CN1194012C - Sperm generation-related protein, its coding sequence and use - Google Patents

Abstract

The invention provides a new sperm generation-related protein-Sgrg protein, a polynucleotide encoding the Sgrg protein and a method for producing the Sgrg protein through recombinant technology. The invention also discloses the application of the polynucleotide encoding the Sgrg protein. The invention also discloses a method for using the Sgrg protein to treat various diseases, such as infertility and other diseases. The invention also provides a pharmaceutical composition containing Sgrg protein.

Description

Sperm generation-related protein, its coding sequence and use

technical field

The invention belongs to the field of biotechnology and medicine, in particular, the invention relates to a new polynucleotide encoding human spermatogenesis-related protein-Sgrg, and a polypeptide encoded by the polynucleotide. The present invention also relates to the use and preparation of such polynucleotides and polypeptides. Specifically, the polypeptide of the present invention is a protein associated with spermatogenesis and development.

Background technique

There are many male reproductive links, the main ones are the neuroendocrine regulation of the male reproductive system, the spermatogenesis of the testis, the maturation of sperm in the epididymis, the sperm discharge process mixed with seminal vesicles and seminal plasma secreted by the prostate to form semen, and the sperm from the male reproductive tract When the sperm is excreted from the body and imported into the female reproductive tract, the sperm fertilizes the egg in the fallopian tube, etc., if these links are disturbed and affected, fertility disorders may occur.

According to the statistics of the World Health Organization, 5%-8% of couples of childbearing age in developed countries may have infertility problems, while in some areas of developing countries, it may be as high as 30%. According to the standards recommended by WHO, couples living together for more than one year after marriage without using any contraceptive measures, and the female infertility due to male reasons are called male infertility. The causes of male infertility are clinically divided into: sexual dysfunction, immune infertility, abnormal seminal plasma, congenital diseases, reproductive tract infection, varicocele, endocrine abnormalities, vas deferens obstruction, abnormal sperm, testicular dysfunction and environment factors and so on.

In recent years, with the continuous improvement of medical level, a part of male infertility has found more effective treatment methods clinically. However, due to the lack of understanding of the mechanism of male reproduction, male infertility, especially the infertility caused by abnormal spermatogenesis, is still at the stage of empirical treatment. The cloning of genes related to spermatogenesis is also rarely reported. Therefore, it is of great practical significance to understand the biochemical events in spermatogenesis at the molecular level.

Therefore, there is an urgent need in the art to develop new proteins related to spermatogenesis and/or production.

Contents of the invention

The purpose of the present invention is to provide a new human spermatogenesis-related protein-Sgrg protein and its fragments, analogues and derivatives.

Another object of the present invention is to provide polynucleotides encoding these polypeptides.

Another object of the present invention is to provide methods for producing these polypeptides and uses of the polypeptides and coding sequences.

In the first aspect of the present invention, a novel isolated Sgrg polypeptide is provided, which is human-derived, and it comprises: a polypeptide having an amino acid sequence of SEQ ID NO: 2 or 3, or a conservative variant polypeptide thereof, or an activity thereof Fragments, or their active derivatives. Preferably, the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2 or 3.

In a second aspect of the present invention, polynucleotides encoding isolated polypeptides are provided, the polynucleotides comprising a nucleotide sequence having at least 70 degrees to a nucleotide sequence selected from the group consisting of % identity: (a) a polynucleotide encoding the human Sgrg polypeptide described above; and (b) a polynucleotide complementary to the polynucleotide (a). Preferably, the polynucleotide encodes a polypeptide having the amino acid sequence shown in SEQ ID NO:2. More preferably, the sequence of the polynucleotide is one selected from the following group: (a) has the sequence of positions 561-1364 in SEQ ID NO: 1 (the coding region of the Sgrg protein); (b) has the sequence of SEQ ID NO : the sequence of 781-1364 positions in 1 (the coding region of the mature Sgrg protein): (c) has the sequence of 1-1640 positions in SEQ ID NO: 1.

In the third aspect of the present invention, there are provided vectors containing the above-mentioned polynucleotides, and host cells transformed or transduced by the vectors or host cells directly transformed or transduced by the above-mentioned polynucleotides.

In the fourth aspect of the present invention, there is provided a method for preparing a polypeptide having human Sgrg protein activity, the method comprising: (a) cultivating the above-mentioned transformed or transduced host cell under conditions suitable for expressing the human Sgrg protein; ( b) isolating a polypeptide having human Sgrg protein activity from the culture.

In the fifth aspect of the present invention, an antibody specifically binding to the above-mentioned human Sgrg polypeptide is provided. Also provided is a nucleic acid molecule useful for detection, which contains consecutive 10-1640 nucleotides of the above-mentioned polynucleotides.

In the sixth aspect of the present invention, compounds that mimic, promote, and antagonize the activity of human Sgrg polypeptides, and compounds that inhibit the expression of human Sgrg polypeptides are provided. Methods of screening and/or preparing these compounds are also provided. Preferably, the compound is the antisense sequence of the coding sequence of human Sgrg polypeptide or a fragment thereof.

In the seventh aspect of the present invention, there is provided a method for detecting whether there is Sgrg protein in a sample, which includes: contacting the sample with a specific antibody for Sgrg protein, and observing whether an antibody complex is formed, and the formation of an antibody complex means that the Sgrg protein is present in the sample. Sgrg protein is present.

In the eighth aspect of the present invention, a method for detecting a disease or disease susceptibility related to abnormal expression of a human Sgrg polypeptide is provided, the method comprising: detecting whether there is a mutation in the nucleic acid sequence encoding the polypeptide.

In the ninth aspect of the present invention, uses of the polypeptides and coding sequences of the present invention are provided. For example, the polypeptide of the present invention can be used to screen for agonists that promote the activity of human Sgrg polypeptides, or to screen for antagonists that inhibit the activities of human Sgrg polypeptides, or to identify peptide fingerprints. The human Sgrg protein coding sequence or its fragments of the present invention can be used as primers for PCR amplification reactions, or as probes for hybridization reactions, or for making gene chips or microarrays.

In the tenth aspect of the present invention, a pharmaceutical composition is provided, which contains a safe and effective amount of the human Sgrg polypeptide of the present invention or its agonist, antagonist, and a pharmaceutically acceptable carrier. These pharmaceutical compositions can treat oligospermia, deformed sperm and other diseases.

Other aspects of the invention will be apparent to those skilled in the art from the technical disclosure herein.

Description of drawings

The following drawings are used to illustrate specific embodiments of the present invention, but not to limit the scope of the present invention defined by the claims.

Fig. 1 is a comparison diagram of amino acid sequence homology between human Sgrg protein of the present invention and human polycythemia-related gene PRV-1 (GI9857661). The upper sequence is human Sgrg, and the lower sequence is human PRV-1 protein. Identical amino acids are marked with an amino acid letter between the two sequences, and similar amino acids are marked with "+".

Fig. 2 is an electrophoresis diagram of the human Sgrg protein expression product of the present invention.

Detailed ways

In the present invention, the terms "Sgrg protein", "Sgrg polypeptide" or "sperm generation-related protein Sgrg" are used interchangeably, and all refer to proteins having the amino acid sequence of human spermatogenesis-related protein Sgrg (SEQ ID NO: 2 or 3) or peptides. They include Sgrg proteins with or without an initial methionine, and Sgrg proteins with or without a signal peptide.

As used herein, "isolated" means that the material is separated from its original environment (if the material is native, the original environment is the natural environment). For example, polynucleotides and polypeptides in the natural state in living cells are not isolated and purified, but the same polynucleotides or polypeptides are isolated and purified if they are separated from other substances that exist together in the natural state .

As used herein, "isolated Sgrg protein or polypeptide" means that the Sgrg polypeptide is substantially free of other proteins, lipids, carbohydrates or other substances with which it is naturally associated. Those skilled in the art can purify Sgrg protein using standard protein purification techniques. Substantially pure polypeptides yield a single major band on non-reducing polyacrylamide gels. The purity of the Sgrg polypeptide can be analyzed by amino acid sequence.

The polypeptide of the present invention can be a recombinant polypeptide, a natural polypeptide, a synthetic polypeptide, preferably a recombinant polypeptide. Polypeptides of the present invention may be naturally purified, or chemically synthesized, or produced using recombinant techniques from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insect and mammalian cells). Depending on the host used in the recombinant production protocol, the polypeptides of the invention may be glycosylated, or may be non-glycosylated.

The present invention also includes fragments, derivatives and analogs of human Sgrg protein. As used herein, the terms "fragment", "derivative" and "analogue" refer to a polypeptide that substantially maintains the same biological function or activity of the natural human Sgrg protein of the present invention. The polypeptide fragments, derivatives or analogs of the present invention may be (i) polypeptides having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues It may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a mature polypeptide in combination with another compound (such as a compound that extends the half-life of the polypeptide, e.g. polyethylene glycol), or (iv) an additional amino acid sequence fused to the polypeptide sequence (such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify the polypeptide, or with Formation of fusion proteins of antigen IgG fragments). Such fragments, derivatives and analogs are within the purview of those skilled in the art in light of the teachings herein.

In the present invention, the term "human Sgrg polypeptide" refers to a polypeptide of SEQ ID NO. 2 or 3 sequence having human Sgrg protein activity. The term also includes variants of SEQ ID NO. 2 or 3 that have the same function as the human Sgrg protein. These variations include (but are not limited to): deletions and insertions of several (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acids and/or substitution, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminal and/or N-terminal. For example, in the art, substitutions with amino acids with similar or similar properties generally do not change the function of the protein. As another example, adding one or several amino acids at the C-terminus and/or N-terminus usually does not change the function of the protein. The term also includes active fragments and active derivatives of the human Sgrg protein.

Variants of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with human Sgrg DNA under high or low stringency conditions , and the polypeptide or protein obtained by using antiserum against human Sgrg polypeptide. The present invention also provides other polypeptides, such as fusion proteins comprising human Sgrg polypeptide or fragments thereof. In addition to nearly full-length polypeptides, the present invention also includes soluble fragments of human Sgrg polypeptides. Typically, the fragment has at least about 10 contiguous amino acids, usually at least about 30 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 80 contiguous amino acids of the human Sgrg polypeptide sequence. 100 consecutive amino acids.

The invention also provides analogs of human Sgrg protein or polypeptide. The difference between these analogues and the natural human Sgrg polypeptide may be the difference in the amino acid sequence, or the difference in the modified form that does not affect the sequence, or both. These polypeptides include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by radiation or exposure to mutagens, but also by site-directed mutagenesis or other techniques known in molecular biology. Analogs also include analogs with residues other than natural L-amino acids (eg, D-amino acids), and analogs with non-naturally occurring or synthetic amino acids (eg, β, γ-amino acids). It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.

Modified (usually without altering primary structure) forms include: chemically derivatized forms of polypeptides such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those resulting from polypeptides that are modified by glycosylation during synthesis and processing of the polypeptide or during further processing steps. Such modification can be accomplished by exposing the polypeptide to an enzyme that performs glycosylation, such as a mammalian glycosylase or deglycosylation enzyme. Modified forms also include sequences with phosphorylated amino acid residues (eg, phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides that have been modified to increase their resistance to proteolysis or to optimize solubility.

In the present invention, "human Sgrg protein conservative variant polypeptide" means that compared with the amino acid sequence of SEQ ID NO: 2 or 3, there are at most 10, preferably at most 8, more preferably at most 5, and optimally Up to 3 amino acids are replaced by amino acids with similar or similar properties to form a polypeptide. These conservative variant polypeptides are preferably produced by amino acid substitutions according to Table 1.

Table 1 initial residue representative replacement preferred substitution Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala His(H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Leu

A polynucleotide of the invention may be in the form of DNA or RNA. Forms of DNA include cDNA, genomic DNA or synthetic DNA. DNA can be single-stranded or double-stranded. DNA can be either the coding strand or the non-coding strand. The coding region sequence encoding the mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant. As used herein, "degenerate variant" in the present invention refers to a nucleic acid encoding a protein having an amino acid sequence shown in SEQ ID NO: 2 or 3, but differing from the sequence of the coding region shown in SEQ ID NO: 1 sequence.

A polynucleotide encoding a mature polypeptide includes: a coding sequence that encodes only the mature polypeptide; a coding sequence for the mature polypeptide and various additional coding sequences; a coding sequence for the mature polypeptide (and optionally additional coding sequences) and non-coding sequences.

The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, or may also include additional coding and/or non-coding sequences.

The present invention also relates to variants of the above-mentioned polynucleotides, which encode polypeptides or polypeptide fragments, analogs and derivatives having the same amino acid sequence as the present invention. Variants of this polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is an alternative form of a polynucleotide which may be a substitution, deletion or insertion of one or more nucleotides without substantially altering the function of the polypeptide it encodes .

The present invention also relates to polynucleotides that hybridize to the above-mentioned sequences and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The invention particularly relates to polynucleotides which are hybridizable under stringent conditions to the polynucleotides of the invention. In the present invention, "stringent conditions" refers to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with There are denaturing agents, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, etc.; or (3) only if the identity between the two sequences is at least 90%, more Preferably, hybridization occurs above 95%. Moreover, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO:2.

The present invention also relates to nucleic acid fragments that hybridize to the above-mentioned sequences. As used herein, a "nucleic acid fragment" is at least 15 nucleotides in length, preferably at least 30 nucleotides in length, more preferably at least 50 nucleotides in length, most preferably at least 100 nucleotides in length. Nucleic acid fragments can be used in nucleic acid amplification techniques (such as PCR) to identify and/or isolate polynucleotides encoding Sgrg proteins.

The polypeptides and polynucleotides of the invention are preferably provided in isolated form, more preferably purified to homogeneity.

The full-length human Sgrg nucleotide sequence or its fragments of the present invention can usually be obtained by PCR amplification, recombination or artificial synthesis. For the PCR amplification method, primers can be designed according to the relevant nucleotide sequences disclosed in the present invention, especially the open reading frame sequence, and the cDNA prepared by a commercially available cDNA library or a conventional method known to those skilled in the art can be used. The library is used as a template to amplify related sequences. When the sequence is long, it is often necessary to carry out two or more PCR amplifications, and then splice together the amplified fragments in the correct order.

Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. Usually, it is cloned into a vector, then transformed into a cell, and then the relevant sequence is isolated from the proliferated host cell by conventional methods.

In addition, related sequences can also be synthesized by artificial synthesis, especially when the fragment length is relatively short. Often, fragments with very long sequences are obtained by synthesizing multiple small fragments and then ligating them.

At present, the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) can be obtained completely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or eg vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.

A method of amplifying DNA/RNA using the PCR technique (Saiki, et al. Science 1985; 230: 1350-1354) is preferably used to obtain the gene of the present invention. Especially when it is difficult to obtain full-length cDNA from the library, the RACE method (RACE-cDNA terminal rapid amplification method) can be preferably used, and the primers used for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein, And can be synthesized by conventional methods. Amplified DNA/RNA fragments can be separated and purified by conventional methods such as by gel electrophoresis.

The present invention also relates to a vector containing the polynucleotide of the present invention, a host cell produced by genetic engineering using the vector or Sgrg protein coding sequence of the present invention, and a method for producing the polypeptide of the present invention by recombinant technology.

By conventional recombinant DNA techniques (Science, 1984; 224:1431), the polynucleotide sequences of the present invention can be used to express or produce recombinant Sgrg polypeptides. Generally speaking, there are the following steps:

(1). Transform or transduce a suitable host cell with the polynucleotide (or variant) encoding the human Sgrg polypeptide of the present invention, or with a recombinant expression vector containing the polynucleotide;

(2). Host cells cultured in a suitable medium;

(3). Isolate and purify protein from culture medium or cells.

In the present invention, the human Sgrg polynucleotide sequence can be inserted into the recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus or other vectors well known in the art. Vectors applicable in the present invention include, but are not limited to: T7-based expression vectors (Rosenberg, et al. Gene, 1987, 56: 125) expressed in bacteria; pMSXND expression vectors expressed in mammalian cells (Lee and Nathans, J Bio Chem.263:3521, 1988) and vectors derived from baculovirus expressed in insect cells. In short, any plasmid and vector can be used as long as it can be replicated and stabilized in the host. An important feature of expression vectors is that they usually contain an origin of replication, a promoter, marker genes, and translational control elements.

Methods well known to those skilled in the art can be used to construct an expression vector containing the human Sgrg coding DNA sequence and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. (Sambroook, et al. Molecular Cloning, a Laboratory Manual, cold Spring Harbor Laboratory. New York, 1989). Said DNA sequence can be operably linked to an appropriate promoter in the expression vector to direct mRNA synthesis. Representative examples of these promoters are: E. coli lac or trp promoter; lambda phage _PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, LTRs of retroviruses and other promoters known to control gene expression in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

In addition, the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.

Vectors containing the above-mentioned appropriate DNA sequences and appropriate promoters or control sequences can be used to transform appropriate host cells so that they can express proteins.

The host cell may be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: Escherichia coli, Streptomyces spp; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; plant cells; insect cells of Drosophila S2 or Sf9; CHO, COS, 293 cells, or Bowes melanoma cells animal cells, etc.

When the polynucleotide of the present invention is expressed in higher eukaryotic cells, if an enhancer sequence is inserted into the vector, the transcription will be enhanced. Enhancers are cis-acting elements of DNA, usually about 10 to 300 base pairs in length, that act on promoters to enhance gene transcription. Examples include the SV40 enhancer of 100 to 270 base pairs on the late side of the replication origin, the polyoma enhancer on the late side of the replication origin, and the adenovirus enhancer.

Those of ordinary skill in the art will know how to select appropriate vectors, promoters, enhancers and host cells.

Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as E. coli, competent cells capable of taking up DNA can be harvested after the exponential growth phase and treated with the _CaCl2 method using procedures well known in the art. Another method is to use _MgCl2 . Transformation can also be performed by electroporation, if desired. When the host is eukaryotic, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

The obtained transformant can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. The medium used in the culture can be selected from various conventional media according to the host cells used. The culture is carried out under conditions suitable for the growth of the host cells. After the host cells have grown to an appropriate cell density, the selected promoter is induced by an appropriate method (such as temperature shift or chemical induction), and the cells are cultured for an additional period of time.

The recombinant polypeptide in the above method can be expressed inside the cell, or on the cell membrane, or secreted outside the cell. The recombinant protein can be isolated and purified by various separation methods by taking advantage of its physical, chemical and other properties, if desired. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitating agents (salting out method), centrifugation, osmotic disruption, supertreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

The recombinant human Sgrg protein or polypeptide has many uses. These uses include (but are not limited to): direct use as drugs to treat diseases caused by low or loss of Sgrg protein function (such as azoospermia, or low sperm motility and other diseases), and for screening antibodies that promote or resist Sgrg protein function, peptides or other ligands. Screening the polypeptide library with the expressed recombinant human Sgrg protein can be used to find therapeutically valuable polypeptide molecules that can inhibit or stimulate the function of the human Sgrg protein.

On the other hand, the present invention also includes polyclonal antibodies and monoclonal antibodies specific to human Sgrg DNA or polypeptides encoded by its fragments, especially monoclonal antibodies. Here, "specificity" means that the antibody can bind to human Sgrg gene product or fragment. Preferably, it refers to those antibodies that can bind to human Sgrg gene products or fragments but do not recognize and bind to other irrelevant antigen molecules. Antibodies in the present invention include those molecules capable of binding and inhibiting human Sgrg protein, as well as those antibodies that do not affect the function of human Sgrg protein. The invention also includes antibodies that bind to modified or unmodified forms of the human Sgrg gene product.

The present invention includes not only complete monoclonal or polyclonal antibodies, but also immunologically active antibody fragments, such as Fab' or (Fab) ₂ fragments; antibody heavy chains; antibody light chains; genetically engineered single-chain Fv molecules ( Ladner et al., US Patent No. 4,946,778); or chimeric antibodies, such as antibodies that have the binding specificity of a murine antibody but retain portions of the antibody from humans.

Antibodies of the present invention can be prepared by various techniques known to those skilled in the art. For example, purified human Sgrg gene products, or antigenic fragments thereof, can be administered to animals to induce polyclonal antibody production. Similarly, cells expressing human Sgrg protein or antigenic fragments thereof can be used to immunize animals to produce antibodies. Antibodies of the invention may also be monoclonal antibodies. Such monoclonal antibodies can be prepared using hybridoma technology (see Kohler et al., Nature 256; 495, 1975; Kohler et al., Eur.J. Immunol. 6:511, 1976; Kohler et al., Eur.J. Immunol . 6:292, 1976; Hammerling et al., In Monoclonal Antibodies and T Cell Hybridomas , Elsevier, NY, 1981). The antibodies of the present invention include antibodies capable of blocking the function of human Sgrg protein and antibodies that do not affect the function of human Sgrg protein. Various antibodies of the present invention can be obtained by conventional immunization techniques using fragments or functional regions of human Sgrg gene products. These fragments or functional regions can be prepared using recombinant methods or synthesized using a polypeptide synthesizer. Antibodies that bind to unmodified forms of the human Sgrg gene product can be produced by immunizing animals with gene products produced in prokaryotic cells (e.g., E. coli); antibodies that bind to post-translationally modified forms (such as glycosylated or phosphorylated Proteins or polypeptides), which can be obtained by immunizing animals with gene products produced in eukaryotic cells (such as yeast or insect cells).

Antibodies against human Sgrg protein can be used in immunohistochemical techniques to detect human Sgrg protein in biopsy specimens. In addition, monoclonal antibodies combined with human Sgrg protein can also be labeled with radioactive isotopes, and injected into the body to track its location and distribution. This radiolabeled antibody can be used as a non-invasive diagnostic method for localization of tumor cells and judgment of metastasis.

The antibody of the present invention can be used to treat or prevent diseases related to human Sgrg protein. Administration of appropriate doses of antibodies can stimulate or block the production or activity of human Sgrg protein.

Antibodies can also be used to design immunotoxins to target a particular part of the body. For example, monoclonal antibodies with high affinity to human Sgrg protein can be covalently combined with bacterial or plant toxins (such as diphtheria toxin, ricin, rhizidine, etc.). A common method is to use a sulfhydryl cross-linking agent such as SPDP to attack the amino group of the antibody, and bind the toxin to the antibody through the exchange of disulfide bonds. This hybrid antibody can be used to kill cells positive for human Sgrg protein (such as lymph node cells, etc.).

For the production of polyclonal antibodies, human Sgrg protein or polypeptide can be used to immunize animals, such as rabbits, mice, rats, etc. Various adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant and the like.

Utilizing the protein of the present invention, substances that interact with the Sgrg protein, such as receptors, inhibitors, agonists or antagonists, can be screened out through various conventional screening methods.

When the protein of the present invention and its antibody, inhibitor, agonist, antagonist or receptor are administered (administered) therapeutically, various effects can be provided. Generally, these materials can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is usually about 5-8, preferably about 6-8, although the pH value can be changed according to the Depending on the nature of the substance formulated and the condition to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): oral, intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, or topical administration.

The polypeptide of the present invention can be directly used for disease treatment, for example, for the treatment of testicular spermatogenesis dysfunction. When using the Sgrg protein of the present invention, other therapeutic agents for the same disease, such as testosterone, can also be used at the same time.

The present invention also provides a pharmaceutical composition, which contains a safe and effective amount of the Sgrg polypeptide of the present invention or its agonist, antagonist, and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to: saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of injection, for example, by conventional methods using physiological saline or aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as tablets and capsules can be prepared by conventional methods. Pharmaceutical compositions such as injections, solutions, tablets and capsules are preferably manufactured under sterile conditions. The active ingredient is administered in a therapeutically effective amount, for example about 1 microgram/kg body weight to about 5 mg/kg body weight per day. In addition, the polypeptides of the invention can also be used with other therapeutic agents.

When using the pharmaceutical composition, a safe and effective amount of the Sgrg protein or its antagonist, agonist is administered to the mammal, wherein the safe and effective amount is usually at least about 10 μg/kg body weight, and in most cases no more than about 8 mg/kg body weight, preferably the dose is about 10 micrograms/kg body weight to about 1 mg/kg body weight. Of course, factors such as the route of administration and the health status of the patient should also be considered for the specific dosage, which are within the skill of skilled physicians.

Polynucleotides of human Sgrg protein can also be used for various therapeutic purposes. Gene therapy technology can be used to treat cell proliferation, development or metabolic abnormalities caused by non-expression of Sgrg protein or expression of abnormal/inactive Sgrg protein. Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated Sgrg proteins to inhibit the activity of endogenous Sgrg proteins. Expression vectors derived from viruses such as retrovirus, adenovirus, adeno-associated virus, herpes simplex virus, parvovirus, etc. can be used to transfer the Sgrg gene into cells. The method for constructing a recombinant viral vector carrying the Sgrg gene can be found in existing literature (Sambrook, et al.). In addition, the recombinant human Sgrg gene can be packaged into liposomes and then transferred into cells.

Oligonucleotides (including antisense RNA and DNA) and ribozymes that inhibit human Sgrg mRNA are also within the scope of the invention. A ribozyme is an enzyme-like RNA molecule that can specifically decompose a specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA to perform an endonucleic cut. Antisense RNA, DNA and ribozyme can be obtained by any existing RNA or DNA synthesis technology, such as solid-phase phosphoamide chemical synthesis of oligonucleotides, which has been widely used. Antisense RNA molecules can be obtained by in vitro or in vivo transcription of the DNA sequence encoding the RNA. This DNA sequence has been integrated into the vector downstream of the RNA polymerase promoter. In order to increase the stability of nucleic acid molecules, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the connection between ribonucleosides should use phosphothioester bonds or peptide bonds instead of phosphodiester bonds.

The methods for introducing polynucleotides into tissues or cells include: directly injecting polynucleotides into tissues in the body; or first introducing polynucleotides into cells in vitro through vectors (such as viruses, phages, or plasmids, etc.) , and then transplant the cells into the body, etc.

The polypeptide molecule capable of binding to the human Sgrg protein can be obtained by screening a random polypeptide library composed of various possible combinations of amino acids bound to solid phases. During screening, the human Sgrg protein molecule must be labeled.

The invention also relates to a diagnostic test method for quantitative and localized detection of human Sgrg protein level. These assays are well known in the art and include FISH assays and radioimmunoassays. The human Sgrg protein level detected in the test can be used to explain the importance of the human Sgrg protein in various diseases and to diagnose diseases in which the Sgrg protein plays a role.

A method for detecting whether there is an Sgrg protein in a sample is to use a specific antibody for the Sgrg protein to detect, which includes: contacting the sample with an Sgrg protein specific antibody; observing whether an antibody complex is formed, which means that the antibody complex is formed Sgrg protein is present in the sample.

The polynucleotide of Sgrg protein can be used for diagnosis and treatment of Sgrg protein-related diseases. In terms of diagnosis, the polynucleotide of Sgrg protein can be used to detect the expression of Sgrg protein or the abnormal expression of Sgrg protein in a disease state. For example, the Sgrg DNA sequence can be used for hybridization of biopsy specimens to determine the abnormal expression of Sgrg protein. Hybridization techniques include Southern blotting, Northern blotting, in situ hybridization, and the like. These technical methods are all open and mature technologies, and relevant kits are available from commercial sources. Part or all of the polynucleotides of the present invention can be immobilized as probes on microarrays or DNA chips (also known as "gene chips") for analysis of differential expression of genes in tissues and gene diagnosis. RNA-polymerase chain reaction (RT-PCR) in vitro amplification with Sgrg protein-specific primers can also detect the transcripts of Sgrg protein.

Detection of Sgrg gene mutations can also be used to diagnose Sgrg protein-related diseases. The form of Sgrg protein mutation includes point mutation, translocation, deletion, recombination and any other abnormality compared with normal wild-type Sgrg DNA sequence. Mutations can be detected using established techniques such as Southern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect protein expression, so Northern blotting and Western blotting can be used to indirectly determine whether a gene has a mutation.

The sequences of the invention are also valuable for chromosome identification. In short, PCR primers (preferably 15-35bp) are prepared according to the cDNA of the Sgrg protein of the present invention, and the sequence can be positioned on the chromosome. These primers were then used for PCR screening of somatic heterozygous cells containing individual human chromosomes. Only those cells heterozygous for the human gene corresponding to the primer will produce an amplified fragment.

Once a sequence has been mapped to an exact chromosomal location, the physical location of the sequence on the chromosome can be correlated with gene map data. These data can be found, for example, in V. Mckusick, Mendelian Inheritance in Man (available online through Johns Hopkins University Welch Medical Library). Linkage analysis can then be used to determine the relationship between the gene and the disease that has been mapped to the chromosomal region.

In one example of the present invention, an isolated polynucleotide encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 2 is provided. The polynucleotides of the present invention were isolated from a cDNA library of human peripheral lymphocytes. Its sequence is shown in SEQ ID NO: 1, and the polynucleotide sequence that it comprises is full-length 1640 bases, and its open reading frame is positioned at 561-1364 positions, and coding is the human Sgrg protein (SEQ ID NO :2). It can provide a new therapeutic approach for treating diseases such as azoospermia or low sperm motility, and thus has great application prospects.

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods not indicating specific conditions in the following examples are usually according to conventional conditions such as Sambrook et al., Molecular cloning: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions specified by the manufacturer. suggested conditions.

Example 1: Obtaining EST

Using mouse Ehrlich ascites carcinoma cells and mouse L929 cells in different division phases as materials, antiserum from patients with scleroderma was used to detect the distribution of antigens in the cells by indirect immunofluorescence, and a case of antiserum was obtained. It can specifically surround the chromosomes of cells in the middle and late stages of division. In order to search for these antigenic proteins, the inventors used the serum to screen a human testis λ phage expression library. Several clones were obtained, and the DNA was extracted and purified according to conventional methods. Sequenced with λgt11 primers, one of them was found to be a new EST, and this EST was named YN11.

Example 2: Obtaining the full-length sequence

(1) Primer synthesis

In order to obtain the full-length cDNA comprising YN11 EST, the following primers were designed and synthesized with reference to the sequence of the EST:

YN11 XF: 5′-TGG ATC TGC TTC CAC AGT CAT GGA CA-3′ (SEQ ID NO: 6)

YN11 NF: 5′-TAC AGC TCT AGG CCC GAG GTC AGG A-3′ (SEQ ID NO: 7)

YN11 NR: 5′-AGA AGC CAT GGG AAC CCT CGT ATC C-3′ (SEQ ID NO: 8)

YN11 R2: 5′-ATT GGC TGC AGG CTG ATG TCT GGA AT-3′ (SEQ ID NO: 9)

(2) Obtaining the 5′-end cDNA sequence:

Using Marathon-Ready-cDNA Human Leukocyte (Clontech-7406-1) as a template, use primers YN11 XF and AP1 (AP1 is the primer provided in the kit) to carry out RACE-PCR according to the conditions suggested by the manufacturer. After the first-round PCR product was diluted 10 times, it was amplified again with nested primers YN11NF and AP2 (AP2 is a primer provided in the kit) as a template. The resulting PCR products were amplified, tapped, purified, cloned into a T-A vector (Sangon), and sequenced using M13 forward and reverse primers.

(3) Acquisition of 3′-end cDNA sequence

Use exactly the same strategy as the 5'-end, the only difference is: the primer is YN11NR.+AP1 and then YN11R2+AP2.

(4) Acquisition of full-length sequence

The 3'-end and 5'-end sequences were spliced to obtain a complete full-length cDNA (GENE BANK accession number: AF241268) (due to the patent application, it was kept confidential before the submission of this application). Sgrg cDNA is 1640bp (SEQ ID NO:1), contains a complete open reading frame of 801bp, and encodes a polypeptide (SEQ ID NO:2) containing 267 amino acid residues.

It was predicted by SignaIP analysis that amino acids 1-40 in SEQ ID NO: 2 are signal peptides pointing to the endoplasmic reticulum. The mature polypeptide molecule consists of 227 amino acid residues (SEQ ID NO: 3), and the homology comparison with human polycythemia-related gene PRV-1 is shown in Figure 1. PRV-1 consists of two highly similar domains forming a repeat structure. So BLAST results showed that SGRG has homology with both fragments of PRV-1. The identity is 32%, and the similarity is 49%.

Example 5: Chromosomal location of Sgrg

According to the conditions suggested by the manufacturer, the RH panel Assay (Research Genetics) was used to carry out the chromosome mapping of Sgrg. The gene was located on chromosome 19q13.2.

PCR conditions: 95°C for 10 minutes → 94°C for 30 seconds; 64°C for 30 seconds; 72°C for 23 seconds, a total of 30 cycles → 72°C for 3 minutes and 30 seconds.

Primers: YN11 XF and NR (this PCR condition is part of the conditions for RH panel Assay)

The amplified product was analyzed by rhserver@shgc.stanford.edu, and the results showed that the genetic marker most closely linked to it was SHGC 3096.

Example 6: Tissue expression profile of Sgrg

A section of cDNA (615-1364 in SEQ ID NO: 1) containing ORF of Sgrg was used as a probe to hybridize with MTE membrane (Clontech-7775-1). The results showed that the Sgrg was very specifically expressed in testis tissue, while in other There is only very low expression in tissues (such as liver, heart, etc.).

Embodiment 7: Gene structure and protein structure of Sgrg

(1) Gene structure

BLAST the full-length sequence of Sgrg cDNA in DATABASE-htgs of http://www.ncbi.nlm.nih.gov/BLAST/, and found that human BAC: chromosome 19, CIT978SKB-61I7 contains the Sgrg gene, the span of the entire gene About 23kb. The gene structure is as follows:

Table 2 Gene structure of Sgrg cDNA sequence genome sequence splice site Exon 1 1-119 20106-20634 exon 2 118-185 33407-33474 /gtggg...tttac/ exon 3 184-280 34677-34773 /gtgcgt...ttttt/ Exon 4 277-575 38133-38432 /tatgag...ctgtg/ Exon 5 573-678 47791-47896 /gtacag...aattca/ exon 6 679-823 48012-48156 /gtgcgt...cctcag/ exon 7 820-1005 48283-48468 /tgaaa...cataa/ Exon 8 1003-1136 49788-49921 /gtacc...ccctc/ Exon 9 1133-1563 50079-50509 /aacct...tctat/

(2) Protein structure

Using the ORF Finder program (http://www.ncbi.nlm.nih.gov/gorf/gorf.html) to predict the Sgrg protein sequence, an open reading frame of 267 amino acids was obtained. After analysis, it may contain the following structural features:

(A) Motif:

1. Amino acids 63-66; 177-180: N-glycosylation site

2. Amino acids 21-23; 179-181: PKC-phosphorylation sites

3. Amino acids 13-16; 40-43; 65-68; 196-199: Casein kinase 2 phosphorylation site

(B) Gene familial analysis: Gene familial analysis was performed at http://fps.sdsc.edu. The results showed that Sgrg protein has uPAR domain (C-rich domain) including uPAR signature sequence (CXDXXCN) and belongs to the urokinase receptor superfamily.

(C) Homology analysis: BLAST (http://www.ncbi.nlm.nih.gov/BLAST/):

As shown in Figure 1, the Sgrg protein has relatively high homology with the human polycythemia-related gene PRV-1 (GI9857661), especially in the conserved region of the uPAR superfamily.

PRV-1 belongs to a member of the uPAR superfamily, and its overexpression corresponds to excessive proliferation of erythrocytes. The PRV-1 gene is also located at the position of 19q13.2, which is only about 9kb away from the position of Sgrg.

Example 8: In situ hybridization of Sgrg

Since the Sgrg gene is specifically expressed in the testis tissue, in order to further understand its function in the male reproductive system, the rhesus monkey testis was used as the material, and the in situ hybridization was carried out according to the conventional method. It was found that it was expressed in the spermatogenic cells of the seminiferous tubules more singularly. This indicates that the function of Sgrg is closely related to the development and/or maturation of sperm.

Embodiment 9: Expression of Sgrg fragments in prokaryotic cells

With primers YN11E-C-F (5'-CGC GGA TCC TTG GGG ACC TGT TTC AGT G-3') (SEQ ID NO: 10) and YN11E-R (5'-ATAAAGCTTAAATGATGGC AGCAGCAATGG-3') (SEQ ID NO: 5 ), using human peripheral blood cDNA as a template to amplify the DNA encoding 167-267a.a of the Sgrg protein.

The PCR amplified product was cloned into PET32(a) (Novagen) using EcoRI and Hind III sites, and then transformed into host E. Coli: BL21(DE3). Transformed host cells express under the induction of IPTG. The results are shown in Figure 2, the molecular weight of the expression product in SDS-PAGE gel is about 33Kda (containing the fusion protein Thioredoxin).

Example 10: Preparation of Antibodies

The fusion protein expressed in the example was purified with Ni-NTA Agarose (Clontech), and the fusion protein was used as an antigen to prepare a polyclonal antibody in rabbits. The specific procedure is as follows: the first immunization of purebred New Zealand rabbits, the protein dosage is 240ug/rat, and Freund's complete adjuvant is used. After 4 weeks, the second immunization was carried out, the dosage of protein was 140ug/only, and incomplete adjuvant was used. After 2 weeks, the third immunization, the protein dosage was 120ug/monkey, with incomplete adjuvant.

Example 11: The promoting effect of Sgrg on cell proliferation

In order to understand the possible significance of Sgrg in terms of cell dynamics, colony formation experiments were carried out in this example.

Primers YN11E-F-2 (5′-TCT AAG CTT ATG GGG GCG AGG CAG ATC CAGATC CAG ACC AGC TCC TCC TCC CAG AC-3′) (SEQ ID NO: 11) and YN11E-R-2 (5′-CGTCTA GAT TAG GAA AAG TGA ATA AAT GAT GGC AGC AGC A-3' (SEQ ID NO: 12), using human peripheral lymphatic blood cDNA as a template, the coding region of Sgrg (SEQ ID NO: 561 in 1) was amplified by RT-PCR -1364 bits).

Then, the amplified product was cloned into the eukaryotic expression vector pRC/CMV (Invitrogen) using the restriction sites (Xba I and Hind III) on the primers to form the plasmid pRC/CMV-Sgrg. The constructed plasmid pRC/CMV-Sgrg was transfected into mouse fibroblast 3T3 line and human liver cancer cell line 7721 using Lipofectamine (18324-020) liposome transfection technology of Gibco Company. Stably transformed cell lines were inoculated at low density, and the empty vector pRC/CMV was used as a negative control, and JCL/L (a known pro-cell proliferation factor) was used as a positive control. The results are shown in Table 3:

Table 3 The promoting effect of Sgrg on cell proliferation 3T3 cells (number of clones) 7721 cells (number of clones) Sgrg (overexpressed) 42 17 empty vector 3 2 JCL/L (positive control) 38 none

It can be seen that overexpression of Sgrg gene in eukaryotic cells will affect cell proliferation. This is also consistent with the characteristics of the uPAR family.

Embodiment 12: RT-PCR method obtains Sgrg coding sequence

Total RNA was isolated from human peripheral blood using RNeasy (Qiagen). Then use SupertScript II RNaseH-Reverse Transcriptase (Gibco) to reverse transcribe to obtain cDNA (according to the conditions provided by the manufacturer). .Using cDNA as a template, use primers YN11 RT-F and YN11 RT-R:

YN11RT-F: 5'AGA TCC AGA CCA GCT CCT CCC-3' (SEQ ID NO: 14)

YN11RT-R: 5′-AAATGATGGC AGCAGCAATG G-3′ (SEQ ID NO: 5)

Perform PCR under the following conditions: 95°C for 10 minutes → 94°C for 30 seconds; 60°C for 30 seconds; 72°C for 90 seconds, a total of 45 cycles → 72°C for 3 minutes and 30 seconds.

The amplified PCR product was sequenced, and the results showed that it was consistent with the sequence of the coding region of Sgrg shown in SEQ ID NO:1.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Sequence Listing

(1) General information:

(ii) Title of invention: new spermatogenesis-related protein, its coding sequence and use

(iii) Number of sequences: 14

(2) Information on SEQ ID NO: 1:

(i) Sequence features:

(A) Length: 1640bp

(B) type: nucleic acid

(C) chain: double chain

(D) Topology: Linear

(ii) Molecular type: cDNA

(xi) Sequence description: SEQ ID NO: 1:

ATTTAGGATG AACTATGTGT GACAAATGGT GCCGTTGAGT CCTTCCAACT GGGAACGAGT 60

CACCGTGTAT CTGGAGACCA TGTGTGTAAC GGGTTGCACC TGCTTGGCTG GATACAAAGA 120

ACCATCCATC CCATCAGGGA AATATGCAAC ATGCTTCAAA GAATAAGAAG AGAACTTTGG 180

GCCAGTAGAG ACGGGGTTTC ACCGTGTTAG CCAGGATGGT CTCGATCTCC TGACCTCGTG 240

ATCCGCCTGC CTTGGCCTCC CAAAGTGCTG GGATTACAGG GCACTGAATG TCAAAGTGAA 300

GGAATTCAAT GAAGCCCGGA TCAAGGGCAG GAGCAACCGT GACCCTCTTA AAAGGCCAA 360

TGCCCCATGT AATTAGTGAC GCGCGCGAAT GGATAGACGC TATTCCCACC GTCCCTACAT 420

AGCATCCAGC GACACCACAG CCAAGGGACA GGCTTGGCGG AACTGCGGGA GAGAAGAACC 480

CTCTGAGCCT GATTGAAAGG CGGTGAAGAG ACAGGAGAGG GGGATCGGTG GGCGGTCCCG 540

CCTCCATCTT CAGTTCCCGC ATGGGGGCGA GGCAGATCCA GACCAGCTCC TCCCAGACCT 600

CTCCAGAAGA AGCCATGGGA ACCCCTCGTA TCCAGCATTT GCTGATCCTC CTGGTCCTAG 660

GAGCCTCCCT CCTGACCTCG GGCCTAGAGC TGTATTGTCA AAAGGGTCTG TCCATGACTG 720

TGGAAGCAGA TCCAGCCAAT ATGTTTAACT GGACCCAGA GGAAGTGGAG ACTTGTGACA 780

AAGGGGCACT TTGCCAGGAA ACCATACTAA TAATTAAAGC AGGGACTGAG ACAGCCATTT 840

TGGCCACGAA GGGCTGCATC CCGGAAGGGG AGGAGGCCAT AACAATTGTC CAGCACTCTT 900

CACCTCCCGG CCTGATCGTG ACCTCCTACA GTAACTACTG TGAGGATTCC TTCTGTAATG 960

ACAAAGACAG CCTGTCTCAG TTTTGGGAGT TCAGTGAGAC CACAGCTTCC ACTGTGTCAA 1020

CAACCCTCCA TTGTCCAACC TGTGTGGCTT TGGGGACCTG TTTCAGTGCT CCTTCTCTTC 1080

CCTGTCCCAA TGGTACAACT CGATGCTATC AAGGAAAACT TGAGATCACT GGAGGTGGCA 1140

TTGAGTCGTC TGTGGAGGTC AAAGGCTGTA CAGCCATGAT TGGCTGCAGG CTGATGTCTG 1200

GAATCTTAGC AGTAGGACCC ATGTTTGTGA GGGAAGCGTG CCCACATCAG CTGCTCACTC 1260

AACCTCGAAA GACTGAAAAT GGGGCCACCT GTCTTCCCAT TCCTGTTTGG GGGTTACAGC 1320

TACTGCTGCC ATTGCTGCTG CCATCATTTA TTCACTTTTC CTAAGAAGGC ACTTCTGGGC 1380

CTGGGTCTGA GGACATCTTT TTTGACTGGG AGCCTTCTTA CTGTTGAGGT TCAACAAGCT 1440

GAGGAGTAGA TGGGAATTTG AGGGAGAATA CAGAGATACT ATGAACGTAT TTGACATTTT 1500

TAATACAATT TCTGCTATAA TTTTTGTATG CAGTAGGCGT TACTAATAAA CATTTCTGCT 1560

GTGAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 1620

AAAAAAAAAA AAAAAAAAAA 1640

(2) Information on SEQ ID NO: 2:

(i) Sequence features:

(A) Length: 267 amino acids

(B) type: amino acid

(D) Topology: linear

(ii) Molecular type: polypeptide

(xi) Sequence description: SEQ ID NO: 2:

MGARQIQTSS SQTSPEEAMG TPRIQHLLIL LVLGASLLTS XLELYCQKGL 50

SMTVEADPAN MFNWTTEEVE TCDKGALCQE TILIIKAGTE TAILATKGCI 100

PEGEEAITIV QHSSPPGLIV TSYSNYCEDS FCNDKDSLSQ FWEFSETTAS 150

TVSTTLHCPT CVAXGTCFSA PSLPYPNGTT RCYQGKLEIT GGXIESSVEV 200

KGCTAMIGCR LMSGILAVGP MFVREACPHQ LLTQPRKTEN GATCLPIPVW 250

GLQLLLPLLL PSFIHFS 267

(2) Information on SEQ ID NO: 3:

(i) Sequence features:

(A) Length: 227 amino acids

(B) type: amino acid

(D) Topology: Linear

(ii) Molecular type: polypeptide

(xi) Sequence description: SEQ ID NO: 3:

XLELYCQKGL SMTVEADPAN MFNWTTEEVE TCDKGALCQE TILIIKAGTE 50

TAILATKGCI PEGEEAITIV QHSSPPGLIV TSYSNYCEDS FCNDKDSLSQ 100

FWEFSETTAS TVSTTLHCPT CVAXGTCFSA PSLPYPNGTT RCYQGKLEIT 150

GGXIESSVEV KGCTAMIGCR LMSGILAVGP MFVREACPHQ LLTQPRKTEN 200

GATCLPIPVW GLQLLLPLLL PSFIHFS 227

() Information on SEQ ID NO: 4

(i) Sequential features

(A) length: 21 bases

(B) type: nucleic acid

(C) chain: single chain

(D) Topology: linear

(ii) Molecular type: oligonucleotide

(xi) Sequence description: SEQ ID NO: 4:

AGATCCAGAC CAGCTCCTCC C 21

(2) Information on SEQ ID NO: 5

(i) Sequential features

(A) length: 21 bases

(B) type: nucleic acid

(C) chain: single chain

(D) Topology: Linear

(ii) Molecular type: oligonucleotide

(xi) Sequence description: SEQ ID NO: 5:

AAATGATGGC AGCAGCAATG G 21

(2) Information on SEQ ID NO: 6

(i) Sequential features

(A) Length: 26 bases

(B) type: nucleic acid

(C) chain: single chain

(D) Topology: linear

(ii) Molecular type: oligonucleotide

(xi) Sequence description: SEQ ID NO: 6:

TGGATCTGCT TCCACAGTCA TGGACA 26

(2) Information on SEQ ID NO: 7

(i) Sequential features

(A) Length: 25 bases

(B) type: nucleic acid

(C) chain: single chain

(D) Topology: Linear

(ii) Molecular type: oligonucleotide

(xi) Sequence description: SEQ ID NO: 7:

TACAGCTCTA GGCCCGAGGT CAGGA 25

(2) Information on SEQ ID NO: 8

(i) Sequential features

(A) Length: 25 bases

(B) type: nucleic acid

(C) chain: single chain

(D) Topology: Linear

(ii) Molecular type: oligonucleotide

(xi) Sequence description: SEQ ID NO: 8:

AGAAGCCATG GGAACCCTCG TATCC 25

(2) Information on SEQ ID NO: 9

(i) Sequential features

(A) Length: 26 bases

(B) type: nucleic acid

(C) chain: single chain

(D) Topology: linear

(ii) Molecular type: oligonucleotide

(xi) Sequence description: SEQ ID NO: 9:

ATTGGCTGCA GGCTGATGTC TGGAAT 26

(2) Information on SEQ ID NO: 10

(i) Sequential features

(A) Length: 28 bases

(B) type: nucleic acid

(C) chain: single chain

(D) Topology: Linear

(ii) Molecular type: oligonucleotide

(xi) Sequence description: SEQ ID NO: 10:

CGCGGATCCT TGGGGACCTG TTTCAGTG 28

(2) Information on SEQ ID NO: 11

(i) Sequential features

(A) Length: 53 bases

(B) type: nucleic acid

(C) chain: single chain

(D) Topology: linear

(ii) Molecular type: oligonucleotide

(xi) Sequence description: SEQ ID NO: 11:

TCTAAGCTTA TGGGGGCGAG GCAGATCCAG ATCCAGACCA GCTCCTCCCA 50

GAC 53

(2) Information on SEQ ID NO: 12

(i) Sequential features

(A) Length: 40 bases

(B) type: nucleic acid

(C) chain: single chain

(D) Topology: linear

(ii) Molecular type: oligonucleotide

(xi) Sequence description: SEQ ID NO: 10:

CGTCTAGATT AGGAAAAGTG AATAAATGAT GGCAGCAGCA 40

(2) Information on SEQ ID NO: 13

(i) Sequential features

(A) Length: 30 bases

(B) type: nucleic acid

(C) chain: single chain

(D) Topology: Linear

(ii) Molecular type: oligonucleotide

(xi) Sequence description: SEQ ID NO: 13:

ATAAAGCTTA AATGATGGCA GCAGCAATGG 30

(2) Information on SEQ ID NO: 14

(i) Sequential features

(A) length: 21 bases

(B) type: nucleic acid

(C) chain: single chain

(D) Topology: Linear

(ii) Molecular type: oligonucleotide

(xi) Sequence description: SEQ ID NO: 14:

AGATCCAGAC CAGCTCCTCC C 21

Claims (10)

1. An isolated human Sgrg polypeptide, characterized in that it comprises: a polypeptide having an amino acid sequence of SEQ ID NO: 2, or a conservative variant polypeptide thereof, or an active fragment thereof, or an active derivative thereof. 2. The polypeptide according to claim 1, wherein the polypeptide is a polypeptide having a SEQ ID NO: 2 or 3 amino acid sequence. 3. An isolated polynucleotide encoding a human Sgrg polypeptide, characterized in that it comprises a nucleotide sequence having at least 70% identity to a nucleotide sequence selected from the following group : (a) polynucleotide encoding polypeptide as claimed in claim 1 or 2; (b) A polynucleotide complementary to polynucleotide (a). 4. The polynucleotide of claim 3, wherein the polynucleotide encodes a polypeptide having an amino acid sequence shown in SEQ ID NO:2. 5. The polynucleotide of claim 3, wherein the sequence of the polynucleotide is selected from one of the following groups: (a) has the sequence of positions 561-1364 in SEQ ID NO: 1; (b) has the sequence of positions 781-1364 in SEQ ID NO: 1; (c) having the sequence of positions 1-1640 in SEQ ID NO:1. 6. A vector comprising the polynucleotide according to claim 3. 7. A genetically engineered host cell, characterized in that it contains the vector according to claim 6. 8. A method for preparing a polypeptide having human Sgrg protein activity, characterized in that the method comprises: (a) cultivating the host cell according to claim 7 under conditions suitable for expressing the human Sgrg protein; (b) Isolating a polypeptide having human Sgrg protein activity from the culture. 9. An antibody capable of specifically binding to the human Sgrg polypeptide according to claim 1. 10. A pharmaceutical composition, characterized in that it contains a safe and effective amount of the polypeptide according to claim 1 and a pharmaceutically acceptable carrier.

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