CN1194012C - Sperm formation relative protein and its coding sequence and use - Google Patents
Sperm formation relative protein and its coding sequence and use Download PDFInfo
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- CN1194012C CN1194012C CNB001273140A CN00127314A CN1194012C CN 1194012 C CN1194012 C CN 1194012C CN B001273140 A CNB001273140 A CN B001273140A CN 00127314 A CN00127314 A CN 00127314A CN 1194012 C CN1194012 C CN 1194012C
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Abstract
The present invention provides a new spermatogenesis associated protein-Sgrg protein, polynucleotide for coding the Sgrg protein and a method for generating the Sgrg protein by recombination technology. The present invention also discloses the application of the polynucleotide for coding the Sgrg protein. The present invention also discloses a method for treating various diseases such as sterility disease, etc., by the Sgrg protein. The present invention also provides a medical composition containing the Sgrg protein.
Description
Technical field
The invention belongs to biotechnology and medical field, specifically, the present invention relates to the polynucleotide of new coding people sperm formation relative protein-Sgrg, and the polypeptide of this polynucleotide encoding.The invention still further relates to the purposes and the preparation of these polynucleotide and polypeptide.Specifically, polypeptide of the present invention is a kind of albumen relevant with growth with spermatogenesis.
Background technology
The male genetic link is a lot, the main neuroendocrine that male reproductive system is arranged is regulated, the spermatogeny of testis, the maturation of sperm in epididymis, with seminal vesicle, prostate gland excretory refining mixes seminal fluid in the sperm discharge process, sperm excretes and is input in the female genital tract from the male genetic road, sperm in women's uterine tube with links such as ovum fertilization, in these links, be interfered and influence, all growing barrier can take place.
According to World Health Organization's statistics, the couple at child-bearing age of the 5%-8% of world developed country has sterile problem, and the certain areas of developing country can be up to 30%.Press the standard that WHO recommends, Mr. and Mrs live together more than 1 year after marriage, do not use any contraceptives, owing to the reason of male sex aspect causes the infertile person in the wife's side, are called male infertility.The cause of disease of male sterility is divided into clinically: sexual dysfunction, and immunological infertility, refining is unusual, congenital disorders, genital tract infection, varicocele, cryptorrhea, obstruction of vas deferen, cacospermia, testicular dysfunction and environmental factors or the like.
In recent years, along with the continuous progress of medical level, a part of male infertility has found comparatively effectively treatment means clinically.But because still very not enough for the understanding of male genetic mechanism, male sterility, particularly spermatogeny are unusual caused sterile, also rest on the stage of empirical treatment.The clone of the gene relevant with spermatogeny also reports seldom.Therefore, the biochemical event of understanding in the spermatogeny on molecular level has crucial practical application meaning.
Therefore, to press for exploitation new new with spermatogenesis and/or generate proteins associated in this area.
Summary of the invention
The purpose of this invention is to provide a kind of new people sperm formation relative protein-Sgrg albumen with and fragment, analogue and derivative.
Another object of the present invention provides the polynucleotide of these polypeptide of coding.
Another object of the present invention provides the method for these polypeptide of production and the purposes of this polypeptide and encoding sequence.
In a first aspect of the present invention, novel isolated Sgrg polypeptide is provided, this polypeptide is the people source, it comprises: polypeptide or its conservative property variation polypeptide or its active fragments or its reactive derivative with SEQ ID NO:2 or 3 aminoacid sequences.Preferably, this polypeptide is the polypeptide with SEQ ID NO:2 or 3 aminoacid sequences.
In a second aspect of the present invention, the polynucleotide of isolating these polypeptide of coding are provided, these polynucleotide comprise a nucleotide sequence, and this nucleotide sequence is shown at least 70% homogeny with a kind of nucleotides sequence that is selected from down group: (a) polynucleotide of the above-mentioned people Sgrg polypeptide of coding; (b) with polynucleotide (a) complementary polynucleotide.Preferably, this polynucleotide encoding has the polypeptide of aminoacid sequence shown in the SEQ ID NO:2.More preferably, the sequence of these polynucleotide is be selected from down group a kind of: the sequence (the proteic coding region of Sgrg) that (a) has 561-1364 position among the SEQ ID NO:1; (b) has the sequence (the proteic coding region of sophisticated Sgrg) of 781-1364 position among the SEQ ID NO:1: the sequence that (c) has 1-1640 position among the SEQ ID NO:1.
In a third aspect of the present invention, the carrier that contains above-mentioned polynucleotide is provided, and has been transformed or host cell of transduceing or the host cell that is directly transformed or transduce by above-mentioned polynucleotide by this carrier.
In a fourth aspect of the present invention, provide preparation to have the method for the polypeptide of people Sgrg protein-active, this method comprises: (a) under the proteic condition of suitable expressing human Sgrg, cultivate the above-mentioned host cell that is transformed or transduce; (b) from culture, isolate polypeptide with people Sgrg protein-active.
In a fifth aspect of the present invention, provide and above-mentioned people Sgrg polypeptid specificity bonded antibody.The nucleic acid molecule that can be used for detecting also is provided, and it contains a successive 10-1640 Nucleotide in the above-mentioned polynucleotide.
In a sixth aspect of the present invention, the compound of simulation, promotion, antagonism people Sgrg polypeptide active is provided, and the compound that suppresses people Sgrg polypeptide expression.The method of screening and/or prepare these compounds also is provided.Preferably, this compound is encoding sequence or its segmental antisense sequences of people Sgrg polypeptide.
In a seventh aspect of the present invention, provide and whether had the proteic method of Sgrg in the test sample, it comprises: sample is contacted with the proteic specific antibody of Sgrg, observe whether form antibody complex, formed antibody complex and just represented to exist in the sample Sgrg albumen.
In a eighth aspect of the present invention, a kind of disease relevant with people Sgrg polypeptide unconventionality expression or method of disease susceptibility of detecting is provided, this method comprises: whether have sudden change in the nucleotide sequence of detection coding said polypeptide.
In a ninth aspect of the present invention, provide the purposes of polypeptide of the present invention and encoding sequence.Polypeptide for example of the present invention can be used to screen the agonist that promotes people Sgrg polypeptide active, and perhaps screening suppresses the antagonist of people Sgrg polypeptide active or is used to the peptide finger print identification.The proteic encoding sequence of people Sgrg of the present invention or its fragment can be used as primer and be used for pcr amplification reaction, perhaps are used for hybridization as probe, perhaps are used to make gene chip or microarray.
In a tenth aspect of the present invention, a kind of pharmaceutical composition is provided, it contains people Sgrg polypeptide of the present invention or its agonist, antagonist and the pharmaceutically acceptable carrier of safe and effective amount.These pharmaceutical compositions can be treated illnesss such as aspermia or oligospermia, teratospermia.
Others of the present invention are because disclosing of the technology of this paper is conspicuous to those skilled in the art.
Description of drawings
Following accompanying drawing is used to illustrate specific embodiments of the present invention, and is not used in qualification by the scope of the invention that claims defined.
Fig. 1 is the amino acid sequence homology comparison diagram of people Sgrg albumen of the present invention and human erythrocytosis genes involved PRV-1 (GI9857661).The top sequence is people Sgrg, and the below sequence is a people PRV-1 albumen.Identical amino acid marks with the amino acid letter between two sequences, and similar amino acid is used "+".
Fig. 2 is the electrophorogram of people Sgrg protein expressioning product of the present invention.
Embodiment
In the present invention, term " Sgrg albumen ", " Sgrg polypeptide " or " sperm formation relative protein Sgrg " are used interchangeably, and all refer to have the albumen or the polypeptide of people's sperm formation relative protein Sgrg aminoacid sequence (SEQ ID NO:2 or 3).They comprise the Sgrg albumen that contains or do not contain initial methionine, also comprise the Sgrg albumen that contains signal peptide or do not contain signal peptide.
As used herein, " isolating " is meant that material separates (if natural substance, primal environment promptly is a natural surroundings) from its primal environment.Do not have separation and purification as polynucleotide under the native state in the active somatic cell and polypeptide, but same polynucleotide or polypeptide as from native state with in other materials that exist separately, then for separation and purification.
As used herein, " isolating Sgrg albumen or polypeptide " is meant that the Sgrg polypeptide is substantially free of natural relative other albumen, lipid, carbohydrate or other material.Those skilled in the art can use the purified technology of protein purifying Sgrg albumen of standard.Basically pure polypeptide can produce single master tape on non-reduced polyacrylamide gel.The purity of Sgrg polypeptide can be used amino acid sequence analysis.
Polypeptide of the present invention can be recombinant polypeptide, natural polypeptides, synthetic polypeptide, preferred recombinant polypeptide.Polypeptide of the present invention can be the product of natural purifying, or the product of chemosynthesis, or uses recombinant technology to produce from protokaryon or eucaryon host (for example, bacterium, yeast, higher plant, insect and mammalian cell).The host used according to the recombinant production scheme, polypeptide of the present invention can be glycosylated, maybe can be nonglycosylated.
The present invention also comprises the proteic fragment of people Sgrg, derivative and analogue.As used herein, term " fragment ", " derivative " are meant with " analogue " and keep identical biological function of natural human Sgrg albumen of the present invention or active polypeptide basically.Polypeptide fragment of the present invention, derivative or analogue can be that (i) has one or more conservative or substituted polypeptide of non-conservation amino-acid residue (preferred conservative amino acid residue), and the amino-acid residue of such replacement can be also can not encoded by genetic code, or (ii) in one or more amino-acid residues, has a polypeptide of substituted radical, or (iii) mature polypeptide and another compound (such as the compound that prolongs the polypeptide transformation period, polyoxyethylene glycol for example) merges formed polypeptide, or (iv) additional aminoacid sequence is fused to this peptide sequence and the polypeptide that forms (as leader sequence or secretion sequence or be used for the sequence or the proteinogen sequence of this polypeptide of purifying, or with the fusion rotein of the segmental formation of antigen I gG).According to the instruction of this paper, these fragments, derivative and analogue belong to the known scope of those skilled in the art.
In the present invention, term " people Sgrg polypeptide " refers to have the SEQ ID NO.2 or 3 polypeptide of sequence of people Sgrg protein-active.This term also comprises having and variant forms people Sgrg albumen identical function, SEQ ID NO.2 or 3 sequences.These variant forms comprise (but being not limited to): several (are generally 1-50, preferably 1-30, more preferably 1-20,1-10 best) amino acid whose disappearance, insertion and/or replacement, and add one or several at C-terminal and/or N-terminal and (be generally in 20, preferably being in 10, more preferably is in 5) amino acid.For example, in the art, when replacing, can not change proteinic function usually with the close or similar amino acid of performance.Again such as, add one or several amino acid at C-terminal and/or N-terminal and also can not change proteinic function usually.This term also comprises proteic active fragments of people Sgrg and reactive derivative.
The variant form of this polypeptide comprises: homologous sequence, conservative property varient, allelic variant, natural mutation, induced mutation body, under high or low tight degree condition can with the coded albumen of the DNA of people Sgrg DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-people Sgrg polypeptide to obtain.The present invention also provides other polypeptide, as comprises people Sgrg polypeptide or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention has also comprised the soluble fragments of people Sgrg polypeptide.Usually, this fragment have people Sgrg peptide sequence at least about 10 continuous amino acids, usually at least about 30 continuous amino acids, preferably at least about 50 continuous amino acids, more preferably at least about 80 continuous amino acids, best at least about 100 continuous amino acids.
Invention also provides the analogue of people Sgrg albumen or polypeptide.The difference of these analogues and natural human Sgrg polypeptide can be the difference on the aminoacid sequence, also can be the difference that does not influence on the modified forms of sequence, perhaps haves both at the same time.These polypeptide comprise natural or the inductive genetic variant.The induce variation body can obtain by various technology, as by radiation or be exposed to mutagenic compound and produce random mutagenesis, also can pass through site-directed mutagenesis method or the biological technology of other known moleculars.Analogue also comprises having the analogue that is different from the amino acid whose residue of natural L-(as D-amino acid), and has non-natural analogue that exist or synthetic amino acid (as β, gamma-amino acid).Should be understood that polypeptide of the present invention is not limited to the above-mentioned representational polypeptide that exemplifies.
(the not changing primary structure usually) form of modification comprises: the chemically derived form such as the acetylize or carboxylated of the polypeptide that body is interior or external.Modification also comprises glycosylation, carries out glycosylation modified and polypeptide that produce in the procedure of processing as those in the synthetic and processing of polypeptide or further.This modification can be carried out glycosylated enzyme (as mammiferous glycosylase or deglycosylating enzyme) and finishes by polypeptide is exposed to.Modified forms also comprises have the phosphorylated amino acid residue sequence of (as Tyrosine O-phosphate, phosphoserine, phosphothreonine).Thereby also comprise the polypeptide that has been improved its anti-proteolysis performance or optimized solubility property by modifying.
In the present invention, " people Sgrg albumen conservative property variation polypeptide " refers to compare with the aminoacid sequence of SEQ ID NO:2 or 3, has 10 at the most, preferably at the most 8, more preferably at the most 5,3 amino acid is replaced by similar performance or close amino acid and is formed polypeptide at the most best.These conservative property variation polypeptide preferably carry out the amino acid replacement according to table 1 and produce.
Table 1
| Initial residue | Representational replacement | The preferred replacement |
| 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 | 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；Ala | Leu |
Polynucleotide of the present invention can be dna form or rna form.Dna form comprises the DNA of cDNA, genomic dna or synthetic.DNA can be strand or double-stranded.DNA can be coding strand or noncoding strand.The coding region sequence of encoding mature polypeptide can be identical with the coding region sequence shown in the SEQ ID NO:1 or the varient of degeneracy.As used herein, " varient of degeneracy " is meant that in the present invention coding has the protein of aminoacid sequence shown in SEQ ID NO:2 or 3, but with the differentiated nucleotide sequence of coding region sequence shown in the SEQ ID NO:1.
The polynucleotide of encoding mature polypeptide comprise: the encoding sequence of an encoding mature polypeptide; The encoding sequence of mature polypeptide and various additional code sequence; Encoding sequence of mature polypeptide (with optional additional code sequence) and non-coding sequence.
Term " polynucleotide of coded polypeptide " can be the polynucleotide that comprise this polypeptide of encoding, and also can be the polynucleotide that also comprise additional code and/or non-coding sequence.
The invention still further relates to the varient of above-mentioned polynucleotide, its coding has the polypeptide of identical aminoacid sequence or fragment, analogue and the derivative of polypeptide with the present invention.The varient of these polynucleotide can be the allelic variant of natural generation or the varient that non-natural takes place.These nucleotide diversity bodies comprise and replace varient, deletion mutation body and insert varient.As known in the art, allelic variant is the replacement form of polynucleotide, and it may be replacement, disappearance or the insertion of one or more Nucleotide, but can be from not changing the function of its encoded polypeptides in fact.
The invention still further relates to and above-mentioned sequence hybridization and two sequences between have at least 50%, preferably at least 70%, the polynucleotide of at least 80% homogeny more preferably.The present invention be more particularly directed under stringent condition and the interfertile polynucleotide of polynucleotide of the present invention.In the present invention, " stringent condition " is meant: (1) than hybridization under low ionic strength and the comparatively high temps and wash-out, as 0.2 * SSC, and 0.1%SDS, 60 ℃; Or (2) hybridization the time is added with denaturing agent, as 50% (v/v) methane amide, 0.1% calf serum/0.1%Ficoll, 42 ℃ etc.; Or (3) only at the homogeny between the two sequences at least more than 90%, be more preferably 95% and just hybridize when above.And the polypeptide of interfertile polynucleotide encoding has identical biological function and activity with the mature polypeptide shown in the SEQ ID NO:2.
The invention still further relates to nucleic acid fragment with above-mentioned sequence hybridization.As used herein, the length of " nucleic acid fragment " contains 15 Nucleotide at least, better is at least 30 Nucleotide, is more preferably at least 50 Nucleotide, preferably more than at least 100 Nucleotide.Nucleic acid fragment can be used for the amplification technique (as PCR) of nucleic acid to determine and/or the proteic polynucleotide of separation coding Sgrg.
Polypeptide among the present invention and polynucleotide preferably provide with isolating form, more preferably are purified to homogeneous.
People Sgrg Nucleotide full length sequence of the present invention or its fragment can obtain with the method for pcr amplification method, recombination method or synthetic usually.For the pcr amplification method, can be disclosed according to the present invention about nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the prepared cDNA storehouse of ordinary method well known by persons skilled in the art as template, amplification and must relevant sequence.When sequence is longer, usually needs to carry out twice or pcr amplification repeatedly, and then the fragment that each time amplifies is stitched together by proper order.
In case obtained relevant sequence, just can obtain relevant sequence in large quantity with recombination method.This normally is cloned into carrier with it, changes cell again over to, separates obtaining relevant sequence then from the host cell after the propagation by ordinary method.
In addition, also the method for available synthetic is synthesized relevant sequence, especially fragment length more in short-term.Usually, by first synthetic a plurality of small segments, and then connect and to obtain the very long fragment of sequence.
At present, can be fully obtain the dna sequence dna of code book invention albumen (or its fragment, or derivatives thereof) by chemosynthesis.This dna sequence dna can be introduced in various existing dna moleculars as known in the art (or as carrier) and the cell then.In addition, also can will suddenly change and introduce in the protein sequence of the present invention by chemosynthesis.
Use method (Saiki, the et al.Science 1985 of round pcr DNA amplification/RNA; 230:1350-1354) be optimized for acquisition gene of the present invention.When particularly being difficult to obtain the cDNA of total length from the library, can preferably use RACE method (the terminal rapid amplifying method of RACE-cDNA), the primer that is used for PCR can suitably be selected according to sequence information of the present invention disclosed herein, and available ordinary method is synthetic.Available ordinary method is as the DNA/RNA fragment by gel electrophoresis separation and purifying amplification.
The present invention also relates to comprise the carrier of polynucleotide of the present invention, and the host cell that produces through genetically engineered with carrier of the present invention or Sgrg albumen coded sequence, and the method that produces polypeptide of the present invention through recombinant technology.
Recombinant DNA technology (Science, 1984 by routine; 224:1431), can utilize polymerized nucleoside acid sequence of the present invention to can be used to express or produce the Sgrg polypeptide of reorganization.In general following steps are arranged:
(1). with the polynucleotide (or varient) of coding of the present invention people Sgrg polypeptide, or with the recombinant expression vector that contains these polynucleotide proper host cell that transforms or transduce;
(2). the host cell of in suitable medium, cultivating;
(3). separation, protein purification from substratum or cell.
Among the present invention, people Sgrg polynucleotide sequence can be inserted in the recombinant expression vector.Term " recombinant expression vector " refers to that bacterial plasmid well known in the art, phage, yeast plasmid, vegetable cell virus, mammalian cell virus are as adenovirus, retrovirus or other carriers.The carrier of Shi Yonging includes but not limited in the present invention: and the expression vector based on T7 of in bacterium, expressing (Rosenberg, et al.Gene, 1987,56:125); The pMSXND expression vector of in mammalian cell, expressing (Lee and Nathans, J Bio Chem.263:3521,1988) and at the carrier that derives from baculovirus of expressed in insect cells.In a word, as long as can duplicate in host and stablize, any plasmid and carrier can be used.A key character of expression vector is to contain replication orgin, promotor, marker gene and translation controlling elements usually.
Method well-known to those having ordinary skill in the art can be used to make up and contains people Sgrg DNA sequences encoding and suitable transcribing/the translate expression vector of control signal.These methods comprise (Sambroook, et al.Molecular Cloning, a Laboratory Manual, coldSpring Harbor Laboratory.New York, 1989) such as extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technologys of body.Described dna sequence dna can effectively be connected on the suitable promotor in the expression vector, and is synthetic to instruct mRNA.The representative example of these promotors has: colibacillary lac or trp promotor; Lambda particles phage P
LPromotor; Eukaryotic promoter comprises LTRs and some other known may command gene expression promoter in protokaryon or eukaryotic cell or its virus of CMV immediate early promoter, HSV thymidine kinase promoter, early stage and late period SV40 promotor, retrovirus.Expression vector also comprises ribosome bind site and the transcription terminator that translation initiation is used.
In addition, expression vector preferably comprises one or more selected markers, to be provided for selecting the phenotypic character of transformed host cells, cultivate Tetrahydrofolate dehydrogenase, neomycin resistance and the green fluorescent protein (GFP) of usefulness as eukaryotic cell, or be used for colibacillary tsiklomitsin or amicillin resistance.
Comprise the carrier of above-mentioned suitable dna sequence dna and suitable promotor or control sequence, can be used to transform appropriate host cell, so that it can marking protein.
Host cell can be a prokaryotic cell prokaryocyte, as bacterial cell; Or eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as mammalian cell.Representative example has: intestinal bacteria, streptomyces; The bacterial cell of Salmonella typhimurium; Fungal cell such as yeast; Vegetable cell; The insect cell of fruit bat S2 or Sf9; The zooblast of CHO, COS, 293 cells or Bowes melanoma cells etc.
When polynucleotide of the present invention are expressed in higher eucaryotic cells, be enhanced if will make to transcribe when in carrier, inserting enhancer sequence.Enhanser is the cis acting factor of DNA, and nearly 10 to 300 base pairs act on promotor transcribing with enhancing gene usually.Can for example be included in the SV40 enhanser of 100 to 270 base pairs of replication origin side in late period one, at the polyoma enhanser of replication origin side in late period one and adenovirus enhanser etc.
Persons skilled in the art all know how to select appropriate carriers, promotor, enhanser and host cell.
Can carry out with routine techniques well known to those skilled in the art with the recombinant DNA transformed host cell.When the host was prokaryotic organism such as intestinal bacteria, the competent cell that can absorb DNA can be used CaCl in exponential growth after date results
2Method is handled, and used step is well-known in this area.Another kind method is to use MgCl
2If desired, transforming also the method for available electroporation carries out.When the host is an eukaryote, can select following DNA transfection method for use: coprecipitation of calcium phosphate method, conventional mechanical method such as microinjection, electroporation, liposome packing etc.
The transformant that obtains can be cultivated with ordinary method, expresses the polypeptide of coded by said gene of the present invention.According to used host cell, used substratum can be selected from various conventional substratum in the cultivation.Under the condition that is suitable for the host cell growth, cultivate.After host cell grows into suitable cell density, induce the promotor of selection with suitable method (as temperature transition or chemical induction), cell is cultivated for some time again.
The extracellular can be expressed or be secreted into to recombinant polypeptide in the above methods in cell or on cytolemma.If desired, can utilize its physics, the separating by various separation methods with other characteristic and the albumen of purification of Recombinant of chemistry.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation handles, with protein precipitant handle (salt analysis method), centrifugal, the broken bacterium of infiltration, superly handle, the combination of super centrifugal, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography (LC) technology and these methods.
The people Sgrg albumen or the polypeptide of reorganization are of use in many ways.These purposes include, but is not limited to: directly as pharmacological agent Sgrg protein function the disease due to the low or forfeiture (as no sperm, or disease such as low sperm activity) and be used to screen and promote or antibody, polypeptide or other part of antagonism Sgrg protein function.The peptide molecule that can suppress or stimulate people Sgrg protein function that can be used for seeking therapeutic value with the recombinant human Sgrg protein screening peptide library of expressing.
On the other hand, the present invention also comprises people Sgrg DNA or the polypeptide of its fragment coding has specific polyclonal antibody and monoclonal antibody, especially monoclonal antibody.Here, " specificity " is meant that antibody capable is incorporated into people Sgrg gene product or fragment.Preferably, refer to that those can combine with people Sgrg gene product or fragment but nonrecognition and be incorporated into the antibody of other irrelevant antigen molecule.Among the present invention antibody comprise those can in conjunction with and suppress the proteic molecule of people Sgrg, comprise that also those do not influence the antibody of people Sgrg protein function.The present invention also comprise those can with modify or without the people Sgrg gene product bonded antibody of modified forms.
The present invention not only comprises complete mono-clonal or polyclonal antibody, but also comprises having immunocompetent antibody fragment, as Fab ' or (Fab)
2Fragment; Heavy chain of antibody; Light chain of antibody; Genetically engineered strand Fv molecule (people such as Ladner, U.S. Patent No. 4,946,778); Or chimeric antibody, as have the murine antibody binding specificity but still keep antibody from people's antibody moiety.
Antibody of the present invention can be prepared by the known various technology of those skilled in that art.For example, the people Sgrg gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing human Sgrg albumen or its have antigenic segmental cell and can be used to immune animal and produce antibody.Antibody of the present invention also can be monoclonal antibody.This type of monoclonal antibody can utilize hybridoma technology prepare (see people such as Kohler,
Nature256; 495,1975; People such as Kohler,
Eur.J.Immunol.6:511,1976; People such as Kohler,
Eur.J.Immunol.6:292,1976; People such as Hammerling,
In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981).Antibody of the present invention comprises the antibody that can block people Sgrg protein function and the antibody that does not influence people Sgrg protein function.Each antibody-like of the present invention can utilize the fragment or the functional zone of people Sgrg gene product, obtains by the routine immunization technology.These fragments or functional zone can utilize recombinant methods or utilize Peptide synthesizer synthetic.Can come immune animal and produce with the gene product of producing in the prokaryotic cell prokaryocyte (for example E.Coli) with the unmodified form bonded antibody of people Sgrg gene product; With posttranslational modification form bonded antibody (as the albumen or the polypeptide of glycosylation or phosphorylation), can come immune animal and obtain with the gene product that produces in the eukaryotic cell (for example yeast or insect cell).
The proteic antibody of anti-people Sgrg can be used in the immunohistochemistry technology, detects the people Sgrg albumen in the biopsy specimen.In addition, with the also available labelled with radioisotope of the protein bound monoclonal antibody of people Sgrg, inject in the body and can follow the tracks of its position and distribution.This radiolabeled antibody can be used as a kind of atraumatic diagnostic method and is used for the location of tumour cell and has judged whether transfer.
Antibody among the present invention can be used for treating or prevention and the relevant disease of people Sgrg albumen.The antibody that gives suitable dosage can stimulate or block proteic generation of people Sgrg or activity.
Antibody also can be used for being designed to the immunotoxin at a certain privileged sites in the body.As the monoclonal antibody of people Sgrg albumen high-affinity can with bacterium or plant poison (as diphtheria toxin, ricin, abrine etc.) covalent attachment.A kind of usual method is with sulfydryl linking agent such as SPDP, attacks the amino of antibody, by the exchange of disulfide linkage, toxin is incorporated on the antibody, and this hybrid antibody can be used for killing the cell (for example lymph-node cell etc.) of people Sgrg protein positive.
The production of polyclonal antibody can choose Sgrg albumen or polypeptide immune animal, as rabbit, mouse, rat etc.Multiple adjuvant can be used for the enhancing immunity reaction, includes but not limited to freund's adjuvant etc.
Utilize albumen of the present invention,, can filter out with Sgrg albumen interactional material takes place, as acceptor, inhibitor, agonist or antagonist etc. by various conventional screening methods.
Albumen of the present invention and antibody thereof, inhibitor, agonist, antagonist or acceptor etc. when using (administration) in treatment, can provide different effects.Usually, can these materials are formulated in nontoxic, inert and the pharmaceutically acceptable aqueous carrier medium, wherein pH is about 5-8 usually, and preferably pH is about 6-8, although the pH value can be with being changed to some extent by preparation Substance Properties and illness to be treated.The pharmaceutical composition for preparing can carry out administration by conventional route, comprising (but being not limited to): oral, intramuscular, intraperitoneal, intravenously, subcutaneous, intracutaneous or topical.
Polypeptide of the present invention can be directly used in disease treatment, for example, is used for the treatment of testicular spermatogenic function obstacle aspect.When using Sgrg albumen of the present invention, also can use other to be used for same treatment of conditions agent simultaneously, as Testosterone etc.
The present invention also provides a kind of pharmaceutical composition, and it contains Sgrg polypeptide of the present invention or its agonist, antagonist and the pharmaceutically acceptable carrier or the vehicle of safe and effective amount.This class carrier comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol and combination thereof.Pharmaceutical preparation should be complementary with administering mode.Pharmaceutical composition of the present invention can be made into the injection form, for example is prepared by ordinary method with the physiological saline or the aqueous solution that contains glucose and other assistant agents.Pharmaceutical composition such as tablet and capsule can be prepared by ordinary method.Pharmaceutical composition such as injection, solution, tablet and capsule should be made under aseptic condition.The dosage of activeconstituents is the treatment significant quantity, for example every day about 1 microgram/kg body weight-Yue 5 mg/kg body weight.In addition, polypeptide of the present invention also can use with the other treatment agent.
When making pharmaceutical composition, be that the Sgrg albumen of safe and effective amount or its antagonist, agonist are applied to Mammals, wherein this safe and effective amount is usually at least about 10 micrograms/kg body weight, and in most of the cases be no more than about 8 mg/kg body weight, preferably this dosage is about 10 micrograms/kg body weight-Yue 1 mg/kg body weight.Certainly, concrete dosage also should be considered factors such as route of administration, patient health situation, and these all are within the skilled practitioners skill.
The proteic polynucleotide of people Sgrg also can be used for multiple therapeutic purpose.Gene therapy technology can be used for treating because cell proliferation, growth or the metabolic disturbance due to the proteic expression of Sgrg of the proteic nothing expression of Sgrg or unusual/non-activity.The Sgrg albumen that the gene therapy vector (as virus vector) of reorganization can be designed to express variation is to suppress endogenic Sgrg protein-active.Deriving from viral expression vector such as retrovirus, adenovirus, adeno-associated virus (AAV), hsv, parvovirus etc. can be used for the Sgrg transgenosis to cell.The method that structure carries the recombinant viral vector of Sgrg gene is found in existing document (Sambrook, et al.).Recombinant human Sgrg gene can be packaged in the liposome in addition, and then is transferred in the cell.
Suppress the oligonucleotide (comprising sense-rna and DNA) of people Sgrg mRNA and ribozyme also within the scope of the invention.Ribozyme is the enzyme sample RNA molecule that a kind of energy specificity is decomposed specific RNA, and its mechanism of action is to carry out the endonuclease effect after ribozyme molecule and the hybridization of complementary target RNA-specific.The RNA of antisense and DNA and ribozyme can obtain with existing any RNA or DNA synthetic technology, as the technology widespread use of solid phase phosphoamide chemical synthesis synthetic oligonucleotide.Antisense rna molecule can be transcribed acquisition by the dna sequence dna of this RNA that encodes in external or body.This dna sequence dna has been incorporated into the downstream of rna polymerase promoter of carrier.In order to increase the stability of nucleic acid molecule, available several different methods is modified it, and as increasing the sequence length of both sides, the connection between the ribonucleoside is used phosphoric acid thioester bond or peptide bond but not phosphodiester bond.
Polynucleotide imports tissue or intracellular method comprises: directly be injected into polynucleotide in the in-vivo tissue; Or external by carrier (as virus, phage or plasmid etc.) earlier with the polynucleotide transfered cell in, again cell is transplanted in the body etc.
Can be incorporated into the rondom polypeptide storehouse that solid formation forms by the various amino acid that may make up by screening with the protein bound peptide molecule of people Sgrg obtains.During screening, must carry out mark to people Sgrg protein molecular.
The invention still further relates to the diagnostic testing process of quantitative and detection and localization people Sgrg protein level.These tests are known in the art, and comprise that FISH measures and radioimmunoassay.The people Sgrg protein level that is detected in the test can be with laying down a definition the importance of people Sgrg albumen in various diseases and be used to the disease of diagnosing Sgrg albumen to work.
Whether having the proteic method of Sgrg in a kind of detection test sample is to utilize the proteic specific antibody of Sgrg to detect, and it comprises: sample is contacted with the Sgrg protein specific antibody; Observe whether form antibody complex, formed antibody complex and just represented to exist in the sample Sgrg albumen.
The proteic polynucleotide of Sgrg can be used for the diagnosis and the treatment of Sgrg protein related diseases.Aspect diagnosis, the proteic polynucleotide of Sgrg can be used for detecting the proteic expression of Sgrg Sgrg abnormal exprssion whether or under morbid state.Can be used for the hybridization of biopsy specimen to judge the proteic abnormal expression of Sgrg as the Sgrg dna sequence dna.Hybridization technique comprises the Southern blotting, Northern blotting, in situ hybridization etc.These technological methods all are disclosed mature technologies, and relevant test kit all can obtain from commercial channels.Part or all of polynucleotide of the present invention can be used as probe stationary on microarray (microarray) or DNA chip (being called " gene chip " again), is used for analyzing the differential expression analysis and the gene diagnosis of tissue gene.Carry out RNA-polymerase chain reaction (RT-PCR) amplification in vitro with the special primer of Sgrg albumen and also can detect the proteic transcription product of Sgrg.
The sudden change that detects the Sgrg gene also can be used for the disease of diagnosing Sgrg albumen relevant.The form of Sgrg protein mutation comprises that the point mutation compared with normal wild type Sgrg dna sequence dna, transposition, disappearance, reorganization and other are any unusual etc.Available existing technology such as Southern blotting, dna sequence analysis, PCR and in situ hybridization detect sudden change.In addition, sudden change might influence proteic expression, therefore can judge indirectly that with Northern blotting, Western blotting gene has or not sudden change.
Sequence of the present invention identifies it also is valuable to karyomit(e).In brief, the proteic cDNA of Sgrg prepares PCR primer (preferred 15-35bp) according to the present invention, sequence can be positioned on the karyomit(e).Then, these primers are used for the somatocyte hybrid cell that the PCR screening contains each bar human chromosome.Have only those hybrid cells that contain corresponding to the people's gene of primer can produce the fragment of amplification.
In case sequence is positioned to chromosome position accurately, the physical location of this sequence on karyomit(e) just can be associated with the gene map data.These data for example are found in, V.Mckusick, Mendelian Inheritance inMan (can by with the online acquisition of Johns Hopkins University Welch Medical Library).Can pass through linkage analysis then, determine gene and navigated to relation between the disease on the chromosomal region already.
In an example of the present invention, a kind of isolating polynucleotide are provided, its coding has the polypeptide of aminoacid sequence shown in the SEQ ID NO:2.Polynucleotide of the present invention are isolated from people's periphery lymph hemocyte cDNA library.Its sequence is shown in SEQ ID NO:1, and the polynucleotide sequence total length that it comprises is 1640 bases, and its open reading frame is positioned at the 561-1364 position, and the coding total length is 267 amino acid whose people Sgrg albumen (SEQ IDNO:2).Can be the no sperm of treatment, or disease such as low sperm activity provides new treatment approach, thereby have great application prospect.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York:Cold SpringHarbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
The acquisition of embodiment 1:EST
The mouse ehrlich's ascite cell of phase and mouse L929 cell are material when dividing with difference, with scleroderma patient's antiserum(antisera) and its effect, utilize indirect immunofluorescence to detect the distribution situation of antigen in born of the same parents, have obtained a routine antiserum(antisera).It can be centered around around the karyomit(e) of division middle and later periods cell specifically.In order to seek these antigen proteins, the inventor is with this serum screening people testis lambda particles phage expression library.Obtain some clones, DNA extraction according to a conventional method, purifying.With the order-checking of λ gt11 primer, find that one of them is new EST, be YN11 with this EST called after.
Embodiment 2: the acquisition of full length sequence
(1) primer is synthetic
In order to obtain comprising the full-length cDNA of YN11 EST, with reference to the sequences Design of this EST with synthesized following primer:
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) 5 '-acquisition of end cDNA sequence:
With Marathon-Ready-cDNA Human Leukocyte (Clontech-7406-1) is template, with the condition that primer YN11 XF and AP1 (AP1 is the primer that provides in the test kit) advise with reference to manufacturer, carries out RACE-PCR.After 10 times of the first round PCR product dilutions, increase once more with nested primer YN11NF and AP2 (AP2 is the primer that provides in the test kit) as template.The PCR product that amplification obtains, rubber tapping, purifying is cloned into T-A carrier (Sangon), with M13 forward and reverse primer order-checking.
(3) 3 '-acquisition of end cDNA sequence
Employing and 5 '-the identical strategy of end employing, difference only is: primer is YN11NR.+AP1 YN11R2+AP2 then.
(4) acquisition of full length sequence
With 3 '-end, 5 '-the terminal sequence splicing obtains complete full-length cDNA (GENE BANK accession number: AF241268) (because of applying for a patent, so be in confidential state before the application's submission).Sgrg cDNA is 1640bp (SEQ ID NO:1), contains complete open frame 801bp, and coding contains the polypeptide (SEQ ID NO:2) of 267 amino-acid residues.
Through the SignaIP analyses and prediction, the 1-40 amino acids is for pointing to the signal peptide of endoplasmic reticulum among the SEQ ID NO:2.Sophisticated peptide molecule is formed (SEQ ID NO:3) by 227 amino-acid residues, wherein relatively sees Fig. 1 with the homology of human erythrocytosis genes involved PRV-1.PRV-1 is made up of two highly similar structural domains, forms a repeating structure.So BLAST result shows two fragments of SGRG and PRV-1 homology is arranged all.Homogeny is 32%, and similarity is 49%.
The chromosomal localization of embodiment 5:Sgrg
By the condition of manufacturer's suggestion, utilize RH panel Assay (Research Genetics) to carry out the chromosomal localization of Sgrg.This gene is positioned 19q13.2 on karyomit(e).
The PCR condition: 95 ℃ 10 minutes → 94 ℃ 30 seconds; 64 ℃ 30 seconds; 72 ℃ 23 seconds, totally 30 the circulation → 72 ℃ 3 minutes 30 seconds.
Primer: YN11 XF and NR (this PCR condition is a part of condition of carrying out RH panel Assay)
Amplified production is analyzed through rhserver@shgc.standford.edu, and the result shows, with its most closely linked genetic marker be SHGC 3096.
The distribution expression pattern of embodiment 6:Sgrg
The one section cDNA (615-1364 among the SEQ ID NO:1) that comprises ORF with Sgrg is a probe, hybridize with MTE film (Clontech-7775-1), the result shows that this Sgrg very expresses specifically in testis tissue, and has only low-down expression in its hetero-organization (as liver, the heart etc.).
The gene structure of embodiment 7:Sgrg and protein structure
(1) gene structure
Sgrg cDNA full length sequence is BLAST at the DATABASE-htgs of http://www.ncbi.nlm.nih.gov/BLAST/, found that people BAC: karyomit(e) 19, CIT978SKB-61I7 have comprised the Sgrg gene, and the span of whole gene is about 23kb.Gene structure is as follows:
The gene structure of table 2 Sgrg
| The CDNA sequence | Genome sequence | Splice site | |
| Exons 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
With ORF Finder program (http://www.ncbi.nlm.nih.gov/gorf/gorf.html) prediction Sgrg protein sequence, obtain one 267 amino acid whose open reading frame.It may contain following constructional feature by analysis:
(A) motif:
1. amino acid 63-66; The 177-180:N-glycosylation site
2. amino acid 21-23; The 179-181:PKC-phosphorylation site
3. amino acid/11 3-16; 40-43; 65-68; 196-199: casein kinase 2 phosphorylation sites
(B) gene family analysis: on http://fps.sdsc.edu, carry out the gene family analysis.The result shows that Sgrg albumen has the uPAR structural domain (C is rich in structural domain) that comprises uPAR signature sequence (CXDXXCN), belongs to the urokinase receptor superfamily.
(C) homology analysis: BLAST (http://www.ncbi.nlm.nih.gov/BLAST/):
As shown in Figure 1, Sgrg albumen and human erythrocytosis genes involved PRV-1 (GI9857661) have than higher homology, and especially the homology in uPAR superfamily conservative region is stronger.
PRV-1 belongs to a member of uPAR superfamily, and too propagation is corresponding with red corpuscle for its high expression level of crossing.The PRV-1 gene also is positioned the position of 19q13.2, only is separated by about 9kb with the position of Sgrg.
The in situ hybridization of embodiment 8:Sgrg
Because the Sgrg gene is expressed specifically,, carry out in situ hybridization according to a conventional method so, be material with the rhesus monkey testis in order further to understand its effect in male reproductive system in testis tissue.Found that it expresses in the androgone of convoluted seminiferous tubule comparatively singlely.This shows that the function of Sgrg and the growth of sperm and/or maturation have very close getting in touch.
The expression of embodiment 9:Sgrg fragment in prokaryotic cell prokaryocyte
With primer 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), be the DNA of the proteic 167-267a.a of template amplification coding Sgrg with human peripheral cDNA.
Utilize EcoRI and Hind III site with pcr amplification product be cloned into PET32 (a) (Novagen) in, transform host E.Coli:BL21 (DE3) then.Transformed host cells is expressed under the inducing of IPTG.The result as shown in Figure 2, expression product molecular weight in SDS-PAGE glue is approximately 33Kda (containing fusion rotein sulphur hydrogen reduction albumen (Thioredoxin)).
Embodiment 10: the preparation of antibody
With the fusion rotein of expressing among Ni-NTA Agarose (Clontech) the purifying embodiment, this fusion rotein as antigen, is prepared polyclonal antibody in rabbit.Specific procedure is as follows: immune purebred new zealand rabbit for the first time, albumen consumption be 240ug/ only, use Freund's complete adjuvant.After 4 weeks, immunity for the second time, the albumen consumption is 140ug/, full adjuvant toos many or too much for use.Afterwards 2 weeks, immune for the third time, the albumen consumption is 120ug/, and full adjuvant toos many or too much for use.
The promoter action of embodiment 11:Sgrg on cell proliferation
For understanding Sgrg in the meaning that may have aspect the cytokinetics, the colony that has carried out in the present embodiment forms experiment.
With primer YN11E-F-2 (5 '-TCT AAG CTT ATG GGG GCG AGG CAG ATC CAGATC CAG ACC AGC 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), cDNA is a template with people's periphery lymph blood, obtains the coding region (561-1364 position among the SEQ IDNO:1) of Sgrg by the RT-PCR amplification.
Utilize the restriction enzyme site (Xba I and Hind III) on the primer then, extension amplification outcome to carrier for expression of eukaryon pRC/CMV (Invitrogen), is formed plasmid pRC/CMV-Sgrg.Lipofectamine (18324-020) liposome transfection technology with Gibco company with the plasmid pRC/CMV-Sgrg transfection l cell 3T3 that builds is and Bel7402 7721.The cell strain of stable conversion is with after the low density inoculation, and with empty carrier pRC/CMV as negative control, with JCL/L (a kind of known short growth factor) as positive control.The result is as shown in table 3:
The promoter action of table 3 Sgrg on cell proliferation
| 3T3 cell (clone's number) | 7721 cells (clone's number) | |
| Sgrg (cross and express) | 42 | 17 |
| Empty carrier | 3 | 2 |
| JCL/L (positive control) | 38 | Do not have |
As can be seen, overexpression Sgrg gene can influence the propagation of cell in eukaryotic cell.The feature of this and uPAR family is also more identical.
Embodiment 12:RT-PCR method obtains the Sgrg encoding sequence
From human peripheral, separate total RNA with RNeasy (Qiagen).Use SupertScript II RNaseH-Reverse Transcriptase (Gibco) then, reverse transcription obtains cDNA (providing conditioned response by manufacturer).. with cDNA is template, uses primer 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)
By following condition PCR::95 ℃ 10 minutes → 94 ℃ 30 seconds; 60 ℃ 30 seconds; 72 ℃ 90 seconds, totally 45 the circulation → 72 ℃ 3 minutes 30 seconds.
The PCR product that amplification obtains is through order-checking, and the result shows with the coding region sequence of the Sgrg shown in the SEQ ID NO:1 and conforms to.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Sequence table
(1) general information:
(ii) denomination of invention: new sperm formation relative protein, its encoding sequence and purposes
(iii) sequence number: 14
(2) information of SEQ ID NO:1:
(i) sequence signature:
(A) length: 1640bp
(B) type: nucleic acid
(C) chain: two strands
(D) topological framework: linearity
(ii) molecule type: cDNA
(xi) sequence description: SEQ ID NO:1:
ATTTAGGATG AACTATGTGT GACAAATGGT GCCGTTGAGT CCTCTCAACT 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 AAAGAGCCAA 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 GGACCACAGA 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 of SEQ ID NO:2:
(i) sequence signature:
(A) length: 267 amino acid
(B) type: amino acid
(D) topological framework: linearity
(ii) molecule 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 of SEQ ID NO:3:
(i) sequence signature:
(A) length: 227 amino acid
(B) type: amino acid
(D) topological framework: linearity
(ii) molecule 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
The information of () SEQ ID NO:4
(i) sequence signature
(A) length: 21 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: oligonucleotide
(xi) sequence description: SEQ ID NO:4:
AGATCCAGAC CAGCTCCTCC C 21
(2) information of SEQ ID NO:5
(i) sequence signature
(A) length: 21 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: oligonucleotide
(xi) sequence description: SEQ ID NO:5:
AAATGATGGC AGCAGCAATG G 21
(2) information of SEQ ID NO:6
(i) sequence signature
(A) length: 26 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: oligonucleotide
(xi) sequence description: SEQ ID NO:6:
TGGATCTGCT TCCACAGTCA TGGACA 26
(2) information of SEQ ID NO:7
(i) sequence signature
(A) length: 25 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: oligonucleotide
(xi) sequence description: SEQ ID NO:7:
TACAGCTCTA GGCCCGAGGT CAGGA 25
(2) information of SEQ ID NO:8
(i) sequence signature
(A) length: 25 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: oligonucleotide
(xi) sequence description: SEQ ID NO:8:
AGAAGCCATG GGAACCCTCG TATCC 25
(2) information of SEQ ID NO:9
(i) sequence signature
(A) length: 26 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: oligonucleotide
(xi) sequence description: SEQ ID NO:9:
ATTGGCTGCA GGCTGATGTC TGGAAT 26
(2) information of SEQ ID NO:10
(i) sequence signature
(A) length: 28 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: oligonucleotide
(xi) sequence description: SEQ ID NO:10:
CGCGGATCCT TGGGGACCTG TTTCAGTG 28
(2) information of SEQ ID NO:11
(i) sequence signature
(A) length: 53 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: oligonucleotide
(xi) sequence description: SEQ ID NO:11:
TCTAAGCTTA TGGGGGCGAG GCAGATCCAG ATCCAGACCA GCTCCTCCCA 50
GAC 53
(2) information of SEQ ID NO:12
(i) sequence signature
(A) length: 40 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: oligonucleotide
(xi) sequence description: SEQ ID NO:10:
CGTCTAGATT AGGAAAAGTG AATAAATGAT GGCAGCAGCA 40
(2) information of SEQ ID NO:13
(i) sequence signature
(A) length: 30 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: oligonucleotide
(xi) sequence description: SEQ ID NO:13:
ATAAAGCTTA AATGATGGCA GCAGCAATGG 30
(2) information of SEQ ID NO:14
(i) sequence signature
(A) length: 21 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: oligonucleotide
(xi) sequence description: SEQ ID NO:14:
AGATCCAGAC CAGCTCCTCC C 21
Claims (10)
1. an isolating people Sgrg polypeptide is characterized in that it comprises: polypeptide or its conservative property variation polypeptide or its active fragments or its reactive derivative with SEQ ID NO:2 aminoacid sequence.
2. polypeptide as claimed in claim 1 is characterized in that, this polypeptide is the polypeptide with SEQ ID NO:2 or 3 aminoacid sequences.
3. the polynucleotide of a separated coding people Sgrg polypeptide is characterized in that it comprises a nucleotide sequence, and this nucleotide sequence is shown at least 70% homogeny with a kind of nucleotides sequence that is selected from down group:
(a) polynucleotide of polypeptide as claimed in claim 1 or 2 of encoding;
(b) with polynucleotide (a) complementary polynucleotide.
4. polynucleotide as claimed in claim 3 is characterized in that, this polynucleotide encoding has the polypeptide of aminoacid sequence shown in the SEQ ID NO:2.
5. polynucleotide as claimed in claim 3 is characterized in that, the sequence of these polynucleotide is selected from down a kind of of group:
(a) has the sequence of 561-1364 position among the SEQ ID NO:1;
(b) has the sequence of 781-1364 position among the SEQ ID NO:1;
(c) has the sequence of 1-1640 position among the SEQ ID NO:1.
6. a carrier is characterized in that, it contains the described polynucleotide of claim 3.
7. a genetically engineered host cell is characterized in that, it contains the described carrier of claim 6.
8. preparation method with polypeptide of people Sgrg protein-active is characterized in that this method comprises:
(a) under the proteic condition of suitable expressing human Sgrg, cultivate the described host cell of claim 7;
(b) from culture, isolate polypeptide with people Sgrg protein-active.
9. energy and the described people Sgrg of claim 1 polypeptid specificity bonded antibody.
10. a pharmaceutical composition is characterized in that, it contains the described polypeptide of claim 1 and the pharmaceutically acceptable carrier of safe and effective amount.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB001273140A CN1194012C (en) | 2000-11-09 | 2000-11-09 | Sperm formation relative protein and its coding sequence and use |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB001273140A CN1194012C (en) | 2000-11-09 | 2000-11-09 | Sperm formation relative protein and its coding sequence and use |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1352138A CN1352138A (en) | 2002-06-05 |
| CN1194012C true CN1194012C (en) | 2005-03-23 |
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| CNB001273140A Expired - Lifetime CN1194012C (en) | 2000-11-09 | 2000-11-09 | Sperm formation relative protein and its coding sequence and use |
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| CN101775371B (en) * | 2009-12-02 | 2012-03-14 | 唐爱发 | Anti-testicle-protein hTSC29 antibody or antibody fragment and application thereof |
| CN101775372B (en) * | 2009-12-02 | 2012-01-11 | 桂耀庭 | Anti-testicle-protein hT279 antibody or antibody fragment and application thereof |
| CN114874309B (en) * | 2022-06-07 | 2023-11-14 | 普迪特(泰州)生物科技有限公司 | TEX101 recombinant protein and application thereof in preparation of monoclonal antibody |
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