotting. Proteins were preincubator with antibody Anti-His (the term is created by the AR (Invitrogen). Lane 1: Standard protein; Lane 2: Total protein; Lane 3: Soluble protein; Lane 4: Insoluble protein. Dog protein MONDAY was detected in total protein and insoluble protein (as marked by arrows).

Figv illustrates the analysis of the protein PI is eroticheskih cells system for the synthesis of the free protein, expressing the protein dog MONDAY - way Western blotting. Proteins were preincubator with rat monoclonal antibodies specific for human MONDAY. Lane 1: Standard protein; Lane 2: Total protein; Lane 3: Soluble protein; Lane 4: Insoluble protein. Dog protein MONDAY was detected in total protein and insoluble protein (as marked by arrows).

Figa illustrates the expression and purification MONDAY from the host cells E. coli and shows the band 61 kDa, which is present in the soluble fraction of E. coli, which is a fusion protein dog MONDAY, protein GST and terminal peptide of 6 histidine residues. "M" oboznachat standard protein (the same on Figa-3D). Track 1 and track 2 are soluble fractions of E. coli B121(DE3)pLysS containing plasmid 1265-93 without and with induction IPTG, respectively. Arrows indicate the band of the fusion protein GST-TSL-His (one and the same on Figa-3D).

Figure 3 b shows that the labeled fusion protein GST-TSL-His can be purified by resin-based glutathione Sepharose 4B. Tracks 1 through 3 show different fractions after elution on glutathione-Sepharose 4V.

Pigs shows that the protein fusion on the track can be further purified using resin Ni-NTA. This figure illustrates a second protein purification merge GS-TSL-His resin Ni-NTA after purification on glutathione-Sepharose 4B. Track 1 denotes the eluate, lane 2 indicates the elution of the resin Ni-NTA.

Fig.3D illustrates the analysis of the fusion protein GST-TSL-His by Western blotting and confirmed that the fusion protein is recognized by the antibody to GST (GE Health Care Cat No.27457701).

Figure 4 illustrates the staining of FITC section sealed in paraffin parts damaged skin, obtained from dogs 10197 diagnosed with atopic dermatitis". This site was preincubation with rabbit polyclonal antibodies to human MONDAY and the reaction was visualized using the Streptavidin-FI (Fluorescein isothiocyanate). The fluorescence intensity (bright areas) determined the binding of rabbit polyclonal antibody to human MONDAY with MONDAY presented in tissue.

Figa illustrates staining of FITC section sealed in paraffin parts damaged skin, obtained from dogs 10197 diagnosed with atopic dermatitis". In this part there is diffuse staining (dark areas) epidermal region of the skin labeled mouse monoclonal antibody to human MONDAY.

Figv illustrates the control plot. This site was sealed in paraffin parts damaged skin, obtained from dogs 10197 with a diagnosis of atopic dermatitis", which is processed only by the control in the form of phosphate buffer.

6 illustriou the t mapping epitopes of canine protein MONDAY with rat monoclonal antibody to human MONDAY. Peaks that cause particular interest, is the peaks of epitopes number 22-26 (SEQ ID NO: 29-33).

Epitopes 22-26 were also preincubator with N-terminal derivative (peak 55 and above) in order to confirm that bind epitopes do not require the N-terminal residue.

7 illustrates a comparison of the dog (SEQ ID NO: 32) and human similar epitope 25 (SEQ ID NO: 3) peptide sequence MONDAY.

Figa illustrates the DNA sequence of the gene of canine MONDAY (SEQ ID NO: 1).

Figv illustrates the predicted polypeptide MONDAY, expressed from the DNA sequence shown in Figa (SEQ ID NO: 2). The asterisk marks the N end of the initial signal sequence (residues 1-28) and the underlined residues 71-92 (SEQ ID NO: 118), representing the area, which were identified overlapping epitopes.

Detailed description of the invention

Atopic dermatitis (AD) is a Th2 dysbalance-mediated allergic inflammatory disease. This disease is manifested many similar clinical signs in humans and in dogs. It is likely that the immunopathogenesis of AD in dogs is similar to HELL in relation to the type of cells and cytokines involved in skin damage.

The binding of the ligand TARC (CCL22) receptor 4 chemokine CC (CCR4), which is selectively expressed on T-lymphocytes, causes selective migration of these cells to Allergy is a mini-injury. It was reported that R and its receptor CCR4 is governed by efferent pathways in skin lesions in dogs with AD. Since MONDAY is a potent inducer of R in humans, it has been suggested that MONDAY may be present in injuries in dogs with AD. Antibodies obtained for human MONDAY, were therefore investigated the damaged skin of dogs with AD. Immunohistochemistry of these skin samples confirmed the presence of antigen reactive with respect to antibodies to human MONDAY when damaged, as shown in Figure 4. However, the task of determining the canine homologue genes encoding mouse and human MONDAY, as it was proved especially difficult because of the high degree of divergence of the sequences of the nucleic acid and amino acid sequences MONDAY in mammals, as found here.

Immunization domestic dog MONDAY the present invention and/or one or more antigenic fragments should serve to reduce the activity levels of endogenous MONDAY and so to keep, remove or prevent one or more symptoms of atopy, such that occur in asthma and/or atopic dermatitis, the immunized dogs. In addition, protein dog MONDAY may be used as the immunogen for cleavage of antibodies to canine MONDAY for used what I as a research or diagnostic reagent in domestic dogs or other mammals. Alternatively, in particular examples, protein dog MONDAY and/or nucleic acids that encode dog MONDAY, may serve to regulate elements of the efferent pathways of the immune system in dogs with a weakened immune system, for example, by the activation of STAT or expression R, for example, hematopoietic cells.

In order to more fully understand the present invention, offers the following definitions.

The use of unique terms for convenience, the description is in no way limited. So, for example, reference to a composition containing "a polypeptide"includes reference to one or more of these polypeptides. Used herein, the term "approximately" is used interchangeably with the term "approximately" and indicates that the value is inside the interval with a 20% deviation from a certain value, i.e. a peptide containing "approximately" 50 amino acid residues may contain from 40 to 60 amino acid residues.

The term "binding composition" refers to molecules that are specific contact dog MONDAY, for example, in the interaction of antigen-antibody. Specificity may be more or less include, for example, specific to private options for performing, or for a group of related embodiments, for example, dog MONDAY and/or dog ant the body. Used herein, the term "dog" includes all domestic dogs, Canis lupus familiaris or Canis familiaris, unless otherwise noted.

Used herein, the term "polypeptide" is used interchangeably with the term "protein" and "peptide" and refers to a polymer that contains 2 or more amino acids connected by peptide bonds. The term "polypeptide"used herein includes a significant fragment or segment and contains a number of amino acid residues approximately at least 8 amino acids, generally from about 12 amino acids, usually at least 16 amino acids, preferably at least 20 amino acids, and in particularly preferred embodiments, performing at least 30 or more amino acids, for example, 35, 40, 45, 50, etc. Such fragments may be all that begin and/or end at virtually every position, for example, beginning at residues 1, 2, 3 and end on, for example, 155, 154, 153, etc. in all practical combinations.

Optionally, the polypeptide may lack certain amino acid residues that are encoded gene or mRNA. For example, a gene or mRNA molecule can encode the sequence of amino acid residues at the N end of the polypeptide (i.e. the signal sequence), which is then cleaved and therefore may not be part of the final protein.

Use what has been created here amino acid sequence 100% is "homologous" to a second amino acid sequence, if two amino acid sequences are identical and/or differ only in neutral or conservative substitutions as defined below. Accordingly amino acid sequence of approximately 80% is "homologous" to a second amino acid sequence, if approximately 80% of the two amino acid sequences are identical and/or differ only in neutral or conservative substitutions.

Functionally equivalent amino acid residues may often substituted residues within the sequence resulting in a conservative amino acid substitution. These changes define the term "conservative substitution"as used here. For example, one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity, which acts as a functional equivalent, resulting in a "silent" changes. Replacement of the amino acid within the sequence may be selected from other members of the class to which belongs the amino acid. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, Proline, phenylalanine, tryptophan and tyrosine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. Positively for agenie (basic) amino acids include arginine, lysine and histidine. Negatively charged (acidic) amino acids include aspartame acid and glutamic acid. It is not expected that these changes will affect the molecular weight as determined by polyacrylamide gel electrophoresis or isoelectric point.

Particularly preferred conservative substitutions are: lysine to arginine, and Vice versa, which in any case provides a positive charge; a glutamic acid to aspartic, and Vice versa, which in any case provides a negative charge; serine to threonine, which in any case provides free group; and glutamine for asparagine, which in any case provides a free amino group. Amino acids can also be substituted within the following similar groups: (1) Proline, alanine, glycine, serine and threonine; (2) glutamine, asparagine, glutamic acid and aspartic acid; (3) histidine, lysine and arginine; (4) cysteine; (5) valine, leucine, isoleucine, methionine; and (6) phenylalanine, tyrosine and tryptophan.

In a related embodiment, the two vysokomolochnye DNA sequences can be identified by its own homology or homology of amino acids they encode. This comparison of sequences can be represented using a standard software package, access the Phnom data banks such sequences. In the private embodiment, the two vysokomolochnye DNA sequences encode amino acid sequences having about 80% identity, more preferably about 90% identity, and even more preferably about 95% identity. In the more particular case of two vysokomolochnye amino acid sequences have approximately 80% identity, more preferably about 90% identity, and even more preferably about 95% identity.

As used here, a percent identity of protein and DNA sequences can be determined using a software package such as MacVector v9, which is available commercially from a company Accelrys (Burlington, Massachusetts) and the algorithm Clustal W for default settings for mapping and default settings for identity. See, e.g., Thompson, et al., 1994, Nucleic Acids Res. 22:4673-4680. Clustal W is a free downloadable program for platforms Dos, Macintosh and Unix, for example, with EMBLI, the European Bioinformatics Institute. The real link for downloading found on These and other available programs can also be used to determine the similarity of the sequences, using the same or similar to the default settings.

"Polynucleotide" or "nucleic acid molecule" is a Molek is Oh, contains nucleotides, including without limitation, RNA, cDNA, genomic DNA, and even synthetic DNA sequences. These terms are also intended to address nucleic acid molecules that comprise any of the known science analogs of DNA and RNA.

The present invention provides nucleic acids which hybridize with a nucleotide sequence encoding a protein MONDAY the present invention. The molecule is a nucleic acid capable of hybridizing to another nucleic acid molecule, such as a cDNA, genomic DNA, or RNA, when a single stranded form of the nucleic acid molecules can otjihase with the other nucleic acid molecule under the appropriate values of temperature and ionic strength of the solution (see Sambrook and Russel Molecular Cloning: A Laboratory Manual. 3rded. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, L.I, (2000)).

Very stringent hybridization conditions correspond to, for example, the highest temperature Tm, 50% formamide, 5X or 6XSSC. When hybridization requires that the two molecules of nucleic acids contain complementary sequences, although depending on the stringency of hybridization, it is possible unpaired lots (mismatch) between the bases. Appropriate stringency hybridization of nucleic acids depends on the length of nucleic sour and the degree of complementarity, varieties which are well known in this field. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of Tmfor hybrids of nucleic acids having those sequences. The relative stability (corresponding more high-Tm) hybridization of nucleic acids is reduced in the following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids longer than 100 nucleotides was obtained the equation for calculation of Tm(Sambrook and Russel Molecular Cloning: A Laboratory Manual. 3rded. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, L.I. (2000)). For hybridization with shorter nucleic acid molecules, i.e. oligonucleotides, plot with unpaired bases (mistletoe) becomes more important and the length of the oligonucleotide determines its specificity.

Preferably, the minimum length of a nucleic acid molecule capable of hybridization is at least about 12 nucleotides; more preferably at least about 18 nucleotides; more preferably the length is at least about 24 nucleotides, and most preferred at least 36 nucleotides. In a specific embodiment, the term "standard hybridization conditions" refers Tm55°C, and conditions of use of the Oia, previously established. In another specific embodiment, the stringent conditions indicate Tm65°C conditions for hybridization and washing, respectively.

"Coding sequence" is a DNA or a sequence encoding a" private protein or peptide, is a DNA sequence which is transcribed and translated into a polypeptide in vitro and in vivo under the control of appropriate regulatory elements.

The boundaries of the coding sequence are determined by a start codon at the 5'end and a stop codon at the 3'-end. The coding sequence can include, without limitation, prokaryotic sequences, cDNA from eukaryotic mRNA, the sequence of genomic DNA from eukaryotic (e.g. mammalian), and even synthetic DNA sequences. The sequence of the transcription termination will usually be located on the 3'-end of the coding sequence.

The term "reasonably linked" refers to the location of elements, which describes the components are placed so as to perform their usual functions. Thus, the control elements are actually associated with the coding sequence that may affect expression of the coding sequence. It is necessary that the control elements are not located adjacent to the coding sequence as long as the any act, controlling their expression. For example, intermediate, even netransliruemye, transcribed sequences can be present between the promoter and the coding sequence and the promoter can still be regarded as "reasonably linked" to the coding sequence.

The term "heterologous nucleotide sequence"as used here, refers to a nucleotide sequence that is added to the nucleotide sequence of the present invention recombination ways to form a molecule of nucleic acid, which is not actually formed in nature. Such nucleic acid molecules may encode fusion proteins (i.e. chimeric). Thus, the heterologous nucleotide sequence can encode the peptides and/or proteins that have regulatory and/or structural properties. In another such embodiment, the heterologous nucleotide sequence can encode a protein or peptide that functions as a means of detecting the protein or peptide encoded by a nucleotide sequence of the present invention, after the molecule is expressed recombinant nucleic acid. In another embodiment, the heterologous nucleotide sequence can function is to operate as a means for detecting the nucleotide sequence of the present invention. Heterologous nucleotide sequence may contain non-coding sequence, including the restriction sites, sites regulation, promoters and the like.

Used herein, the term "fusion protein" or "peptide merge" is used interchangeably and encompass "chimeric proteins and/or chimeric peptides and fusion "indinavir proteins/peptides. Protein fusion contains at least one part protein dog MONDAY the present invention, coupled peptide bond to at least a section of another protein, for example, nanobacteria protein MONDAY, and/or contains a combination of two or more non-adjacent parcels dog squirrel MONDAY, for example, epitopes that are not commonly found in adjacent sequential order in dog polypeptide MONDAY (for example, protein a merger of 10 amino acid residues, which consists of amino acid residues 71-75 and 101-105 of SEQ ID NO: 2, connected by a peptide bond). In preferred versions of the invention, the section(s) of dog protein MONDAY are functional, for example, have their antigenicity. Proteins mergers can also contain protein marker, or a protein that facilitates the isolation and/or purification (e.g., an end tag FLAG, see the examples below) and/or antigenicity protein dog MONDAY this is subramania. Sequence nanobacteria MONDAY can be at the N - or C-end sequences dog MONDAY.

Recombinant DNA molecule encoding a fusion protein of the present invention, for example, can contain a sequence encoding at least the plot nanobacteria protein MONDAY, the joint reading frame with the coding sequence of the canine protein MONDAY, may further encode a cleavage site for a specific protease, such as thrombin or Factor XA, preferably at or near the junction sequences dog MONDAY and nanobacteria MONDAY. In a specific embodiment, a fusion protein is expressed prokaryotic cells. Such a fusion protein can be used to highlight dog MONDAY the present invention through the use of columns for affinity chromatography, specific protein and/or end tag, "sewn" to the dog MONDAY (see examples below).

Purified protein dog MONDAY, for example, may be separated from the fusion protein through the use of proteolytic enzymes and cleavage site, which, for example, referred to above.

"Vector" or "vector for replication" is a replicon, such as plasmid, virus, phage or cosmid, to which may be attached or vstall the n different segment of DNA, so that it will lead to the replication of the attached segment. The term also includes a replicon, which includes inserted or attached segment of DNA of interest.

Vectors that can be used in this invention include bacterial plasmids, viruses, bacteriophages, DNA fragments for insertion and other vectors that can carry out the integration of nucleic acid into the host genome. Plasmids are the most commonly used vector, but all other vectors that have equivalent functions and known or become known in science and technology, suitable for use in the present invention (see, e.g., Pouwels et al. Cloning Vectors: A Laboratory Manual, 1985, Supplement, Elsevier, N.Y.; Rodriguez et al., (eds.). Vectors: A Survey of Molecular Cloning Vectors and Their Uses, 1988, Buttersworth, Boston, MA).

The insert DNA that encodes a protein dog MONDAY the present invention, the vector easily occurs when the ends of the DNA molecule and the vector comprise compatible restriction sites. If this cannot be done, then it may be necessary to modify the ends of the DNA and the vector by cutting the single-stranded DNA using restriction endonucleases, so as to form blunt ends, or to reach the same result through completion of single-stranded DNA using a suitable DNA polymerase. As an alternative what about the option desired sites can be obtained, for example, by ligating nucleotide sequences (linkers) onto the ends. Such linkers may comprise specific oligonucleotide sequences that define the desired restriction sites. The restriction sites can also be obtained by use of the polymerase chain reaction (PCR) (see, for example, Saiki et al., Science 239:487 (1988)). Split vector and the DNA fragments can also be modified, if required, through a homopolymer lengthening of the ends of the molecule. Recombinant vector for the expression used in this invention are typically self-replicating constructs DNA or RNA containing a nucleic acid encoding the protein dog MONDAY the present invention and/or antigenic fragment, usually really linked to suitable genetic control elements that are able to regulate the expression of a nucleic acid in a compatible host cells. Control of genetic elements may include system prokaryotic promoter or a eukaryotic promoter to control expression and usually include a transcriptional promoter, an optional operator to control the start of transcription, transcription enhancers to increase the level of expression of mRNA sequence, which is derouet a suitable binding site of the ribosome, and sequences that terminorum transcription and translation. Vector for expression may also contain the replication origin, which allows the vector to replicate independently of the host cell.

Expression of nucleic acids encoding protein dog MONDAY according to the invention can be carried out using conventional methods in prokaryotic and eukaryotic cells.

"Host cell" is a cell that contains or can contain and Express the exogenous nucleic acid molecule, either temporarily or permanently. The cell has been "transformed" by exogenous DNA by the introduction of such exogenous DNA for the cell membrane. Exogenous DNA can be included or not included (covalently to contact) in the chromosomal DNA, altering the host cell genome. For example, in prokaryotes and yeast exogenous DNA can be inserted in the episomal element, such as a plasmid. As for eukaryotic cells, the stable transformed cell is a cell in which the exogenous DNA has been inserted into the chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of eukaryotic cells to the formation of cell lines or clones contained in the population of daughter cells containing ekzohenno the DNA.

Prokaryotes include gram-negative and gram-positive organisms, for example E. coli and .subtilis. The eukaryotes include cell lines derived from tissues and cells of animals, from animals that are not relevant to mammals, such as insects and birds, and mammals, such as humans, primates, rodents.

Prokaryotic system the host - vector" includes a large variety of vectors for many different species. Vectors for DNA amplification include pBR322 and many of its derivatives, or 42b(+)vector for expression (Novagen).

Commonly used prokaryotic sequences control gene expression include promoters, including promoters derived from β-lactamase and lactose promoter (Chang et al., Nature, 198:1056 (177)), for example, the pUC series, system tryptophan promoter (trp) (Goeddel et al., Nucleic Acids Res. 8:4057 (1980)), for example, pBR322-trp, promoter λ PL(Shimatake et al., Nature, 292:128 (181)), promoters λ PP or pR (pOTS), arabinose-inducible promoter (Invitrogen), tac promoter (De Boer et al., Proc. Natl. Acad. Sci. USA 292:128 (1983)), the promoters ipp (the pIN-series); or hybrid promoters such as ptac (pDR540). Numerous other vectors for expression containing such control sequences, also known in this area and are available commercially (see, for example, Brosius et al., "Expression Vectors Emplying Lambda, trp-, lac - and lpp-derived Promoters", Rodriguez and Denhardt (eds.) Vectors: A Survey of Molecular Cloning Vectors and Their Uses, 1988, Buttersworth, Boston, p.205-236).

In General, the equivalent of a suitable E. coli vector, which can be used in other prokaryotes, can also be used for protein expression MONDAY the present invention.

Yeast, as well as the culture of cells of higher eukaryotes, are also considered as hosts for production of recombinant canine protein MONDAY according to the invention and/or antibodies to canine MONDAY, and/or fragments of these antibodies. Although it can be used to culture cells of any eukaryotic, including baculovirus systems for the expression in insect, preferred are mammals. Transformation or transfection and propagation of such cells has become a routine procedure. Examples of the used cell lines are HeLa cells, cell lines Chinese hamster ovary (Cho cell line kidney of young rats (BRK), lines, insect cells (e.g., SF9), cell line birds (e.g., DF-11), cells of the kidneys of cattle Madine-Darby (MDBK)cells, kidney dogs Madine-Darby (MDCK)cells, Vero, SOME 23 cell lines and cells of the monkeys (COS).

Vector for expression in such cells typically include, for example, a replication origin, a promoter, a site of translation initiation sites of RNA splicing (ISOE is lovanii genomic DNA), the site of polyadenylation and termination of transcription. These vectors typically contain gene selection or gene amplification. Suitable vectors for the expression can be plasmids, viruses or retroviruses carrying derived from the promoter, for example, from sources such as adenovirus, SV40, parvovirus, a virus for vaccine or cytomegalovirus. Examples of suitable vectors for expression include pCR®3.1, pCDNAl, pCD (Okayama et al., Mol Cell Biol. 5:1136 (1985)), pMC1neo Poly-A (Thomas et al., Cell 5:503 (1987)), pUC19, pREP8, pSVSPORT and their derivatives, and baculovirus vectors, such as RAS and RAS.

After the expression of protein dog MONDAY according to the invention can be purified according to standard procedures in this area, including pereosazhdeniya ammonium sulfate, affinity chromatography, column chromatography and the like (see, generally, R.Scorpes. Protein Purification, Springer-Verlag, N.Y. (1982)). Clean enough mixture for at least 90 to 95% homogeneity are preferred, and 98 to 100% or more homogeneity most preferred for use in pharmacology. Clearance may be partial or until the desired homogeneity. If the dog's MONDAY must be used therapeutically, protein should be sufficiently free of endotoxin. The strategy of selective purification MONDAY after the expression on the affinity column with antibodies to MONDAY or receptors available for MONDAY received what I purified protein dog MONDAY.

The purification methods are well known in this area. For example, nucleic acids can be purified by presidenial, chromatography, ultracentrifugation, and other Proteins and polypeptides, and peptides can be purified in a variety of ways, including without limitation, preparative disc-gel electrophoresis, isoelectric focusing, HPLC, HPLC with reversed phase, gel filtration, ion exchange and substitution chromatography, pereosazhdeniya and vasilevousa chromatography, extraction and countercurrent distribution. For several reasons it is preferable to obtain the polypeptide in a recombinant system in which the protein contains an additional end tag that facilitates purification, such as, for example, but without limitation, as polyhistidine sequence or a sequence that specifically binds with the antibody, such as FLAG® and GST. The polypeptide can then be isolated from the crude lysate of the host cells using chromatography with a suitable matrix for the solid phase. Alternatively, as agents for cleaning can be used antibodies or their binding fragments of the polypeptide.

The solvent and the electrolyte are usually in a biologically acceptable buffer, of the type which is used to preserve biological activity, is usually in the approximation contain physiological solvent is water based. Typically, the solvent has a neutral pH, typically between 5 and 10, and preferably only about 7.5. in some cases, there may be added one or more detergents, usually soft and adenocarinoma, for example, CHS (hemisuccinate cholesterol) or CHAPS (3-(3-cholamidopropyl)dimethylammonio-)1-propane sulfonate, or a sufficiently low concentration to avoid considerable damage to the structural or physiological properties of the protein. In other examples, can be used harsh detergent to achieve significant denaturation.

Alternatively, functional heterologous proteins from E. coli or other bacteria can be isolated from Taurus inclusion through solubilization using hard denaturing agents, and subsequent refolding. Known in this area denaturing agents include, for example, urea, potassium thiocyanate, guanidine chloride (GuHCl), potassium Iodate, sodium iodide, and combinations of them. Preferably, GuHCl is used as a reducing agent, for example, at a concentration of approximately 6 to 8 M, under alkaline conditions, for example, a pH of approximately 8. Optional uses a different reducing agent, dithiothreitol (DTT), either alone or in combination with GuHCl. When using the DTT concentration range simply by example is approximately 5 mm to 0.5 mm DTT. During phase solubilization, well known in this field, the reducing agent must be present to separate or denaturation disulfide bonds. One buffer for sample contains 0.1 M Tris pH 8.0, 8 M guanidine, 2 mm EDTA and 0.3 M DTE (dithioerythritol).

Resaturate usually accompanied by breeding (for example, 100 times) of denatured and reduced protein in the buffer for refolding in the presence of an oxidizing agent. Can be used any known in this area suitable oxidizing agent, if it allows you to place the correct refolding with high yield. For example, oxidation and refolding can be achieved with low molecular weight thiol reagents in reduced and oxidized form, as described by Saxena et al., 1970, Biochemistry 9:5015-5021, incorporated herein by reference, and especially as described in Buchner, et al., above. Resaturate usually accompanied by dilution (e.g., 100 times) of denatured and reduced protein in the buffer for refolding. One of the sample buffers for refolding is a Tris HC 1100 mm, pH 10.0, 25 mm EDTA, NaCl 0.1 M, GSSG 551 mg/l, 0.5 M arginine. GSSG is the oxidized form of glutathione.

The size and structure of the polypeptide will be substantially stable condition and is usually not in the denatured state. The polypeptide may be associated with other polypeptides in quadratics the th structure, for example, in order to provide the solubility or lipids or detergents.

Substantially pure, e.g., in relation to protein, usually indicates that the protein does not contain other contaminating proteins, nucleic acids or other biological objects, obtained from the original organism. Purity can be analysed using standard methods, typically by weight, and should be at least 40% purity, generally approximately 50% purity, often at least 60% purity, typically at least about 80% purity, preferably at least about 90% purity and in the most preferred embodiments, performing at least 95% purity. Often added carriers or fillers. Purity can be assessed by using chromatography, gel electrophoresis, immunoassay, composition analysis, biological analysis, and other methods known in this field. From the functional aspects of selected protein dog MONDAY according to the invention is sufficiently separated from other proteins, including precursor protein dog MONDAY and/or Mature protein dog MONDAY, so he is able to induce an immune response that is specific for protein dog MONDAY.

The solubility of the polypeptide or fragment depends on the environment and the polyp is Chida. Many parameters affect the solubility of the polypeptide, including temperature, electrolytic environment, size, and molecular characteristics of the polypeptide, and the nature of the solvent. Typically, the temperature at which the polypeptide is used, lies in the range from 4 to 65°C. Typically, the temperature is more than 18°C. For diagnostic purposes, the temperature is usually room or warmer, but less than the denaturation temperature of the components of the test. For therapeutic purposes the temperature is usually equal to the body temperature, usually from 36 to 40°C (e.g., 39°C for dogs), although under certain conditions the temperature may be raised or lowered in situ or in vitro.

Used herein, the term "antigenic fragment" in relation to a specific protein is a fragment of this protein (including large fragments, which missed only one amino acid of a full length protein), which is antigenic, i.e. capable of specific interaction with the antigen-recognizing molecule of the immune system, such as immunoglobulin (antibody) or T-cell antigen receptor.

For example, antigenic fragment of dog MONDAY the present invention is an antigenic fragment of a dog MONDAY. Such fragments are not immunogenic themselves, is able to induce an immune response in the absence of the carrier, so that they can the be used to produce antibodies to the protein MONDAY after conjugation fragment with a carrier molecule for immunization. Preferably, however, to antigenic fragment of the present invention was immunodominant for antibodies and/or for detection of T-cell receptor.

In the private embodiment, the antigenic fragment of the protein dog MONDAY contains from 5 to 150 amino acid residues. In a particular embodiment, the antigenic fragment of dog MONDAY contains more than 120 amino acid residues. In another embodiment, the antigenic fragment of dog MONDAY contains from 10 to 120 amino acid residues. In another embodiment, the antigenic fragment of dog MONDAY contains from 20 to 100 amino acid residues. In another embodiment, the antigenic fragment of dog MONDAY contains from 25 to 75 amino acid residues.

Antigenic fragment of dog MONDAY can be obtained from a recombinant source of protein obtained from a natural source, or by chemical synthesis. Moreover, antigenic fragment can be obtained by subsequent proteolytic cleavage, dog MONDAY or its fragment by recombination expression or, alternatively, it can be synthesized de novo, for example, by peptide synthesis.


The present invention further provides vaccines, which include an effective amount of proteins is MONDAY the present invention, one or more antigenic fragments, or a combination of full-size protein and one or more of these fragments. For example, protein dog MONDAY and/or its fragments, such as numbered in table 2 below, can be included in compatible with a protein or peptide vaccine of any composition. Vaccines such structure is well known in this field and can, optionally, include, for example, a physiologically compatible buffer and salt solutions and the like, as well as pharmacologically acceptable adjuvants, such as CARBOPOL® or Emulsigen®.

Mixture for vaccination can be used for the purpose of endogenous antibodies to MONDAY the dog that needs it, for example, in order to treat the clinical manifestations of the disease or disorder, which are responsible for regulating the activity of afferent pathways MONDAY in dogs. Alternatively, or in conjunction with this, the vaccine of the present invention can also be used to identify anticigarette for screening and/or identification of a dog's MONDAY, for example, as an auxiliary agent in China to identify dogs with overexpression MONDAY.

Peptides from MONDAY, such as shown in table 2 below, and their variants, can be used as immunogens both individually and in various who's combinations. Such peptides can be optionally linked to each other and/or with larger proteins, known as carriers, as by chemical methods, or by using recombinant DNA. Action media aimed at increasing recognition of the peptide by the cell of an animal host as targets for the immune response and increase the immunogenicity of peptides MONDAY. Separate carriers known in this area and include tetanus toxoid or non-toxic C-terminal fragment of tetanus toxoid, diphtheria toxoid, PhoP protein, hemocyanin lymph snails, beta-galactosidase, the gD protein of the virus, BHV-1, the G protein of rabies virus, the F protein of a virus of canine plague and synthetic media, the resulting polymerization known universal epitopes T-cells.

Peptides MONDAY used as immunogen, can be selected from table 2, and their variants, using known algorithms, which evaluate properties such as the availability of surface natural protein MONDAY, hydrophilicity, the mobility of atoms and antigenicity. Epitopes on the peptides listed in table 2, and their variants can also be selected on the basis of their reactivity when interacting with polyclonal or monoclonal antibodies that react with native proteins MONDAY, and especially those antibodies to the e can neutralize the bioactivity MONDAY. Such antigens may include synthetic peptides derived from sequences that are detected using standard peptide synthesis Protocol and/or alternatively, can be fragments derived from recombinant or natural protein MONDAY.

Pharmacologically acceptable adjuvants of the present invention can be obtained from any source, including natural, recombinant and/or synthetic chemically etc. are examples of chemical compounds used as adjuvants include, but without limitation, aluminum compounds, converted or not converted in the course of metabolism, blakolmer, ISCOM (immunostimuliruyushchie complexes), vitamins and minerals (including, but without limitation, vitamin E, vitamin a, selenium and vitamin B12) and Quil A (saponins), complete Freund adjuvant, polymers of acrylic acid cross sewn together with polyalkylimide esters or diphenylglycine, as sold under the trademark CARBOPOL® (e.g., CARBOPOL® 941), and was uniformly dispersibility in the form of drops of oil micron size in water (e.g., as sold under the trademark Emulsigen®). Additional examples of adjuvants, which are sometimes referred to as a specific Immunostimulants include components of the cell wall of bacteria and fungi (EmOC is emer, lipopolysaccharides, lipoproteins, glycoproteins, muramylpeptide, beta-1,3/1,6-glucan), a variety of complex hydrocarbons derived from plants (e.g., glucan, acemannan), various proteins and peptides derived from animals (e.g., hormones, cytokines, co-stimulatory factors) and new nucleic acids derived from viruses and other sources (e.g., double-stranded RNA, CpG). In addition, any number of combinations of the above compounds may provide an adjuvant effect, and therefore can form the adjuvant of the present invention.

Vaccines of the present invention may be administered by any route, including intramuscular, subcutaneous injection, intravenous injection, intradermal injection, oral, intranasal purpose, and combinations thereof.


The present invention also includes polyclonal and monoclonal (mAb) antibodies that are specifically associated with a dog's protein MONDAY according to the invention. Used herein, the term "antibody" means an immunoglobulin and/or its fragments. Naturally occurring immunoglobulin consists of one or more polypeptides substantially encoded in the genes of immunoglobulins. The recognized immunoglobulin genes include the genes Kappa, lambda, alpha, gamma, Delta, Epsilon and the Yu constant regions as well as numerous gene variable regions of immunoglobulin. The antibody or antibody according to the present invention also include frumenty antibodies, or antigen-binding fragments, e.g., constructed of single-chain Fv, Fab and F(ab')2 binding proteins (e.g., Huston et al., Proc. Natl. Acad. Sci. USA, 85, 5879-5883 (1988), Bird et al., Science, 242, 423-426 (1988), included here as a reference), as well as bifunctional hybrid antibodies (e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)). See, mostly. Hood et al., Immunology, Benjamin, N.Y., 2nded. (1984), Harlow and Lane, Antibodies. A Laboratory Manual, Cold Spring Harbor Laboratory (1988), Hunkapiller and Hood, Nature, 323,15-16 (1986), all included here as a reference.

For example, serum obtained from animals immunized with canine protein MONDAY according to the invention, using standard methods, can be used directly, or the IgG fraction can be isolated from serum using standard methods, such as plasmapheresis or adsorption chromatography with specific to IgG adsorbents, such as immobilized protein a or protein G. alternatively, can be prepared monoclonal antibodies and of these mAb may not be necessarily obtained antigennegative fragments or recombinant binding proteins. Such mAb or fragments thereof may not necessarily be humanitarian or "osabutey" by means known in the field of methods or direct modification.

Used herein, the term "epitope-specific" dog antibody to MONDAY is an antibody that interacts with a fragment of a dog's MONDAY, which contains one or more of the five following amino acid sequences: SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 or SEQ ID NO: 34; and which further bind to the protein having the amino acid sequence of SEQ ID NO:2, and/or a protein having the amino acid sequence of SEQ ID NO: 2, excluding the signal sequence 28 amino acid residue. In the private embodiment, the epitope-specific antibody to canine MONDAY is a monoclonal antibody.

Hybridoma producing mAb that selectively bind protein dog MONDAY according to the invention are obtained using well known methods. Usually the process involves merging immortalizing cell lines with b-lymphocyte that produces the desired antibody. In the alternative, can be used in ways not involving the merger, to receipt of the immortalized antibody-producing cells, for example, virus-induced transformation (Casali et al., Science 234:476 (1986)). Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human. Most hour what about the use of rat or mouse myeloma cells for convenience and availability.

Ways to get antelo-producing lymphocytes mammals injected antigens, are well known. Commonly used peripheral blood lymphocytes, if you are using cells of human origin, or spleen cells or lymph nodes using antibodies from mammalian cells, but not human. Pet-owner is injected repeated doses of purified antigen (human cells sencibilisiruet in vitro) and animal gets the opportunity to produce the desired antibody-producing cells before they will grow to confluence using immortalizing cell line. How to merge is also well known in this area, and mainly includes mixing the cells with the merge agent, such as polyethylene glycol.

Hybridoma are selected by standard procedures, such as HAT (gipoksantin-aminopterin-thymidine) selection. Those that secrete the desired antibody are selected using the standard immune tests, such as Western blotting, ELISA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay), and the like. Antibodies are recovered from the medium using standard protein purification protocols (Tijssen, Practice and Theory of Enzyme Immunoassays (Elsevier, Amsterdam, 1985)). There are many references to provide guidance for the application of the above who's methods (Kohler et al., Hybridoma Techniques (Cold Spring Harbor Laboratory, New York, 1980), Tijssen, Practice and Theory of Enzyme Immunoassays (Elsevier, Amsterdam, 1985), Campbell, Monoclonal Antibody Technologies (Elsevier, Amsterdam, 1984), Hurrel, Monoclonal Hybridoma Antibodies: Techniques and Applications (CRC Press, Boca Raton, FL, 1982)). Monoclonal antibodies can also be produced using well-known phage libraries (see, e.g., Huse et al., Science 246:1275 (1989); Ward et al., Nature, 341:544 (1989)).

The antibodies thus obtained, polyclonal or monoclonal, can be used, for example, in an immobilized form, associated with the solid substrate is well known methods for purification of protein dog MONDAY through immunoaffinity chromatography.

Antibodies to canine squirrel MONDAY can also be used, labeled or unlabeled standard methods, as the basis for immunological tests for the detection or determination of the quantity of protein dog MONDAY. In particular label used depends on the type of immune test. Examples of labels may serve as, but not limited to, a radioactive label, such as32P,125I3H and14C; fluorescent labels, such as fluorescein and its derivatives, rhodamine and its derivatives, dansyl and umbelliferone; chemiluminescent labels such as luciferin, and 2,3-dihydropteridine; and enzymes, such as horseradish peroxidase, alkaline phosphatase, lysozyme, and glucose-6-phosphate dehydrogenase.

Antibodies which may be marked by such marks by using known methods. For example, cross-linking agents such as aldehydes, carbodiimides, timelimit, imidate, suktinimida, bis-diazobenzene and the like, can be used for labeling antibodies, fluorescent, chemiluminescent, or enzymatic labels. The main methods included in this process, well known in this field and are described, for example, Immunoassay: A Practical Guide, 1987, Chan (Ed.), Academic Press, Inc., Orlando, FL. Such immune tests can be conducted, for example, the fractions obtained during purification of receptors.

Antibodies of the present invention can also be used to identify specific cDNA clones expressing the protein dog MONDAY in systems for cloning and expression. Neutralizing antibodies specific to the ligand-binding site of the receptor, can also be used as antagonists (inhibitors) to block or weaken the function of the protein dog MONDAY. Such neutralizing antibodies can be easily identified by conventional experiments.

The antagonistic activity of the protein dog MONDAY may be accompanied by the use of whole antibody molecules or well-known antigen-binding fragments, such as Fv, Fab and F(ab')2 fragments. The definition of such fragments can be found in the above-described or, for example, in Klein, Immunology (John Wiley, New York, 1982), Parham, Chapter 12, Weir ed. Immunochemistry, 4thEd. (Blackwell Scientific Publishers, Oxford, 1986). Applying and getting fragments of antibodies has also been described, for example, Fab fragments (Tijssen, Practice and Theory of Enzyme Immunoassays (Elsevier, Amsterdam, 1985), Fv fragments (Hochman et al., Biochemistry 12:1130 (1973); Sharon et al., Biochemistry 15:1591 (1976); Ehrich et al., U.S. Patent No. 4355023) and complete antibody molecules (Auditore-Hargreaves, U.S. Patent No. 4470925). Methods for obtaining recombinant Fv fragments, based on the sequences of the heavy and light chain variable regions of known antibodies, have been described, for example, the Moog et al. (U.S. patent No. 4642334) and Plückthun (Biotechnology 9:545 (1991)). Alternatively, they can be synthesized chemically using standard methods.

The present invention also encompasses anti-idiotypical antibodies as polyclonal and monoclonal, which are produced when using the above antibodies as antigens. These antibodies are useful because they can mimic the structures of ligands. Antibodies nanobacteria origin or from nedobachii hybridoma systems can be optionally designed in order to translate them into substantially panthenol form with the introduction of the dog, i.e. can be "osabutey". The process of modification of monoclonal antibodies from the animal, in order to translate it in less immunogenic form for therapeutic used the I in humans (humanization), actively developed and described in several publications (e.g. Antibody Engineering: A practical Guide. Carl A.K. Borrebaeck, ed. W.H. Freeman and Company, 1992; Reichman, L. et al., "Reshaping human antibodies for therapy". Nature 332:323-327 (1988)). Alternatively, monoclonal antibodies nanobacteria origin, such as murine monoclonal antibodies, modified with chimeric canine antibodies or their sequences in order to obtain antibodies are less immunogenic for the recipient than a standard mouse antibodies. See, for example. U.S. patent No. 5593861, "Dog-Mouse Heterohybridoma and Gene Fragment Coding for the Constant Region of Canine Immunoglobulins", which is included here as a reference.

In addition, Wasserman and Capra (Biochem. 16:3160 (1977)identified amino acid sequence of heavy chain variable regions as dog IgM and canine IgA. These researchers further identified amino acid sequence of the Kappa light chain of canine IgA (Wasserman and Capra, Immunochem. 15:303 (1978)). McCumber and Capra (Mol. Immunol. 16:565 (1979)have determined the complete amino acid sequence of canine mu chain. Tang et al., (Vet. Immunology Immunopathology 80:259 (2001)identified one cDNA sequence of the gamma chain of canine IgG-A and four protein sequence of the gamma chain of canine IgG-A. Tang et al., above, then describe the PCR-amplification of cDNA from a cDNA library of the spleen of the dog with degenerate primers designed on the conservative region of IgG-h the rights, mice, pigs and cattle. Moreover, Krah, et al. (U.S. Publication No. 20040181039 published September 16, 2004 and incorporated here by reference) describes in detail one of the processes "isobutylene" nedobachii antibodies.

Selection of dog gene MONDAY

A. Initial attempts

Initial attempts to identify dog MONDAY were based on the mapping of the cloned cDNA sequences MONDAY of human and mouse cDNA sequences MONDAY rats, chimpanzees and rhesus macaques, which are collected in BLAT (public genomic database of the University of California, Santa Cruz). MONDAY chimpanzee was 100% homologous MONDAY person, while MONDAY rhesus macaques was homologous MONDAY man 90% (12 of 151 amino acid residues did not coincide) in the Mature state. However, protein sequence and cDNA MONDAY humans and primates strongly disagree with sequences MONDAY mouse. Sequence of cDNA MONDAY between human and mouse have only 43% homology that does not allow cloning through lax interspecific hybridization between these species. Moreover, the sequence MONDAY rats showed 39 changes of 121 amino acid residues in the Mature protein compared to murine MONDAY that shows that even in closely related species of rodent sequences MONDAY considerably according to the evidence.

Unfortunately, as defined here, it was proved that the sequence of canine MONDAY at odds with all mouse sequences and sequences in primates. So getting a dog MONDAY through lax interspecific hybridization was considered unsuccessful. Indeed, the primers are designed to attempt the cloning of the canine ortholog MONDAY by the method of "nested" PCR, sequences of human, mouse, rat and monkey, not identified a single band corresponding dog MONDAY.

C. the Successful selection of the gene of canine MONDAY

When searching for dog sequences in the available genomic database (derived from the full original genomic sequence, the open access by the University of California, Santa Cruz) sequence MONDAY of man led to partial identification 1 and 4 exons dog MONDAY. In short, there were identified some successful attempts of finding a significant homology of sequences in the original search (see successful attempts ("hits") 1-6 below). These sequences were collected and used as query expansion and collection of partial sequences dog MONDAY in electronic form.

A comparison of the electron-designed sequence with MONDAY humans, monkeys, rats and mice showed the conservative boundaries of exons/introns and essential identity, leading to the identification of this sequence as part of the canine ortholog of MONDAY. Primers for PCR were subsequently constructed on the basis of this discovery and used for amplification of missed segments of the gene. Using duplicated "nested" PCR were obtained from two partially overlapping clone from a cDNA library of activated peripheral mononuclear blood cells (RVMS) dogs. Additional attempts to detect the complete cDNA sequence of a dog MONDAY by "nested" PCR or attempts to extend the sequence in the direction from 5' to 3' end were unsuccessful. However, repeated rounds of database lookup using extended sequence information obtained from these clones according to the original genomic sequence dogs (University of California, Santa Cruz) in combination with manual collection of raw DNA sequences from this library led to the electronic set of full-size cDNA dog MONDAY. Physical clone of this sequence cDNA was then synthesized in vitro using DNA synthesizer. the conclusion, using current and installed in the field techniques of molecular cloning was not possible to obtain a sequence of canine MONDAY directly from sequences of human, mouse, rat or monkey. Only exact duplicate searches the database using accumulated MONDAY genes of human, mouse, rat and NHP, using approximations of the boundaries of exons/introns and identity sequence from the genomic database, in combination with the techniques of molecular cloning by PCR, resulted in the identification of the gene encoding canine MONDAY. When getting a dog MONDAY showed 58 changes from 132 compared with the amino acid sequence of the Mature protein MONDAY man (61% identity) and 83 changes from 129 compared to the amino acid sequence of the Mature protein MONDAY mouse (33% identity) (see below).

Thus, overcoming the previously mentioned difficulties, the present invention provides DNA sequences encoding canine MONDAY and encoded protein dog MONDAY. Dog protein MONDAY and some of its fragments are good antigens, i.e. immunogenum, produced antibodies to various epitopes of the protein to both linear and conformational. DNA coding for the maintenance dog MONDAY, also important in ensuring vectors and host cells to obtain protein MONDAY for immunization and/or as an object for study, as well as to obtain vaccines, DNA-based, to generate antibodies to MONDAY, or in the form of "naked" DNA, either in the form of plasmids, either in the form of a viral vector suitable for expression MONDAY cells of the vaccinated animal. Thus obtained gene sequences dog MONDAY is illustrated in FIG. 8A (SEQ ID NO: 1)and predicted downregulation of protein MONDAY is illustrated in FIG. 8B (SEQ ID NO: 2). Residues 1-28 represent a signal sequence, and remains 29-155 represent the Mature protein.

Research to identify homologous MONDAY proteins

The present invention also provides proteins MONDAY, which contain the amino acid sequence of SEQ ID NO: 2, excluding the signal sequence of 28 amino acid residues, which when applied to dogs as a vaccine causes the production of antibodies that bind to canine protein MONDAY, containing the amino acid sequence of SEQ ID NO: 2. there are also antigenic fragments of that protein MONDAY.

Indeed, one of the variants show that the estimated protein MONDAY is a protein MONDAY the present invention is to ensure whether this protein to produce antibodies, associated with the dog MONDAY containing the amino acid sequence of SEQ ID NO: 2. One of such ways is vaccination (i.e. injection) dogs with different doses, lying in the range from 5 to 500 μg of the intended antigen MONDAY-GST. Such antigens can be prepared based adjuvant containing aluminum hydroxide, such as Rehydrogel. Dogs vaccine is then injected intramuscularly three times: at day 0, day 21 and day 42. Serum samples collected from vaccinated and control (non-immunized) dogs on day 0, 21, 42 and 63.

Induction of antibodies in dogs vaccinated with antigens, can be assessed using ELISA in accordance with the following Protocol: protein dog MONDAY, containing the amino acid sequence of SEQ ID NO: 2, was dissolved at a concentration of 5 μg/ml in covering the buffer (sodium bicarbonate pH 9.0) and was uncovered 100 μl/well in 96-well tablet (Pierce).

The plates were incubated at 4°C over night. Then the tablets three times washed with phosphate-saline buffer containing 0.05% Tween-20 (PBST). Then to each well was added 200 μl of blocking buffer (2% skim milk in PBST) and the plates were incubated at room temperature for 60 minutes, the Tablets were then washed three times with PBST. Then add 100 μl/well of a solution of the antisera tested dogs in water 1:100 in the top row of the corresponding Lu is OK. Serum samples were then diluted 10 times to the corresponding position of the tablet. After incubation tablets at room temperature for 60 min tablets were washed three times with PBST.

Then to each well was added a solution of 1:20000 horseradish peroxidase conjugated goat antibody to dog IgG (Bethyl Laboratories). Then the plates were incubated at room temperature for 60 minutes and Then the tablets were washed three times in PBST was added to each well 100 μl of TMB substrate (3,3', 5,5' tetramethylbenzidine, Sigma Chemical Co., St. Louis, MO). Gave to develop qualitative reactions for 10-120 min at room temperature before stopping the reaction by adding 50 µl per well of 0.18 M sulfuric acid.

The optical density (OD) of all wells was determined at a wavelength of 450 nm using ELISA reader for tablets (Thermo Max; Molecular Devices, Sunnyvale, CA). Serum samples obtained from dogs vaccinated with putative antigens MONDAY, are detectivesyme and, therefore, the antigens are identified MONDAY as proteins of the present invention, when the analysis showed OD values equal to or greater than three times the background obtained from serum samples from dogs before immunization. Similarly, the titles of related antigens MONDAY can be determined based on the highest dilution of serum giving C is achene OD, equal to or greater than three times the background obtained from serum samples from dogs before immunization with these antigens.

Antibodies to specific epitopes of the protein dog MONDAY

Antibodies can be obtained for different epitopes of canine MONDAY proteins, including forms, polymorphs or allelic variants, and fragments thereof, both in naturally occurring form, and in recombinant forms. Additionally, antibodies can be obtained by dog MONDAY, either in their active forms, or inactive, including native or denatured option. Also discusses antiidiotypic antibodies.

Antibodies, including binding fragments and variants with single chain, to certain pre-antigens, can be obtained by immunization of animals dog MONDAY and/or its fragments, together with the standard in this area adjuvants and/or conjugated to immunogenic proteins. Immunized so dogs can be dogs, which were immunized in order to lower the activity of the dog MONDAY.

Suitable host, for example, inbred line of mice such as Balb/c, are subjected to immunization of the selected protein, typically using a standard adjuvant, and a standard immunization Protocol mice (see, Harlow and Lane, Id, above). Adjuvant can be assigned Clevo is the animal before in combination with or after administration of the vaccine.

Alternatively, a synthetic peptide derived from the sequences described herein, and conjugated to protein carrier, can serve as an immunogen. Polyclonal sera are collected and titrated with protein immunogen when the immune analysis, for example, solid-phase immune analysis with the immunogen immobilized on a solid substrate. Polyclonal antisera with a titer of 1*104or more, are selected and tested for their cross reactivity against other members of the family of IL-7, for example a murine IL-7, using immune competitive analysis, as described in Harlow and Lane, Id, above, SCR-573. Preferably, at least one of the other members of the family of IL-7 was used in this definition, for example, IL-7 primates. The members of the family of IL-7 can be produced as recombinant proteins and selected using standard molecular biological and protein chemical methods, as described here. Immune tests in the form of competitive binding can be used to determine cross-reactivity. For example, the protein SEQ ID NO: 2 can be immobilized on a solid substrate. Proteins used in the test, compete for binding of the antisera to the immobilized antigen. The ability to compete of the above proteins Savasana of the antisera to the immobilized protein is compared to the protein, containing the amino acid sequence of SEQ ID NO: 2. The percent cross-reactivity of the above proteins is calculated, using standard calculations. Those antisera that have less than 10% cross-reactivity with each of the proteins listed above, select and group. Cross-reactive antibodies are then removed from grouped sera using immunoabsorption with the above proteins.

Immunoabsorbant and grouped antisera are then used in a competitive binding immunoassay as described above to compare a second protein with the immunogenic protein (e.g., IL-7-like protein of SEQ ID NO: 2). To make this comparison, the two proteins tested in a wide range of concentrations and determined the required number of each protein, capable of 50% inhibition of the binding of the antisera. If the required amount of the second protein is less than twice the amount of protein from a selected protein or proteins than you want, then say that the second protein specifically binds to an antibody obtained by the action of the immunogen.

Antibodies of the present invention can also be used for diagnostic purposes. As a trap or non-neutralizing antibodies, they can be conducted by screening for the ability to bind antigens without ingibirovany the binding of the receptor. As neutralizing antibodies, they can be used in the analysis of competitive binding. They will also be used to detect and quantify protein dog MONDAY or its receptor (see, for example, Chan (ed. 1987) Immunology: A Practical Guide. Academic Press, Orlando, FIa.; Price and Newman (eds. 1991) Principles and Practice of Immunoassay. Stockton Press, N.Y.; and Ngo (ed. 1988) Nonisotopic Immunoassay. Plenum Press, N.Y). Cross-absorption, decomposition or other means provide a certain selectivity, for example, the unique characteristics of the species or found in other species. This can be the basis for tests to determine different groups of antigens.

Further, antibodies, including antigen-binding fragments of the present invention may be potential antagonists that bind to the antigen and inhibit functional linking, for example, with the receptor, which can cause a biological response. Further, these antibodies can be conjugated with drugs or other therapeutic agents, either directly or not directly by the linker, and can affect the targeting of drugs.

Synthetic peptide derived from the sequence described here and conjugated to protein carrier, can be used as an immunogen. In any case, fragments of the antigen can be attached to the other substances, in particular polypeptides, merged or covalently linked, for use as immunogens. Antigen and its fragments can be combined or covalently crosslinked with various immunogenum, such as hemocyanin lymph snails, bovine serum albumin, tetanus toxoid, etc. see Microbiology. Hoeber Medical Division, Harper and Row, 1969; Landsteiner (1962) Specificity of Serological Reactions. Dover Publications, New York; Williams, et al. (1967) Methods in Immunology and Immunochemistrv, vol.1, Academic Press, New York; Harlow and Lane (1988) Antibodies: A Laboratory Manual. CSH Press, NY, for descriptions of methods of preparing polyclonal antisera.

In some cases it is desirable to obtain monoclonal antibodies from various mammals, such as mice, rodents, primates, people, etc. Description of methods for preparing such monoclonal antibodies can be found, e.g., Stites, et al. (eds.) Basic and Clinical Immunology (4th ed.), Lange Medical Publications, Los Altos, Calif, in the references cited here (Harlow and Lane (1988) Antibodies: A Laboratory Manual. CSH Press; Goding (1986) Monoclonal Antibodies: Principles and Practice (2d ed.), Academic Press, New York) and particularly in Kohler and Milstein (1975) in Nature 256:495-497, which describe one method of obtaining monoclonal antibodies.

Other suitable methods include in vitro representation of lymphocytes to the antigenic polypeptides for the selection of libraries of antibodies in phage or similar vectors (see Huse, et al. (1989) "Generation of a Large Combinatorial Library of the Immunoglobulin Repertoire in Phage Lambda," Science 246:1275 - 1281; Ward, et al. (1989) Nature 341:544-546.]). The polypeptides and antibodies of the present invention can be used with or without modification, including the obtaining of chimeric antibodies, "isobutylene" or the humanization of antibodies.

Frequently, the polypeptides and antibodies of the present invention have been labelled compounds, which provide the detected signal. Such attachment can be made both covalently and ecovalence. There are a large number of labels and methods of conjugation and about them a lot and reported in the scientific and patent literature. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent areas, chemiluminescent areas, magnetic particles, and the like. Patents disclosing the use of such labels include U.S. patents№3817837; 3850752; 3939350; 3996345; 4277437; 4275149; and 4366241. Also recombinant or chimeric antibodies can be obtained by Cabilly, U.S. patent No. 4816567; Moore, et al., U.S. patent No. 4642334; and Queen, et al. (1989) Proc. NaVI Acad. Sci. USA 86:10029-10033; or obtained in transgenic mice, see Mendez, et al. (1997) Nature Genetics Vx 146-156. These references are incorporated herein by reference.

Antibodies of the present invention can also be used for affinity chromatography in the allocation of proteins. Columns can be prepared when antibodies are associated with a solid substrate (see Wilchek et al. (1984) Meth. Enzymol. 04:3-55). Alternatively, the antibody associated with the solid substrate, can be used for cleaning the respective antibodies. Antibodies obtained for each dog MONDAY, can also be used to obtain antiidiotypic antibodies. These antibodies will be used for detection or diagnosis of various immunological conditions associated with expression of the corresponding antigen.

Inhibition of RNA

Interference with RNA that encodes a dog MONDAY in the cells that produce dog MONDAY, is an additional means of inhibiting the biological activity MONDAY and, therefore, treatment of a number associated with MONDAY diseases, such as atopic dermatitis. For this purpose, chemically synthesized or cloned using a suitable vector for delivery, such as a plasmid or viral vector, double-stranded RNA molecules can be introduced into the cell, actively producing mRNA MONDAY, with the purpose of lowering the level of endogenous mRNA that encodes a MONDAY. After getting into the cell such RNA molecules (in the case of exogenously delivered molecules or RNA transcription, which is followed by introduction of a plasmid or viral vector in the desired cells), they are processed through the digestive activity of ribonuclease type III short nucleotide fragments, which are called miRNAs. Ethipramine miRNAs are then incorporated into the containing nuclease multiprotein complex, called RISC (PHK-inducing "silent" complex), which comes into an active state as a result of the unwinding of duplex miRNAs under the action of RNA-helicase. Then single-stranded fragments of miRNAs guide the RISC complex to the target mRNA, which is then cleaved and degraded under the action of their endonuclease activity of RISC.

In the more particular case of the plasmid containing the gene for MONDAY or fragments clone in any of a number of commercially available eukaryotic plasmids, where the transcription of the gene MONDAY or its fragments managed by an appropriate promoter, for example, CMV or SV40. Purified plasmid DNA (1-100 μg) and then injected into the injury site on the skin or in the area surrounding the damage to the skin, characteristic of atopic dermatitis. Introduction of plasmid DNA can then be repeated with the frequency required to achieve a significant reduction in mRNA MONDAY. This reduction can be estimated by obtaining a biopsy of skin from the affected areas and determine the level of mRNA MONDAY through methods such as quantitative PCR.

The following embodiments of the present invention are used to provide further understanding of the invention, but do not mean in any way limiting the scope of the invention.


Example 1

DNA and protein sequences dog MONDAY

Dog gene, expr shiroudi dog MONDAY, was identified using the method of successive iterations using the data found in electronic databases, and molecular biological methods, as described in detail above.


The sequence of the gene of canine MONDAY illustrates FIGA (SEQ ID NO: 1) and predicted downregulation of protein MONDAY illustrated FIGV (SEQ ID NO: 2). Residues 1-28 represent a signal sequence, and remains 29-155 represent the Mature protein.

Example 2

Cloning and expression of canine MONDAY

DNA encoding canine MONDAY, was identified, as described here, and cloned into the donor vector pDONR221 (Invitrogen Gateway System) standard methods in this area. The selection gene and cloning it into the donor vector was conducted by the research Corporation under contract, which is called DNA 2.0. and led to the construction of plasmids, named pDONR221.G03276, which contained the identified genomic gene dog MONDAY. DNA encoding the Mature (i.e., without the signal sequence) protein dog MONDAY was amplified by PCR from pDONR221.G03276 using primers that contained the V sites Nco I and EcoR, respectively.



#2: 5'-AAAATAGATATCTGAAATGCGACTGAAACGACG-3' (SEQ ID NO: 5). After receiving Nco I and EcoR V, PC the products were inserted into the sites Nco I and Sma I vector pIVEX 1.3 WG (Roche Applied Sciences, Cat# 3728803). This led to the construction of a plasmid containing a gene that encodes a Mature dog MONDAY, connected with six histidine residues at the C-end ("His6 tail"). The plasmid containing the correct sequence of the insert was named 1265-93 .D. Plasmid 1265-93 .D was used for the expression of MONDAY in the RTS Proteomaster Instrument according to the manufacturer's recommendations (Roche Applied Sciences, Cat# 3064859). As shown in FIG. 1, the band at @16 kDa apparent in tracks 2 and 4 (arrows). Analysis using Western blotting (FIG. 2A and 2B) shows that this band is specifically reacts with antibody to "His tail" (FIG. 2A) and with mouse monoclonal antibody specific for MONDAY man (FIG. 2B).

Example 3

Getting a dog MONDAY from host cells

In order to Express the recombinant protein MONDAY in E. coli, the nucleotide sequence encoding ctsv (i.e. MONDAY without nucleotides encoding the signal sequence)was amplified by PCR using the plasmid 1265-S as the sample together with the forward and reverse primers containing Ncol sites and Hind III, respectively.

Direct primer


Reverse primer


After dissolution Nco I and Hind III PCR products were inserted into the sites Ncol/Hindlll vector for expression pET42b(+) (Novagen). As a result, it is about process received plasmid, which encodes a Mature protein ctsv, coupled with GST-tail at the N-end and 6-His tail-end. The plasmid containing the correct sequence of the insert was named 1265-93 Century expression of the fusion protein GST-TSLP-His was carried out in E. coli BL21(DE3)/pLysS, which contained the gene for T7 RNA polymerase under control of the isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible lacUV5 promoter. The E. coli cells carrying plasmid 1265-93, were grown at 30°C until O.D. 600 of 0.6 and then protein expression was induced by adding 0.5 mm IPTG and further incubated at the temperature at 30°C for 2 hours. SDS-polyacrylamide gel electrophoresis revealed a protein band (arrow) with the correct mass (~61 kDa)present in the soluble fraction of E. coli (FIG. 3A). Analysis by Western blot showed that the expressed protein reacts with antibody to GST (Fig.3D). Protein GST-TSLP-His can be purified using Glutathione resin of Sepharose 4 (FIG. 3B). After additional purification resin Ni-NTA more protein GST-TSLP is contained in the eluate from the column (FIGS).

Example 4

Immunofluorescent detection dog MONDAY

The expression of protein dog MONDAY in the skin of the dog and the tissues of the tonsils was determined by immunohistochemistry tissues using polyclonal rabbit antibodies to the protein of the MONDAY man. The immunohistochemistry was performed on tissue samples the paraffin blocks, obtained from healthy dogs injected with saline, as well as from dogs with a diagnosis includes atopic dermatitis, erythema in multiforme, cutaneous lupus erythematosus and synaptic bullous bullosa. Additionally, expression of the protein MONDAY was determined in frozen tissue sample tonsils obtained from two dogs. The procedure for determining the expression of MONDAY by way of tissues was as follows:

I. sample Preparation

1. Paraffin blocks of prisoners in their skin samples were cut into samples with a thickness of 5-7 microns and attached to the substrate treated with poly-L-lysine in order to cause adhesion.

2. Sections were deparaffinization with xylene and rehydratable through a series of ethanol solutions.

3. Recovery of antigen was performed in citrate buffer [10 mm sodium citrate containing Tween-20 at a concentration of 0.5 ml/l for 25 min using a laboratory microwave installation to achieve 99-100°C]. It is a process that restores the antigenicity of the tissue sections, which are closed during the process of pouring in the wax.

II. Immunoablative:

1. Sections were incubated in 10% normal donkey serum diluted in phosphate buffer (PBS)for 1 hour at room temperature for nonspecific binding with the antibody.

2. Excess savored is carefully removed and sections were treated with rabbit antibody (1:100), diluted in PBS, then incubated either at room temperature for 1 hour or over night at 100°C in a humid atmosphere.

3. Sections were then washed twice for 5 min in PBS, then gently shook.

4. Excess PBS was then carefully removed and sections were processed biotinylating donkey antibody to rabbit IgG, diluted 1:5000 in PBS for 30 min at room temperature in a humid atmosphere.

5. The section then twice washed with PBS, gently shaking.

6. Excess PBS was removed and sections were incubated for 30 min at room temperature with Streptavidin conjugate-fluorescein sottoriahat (Streptavidin-TCP) in PBS at a concentration of 5 micrograms/ml

7. Sections were then washed twice for 5 min in PBS with gentle shaking.

8. Sections were then stained for the second time by hematoxiline within 2-3 minutes

9. Sections were then examined under a fluorescent microscope.

10. Suitable images photographed.

11. The control experiment consisted of omitting the primary antibodies to MONDAY or substitution of the primary antibodies to normal MONDAY antibodies rabbit.

Table 1 summarizes the results from experiments with tissues.

Table 1
Immunol stheme with rabbit antibodies to a MONDAY person. Conducted on sealed in paraffin sections of skin of dogs with various diseases of the skin
The status of the disease (totalPositiveNegative
The damaged skin of AD (* p=10)82
Normal skin, injected PBS (n=5)14
Synaptic bullous bullosa (n=2)20
Canine cutaneous lupus erythematosus (n=3)03
Erythema in multiforme (n=3)21
* n=number of animals

Expression of MONDAY was noted in 80% of samples of skin tissue from dogs with a diagnosis of AD, but only 20% of the norm is lnyh skin tissues, in which made an injection of saline solution. MONDAY was also detected in 66% and 100% tissue of dogs with erythema in multiforme and dogs with a genetic skin disease, synaptic epidermolysis bullosa, respectively. Was not observed in the expression of protein MONDAY in skin tissue of dogs with cutaneous lupus erythematosus. In samples of skin tissue, paraffin embedded expression MONDAY was noted in the sweat glands. Expression of MONDAY in frozen tissue tonsil was discovered in layered squamous epithelium and has been associated with the salivary glands. An example of positive tissues staining tissue samples of dogs shown in FIG. 4, which represents the samples of skin tissue, sealed in wax, from dogs diagnosed with atopic dermatitis".

Example 6

Immunoperoxidase detection dog MONDAY

Expression of protein dog MONDAY in blocks, sealed in paraffin and prepared from the skin of dogs diagnosed with atopic dermatitis", was also determined immunohistochemically when using staining immunoperoxidase as the method of detection. In this way the epitope-specific monoclonal antibody to the protein of the MONDAY man was used as primary antibody. The procedure for determining the expression of MONDAY through staining immunoperoxidase was as follows:

Special reagents:

Normal is the first serum of newborn calves: #N-4762 Sigma

Sealed in paraffin tissue

Primary antibodies: rat monoclonal antibody lgG2a MONDAY to person

Secondary antibodies: rabbit biotinylated antibodies to human IgG: VA-4000 Vector Lab, Burlingame, CA.

Reagent for detection: Streptavidin-HRP: #43-8323 Zymed Labs. San Francisco, CA

Set-based AES: Biogenex #HK129-5K San Ramon, CA

1. Selected samples 4-6 µl.

2. Was dried in air for 10 min at room temperature.

3. 10 min were fixed with acetone.

4. Washed in PBS (0.01 phosphate-saline buffer) 3 minutes

5. Inhibited using incubation in 0.3% hydrogen peroxide with 0.1% sodium azide for 7-10 minutes

6. Washed in PBS for 5 min.

7. Blocked sections with 1% normal serum of newborn calves for 20 min in a humid chamber.

8. Was removed from the slices liquid and acted primary antibodies at a dilution of 1:100 for 2 hours at room temperature.

9. Were washed in 5 minutes

10. Acted secondary antibody (rabbit antibody to rat IgG at a dilution of 1:400) for 30 min in a humid chamber at room temperature.

11. Were washed in 5 minutes

12. Remove the liquid and acted reagent for detection (Streptavidin-HRP at a dilution of 1:400) for 30 min at room temperature.

13. Washed 2X5 minutes

14. Acted AES 2.5 minutes Levelled in accordance with the desired intensity of the or ink into the background staining.

15. Stained again with Hematoxylin and transferred to the substrate. Sample of dermal tissue from dogs with AD was investigated using tissues using epitope-specific canine antibodies to MONDAY. The results presented in FIGURE 5, show that this antibody reacts with a molecule that shares antigenic epitopes with a MONDAY person. Modes staining samples of the skin of dogs with AD were strict in the areas of chronic inflammation, where the epidermis thickens. This model coincides with what is known about the localization of the expression of MONDAY when skin lesions in humans and further assumes that the recognized molecule in skin lesions in dogs is canine MONDAY. No staining was observed in the processing of PBS or by using another rat monoclonal body to another protein (the protein from the cells).

Example 7

Mapping of epitopes dog MONDAY

In order to identify the epitopes in the dog's MONDAY, which are useful for inclusion in a vaccine that is able to neutralize the activity of MONDAY, was synthesized sample overlapping peptides based on the sequence of canine protein TSL, and tested for the presence of the ability to respond, neutralizing monoclonal antibody to a MONDAY person. To this end was synthesized sample overlapping peptides of 15 amino acid is the shaft residues each, and ending with 2 amino acids for the formation of a hairpin, firm MIMOTOPES (Minneapolis, MN). The sequences of these peptides are listed in Table 2. Peptides 1-57 were synthesized with laminirovannyy end configuration NH2-PEPTIDE-HAIRPIN. Peptides 58-94 (duplicates related peptides 1-37) were synthesized with acetylated end configuration ACETYL-PEPTIDE-HAIRPIN.

Stud, bearing a peptide listed in Table 2, were investigated using ELISA in accordance with the methods recommended by the manufacturer (Mimotopes, Minneapolis, MN). As shown in FIG. 6, the peptide 25 (epitope 25) with the amino acid sequence NH2-ARIERLTLHRIRGCA (SEQ ID NO: 32) had the greatest reactivity with respect to the monoclonal antibody RAW. Comparison of the sequence of this peptide with the corresponding expected sequence peptide MONDAY of the person shown in FIG.7.

Table 2
Peptides dog MONDAY, used for epitope mapping
Room epitopeSEQ ID NORoom epitopeSEQ ID NO

The present invention should not be limited to particular embodiments described herein. Indeed, various modifications of the invention in addition to those described here will be obvious to a person skilled in the art from the preceding description. Such modifications will be included in the scope of the attached claims.

Next, you need to understand that all sizes of bases or dimensions of amino acids, all values of molecular weights or molecular mass data for nucleic Ki the lot or polypeptides are approximate, and are available for the description.

Here cited various publications, descriptions of which are included here in the form of a full reference.

1. Selected timony stromal lymphopoiesis protein (MONDAY) or antigenic fragment for the induction of immune responses against timusewo stromal lymphopoietin protein, where this protein MONDAY contains an amino acid sequence having 90% or more identity with the amino acid sequence SEQ ID NO: 2, excluding the signal sequence of 28 amino acid residues, and where the specified protein MONDAY contains the epitope binding antibodies to canine MONDAY.

2. Selected MONDAY according to claim 1, where MONDAY is associated with epitopespecific dog MONDAY antibody.

3. Selected MONDAY according to claim 1, which is a dedicated dog MONDAY.

4. Selected protein dog MONDAY according to claim 3, which contains amino acid residues 29-155 of SEQ ID NO: 2.

5. The selected nucleic acid molecule encoding a protein MONDAY or antigenic fragment according to claim 1.

6. The selected nucleic acid molecule encoding a protein MONDAY according to claim 4.

7. The selected nucleic acid molecule according to claim 6, which contains the nucleotide sequence of SEQ ID NO: 1.


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24 cl, 6 dwg, 12 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to biochemistry and represents versions of monoclonal anti-TAT226 antibodies, their immunoconjugates and pharmaceutical compositions.

EFFECT: antibodies by invention can be efficiently applied for inhibition of tumour cell proliferation.

41 cl, 22 dwg, 3 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: there are offered versions of antibodies specific to CD22 epitope located from amino acid 22 to amino acid 240 CD22. There are disclosed: a coding polynucleotide, an expression vector, a based host cell and a method of producing an antibody with the use of the cell. There are described versions of a method of CD22 detection on the basis of the antibodies. There are disclosed versions of the CD22 immunoconjugate and based pharmaceutical compositions for treating disturbed B-cell proliferation, and also versions of a method of treating with the use of the pharmaceutical composition. There is disclosed a method of B-cell proliferation inhibition on a basis the immunoconjugate. There are described versions of an engineered cystein-substituted antibody specific to CD22 with one or more free cysteines of thiol reactance within the range 0.6 to 1.0. There are disclosed versions of the "antibody-drug" conjugate, the immunoconjugate and pharmaceutical formulaitons for treating disturbed B-cell proliferation. There are also described a method for protein CD22 detection in a sample on the basis of the immunoconjugate, a method for B-cell detection and a method of treating a malignant tumour on the basis of the "antibody-drug" conjugate. There are disclosed: a product for treating disturbed B-cell proliferation on the basis of the pharmaceutical formulation and a method of producing the "antibody-drug" conjugate.

EFFECT: use of the invention provides new specific CD22 antibodies and the based drugs of acceptable therapeutic efficacy with lower toxicity that can find application in therapy of tumours.

227 cl, 25 dwg, 16 tbl, 14 ex

FIELD: medicine.

SUBSTANCE: by recombinant method obtained is fused protein, which contains natural molecule of human erythropoetine with cysteine residue near its C-end and Fc fragment of humal IgG, containing hinge region, N-end of said Fc fragment is connected to said C-end of said erythropoetine molecule, and said Fc fragment is natural, excluding mutation, consisting in substitution of cysteine residue in said hinge region, located the nearest of all to said erythropoetine molecule, with non-cysteine residue, which resulted in the fact that first cysteine residue of said hinge region, located the nearest of all to said N-end, is separated, by, at least, 12 or 17 amino acids from said cysteine residue of said erythropoetine molecule. Obtained peptide is used for stimulation of erythropoesis in mammal.

EFFECT: invention makes it possible to obtain fused protein, which possesses erythropoetine activity, has prolonged time of half-life in vivo in comparison with native human erythropoetine.

43 cl, 20 dwg, 10 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: claimed invention relates to optimised fused protein for blocking BLyS or APRIL, which contains extracellular region of N-end of truncated TACI (transmembrane activator and CAML-partner) and Fc sequence IgG. TACI segment of fused protein contains sequence of amino-end region of extracellular region, starting with 13-th amino acid residue, complete sequence of stem area from TACI and is obtained from native sequence of TACI between 12-th and 120-th amino acids. Segment Fc of immunoglobulin IgG of fused protein contains hinge region, CH2 region and CH3 region, TACI segment and Fc segment are fused either directly or through linker sequence. In addition, claimed is DNA sequence which codes fused protein, expression vector, host-cell, pharmaceutical composition, containing fused protein, and application of fused protein for blocking BLyS or APRIL. Obtained fused protein does not degrade in process of expression, possesses high biological activity and high level of expression.

EFFECT: fused protein in accordance with claimed invention can be used in treatment of diseases, associated with abnormal immunologic functions and in treatment of diseases caused by abnormal proliferation of B-lymphocytes.

10 cl, 6 dwg, 8 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology. Disclosed is a cell wall-destroying fused protein containing feruloyl esterase and xylanase, which do not contain a C-terminal hydrocarbon-binding molecule (CBM). If necessary, said fused protein contains CBM from a third enzyme, wherein the enzymes and CBM are recombinant proteins corresponding to native fungal proteins. Also disclosed is use of said fused protein to realise methods of destroying plant cell walls when producing compounds of interest from plants or plant by-products. The invention also discloses nucleic acid which codes said fused protein, an expression vector transformed by said nucleic acid and a host cell containing said vector. Described is a method of producing said fused proteins, involving culturing host cells, extraction and, if necessary, cleaning the fused proteins produced by said host cells in the culture. Disclosed is a method of producing desired compounds from plant products, comprising the following steps: 1) enzymatic treatment of plant products with fused proteins or transformed fungus cells, 2) if necessary, treating plant products with steam combined with the action of fused proteins, 3) if necessary, biotransformation of compounds extracted from cell walls during said enzymatic treatment, 4) extraction and, if necessary, cleaning the desired compounds.

EFFECT: invention increases the effect of destroying plant cell walls.

24 cl, 5 dwg, 2 tbl

FIELD: chemistry.

SUBSTANCE: method involves conducting a conjugation reaction by mixing ciprofloxacin and carbodiimide in ratio of 1:1 for 5 minutes at room temperature, adding the reaction mixture obtained as a result of conjugation to a solution of a human alpha-fetoprotein fragment followed by incubation of the obtained solution for 20-30 minutes while stirring and monitoring pH. By-products obtained from adding the reaction mixture to the solution of the human alpha-fetoprotein fragment are removed via dialysis against a phosphate-salt buffer at pH 8.0.

EFFECT: invention widens the field of using methods of producing conjugates of human alpha-fetoprotein with ciprofloxacin.

1 dwg, 1 ex

FIELD: medicine.

SUBSTANCE: chimeric polypeptide contains a first domain which is a first intracellular loop (i1-loop) of chemokine receptor, and a second domain coupled with the first domain where the second domain is a cell-penetrating and membrane-bound natural or artificial hydrophobic residue. The produced polypeptide is used in treating a disease or a condition characterised by pathological activity of chemokine receptor.

EFFECT: invention enables producing chimeric polypeptide which is selectively reacting with related chemokine receptor.

13 cl, 33 dwg, 3 tbl, 15 ex

FIELD: medicine.

SUBSTANCE: invention refers to producing fused proteins. The fused construct consists of an amino acid sequence of glycosyl phosphatidylinositol anchored tissue inhibitor of metalloproteinase.

EFFECT: cictrisation prevention during skin injures treatment when using the fused construct.

10 cl, 33 dwg, 18 ex

FIELD: medicine.

SUBSTANCE: protein is capable to inhibit thrombin activity specifically. Besides, what is offered is a nucleic acid coding said chimeric protein, a vector containing this nucleic acid and a host cell carrying the vector. The present invention also refers to a pharmaceutical composition containing the recombinant chimeric protein of neutrophils and girugen inhibition factor. An effect of the given composition consists in thrombocyte aggregation inhibition or peripheral leukocyte activation inhibition.

EFFECT: composition can be used for treating a cardio-cerebrovascular disease or preventing a cerebral ischemic injury or a cerebral hematoma.

13 cl, 11 dwg, 16 tbl, 17 ex

FIELD: medicine.

SUBSTANCE: compound contains IL-15 indirectly bound by covalent links with a polypeptide containing a sushi domain of an extra-cellular region of the alpha IL-15R subunit.

EFFECT: invention allows inducing and stimulating activation and proliferation of said cells, and effectively treating the conditions and diseases which requires higher activity of IL-15.

44 cl, 42 dwg, 4 tbl, 1 ex