CN102120037A

CN102120037A - Human epididymis secretion sperm bonding protein HEL-S-64C as well as coding gene and application thereof

Info

Publication number: CN102120037A
Application number: CN2009103075085A
Authority: CN
Inventors: 李建远
Original assignee: Individual
Current assignee: Individual
Priority date: 2009-09-23
Filing date: 2009-09-23
Publication date: 2011-07-13

Abstract

The invention discloses a human epididymis secretion sperm bonding protein HEL-S-64C as well as a coding gene and application thereof, in particular disclosing an application of an HEL-S-64C gene or application of a coding protein thereof in regulating sperm motion or sperm penetration. The invention also discloses an application of an HEL-S-64C antagonist or agonist in regulating perm motion capability or sperm penetration capability. In the invention, HEL-S-64C protein is found to be necessary to sperm motion or sperm penetration for the first time, and the fact that the HEL-S-64C antagonist (such as an HEL-S-64C specific antibody) can obviously inhabit the sperm motion or sperm penetration is confirmed.

Description

Human epididymal secretion sperm binding protein HEL-S-64C and encoding gene and application

Technical field

The present invention relates to biotechnology and medical domain, specifically, the present invention relates to a kind of new people's epididymis secretion sperm binding protein HEL-S-64C and encoding gene thereof and wear function and application thereof in the ovum process at sperm motility or sperm.

Background technology

This century, the mankind's development was faced with the problem of two sternnesses: on the one hand, global population is estimated will reach 9,000,000,000 in the year two thousand fifty in rapid expansion; On the other hand, according to World Health Organization's investigation, about 15% Mr. and Mrs exist sterile problem, and the sickness rate of developed country's infertility soared at nearest 10 years, and European developed country can be up to 30%, and wherein male factor accounts for half.World Health Organization's prediction, along with the increase of paathogenic factors such as environmental pollution and sexually transmitted disease (STD), in this century, infertility will become the third-largest disease that is only second to tumor and cardiovascular and cerebrovascular disease.

Because the ripe dysplasia of sperm function can not natural fertilization, needs the doctor to obtain the offspring by making test-tube baby.Utilize this kind technology to obtain the offspring, not only expended valuable time and fund, and have a lot of unhealthful latencies, the natural combination of smart ovum, the advantage in the normal growing process of having lost selected hereditary offspring's chance.Brought heavy spirit and financial burden for family and society.

In addition, the sharp increase of world population has caused resource to exhaust and ecological deterioration, becomes the serious threat of human health and existence.Our times population surpasses 6,000,000,000.Estimate that according to the expert maximum population's capacity that Chinese resource environment can support is 15-16 hundred million.Develop safe and effective, the advanced practical new technique that avoids conception and control birth, new product, requiring with the personalization of the contraception medication of satisfying different crowd, different levels is the general trend of technical development of avoiding conception and controling birth both at home and abroad in recent years.As seen the treatment of contraception and infertility is human two significant problems that are related to national economy to be solved.

(human heat shock protein 90 is the chaperone that a kind of known ATP relies on HSP90) to HEL-S-64C, and the gene accession number is GQ472230.When ATP in conjunction with after the HSP90 conformational change, form dimerization.The major function of HSP is that to keep cell protein stable, improve cell to stress toleration, strengthen antioxidation and make cell keep normal physiological function.In recent years, the effect of HSP90 in tumor tissues also is subjected to extensive attention.At present confirmed that the substrate of HSP90 has kind more than 100, HSP90 can combine with it, changes the effect of functional protein, causes cellular metabolism obstacle or abnormal division propagation, causes the formation of malignant tumor.Find again that recently HSP90 has the important anti-accent effect of dying.Because HSP90 plays an important role in the generation of tumor, development, HSP90 and inhibitor thereof have become the novel targets of antitumor research.Geldanamycin is the 1st a HSP90 inhibitor, is the natural product of a kind of separation from the streptomycin hygroscopin.The biological function of the feasible research of its discovery HSP90, research relies on the effect of HSP90 albumen in cancer development becomes possibility.

Yet people are very few to other functions understandings of HSP90 gene and expression product thereof so far, more do not report the dependency of HSP90 and fertilization.

In sum, in order to practise contraception and the treatment of infertility, this area presses for to be sought with fertilization and becomes pregnant and wait relevant functional gene and albumen, so that further method and the product of developing reproduction or fertility.

Summary of the invention

The purpose of this invention is to provide a kind of HEL-S-64C gene and wear application in the ovum regulating sperm motility or sperm.

Another object of the present invention provides the purposes of antagonist or the agonist of HEL-S-64C, is used to regulate sperm motility or sperm is worn ovum.

In a first aspect of the present invention, the purposes of a kind of HEL-S-64C gene or its encoding proteins is provided, described HEL-S-64C gene or its encoding proteins are used as the promoter that promotion (especially external non-therapeutic promote) sperm motility or sperm are worn ovum.

In a second aspect of the present invention, the purposes of a kind of HEL-S-64C gene or its encoding proteins is provided, described HEL-S-64C gene or its encoding proteins are used to prepare the medicine that promotes sperm motility or sperm to wear ovum.

In a preference of the present invention, described medicine is the progestogenic medicine.

In a third aspect of the present invention, a kind of purposes of HEL-S-64C antagonist is provided, described HEL-S-64C antagonist is used as the inhibitor that inhibition (especially external non-therapeutic suppress) sperm motility or sperm are worn ovum.

In a fourth aspect of the present invention, a kind of purposes of HEL-S-64C antagonist is provided, described HEL-S-64C antagonist is used to prepare and suppresses the medicine that sperm motility or sperm are worn ovum.

In a preference of the present invention, described medicine is a contraceptive.

In another one preference of the present invention, described antagonist is the antisensenucleic acids of HEL-S-64C, the antibody or the geldanamycin of the part of HEL-S-64C, anti-HEL-S-64C polypeptide.

In a fifth aspect of the present invention, a kind of purposes of HEL-S-64C agonist is provided, it is used as the promoter that promotion (especially external non-therapeutic promote) sperm motility or sperm are worn ovum.

In a sixth aspect of the present invention, a kind of purposes of HEL-S-64C agonist is provided, it is used to prepare the medicine that promotes sperm motility or sperm to wear ovum.

In a seventh aspect of the present invention, provide whether a kind of definite test compounds is the method for antagonist or the agonist of HEL-S-64C, it comprises step:

(a) test compounds and the external sperm solution of HEL-S-64C polypeptide adding are organized as test, and the external sperm solution of HEL-S-64C polypeptide adding is organized in contrast;

(b) sperm motility of observation test group and matched group or sperm are worn the ovum situation, if the sperm motility ability of test group or sperm are worn ovum quantity greater than matched group, represent that then test compounds is the agonist of HEL-S-64C; If the sperm motility ability of test group or sperm are worn ovum quantity less than matched group, represent that then test compounds is the antagonist of HEL-S-64C.

In a eighth aspect of the present invention, a kind of pharmaceutical composition is provided, it contains HEL-S-64C polypeptide, its agonist or its antagonist and the pharmaceutically acceptable carrier of safe and effective amount.

In a ninth aspect of the present invention, the method that provides a kind of external non-therapeutic inhibition sperm motility or sperm to wear ovum, described method comprises step: sperm is contacted with the HEL-S-64C antagonist, thereby reduce the motor capacity of sperm and wear the ovum ability.

In another preference, this method also comprises and will contact with ovum with sperm after the HEL-S-64C antagonist contacts.

In another preference, described sperm and ovum are the people.

In another preference, the antibody of described anti-HEL-S-64C polypeptide or geldanamycin.

In a tenth aspect of the present invention, a kind of utensil of regulating fertility is provided, described utensil is the contraceptive device that contains the HEL-S-64C antagonist.

Others of the present invention are because the disclosure of this paper is conspicuous to those skilled in the art.

Description of drawings

Fig. 1 has shown that HEL-S-64C albumen locatees at sperm immunization fluorescence.Wherein, red fluorescence shows sperm nucleus; Green fluorescence shows the location of albumen on sperm; Scale: 5um.

The A group: positive control (GenBank AAG27712 HEL-75), A3. show that the HEL-75 protein binding is in whole sperm.

B group: negative control.

The C group: HEL-S-64c albumen location, C3. shows that the HEL-S-64c protein binding is in the perforatorium back zone.

Fig. 2 has shown that HEL-S-64C albumen is in epididymis and testis tissue immunofluorescence location.The result shows, the epididymis microvillus positioning result positive; The epididymis Interstitial cell positioning result positive; The testis positioning result positive.

Fig. 3 has shown the RT-PCR result of HEL-S-64C.Each swimming lane is respectively among the figure: 1. head of epididymis; 2. body of epididymis; 3. tail of epididymis; 4. testis; 5. the heart; 6. liver; 7. spleen; 8. kidney; 9. stomach; 10. negative control.

Fig. 4 has shown the Western trace result of HEL-S-64C.Among the figure, each swimming lane is respectively 1.HEL-75 (positive control); 2. blank; Fluid: epididymal fluid; Testis: testis.

The specific embodiment

The inventor is through extensive and deep research, and (be also referred to as human epididymal secretion sperm binding protein Li 64c, HEL-S-64C) to be that sperm motility or sperm are worn ovum necessary for albumen to find heat shock protein 90 first; And the antagonist (as the antibody of HEL-S-64C) that confirms HEL-S-64C first can suppress sperm motility significantly or sperm is worn ovum.Finished the present invention based on this.

Particularly, the inventor obtains a clone from normal young people epididymis cDNA library, and according to order called after HEL-S-64C, this albumen is that human epididymal secretion sperm is in conjunction with one newcomer of family.Find that through BLAST comparison back the gene of HSP90 in HEL-S-64C and the ncbi database (NM_005348.3) has 100% homology, belongs to the heat-shock protein family member again.

By HEL-S-64C is discovered, its mRNA all has higher expression at people's head of epididymis, body, tail and testis, expresses lower in other tissue or does not express.Sperm immunization fluorescence shows that this protein binding is at the mature sperm back of head.Histogenic immunity fluorescence shows that this albumen is lower at epididymis and testis tissue expression.The motion of sperm and wear the ovum rate and obviously reduce behind the rHEL-S-64C multi-resistance sealing sperm, this prompting, the proteic antagonist of HEL-S-64C has the motion that suppresses sperm and is subjected to essence function on the one hand, normal on the other hand HEL-S-64C is a sperm proper motion and to wear ovum required, the shortage of HEL-S-64C or quantity reduce, and may cause the decline of sperm motility ability and/or wear the decline of ovum ability.

In the present invention, term " HEL-S-64C albumen ", " HEL-S-64C polypeptide ", " the HEL-S-64C factor ", " HSP90 albumen ", " HSP90 polypeptide ", " heat shock protein 90 " or " HEL-S-64C (human epididymal secretion sperm binding protein Li 64c) " etc. are used interchangeably, all refer to have the heat shock protein 90 aminoacid sequence albumen or the polypeptide of (SEQ ID NO:2).They can contain or not contain initial methionine.Should be understood that these terms had both comprised the albumen in people (source), be also included within homologue with function of the same race or homologous protein in other mammals (as Canis familiaris L., cattle, sheep, monkey, rodent (as mice)).In addition, this term comprises wild type and saltant heat shock protein 90.

(the GenBank accession number: GQ472230) shown in SEQ ID NO:1, its amino acid sequence coded is shown in SEQ ID NO:2 for the cDNA sequence of people HEL-S-64C gene.

Should be understood that because proteic nucleotide sequence of mammiferous HSP90 such as people and aminoacid sequence all are known, can obtain its albumen with the DNA recombinant technique of this area routine.

The particularly preferred albumen of one class is the HEL-S-64C analog, i.e. the homologous protein of HEL-S-64C in other mammals (as cattle, sheep, rabbit, Canis familiaris L., monkey, Mus etc.).The coded sequence of the homologous protein of these other species can obtain by the method for hybridizing or increase, and then obtain these homologous proteins by conventional recombination method according to the sequence of HEL-S-64C.

As used herein, " isolating HEL-S-64C albumen or polypeptide " is meant that the HEL-S-64C polypeptide is substantially free of natural relative other albumen, lipid, saccharide or other material.Those skilled in the art can use the purified technology of protein purification HEL-S-64C polypeptide of standard.Basically pure polypeptide can produce single master tape on non-reduced polyacrylamide gel.

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 purification, or the product of chemosynthesis, or uses recombinant technique to produce from protokaryon or eucaryon host (for example, antibacterial, yeast, higher plant, insecticide and mammalian cell).The host used according to the recombinant production scheme, polypeptide of the present invention can be glycosylated, maybe can be nonglycosylated.Polypeptide of the present invention also can comprise or not comprise initial methionine residues.

The present invention also comprises fragment, derivant and the analog of HEL-S-64C polypeptide.As used herein, term " fragment ", " derivant " are meant biological function or the active polypeptide that keeps the natural HEL-S-64C factor of the present invention identical basically with " analog ".Polypeptide fragment of the present invention, derivant or analog 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 chemical compound (such as the chemical compound that prolongs the polypeptide half-life, Polyethylene Glycol for example) merges formed polypeptide, or (iv) additional aminoacid sequence is fused to this peptide sequence and the polypeptide that forms (as targeting sequencing or secretion sequence or be used for the sequence or the proteinogen sequence of this polypeptide of purification, or with the fusion rotein of the segmental formation of antigen I gG).According to the instruction of this paper, these fragments, derivant and analog belong to the known scope of those skilled in the art.

In the present invention, term " HEL-S-64C polypeptide " refers to have direct or indirect adjusting sperm motility or sperm is worn the active SEQ ID of ovum NO:2 polypeptide of sequence.This term also comprises having and variant form HEL-S-64C factor identical function, SEQ ID NO:2 sequence.These variant forms comprise (but being not limited to): one or more (it is individual to be generally 1-50, preferably 1-30, more preferably 1-20,1-10 best) amino acid whose disappearance, insertion and/or replacement, and C-terminal and/or one or more (being generally in 20 of N-terminal interpolation, preferably being in 10, more preferably is in 5) aminoacid.For example, in the art, when replacing, can not change proteinic function usually with the close or similar aminoacid of performance.Again such as, add one or several aminoacid at C-terminal and/or N-terminal and also can not change proteinic function usually.This term also comprises the active fragment and the reactive derivative of the HEL-S-64C factor.

The variant form of this polypeptide comprises: homologous sequence, conservative variant, allelic variant, natural mutation, induced mutation body, under high or low tight degree condition can with the coded albumen of the DNA of HEL-S-64C DNA hybridization and the polypeptide or the albumen that utilize the antiserum of anti-HEL-S-64C polypeptide to obtain.The present invention also provides other polypeptide, as comprises HEL-S-64C polypeptide or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention has also comprised the soluble fragments of HEL-S-64C polypeptide.Usually, this fragment have the HEL-S-64C peptide sequence at least about 50 continuous amino acids, usually at least about 100 continuous amino acids, preferably at least about 150 continuous amino acids, more preferably at least about 200 continuous amino acids, best at least about 300 continuous amino acids.

The invention still further relates to the analog of HEL-S-64C polypeptide.The difference of these analog and natural HEL-S-64C 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 inductive genetic variant.The induce variation body can obtain by various technology, as by radiation or be exposed to mutagenic agent and produce random mutagenesis, also can pass through direct mutagenesis method or the biological technology of other known moleculars.Analog also comprises having the analog that is different from the amino acid whose residue of natural L-(as D-aminoacid), and has non-natural analog that exist or synthetic aminoacid (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 acetylation 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 phosphotyrosine, phosphoserine, phosphothreonine).Thereby also comprise the polypeptide that has been improved its anti-Proteolytic enzyme performance or optimized solubility property by modifying.

HEL-S-64C 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 conventional method well known by persons skilled in the art as template, amplification and must relevant sequence.When sequence than long time, usually need to carry out twice or pcr amplification repeatedly, and then the fragment that each time amplifies be 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 conventional 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 fragments, and then connect and to obtain the very long fragment of sequence.

At present, can be fully obtain the DNA sequence of code book invention albumen (or its fragment, or derivatives thereof) by chemosynthesis.This DNA sequence 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.

Based on the inventor's new discovery, the HEL-S-64C polypeptide is of use in many ways.These purposes include, but is not limited to: directly as the disease due to Drug therapy HEL-S-64C factor hypofunction or the forfeiture (as low because of the sperm motility ability or wear infertile that the ovum rate lowly causes) be used to screen antibody, polypeptide or other part and the micromolecular compound that promotes or resist HEL-S-64C factor function.Usually, can be used for seeking the peptide molecule that can suppress or stimulate HEL-S-64C factor function of therapeutic value with the reorganization HEL-S-64C factor screening peptide library of expressing.

On the other hand, the present invention also comprises HEL-S-64C 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 HEL-S-64C gene outcome or fragment.Preferably, refer to that those can combine with HEL-S-64C gene outcome 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 molecule of the HEL-S-64C factor, comprise that also those do not influence the antibody of HEL-S-64C factor function.The present invention also comprise those can with modify or without the bonded antibody of HEL-S-64C gene outcome of modified forms.

The present invention not only comprises complete monoclonal 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 HEL-S-64C gene outcome of purification or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing the HEL-S-64C factor or its has 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 be utilized hybridoma technology to prepare (to 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 CellHybridomas, Flsevier, N.Y., 1981).Antibody of the present invention comprises the antibody that can block the HEL-S-64C function and the antibody that does not influence the HEL-S-64C function.Each antibody-like of the present invention can utilize the fragment or the functional areas of HEL-S-64C gene outcome, obtains by the routine immunization technology.These fragments or functional areas can utilize recombinant methods or utilize Peptide synthesizer synthetic.Can come immune animal and produce with the gene outcome of producing in the prokaryotic cell (for example E.Coli) with the bonded antibody of unmodified form of HEL-S-64C gene outcome; With the bonded antibody of post translational modification form (as the albumen or the polypeptide of glycosylation or phosphorylation), can come immune animal and obtain with the gene outcome that produces in the eukaryotic cell (for example yeast or insect cell).

The antibody of anti-HEL-S-64C can be used in the immunohistochemistry technology, detects the HEL-S-64C in the biopsy specimen.

Antibody among the present invention is as the antagonist of HEL-S-64C, also can be used for suppressing sperm motility or sperm is worn ovum, thereby is used for contraception.The antibody that gives suitable dosage can be blocked generation or the activity of HEL-S-64C.

Utilize albumen of the present invention,, can filter out with HEL-S-64C interactional material takes place, as receptor, inhibitor, agonist or antagonist etc. by various conventional screening techniques.For example, can be incorporated into the rondom polypeptide storehouse that solid formation forms by the various aminoacid that may make up by screening with the bonded peptide molecule of HEL-S-64C obtains.

Albumen of the present invention and antibody thereof, inhibitor, agonist, antagonist or receptor etc. when using (administration) in treatment, can provide different effects.Usually, can these materials are formulated in nontoxic, the inert and pharmaceutically acceptable aqueous carrier medium, wherein pH is about 5-8 usually, and preferably pH is about 6-8, although pH value can be with being changed to some extent by preparation Substance Properties and disease to be treated.The pharmaceutical composition for preparing can carry out administration by conventional route, comprising (but being not limited to): oral, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, Intradermal, intravaginal or topical.

The present invention also provides pharmaceutical composition, and it contains HEL-S-64C polypeptide of the present invention or its agonist, antagonist and the pharmaceutically acceptable carrier or the excipient of safe and effective amount.This class carrier comprises (but being not limited to): saline, buffer, glucose, water, glycerol, 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 conventional method with normal saline or the aqueous solution that contains glucose and other adjuvant.Pharmaceutical composition such as tablet and capsule can be prepared by conventional method.Pharmaceutical composition such as injection, solution, tablet and capsule should be made under aseptic condition.The dosage of active component is the treatment effective dose, for example every day about 1 microgram/kg body weight-Yue 10 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 HEL-S-64C albumen of safe and effective amount or its antagonist, agonist are applied to mammal, wherein this safe and effective amount is usually at least about 1 microgram/kg body weight, and in most of the cases be no more than about 10 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, individual health situation, and these all are within the skilled practitioners skill.

The polynucleotide of HEL-S-64C also can be used for multiple therapeutic purposes.Antisense nucleotide can suppress the expression of HEL-S-64C, and then suppresses sperm motility or sperm is worn ovum, thereby is expected to be used for contraception.

The invention still further relates to the diagnostic testing process of quantitative and detection and localization HEL-S-64C level.These tests are known in the art, comprise methods such as radioimmunoassay.The HEL-S-64C level that is detected in the test, can with lay down a definition the HEL-S-64C factor in disease such as infertile importance and be used for diagnosis.

A kind of method that whether has the HEL-S-64C factor in the test sample that detects is to utilize the specific antibody of the HEL-S-64C factor to detect, and it comprises: sample is contacted with HEL-S-64C factor-specific antibody; Observe whether form antibody complex, formed antibody complex and just represented to exist in the sample HEL-S-64C factor.

Compositions is regulated in fertility

The present invention also provides fertility to regulate compositions.

It is composition for contraception that compositions is regulated in one class fertility, and it contains the HEL-S-64C antagonist of the present invention of (a) safe and effective amount and (b) pharmaceutically acceptable carrier or excipient.The dosage of active component is the contraception effective dose, for example every day about 1 microgram/kg body weight-Yue 10 mg/kg body weight.In addition, polypeptide of the present invention also can use with other contraceptives.

It is the progestogenic compositions that compositions is regulated in another kind of fertility, and it contains the HEL-S-64C of the present invention of (a) safe and effective amount and/or its agonist and (b) pharmaceutically acceptable carrier or excipient.The dosage of active component is the progestogenic effective dose, for example every day about 1 microgram/kg body weight-Yue 10 mg/kg body weight.In addition, polypeptide of the present invention also can use with the medicament that other promotions are become pregnant.

The preferred method of application that compositions is regulated in fertility is local application, more preferably is intravaginal or intrauterine local application (for example as powder, ointment or drop).

Contraceptive device

The present invention also provides a kind of utensil of regulating fertility, and described utensil is the contraceptive device that contains the HEL-S-64C antagonist.

In contraceptive device of the present invention, comprise the releasing parts that is used at vagina or intrauterine release HEL-S-64C antagonist.In another preference, described contraceptive device is pessary or intrauterine device.

Should be understood that described releasing parts can be independent, also can make up.For example, releasing parts can be an independent releasing member or the combination type releasing member combined with miscellaneous part, or is bonded in or is integrated in the releasing layer of miscellaneous part.

A kind of optimal way is to add a releasing layer (for example slow release layer) on the pessary of routine or intrauterine device, and this releasing layer can be in intravaginal or in utero discharge the HEL-S-64C antagonist in a period of time, thereby inhibition sperm motility or sperm are worn ovum.

Usually, the releasing member in contraceptive device should contain the 5-5000 milligram, more preferably 10-2500 milligram, the HEL-S-64C antagonist of 20-1000 milligram best.

Major advantage of the present invention is:

(1) it is necessary to find that first HEL-S-64C albumen is that sperm motility or sperm are worn ovum.

(2) antagonist (as HEL-S-64C antibody) that confirms HEL-S-64C first can obviously suppress sperm motility or sperm is worn ovum.

(3) epididymis is given functions such as sperm motility, fertilization and defence, is the male genital vitals of regulation and control.Based on epididymal function gene and proteinic research, help to solve male sterility and contraception two hang-ups.

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 Spring Harbor LaboratoryPress, 1989) condition described in, or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise percentage ratio and umber calculate by weight.

Unless otherwise defined, the same meaning that employed all specialties and scientific words and one skilled in the art are familiar with in the literary composition.In addition, any method similar or impartial to described content and material all can be applicable among the present invention.The usefulness that preferable implementation method described in the literary composition and material only present a demonstration.

Embodiment 1

1.1 the structure of expression vector

According to the gene order of HEL-S-64C, the special primer of the synthetic ripe coding region of a pair of amplification:

Upstream HEL-S-64C-F:5 '-TTGGTACCGACGACGACGACCCTGAGGAAACCCAGACCCAA-3 (SEQ ID NO:3)

Downstream HEL-S-64C-R:5 '-GGCGAATTCTCATTAGTCTACTTCTTCCATGCGTG-3 ' (SEQ ID NO:4)

Upstream and downstream is introduced restriction enzyme site KpnI and EcoRI respectively.With the people's epididymis cDNA library with the conventional method preparation is template, by the direct amplifying target genes of PCR, is cloned into commercially available carrier pGM-T vector (available from the rich biotech firm of the last sea cowry) evaluation of checking order then.

Sequencing result is found that through BLAST comparison back the gene of HSP90 in HEL-S-64C and the ncbi database (NM_005348.3) has 100% homology.

HEL-S-64C gene after will identifying through order-checking is cloned among the expression vector pET32b (+) (available from the rich biotech firm of last sea cowry) by KpnI and EcoRI site, make its reading frame consistent, obtain expression vector pET32b (+)-HEL-S-64C with fusion tag.Change recombinant expression carrier pET32b (+)-HEL-S-64C over to conventional E.coliBL21 (DE3) competent cell, obtain engineering bacteria.

The gained engineered strain through 1mM IPTG 32 ℃ induce 4h after, all components separates with 15%SDS-PAGE, the expression of the bright blue G-250 staining analysis engineering bacteria of Kao Masi.

Strategy by amalgamation and expression prepares recombiant protein.SDS-PAGE the analysis showed that fusion rotein Trx-HEL-S-64C mainly exists with soluble form.

1.2 the purification of reorganization HEL-S-64C

According to the His-Tag in the carrier, utilization " two step nickel affinity chromatography methods " is carried out separation and purification to reorganization HEL-S-64C albumen.Method is as follows:

" first step affinity chromatograph " is used for purification of Recombinant Trx-HEL-S-64C fusion rotein.Fusion rotein recombinant enterokinase cracking then discharges fusion tag.

At last, reorganization HEL-S-64C albumen is reclaimed in utilization " the second step affinity chromatograph ".Recombiant protein behind the purification carries out quantitatively preserving with lyophilization then with Bradford (Bradford 1976) method.

The preparation of embodiment 2. anti-HEL-S-64C polyclonal antiserums (multi-resistance)

Reorganization HEL-S-64C protein immunization BALB/C mice with embodiment 1 preparation prepares multi-resistance.Briefly, every mouse was injected the complete freund adjuvant (CFA) of 50 μ g recombiant proteins and equivalent in the 1st day.Injected incomplete freund adjuvant (IFA) booster immunization of 25 μ g recombiant proteins and equivalent then at the 15th, 30 and 45 day.Got blood from eyeball on the 60th day, tiring and specificity with ELASA and western engram analysis antibody behind the separation of serum.

Elisa assay shows that antibody titer reaches 1: 10000.

The Western engram analysis shows that the natural HEL-S-64C that this antibody extracts to recombiant protein with from people's epididymal fluid has good specificity.

The research of embodiment 3. people HEL-S-64C mRNA distribution expression patterns

In order to determine the expression pattern of HEL-S-64C, adopt sxemiquantitative RT-PCR to analyze the HEL-S-64C gene, testis, the heart, liver, spleen, lung, kidney, the differential expression in the tissue such as stomach at people's head of epididymis, body, tail.With TRIzol (Tiangen, Beijing, China) extracted total RNA, reverse transcription becomes cDNA to the total RNA of 1ug with 0.3ugoligodT18 (Promega) with 20UAMV reverse transcriptase (Promega).Then, the synthetic cDNA of 2 μ l utilizes gene specific primer to carry out pcr amplification.20 μ l reaction systems contain: 2 μ l, 10 * PCRbuffer (withMgCl ₂), 2 μ ldNTPMix (10mmol/L), 1 each primer of μ l (25 μ mol/L), 1ulTaqDNA polymerase (2.5U/ μ l), 2 μ lcDNA templates and 11 μ l ddH ₂O.The program of PCR reaction is: 94 ℃, and 10min; 94 ℃, 1min; 54 ℃ of (to HEL-S-64C)/49 ℃ are (to the 30s of β-actin); 72 ℃, 1min (35 circulation); 72 ℃ are extended 7min.The expression of β-actin is as confidential reference items.All pcr amplification products 1.5% agarose electrophoretic analysis.

The result as shown in Figure 3, HEL-S-64C mRNA all has higher expression at people's head of epididymis, body, tail and testis, expresses lower in other tissue or does not express.

Embodiment 4. people HEL-S-64C albumen are in the immunofluorescence location of tissue

Get people's testis, epididymis tissue freezing section, carry out immunofluorescence dyeing, wherein one anti-is mouse-anti rHEL-S-64C multi-resistance (1: 400), and two anti-ly are FITC-labelling sheep anti-mouse igg (1: 200), and nucleus dyes with PI.Sections all after the dyeing are used Laser Scanning Confocal Microscope (LeicaTCSSP2 AOBS) observed result then with 80% glycerol mounting.Experiment is anti-as negative control with blood serum substituting before the mouse immune one simultaneously.

The result as shown in Figure 2, histogenic immunity fluorescence shows that this albumen expresses lower at epididymis and testis tissue.

Embodiment 5. people's epididymal proteins extracts and Western engram analysis

The total protein extract 20 μ g that will extract from people's epididymal fluid separate with 15%SDS-PAGE, and wet then forwarding to carried out the western trace on the pvdf membrane.Mouse-anti people HEL-S-64C multi-resistance is one anti-(1: 5000), and two anti-ly are HRP-labelling sheep anti-mouse igg (1: 500).The activity of horseradish peroxidase is analyzed with enhancement mode HRP-DAB substrate colour reagent box.

The result all detects a size and is the many protein bands of 70KD as shown in Figure 4 in epididymal fluid and testis, HEL-75 has a more special band as positive controls, and the blank group is without any assorted band.

Embodiment 6. people HEL-S-64C albumen are in the immunofluorescence location of sperm

Collect and coat on the microscope slide of 1% gelatin bag quilt after sperm washs with PBS, dry naturally, fix 10 minutes with methanol then.The microscope slide that contains sperm at room temperature sealed 1 hour with 3%BSA, added mouse-anti rhHEL-S-64C multi-resistance (1: 200) then, and 4 ℃ are spent the night.Mice serum is done negative control before the immunity.With PBST (PBScontaining0.1%Tween-20) washing three times, add corresponding FITC-labelling sheep anti-mouse igg two anti-(1: 200).Microscope slide is used 80% glycerol mounting then with PBST washing three times.With Olympus BX-52 microscopic examination result.

The result shows that the HEL-S-64C protein binding is at the mature sperm back of head as shown in Figure 1.

The relation research of embodiment 7 HEL-S-64C and sperm motility and external fertilization

Utilize HEL-S-64C multi-resistance sealing normal person sperm, carry out sperm motility and external fertilization analysis then.Method is as follows:

Sperm uses SpermRinse-30 in 37 ℃, 5%CO earlier ₂Cultivate capacitation in 1 hour.Added mouse-anti rHEL-S-64C multi-resistance according to 1: 200 then, in 37 ℃, 6%CO ₂Cultivate and sealed in 2 hours.Sperm separated into two parts after the sealing, a part directly utilize computer aided system to analyze the mobility of sperm.Another part is with BWW culture medium washing 2 times, and to be adjusted to final concentration be 1.0 * 10 ⁷Cells/ml.Add 100 μ l sample of sperm in the culture dish of 35mm.The Golden Hamster ovum that removes zona pellucida is added during preprepared fertilization drips, at 37 ℃, 6%CO ₂Cultivate the ovum situation of wearing of observing in the saturated humidity incubator.More than all the experiment use simultaneously the immunity before mice serum as negative control.

The result as shown in Table 1 and Table 2.

Table 1 sperm is worn the ovum experiment

The experiment group	The test with total ovum number (piece)	The germ cell number (piece)	SPA(％)	Wear ovum sperm sum (individual)	All ovum penetrates sperm count (individual)
						Blank	8	7	88.9％	34	4.3
HEL-75*	7	7	100％	28	4
						HEL-S-64C	7	0	0	0	0

Annotate:

1. the blank group is that sperm does not carry out any albumen one anti-sealing treatment

2.* positive matched group is selected HEL-75 for use, behind the anti-sealing sperm of its anti-HEL-75 people such as (, Eur.J.Immunol. 23:2086-2094,2008) Kohler, does not have influence to wearing the ovum result.

The result: after HEL-S-64C one anti-sealing, sperm is not worn ovum, and SPA (wearing the ovum index) is 0.

The experiment of table 2 sperm motility

HEL-S-64C

Sperm motility

Proal sperm count

Average path speed

Average point-to-point speed

The averaged curve movement velocity

Average side-sway amplitude

Whip frequency

Preceding tropism

Rectilinearity

Before the capacitation

P＜0.01

P＜0.05

P＞0.05

P＜0.05

P＞0.05

After the capacitation

P＞0.05

P＜0.05

P＞0.05

P＜0.05

P＞0.05

P＜0.05

P＜0.01

Annotate: * P＞0.05 difference is not remarkable; P＜0.05 significant difference; P＜0.01 difference is extremely remarkable

The result: behind the antibody sealing sperm, sperm motility, proal sperm count are compared difference with matched group extremely remarkable before the capacitation, average path speed, average point-to-point speed, whips frequency and compare significant difference with matched group; After the capacitation behind the antibody sealing sperm, proal sperm, average point-to-point speed, whip frequency, preceding tropism compares significant difference with matched group, it is extremely remarkable that rectilinearity and matched group are compared difference.

The above results shows, utilizes mouse-anti rHEL-S-64C multi-resistance sealing sperm, analyzes to show that this antibody obviously suppresses the motion of sperm.Simultaneously, the external fertilization experiment shows, wears the ovum rate through the sperm after the antibody sealing and obviously reduces.This prompting, the proteic antagonist of HEL-S-64C has the motion that suppresses sperm and is subjected to essence function on the one hand, on the other hand, HEL-S-64C participates in sperm in conjunction with motion and fertilization process, normal HEL-S-64C is a sperm proper motion and to wear ovum required, the shortage of HEL-S-64C or quantity reduce, and may cause the decline of sperm motility ability and/or wear the decline of ovum ability.

Preparation of embodiment 8 monoclonal antibodies and active testing

Immunologic process prepares identical with method and polyclonal antiserum, after the last immunity 3～4 days, separating Morr. cell mixes back adding PEG with the Sp2 myeloma cell strain merge cell each other, cell after merging is suitably diluted, split to cultivate in the plate hole and cultivate, generally be diluted to 0.8 cells/well, cell culture is drawn culture supernatant and is detected antibody content with ELISA at the bottom of cover 20% hole the time.Ascites is collected in the inoculation of the positive cell strain mouse peritoneal that filters out, with staphylococcal protein A prepare affinity column with antibodies after eluting, reclaim monoclonal antibody.

Repeat the external fertilization experiment of embodiment 7 with the monoclonal antibody of preparation.The result shows that after the monoclonal antibody sealing through this anti-HEL-S-64C, sperm is worn the ovum rate and obviously reduced.

The antagonist of embodiment 9 screening HEL-S-64C

Press embodiment 7 described methods, following two groups of materials added in people's sperm solution, observe the effect that sperm motility or sperm are worn ovum then external:

(a) candidate substances+HEL-S-64C factor;

(b) the HEL-S-64C factor.

In addition, also be provided with and neither add the blank group that candidate substances does not add the HEL-S-64C factor yet.

If group sperm motility of (a) or sperm are worn sperm motility or the sperm that the ovum rate significantly is lower than group (b) (more preferably significantly being lower than the blank group) statistically and worn the ovum rate, then pointing out this candidate substances is the antagonist of HEL-S-64C.

In the double-blind method test, successfully filtered out the HEL-S-64C monoclonal antibody and the geldanamycin of preparation among the embodiment 8.

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

＜110〉Lee builds far away

＜120〉human epididymal secretion sperm binding protein HEL-S-64C and encoding gene and application

<130>092989

<160>4

<170>PatentIn?version?3.5

<210>1

<211>3366

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<220>

<221>CDS

<222>(191)..(2389)

<400>1

gcatgcgtag?gcgcgcggcc?gcggcggcgg?ctggggaggg?ttcttccgga?aggttcggga 60

ggcttctgga?aaaagcgccg?cgcgctgggc?gggcccgtcg?ctatataagg?caggcgcggg 120

ggtggcgcgt?cagttgcttc?agcgtcccgg?tgtggctgtg?ccgttggtcc?tgtgcggtca 180

cttagccaag?atg?cct?gag?gaa?acc?cag?acc?caa?gac?caa?ccg?atg?gag 229

Met?Pro?Glu?Glu?Thr?Gln?Thr?Gln?Asp?Gln?Pro?Met?Glu

1 5 10

gag?gag?gag?gtt?gag?acg?ttc?gcc?ttt?cag?gca?gaa?att?gcc?cag?ttg 277

Glu?Glu?Glu?Val?Glu?Thr?Phe?Ala?Phe?Gln?Ala?Glu?Ile?Ala?Gln?Leu

15 20 25

atg?tca?ttg?atc?atc?aat?act?ttc?tac?tcg?aac?aaa?gag?atc?ttt?ctg 325

Met?Ser?Leu?Ile?Ile?Asn?Thr?Phe?Tyr?Ser?Asn?Lys?Glu?Ile?Phe?Leu

30 35 40 45

aga?gag?ctc?att?tca?aat?tca?tca?gat?gca?ttg?gac?aaa?atc?cgg?tat 373

Arg?Glu?Leu?Ile?Ser?Asn?Ser?Ser?Asp?Ala?Leu?Asp?Lys?Ile?Arg?Tyr

50 55 60

gaa?agc?ttg?aca?gat?ccc?agt?aaa?tta?gac?tct?ggg?aaa?gag?ctg?cat 421

Glu?Ser?Leu?Thr?Asp?Pro?Ser?Lys?Leu?Asp?Ser?Gly?Lys?Glu?Leu?His

65 70 75

att?aac?ctt?ata?ccg?aac?aaa?caa?gat?cga?act?ctc?act?att?gtg?gat 469

Ile?Asn?Leu?Ile?Pro?Asn?Lys?Gln?Asp?Arg?Thr?Leu?Thr?Ile?Val?Asp

80 85 90

act?gga?att?gga?atg?acc?aag?gct?gac?ttg?atc?aat?aac?ctt?ggt?act 517

Thr?Gly?Ile?Gly?Met?Thr?Lys?Ala?Asp?Leu?Ile?Asn?Asn?Leu?Gly?Thr

95 100 105

atc?gcc?aag?tct?ggg?acc?aaa?gcg?ttc?atg?gaa?gct?ttg?cag?gct?ggt 565

Ile?Ala?Lys?Ser?Gly?Thr?Lys?Ala?Phe?Met?Glu?Ala?Leu?Gln?Ala?Gly

110 115 120 125

gca?gat?atc?tct?atg?att?ggc?cag?ttc?ggt?gtt?ggt?ttt?tat?tct?gct 613

Ala?Asp?Ile?Ser?Met?Ile?Gly?Gln?Phe?Gly?Val?Gly?Phe?Tyr?Ser?Ala

130 135 140

tat?ttg?gtt?gct?gag?aaa?gta?act?gtg?atc?acc?aaa?cat?aac?gat?gat 661

Tyr?Leu?Val?Ala?Glu?Lys?Val?Thr?Val?Ile?Thr?Lys?His?Asn?Asp?Asp

145 150 155

gag?cag?tac?gct?tgg?gag?tcc?tca?gca?ggg?gga?tca?ttc?aca?gtg?agg 709

Glu?Gln?Tyr?Ala?Trp?Glu?Ser?Ser?Ala?Gly?Gly?Ser?Phe?Thr?Val?Arg

160 165 170

aca?gac?aca?ggt?gaa?cct?atg?ggt?cgt?gga?aca?aaa?gtt?atc?cta?cac 757

Thr?Asp?Thr?Gly?Glu?Pro?Met?Gly?Arg?Gly?Thr?Lys?Val?Ile?Leu?His

175 180 185

ctg?aaa?gaa?gac?caa?act?gag?tac?ttg?gag?gaa?cga?aga?ata?aag?gag 805

Leu?Lys?Glu?Asp?Gln?Thr?Glu?Tyr?Leu?Glu?Glu?Arg?Arg?Ile?Lys?Glu

190 195 200 205

att?gtg?aag?aaa?cat?tct?cag?ttt?att?gga?tat?ccc?att?act?ctt?ttt 853

Ile?Val?Lys?Lys?His?Ser?Gln?Phe?Ile?Gly?Tyr?Pro?Ile?Thr?Leu?Phe

210 215 220

gtg?gag?aag?gaa?cgt?gat?aaa?gaa?gta?agc?gat?gat?gag?gct?gaa?gaa 901

Val?Glu?Lys?Glu?Arg?Asp?Lys?Glu?Val?Ser?Asp?Asp?Glu?Ala?Glu?Glu

225 230 235

aag?gaa?gac?aaa?gaa?gaa?gaa?aaa?gaa?aaa?gaa?gag?aaa?gag?tcg?gaa 949

Lys?Glu?Asp?Lys?Glu?Glu?Glu?Lys?Glu?Lys?Glu?Glu?Lys?Glu?Ser?Glu

240 245 250

gac?aaa?cct?gaa?att?gaa?gat?gtt?ggt?tct?gat?gag?gaa?gaa?gaa?aag 997

Asp?Lys?Pro?Glu?Ile?Glu?Asp?Val?Gly?Ser?Asp?Glu?Glu?Glu?Glu?Lys

255 260 265

aag?gat?ggt?gac?aag?aag?aag?aag?aag?aag?att?aag?gaa?aag?tac?atc 1045

Lys?Asp?Gly?Asp?Lys?Lys?Lys?Lys?Lys?Lys?Ile?Lys?Glu?Lys?Tyr?Ile

270 275 280 285

gat?caa?gaa?gag?ctc?aac?aaa?aca?aag?ccc?atc?tgg?acc?aga?aat?ccc 1093

Asp?Gln?Glu?Glu?Leu?Asn?Lys?Thr?Lys?Pro?Ile?Trp?Thr?Arg?Asn?Pro

290 295 300

gac?gat?att?act?aat?gag?gag?tac?gga?gaa?ttc?tat?aag?agc?ttg?acc 1141

Asp?Asp?Ile?Thr?Asn?Glu?Glu?Tyr?Gly?Glu?Phe?Tyr?Lys?Ser?Leu?Thr

305 310 315

aat?gac?tgg?gaa?gat?cac?ttg?gca?gtg?aag?cat?ttt?tca?gtt?gaa?gga 1189

Asn?Asp?Trp?Glu?Asp?His?Leu?Ala?Val?Lys?His?Phe?Ser?Val?Glu?Gly

320 325 330

cag?ttg?gaa?ttc?aga?gcc?ctt?cta?ttt?gtc?cca?cga?cgt?gct?cct?ttt 1237

Gln?Leu?Glu?Phe?Arg?Ala?Leu?Leu?Phe?Val?Pro?Arg?Arg?Ala?Pro?Phe

335 340 345

gat?ctg?ttt?gaa?aac?aga?aag?aaa?aag?aac?aac?atc?aaa?ttg?tat?gta 1285

Asp?Leu?Phe?Glu?Asn?Arg?Lys?Lys?Lys?Asn?Asn?Ile?Lys?Leu?Tyr?Val

350 355 360 365

cgc?aga?gtt?ttc?atc?atg?gat?aac?tgt?gag?gag?cta?atc?cct?gaa?tat 1333

Arg?Arg?Val?Phe?Ile?Met?Asp?Asn?Cys?Glu?Glu?Leu?Ile?Pro?Glu?Tyr

370 375 380

ctg?aac?ttc?att?aga?ggg?gtg?gta?gac?tcg?gag?gat?ctc?cct?cta?aac 1381

Leu?Asn?Phe?Ile?Arg?Gly?Val?Val?Asp?Ser?Glu?Asp?Leu?Pro?Leu?Asn

385 390 395

ata?tcc?cgt?gag?atg?ttg?caa?caa?agc?aaa?att?ttg?aaa?gtt?atc?agg 1429

Ile?Ser?Arg?Glu?Met?Leu?Gln?Gln?Ser?Lys?Ile?Leu?Lys?Val?Ile?Arg

400 405 410

aag?aat?ttg?gtc?aaa?aaa?tgc?tta?gaa?ctc?ttt?act?gaa?ctg?gcg?gaa 1477

Lys?Asn?Leu?Val?Lys?Lys?Cys?Leu?Glu?Leu?Phe?Thr?Glu?Leu?Ala?Glu

415 420 425

gat?aaa?gag?aac?tac?aag?aaa?ttc?tat?gag?cag?ttc?tct?aaa?aac?ata 1525

Asp?Lys?Glu?Asn?Tyr?Lys?Lys?Phe?Tyr?Glu?Gln?Phe?Ser?Lys?Asn?Ile

430 435 440 445

aag?ctt?gga?ata?cac?gaa?gac?tct?caa?aat?cgg?aag?aag?ctt?tca?gag 1573

Lys?Leu?Gly?Ile?His?Glu?Asp?Ser?Gln?Asn?Arg?Lys?Lys?Leu?Ser?Glu

450 455 460

ctg?tta?agg?tac?tac?aca?tct?gcc?tct?ggt?gat?gag?atg?gtt?tct?ctc 1621

Leu?Leu?Arg?Tyr?Tyr?Thr?Ser?Ala?Ser?Gly?Asp?Glu?Met?Val?Ser?Leu

465 470 475

aag?gac?tac?tgc?acc?aga?atg?aag?gag?aac?cag?aaa?cat?atc?tat?tat 1669

Lys?Asp?Tyr?Cys?Thr?Arg?Met?Lys?Glu?Asn?Gln?Lys?His?Ile?Tyr?Tyr

480 485 490

atc?aca?ggt?gag?acc?aag?gac?cag?gta?gct?aac?tca?gcc?ttt?gtg?gaa 1717

Ile?Thr?Gly?Glu?Thr?Lys?Asp?Gln?Val?Ala?Asn?Ser?Ala?Phe?Val?Glu

495 500 505

cgt?ctt?cgg?aaa?cat?ggc?tta?gaa?gtg?atc?tat?atg?att?gag?ccc?att 1765

Arg?Leu?Arg?Lys?His?Gly?Leu?Glu?Val?Ile?Tyr?Met?Ile?Glu?Pro?Ile

510 515 520 525

gat?gag?tac?tgt?gtc?caa?cag?ctg?aag?gaa?ttt?gag?ggg?aag?act?tta 1813

Asp?Glu?Tyr?Cys?Val?Gln?Gln?Leu?Lys?Glu?Phe?Glu?Gly?Lys?Thr?Leu

530 535 540

gtg?tca?gtc?acc?aaa?gaa?ggc?ctg?gaa?ctt?cca?gag?gat?gaa?gaa?gag 1861

Val?Ser?Val?Thr?Lys?Glu?Gly?Leu?Glu?Leu?Pro?Glu?Asp?Glu?Glu?Glu

545 550 555

aaa?aag?aag?cag?gaa?gag?aaa?aaa?aca?aag?ttt?gag?aac?ctc?tgc?aaa 1909

Lys?Lys?Lys?Gln?Glu?Glu?Lys?Lys?Thr?Lys?Phe?Glu?Asn?Leu?Cys?Lys

560 565 570

atc?atg?aaa?gac?ata?ttg?gag?aaa?aaa?gtt?gaa?aag?gtg?gtt?gtg?tca 1957

Ile?Met?Lys?Asp?Ile?Leu?Glu?Lys?Lys?Val?Glu?Lys?Val?Val?Val?Ser

575 580 585

aac?cga?ttg?gtg?aca?tct?cca?tgc?tgt?att?gtc?aca?agc?aca?tat?ggc 2005

Asn?Arg?Leu?Val?Thr?Ser?Pro?Cys?Cys?Ile?Val?Thr?Ser?Thr?Tyr?Gly

590 595 600 605

tgg?aca?gca?aac?atg?gag?aga?atc?atg?aaa?gct?caa?gcc?cta?aga?gac 2053

Trp?Thr?Ala?Asn?Met?Glu?Arg?Ile?Met?Lys?Ala?Gln?Ala?Leu?Arg?Asp

610 615 620

aac?tca?aca?atg?ggt?tac?atg?gca?gca?aag?aaa?cac?ctg?gag?ata?aac 2101

Asn?Ser?Thr?Met?Gly?Tyr?Met?Ala?Ala?Lys?Lys?His?Leu?GluIle?Asn

625 630 635

cct?gac?cat?tcc?att?att?gag?acc?tta?agg?caa?aag?gca?gag?gct?gat 2149

Pro?Asp?His?Ser?Ile?Ile?Glu?Thr?Leu?Arg?Gln?Lys?Ala?Glu?Ala?Asp

640 645 650

aag?aac?gac?aag?tct?gtg?aag?gat?ctg?gtc?atc?ttg?ctt?tat?gaa?act 2197

Lys?Asn?Asp?Lys?Ser?Val?Lys?Asp?Leu?Val?Ile?Leu?Leu?Tyr?Glu?Thr

655 660 665

gcg?ctc?ctg?tct?tct?ggc?ttc?agt?ctg?gaa?gat?ccc?cag?aca?cat?gct 2245

Ala?Leu?Leu?Ser?Ser?Gly?Phe?Ser?Leu?Glu?Asp?Pro?Gln?Thr?His?Ala

670 675 680 685

aac?agg?atc?tac?agg?atg?atc?aaa?ctt?ggt?ctg?ggt?att?gat?gaa?gat 2293

Asn?Arg?Ile?Tyr?Arg?Met?Ile?Lys?Leu?Gly?Leu?Gly?Ile?Asp?Glu?Asp

690 695 700

gac?cct?act?gct?gat?gat?acc?agt?gct?gct?gta?act?gaa?gaa?atg?cca 2341

Asp?Pro?Thr?Ala?Asp?Asp?Thr?Ser?Ala?Ala?Val?Thr?Glu?Glu?Met?Pro

705 710 715

ccc?ctt?gaa?gga?gat?gac?gac?aca?tca?cgc?atg?gaa?gaa?gta?gac?taa 2389

Pro?Leu?Glu?Gly?Asp?Asp?Asp?Thr?Ser?Arg?Met?Glu?Glu?Val?Asp

720 725 730

tctctggctg?agggatgact?tacctgttca?gtactctaca?attcctctga?taatatattt 2449

tcaaggatgt?ttttctttat?ttttgttaat?attaaaaagt?ctgtatggca?tgacaactac 2509

tttaagggga?agataagatt?tctgtctact?aagtgatgct?gtgatacctt?aggcactaaa 2569

gcagagctag?taatgctttt?tgagtttcat?gttggtttat?tttcacagat?tggggtaacg 2629

tgcactgtaa?gacgtatgta?acatgatgtt?aactttgtgg?tctaaagtgt?ttagctgtca 2689

agccggatgc?ctaagtagac?caaatcttgt?tattgaagtg?ttctgagctg?tatcttgatg 2749

tttagaaaag?tattcgttac?atcttgtagg?atctactttt?tgaacttttc?attccctgta 2809

gttgacaatt?ctgcatgtac?tagtcctcta?gaaataggtt?aaactgaagc?aacttgatgg 2869

aaggatctct?ccacagggct?tgttttccaa?agaaaagtat?tgtttggagg?agcaaagtta 2929

aaagcctacc?taagcatatc?gtaaagctgt?tcaaaaataa?ctcagaccca?gtcttgtgga 2989

tggaaatgta?gtgctcgagt?cacattctgc?ttaaagttgt?aacaaataca?gatgagttaa 3049

aagatattgt?gtgacagtgt?cttatttagg?gggaaagggg?agtatctgga?tgacagttag 3109

tgccaaaatg?taaaacatga?ggcgctagca?ggagatggtt?aaacactagc?tgctccaagg 3169

gttgacatgg?tcttcccagc?atgtactcag?caggtgtggg?gtggagcaca?cgtaggcaca 3229

gaaaacagga?atgcagacaa?catgcatccc?ctgcgtccat?gagttacatg?tgttctctta 3289

gtgtccacgt?tgttttgatg?ttattcatgg?aataccttct?gtgttaaata?cagtcactta 3349

attccttggc?cttaaaa 3366

<210>2

<211>732

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>2

Met?Pro?Glu?Ghr?Thr?Gln?Thr?Gln?Asp?Gln?Pro?Met?Glu?Glu?Glu?Glu

1 5 10 15

Val?Glu?Thr?Phe?Ala?Phe?Gln?Ala?Glu?Ile?Ala?Gln?Leu?Met?Ser?Leu

20 25 30

Ile?Ile?Asn?Thr?Phe?Tyr?Ser?Asn?Lys?Glu?Ile?Phe?Leu?Arg?Glu?Leu

35 40 45

Ile?Ser?Asn?Ser?Ser?Asp?Ala?Leu?Asp?Lys?Ile?Arg?Tyr?Glu?Ser?Leu

50 55 60

Thr?Asp?Pro?Ser?Lys?Leu?Asp?Ser?Gly?Lys?Glu?Leu?His?Ile?Asn?Leu

65 70 75 80

Ile?Pro?Asn?Lys?Gln?Asp?Arg?Thr?Leu?Thr?Ile?Val?Asp?Thr?Gly?Ile

85 90 95

Gly?Met?Thr?Lys?Ala?Asp?Leu?Ile?Asn?Asn?Leu?Gly?Thr?Ile?Ala?Lys

100 105 110

Ser?Gly?Thr?Lys?Ala?Phe?Met?Glu?Ala?Leu?Gln?Ala?Gly?Ala?Asp?Ile

115 120 125

Ser?Met?Ile?Gly?Gln?Phe?Gly?Val?Gly?Phe?Tyr?Ser?Ala?Tyr?Leu?Val

130 135 140

Ala?Glu?Lys?Val?Thr?Val?Ile?Thr?Lys?His?Asn?Asp?Asp?Glu?Gln?Tyr

145 150 155 160

Ala?Trp?Glu?Ser?Ser?Ala?Gly?Gly?Ser?Phe?Thr?Val?Arg?Thr?Asp?Thr

165 170 175

Gly?Glu?Pro?Met?Gly?Arg?Gly?Thr?Lys?Val?Ile?Leu?His?Leu?Lys?Glu

180 185 190

Asp?Gln?Thr?Glu?Tyr?Leu?Glu?Glu?Arg?Arg?Ile?Lys?Glu?Ile?Val?Lys

195 200 205

Lys?His?Ser?Gln?Phe?Ile?Gly?Tyr?Pro?Ile?Thr?Leu?Phe?Val?Glu?Lys

210 215 220

Glu?Arg?Asp?Lys?Glu?Val?Ser?Asp?Asp?Glu?Ala?Glu?Glu?Lys?Glu?Asp

225 230 235 240

Lys?Glu?Glu?Glu?Lys?Glu?Lys?Glu?Glu?Lys?Glu?Ser?Glu?Asp?Lys?Pro

245 250 255

Glu?Ile?Glu?Asp?Val?Gly?Ser?Asp?Glu?Glu?Glu?Glu?Lys?Lys?Asp?Gly

260 265 270

Asp?Lys?Lys?Lys?Lys?Lys?Lys?Ile?Lys?Glu?Lys?Tyr?Ile?Asp?Gln?Glu

275 280 285

Glu?Leu?Asn?Lys?Thr?Lys?Pro?Ile?Trp?Thr?Arg?Asn?Pro?Asp?Asp?Ile

290 295 300

Thr?Asn?Glu?Glu?Tyr?Gly?Glu?Phe?Tyr?Lys?Ser?Leu?Thr?Asn?Asp?Trp

305 310 315 320

Glu?Asp?His?Leu?Ala?Val?Lys?His?Phe?Ser?Val?Glu?Gly?Gln?Leu?Glu

325 330 335

Phe?Arg?Ala?Leu?Leu?Phe?Val?Pro?Arg?Arg?Ala?Pro?Phe?Asp?Leu?Phe

340 345 350

Glu?Asn?Arg?Lys?Lys?Lys?Asn?Asn?Ile?Lys?Leu?Tyr?Val?Arg?Arg?Val

355 360 365

Phe?Ile?Met?Asp?Asn?Cys?Glu?Glu?Leu?Ile?Pro?Glu?Tyr?Leu?Asn?Phe

370 375 380

Ile?Arg?Gly?Val?Val?Asp?Ser?Glu?Asp?Leu?Pro?Leu?Asn?Ile?Ser?Arg

385 390 395 400

Glu?Met?Leu?Gln?Gln?Ser?Lys?Ile?Leu?Lys?Val?Ile?Arg?Lys?Asn?Leu

405 410 415

Val?Lys?Lys?Cys?Leu?Glu?Leu?Phe?Thr?Glu?Leu?Ala?Glu?Asp?Lys?Glu

420 425 430

Asn?Tyr?Lys?Lys?Phe?Tyr?Glu?Gln?Phe?Ser?Lys?Asn?Ile?Lys?Leu?Gly

435 440 445

Ile?His?Glu?Asp?Ser?Gln?Asn?Arg?Lys?Lys?Leu?Ser?Glu?Leu?Leu?Arg

450 455 460

Tyr?Tyr?Thr?Ser?Ala?Ser?Gly?Asp?Glu?Met?Val?Ser?Leu?Lys?Asp?Tyr

465 470 475 480

Cys?Thr?Arg?Met?Lys?Glu?Asn?Gln?Lys?His?Ile?Tyr?Tyr?Ile?Thr?Gly

485 490 495

Glu?Thr?Lys?Asp?Gln?Val?Ala?Asn?Ser?Ala?Phe?Val?Glu?Arg?Leu?Arg

500 505 510

Lys?His?Gly?Leu?Glu?Val?Ile?Tyr?Met?Ile?Glu?Pro?Ile?Asp?Glu?Tyr

515 520 525

Cys?Val?Gln?Gln?Leu?Lys?Glu?Phe?Glu?Gly?Lys?Thr?Leu?Val?Ser?Val

530 535 540

Thr?Lys?Glu?Gly?Leu?Glu?Leu?Pro?Glu?Asp?Glu?Glu?Glu?Lys?Lys?Lys

545 550 555 560

Gln?Glu?Glu?Lys?Lys?Thr?Lys?Phe?Glu?Asn?Leu?Cys?Lys?Ile?Met?Lys

565 570 575

Asp?Ile?Leu?Glu?Lys?Lys?Val?Glu?Lys?Val?Val?Val?Ser?Asn?Arg?Leu

580 585 590

Val?Thr?Ser?Pro?Cys?Cys?Ile?Val?Thr?Ser?Thr?Tyr?Gly?Trp?Thr?Ala

595 600 605

Asn?Met?Glu?Arg?Ile?Met?Lys?Ala?Gln?Ala?Leu?Arg?Asp?Asn?Ser?Thr

610 615 620

Met?Gly?Tyr?Met?Ala?Ala?Lys?Lys?His?Leu?Glu?Ile?Asn?Pro?Asp?His

625 630 635 640

Ser?Ile?Ile?Glu?Thr?Leu?Arg?Gln?Lys?Ala?Glu?Ala?Asp?Lys?Asn?Asp

645 650 655

Lys?Ser?Val?Lys?Asp?Leu?Val?Ile?Leu?Leu?Tyr?Glu?Thr?Ala?Leu?Leu

660 665 670

Ser?Ser?Gly?Phe?Ser?Leu?Glu?Asp?Pro?Gln?Thr?His?Ala?Asn?Arg?Ile

675 680 685

Tyr?Arg?Met?Ile?Lys?Leu?Gly?Leu?Gly?Ile?Asp?Glu?Asp?Asp?Pro?Thr

690 695 700

Ala?Asp?Asp?Thr?Ser?Ala?Ala?Val?Thr?Glu?Glu?Met?Pro?Pro?Leu?Glu

705 710 715 720

Gly?Asp?Asp?Asp?Thr?Ser?Arg?Met?Glu?Glu?Val?Asp

725 730

<210>3

<211>41

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>3

ttggtaccga?cgacgacgac cctgaggaaa?cccagaccca?a 41

<210>4

<211>35

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>4

ggcgaattct?cattagtcta?cttcttccat?gcgtg 35

Claims

1. the purposes of a HEL-S-64C gene or its encoding proteins is characterized in that, described HEL-S-64C gene or its encoding proteins are used as the promoter that promotes that sperm motility or sperm are worn ovum.

2. the purposes of a HEL-S-64C gene or its encoding proteins is characterized in that, described HEL-S-64C gene or its encoding proteins are used to prepare the medicine that promotes sperm motility or sperm to wear ovum.

3. the purposes of a HEL-S-64C antagonist is characterized in that, described HEL-S-64C antagonist is used as and suppresses the inhibitor that sperm motility or sperm are worn ovum.

4. the purposes of a HEL-S-64C antagonist is characterized in that, described HEL-S-64C antagonist is used to prepare and suppresses the medicine that sperm motility or sperm are worn ovum.

5. purposes as claimed in claim 4 is characterized in that, described antagonist is the antisensenucleic acids of HEL-S-64C, the part of HEL-S-64C, the antibody or the geldanamycin of anti-HEL-S-64C polypeptide.

6. the purposes of a HEL-S-64C agonist is characterized in that, it is used as the promoter that promotes that sperm motility or sperm are worn ovum.

7. the purposes of a HEL-S-64C agonist is characterized in that, it is used to prepare the medicine that promotes sperm motility or sperm to wear ovum.

8. whether a definite test compounds is the method for antagonist or the agonist of HEL-S-64C, it is characterized in that it comprises step:

9. a pharmaceutical composition is characterized in that, it contains HEL-S-64C polypeptide, its agonist or its antagonist and the pharmaceutically acceptable carrier of safe and effective amount.

10. a utensil of regulating fertility is characterized in that, described utensil is the contraceptive device that contains the HEL-S-64C antagonist.