CN1795928A - Correlation factor of blastocyst nidation and application - Google Patents

Abstract

A blastocyst nidation associated factor EMO-2 able to promote the nidation of blastocyst is disclosed. Its antisense nucleic acid and its polypeptide antibody can suppress the nidation of blastocyst. A process for screening the compounds to suppress or promote the nidation of blastocyst by use of said EMO-2 factor is also disclosed.

Description

Blastocyst implantation related factor and uses thereof

Technical field

The invention belongs to biotechnology and medical domain, specifically, the present invention relates to blastocyst implantation related factor EMO-2 in the purposes that promotes aspects such as blastocyst implantation.

Background technology

Embryo nidation is a peculiar important step in the mammal reproductive process.Just calculating after finishing real conceivedly because of implantation process, so give birth to regulation and control at the embryo nidation process, do not have ethical issues, is an ideal fertility regulation and control medical instrument effect link.In addition,, transplant back embryo implantation smoothly, become the key that influences artificial Assisted Reproductive Technology ART (ART/IVF) success rate at present along with technology such as the In vitro culture of oocyte and maturation, external fertilization, in-vitro oosperm culture and growth improve constantly.

The embryo is one and half allosome tissues with respect to parent, is subject to parent and repels, and can only (i.e. " implantation window ") could be admitted by the parent endometrial tissue in a certain period of time.The embryo nidation process finish synchronous growth, parent endometrium endometrial receptivity and the formation of " implantation window " and the formation of parent uterine cancer cell local immunity toleration that depends on parent uterus and embryo, relate to the adjusting of the propagation of cell and differentiation, local immunity function.Female, the effect of progestogen in implantation is very clear and definite, their effects in reproductive process grow up existing flute body class contraceptive with regard to being based on.But owing to female, progestogen are endocrine hormones, its effect is a general, also is not optimal fertility regulatory factor therefore.In recent years, the non-hormone factor that the uterine cancer cell part plays a role during embryo nidation more and more obtains people's attention, has become a focus of biology of reproduction research field.

For various reasons, people's pair factor relevant with blastocyst implantation also known little about it, so this area presses for the new blastocyst implantation related factor of exploitation.

Summary of the invention

The purpose of this invention is to provide relevant EMO-2 factor of a kind of new blastocyst implantation and uses thereof.

In a first aspect of the present invention, the purposes of EMO-2 gene (and encoding proteins) is provided, it is used as the promoter that promotes blastocyst implantation.

The present invention also provides another purposes of EMO-2 gene (and encoding proteins), and it is used to prepare the medicine that promotes blastocyst implantation.

In a second aspect of the present invention, the purposes of EMO-2 antagonist is provided, it is used as the inhibitor of vitro inhibition blastocyst implantation.

The present invention also provides another purposes of EMO-2 antagonist, and it is used to prepare the medicine that suppresses blastocyst implantation.

In another preference, described medicine is a contraceptive.

In another preference, described antagonist is the antisensenucleic acids of EMO-2 or the antibody of anti-EMO-2 polypeptide.

In a third aspect of the present invention, the purposes of EMO-2 agonist is provided, it is used as the promoter of external promotion blastocyst implantation.

The present invention also provides another purposes of a kind of EMO-2 agonist, and it is used to prepare the medicine that promotes blastocyst implantation.

In a fourth aspect of the present invention, provide whether a kind of definite test compounds is the method for antagonist or the agonist of EMO-2, it comprises step:

(a) blastocyst of test compounds and EMO-2 polypeptide adding In vitro culture is organized as test, and the blastocyst of EMO-2 polypeptide adding In vitro culture is organized in contrast;

(b) observation test group and matched group blastocyst implantation situation are if the blastocyst implantation quantity of test group, represents then that test compounds is the agonist of EMO-2 greater than matched group; If the blastocyst implantation quantity of test group, represents then that test compounds is the antagonist of EMO-2 less than matched group.

In a fifth aspect of the present invention, a kind of pharmaceutical composition is provided, it contains EMO-2 polypeptide, its agonist or its antagonist and the pharmaceutically acceptable carrier of safe and effective amount (as 0.001-99.9wt%).

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 has shown the full length amino acid sequence of mice EMO-2 polypeptide.

Fig. 2 A and 2B have shown that the antisensenucleic acids of EMO-2 can effectively suppress the blastocyst growth external.

The specific embodiment

The inventor finds by further investigation, the EMO-2 gene behind implantation the expression in the mice embryonic before implantation.Studies show that antisensenucleic acids can be hatched from zona pellucida at the external blastocyst that can suppress effectively by the translation process of blocking-up EMO-2 mRNA, then can reduce the embryo number of normal implantation in vivo significantly.This explanation EMO-2 gene plays important facilitation in the embryo nidation process.

In the present invention, term " EMO-2 albumen ", " EMO-2 polypeptide ", " the EMO-2 factor " or " blastocyst implantation associated protein EMO-2 " etc. are used interchangeably, all refer to the to have blastocyst implantation related factor EMO-2 aminoacid sequence albumen or the polypeptide of (SEQ ID NO:2).They can contain or not contain initial methionine.In addition, these terms also are included in homologue or the homologous protein that promotes the blastocyst implantation function that have in other mammals (as people, Canis familiaris L., cattle, sheep, monkey).

The cDNA gene accession number of the Emo-2 of mice is 556728, AY372183, and total length 5700bp (SEQ IDNO:1), ORF is positioned at the 78-1439 position, and the total length of encoding is 454 amino acid whose albumen (SEQ ID NO:2 and Fig. 1).

Should be understood that because proteic nucleotide sequence of mammiferous EMO-2 such as Mus 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 EMO-2 analog, i.e. the homologous protein of EMO-2 in other mammals (as cattle, sheep, rabbit, Canis familiaris L., monkey, people 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 EMO-2.

As used herein, " isolating EMO-2 albumen or polypeptide " is meant that the EMO-2 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 EMO-2 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 EMO-2 polypeptide.As used herein, term " fragment ", " derivant " are meant biological function or the active polypeptide that keeps the natural EMO-2 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 " EMO-2 polypeptide " refers to have the SEQ ID NO:2 polypeptide of sequence that promotes EMO-2 factor actives such as blastocyst implantation.This term also comprises having and variant form EMO-2 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 EMO-2 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 EMO-2DNA hybridization and the polypeptide or the albumen that utilize the antiserum of anti-EMO-2 polypeptide to obtain.The present invention also provides other polypeptide, as comprises EMO-2 polypeptide or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention has also comprised the soluble fragments of EMO-2 polypeptide.Usually, this fragment have the EMO-2 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 EMO-2 polypeptide.The difference of these analog and natural EMO-2 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.

EMO-2 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.

The EMO-2 polypeptide is of use in many ways.These purposes include, but is not limited to: directly as the disease (infertile as what lowly cause because of the blastocyst implantation ability) due to Drug therapy EMO-2 factor hypofunction or the forfeiture be used to screen antibody, polypeptide or other part that promotes or resist EMO-2 factor function.The peptide molecule that can suppress or stimulate EMO-2 factor function that can be used for seeking therapeutic value with the reorganization EMO-2 factor screening peptide library of expressing.

On the other hand, the present invention also comprises EMO-2DNA 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 EMO-2 gene outcome or fragment.Preferably, refer to that those can combine with EMO-2 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 EMO-2 factor, comprise that also those do not influence the antibody of EMO-2 factor function.The present invention also comprise those can with modify or without the bonded antibody of EMO-2 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 EMO-2 gene outcome of purification or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing the EMO-2 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 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 the EMO-2 function and the antibody that does not influence the EMO-2 function.Each antibody-like of the present invention can utilize the fragment or the functional areas of EMO-2 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 EMO-2 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-EMO-2 can be used in the immunohistochemistry technology, detects the EMO-2 in the biopsy specimen.

Antibody among the present invention also can be used for suppressing blastocyst implantation as the antagonist of EMO-2, thereby is used for contraception.The antibody that gives suitable dosage can be blocked generation or the activity of EMO-2.

Utilize albumen of the present invention,, can filter out with EMO-2 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 EMO-2 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 a kind of pharmaceutical composition, and it contains EMO-2 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 EMO-2 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, patient health situation, and these all are within the skilled practitioners skill.

The polynucleotide of EMO-2 also can be used for multiple therapeutic purposes.Gene therapy technology can be used for treating because the blastocyst implantation level due to the expression of the EMO-2 of the nothing expression of EMO-2 or unusual/non-activity is low.Antisense nucleotide can suppress blastocyst implantation, thereby is used for contraception.

The invention still further relates to the diagnostic testing process of quantitative and detection and localization EMO-2 level.These tests are known in the art, comprise methods such as radioimmunoassay.The EMO-2 level that is detected in the test, can with lay down a definition the EMO-2 factor in disease such as infertile importance and be used for diagnosis.

A kind of method that whether has the EMO-2 factor in the test sample that detects is to utilize the specific antibody of the EMO-2 factor to detect, and it comprises: sample is contacted with EMO-2 factor-specific antibody; Observe whether form antibody complex, formed antibody complex and just represented to exist in the sample EMO-2 factor.

Major advantage of the present invention is, blastocyst implantation to EMO-2 to tangible dependency is arranged, if lack EMO-2 then implantation can not be successful.

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:ColdSpring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

The clone of embodiment 1:EMO-2cDNA

1. laboratory animal: ICR white mice, female mice 28 ± 2g, 7-8 week, period of maturation.Male mice 30 ± 2g, two male female mating are looked into cloudy bolt second day morning, find that cloudy bolt day is pregnant first day.

2. blastocyst is collected: mouse web portion 75% alcohol disinfecting, open the abdominal cavity, take out the uterus, with PBS from the uterus one end go out blastocyst, place on the concave slide, the collection blastocyst places liquid nitrogen in a tubule rapidly under anatomical lens, preserves stand-by.

3. total RNA extracts: use Quickprep Micro mRNA Puriflcation Kit (AmershamPharmacia Biotech) reagent and extract total RNA.

4. reverse transcription (RT): use two kinds of enzyme: AMV and MMLV, obtain cDNA (strand) from mRNA through reverse transcription.

(5.DD-PCR post transcription cloning): strand cDNA obtains double-stranded cDNA through DD-PCR.Use two kinds of primers, random primer and anchor primer in the amplification.

6. difference shows: difference shows uses protein sequencing analytical electrophoresis instrument

(1) loading amount of electrophresis apparatus, encapsulating (6% degeneration sequencing gel)

(2) go up sample: the sample of various DD-PCR products

(3) prerunning and electrophoresis

(4) dried glue and exposure

(5) get differential fragment quarter,, and be numbered,, carve and get differential fragment then with reference to the labelling on glue and the X-ray at punching post analysis differential fragment.Freezing preservation is stand-by.

7.DD-PCR the purification of the recovery of differential fragment and amplification and PCR product.

8. being connected of purpose fragment and carrier Pucm-T (available from Shanghai company of lottery industry biotechnology company), become the Pucm-T-Tar carrier, be transformed into escherichia coli, clone (product description by manufacturer carries out).

9. recombinant clone screening indigo plant/from the speckle screening, recombinant clone is a white.

10. insert the fragments sequence analysis, the insertion fragment that is inserted among the above-mentioned carrier Pucm-T is analyzed with the full-automatic sequenator of ABI377, obtain respectively to insert segmental full length sequence.

The cDNA that found that a differentially expressed protein is named as the EMO-2 gene shown in SEQ ID NO:1.

EMO-2cDNA is 5700bp (SEQ ID NO:1), and the 78-1439 position is complete open reading frame, and coding contains the polypeptide (Fig. 1 and SEQ ID NO:2) of 454 amino acid residues.

The in situ hybridization of embodiment 2:EMO-2

Nucleotide fragments with 207-398 position among the SEQ ID NO:1 is a probe, carries out in situ hybridization in order to following method: got the 5th day endometrium of mouse conceived the 4th day, fix 2.5 hours with 4% paraformaldehyde room temperature, fixing back sample dewaters through gradient, paraffin embedding, section.Section is carried out prehybridization and hybridization through dewaxing and the linear DNA template probe for preparing.

The result shows that the EMO-2 gene has higher blastocyst specificity, its hetero-organization amixia.

Embodiment 3:EMO-2 antisensenucleic acids is to the vitro inhibition effect of blastocyst implantation

Antisensenucleic acids that present embodiment uses or contrast nucleic acid are as follows:

Antisensenucleic acids is the following oligonucleotide of sequence

5’-GTCTTCTGTGGGCTGCTCCTCA-3’(SEQ ID NO：3)

Contrast nucleic acid is the oligonucleotide of random sequence

5’-TGAGGAGCAGCCCACAGAAGA C-3’(SEQ ID NO：4)

With transfection agents LipofectAmin ^TMThe mixed liquor (concentration is 1 μ g/ μ l) of 2000Reagent (Invitrogen company) and above-mentioned antisensenucleic acids or contrast nucleic acid, (every hole adds 100 μ l culture fluid and 6 μ l mixed liquors in the blastocyst culture fluid of adding mice, final concentration is 0.056 μ g/ μ l), observe influence to the blastocyst growth.

Found that the 4th day mice blastaea of In vitro culture gestation when carrying out the function sealing with the antisense RNA of EMO-2, can significantly suppress the growth of blastocyst, suppresses blastocyst from zona pellucida effusion (Fig. 2 A and 2B).

Embodiment 4:EMO-2 antisensenucleic acids is to inhibitory action in the body of blastocyst implantation

Antisensenucleic acids that present embodiment uses or contrast nucleic acid are as follows:

Antisensenucleic acids is the following oligonucleotide of sequence

5’-GTCTTCTGTGGGCTGCTCCTCA-3’(SEQ ID NO：3)

Contrast nucleic acid is the oligonucleotide of random sequence

5’-TGAGGAGCAGCCCACAGAAGA C-3’(SEQ ID NO：4)

With transfection agents Mirus TransIT  Polymer Solution (Madison company, WI, USA) and above-mentioned antisensenucleic acids or the contrast nucleic acid mixture (concentration is 1 μ g/ μ l), the 4th day afternoon antisensenucleic acids is injected directly into every Aconitum carmichaeli Debx. palace by dosage shown in the table 1 in gestation, added up the embryo number of normal implantation then at the 9th day.

The result shows that the EMO-2 antisensenucleic acids can obviously reduce the embryo number (seeing Table 1) of normal implantation.

Table 1 EMO-2 nucleic acid is to the influence (repeating 3 tests) of mice implantation

Group	The laboratory animal number	Dosage (a μ g/ cornua uteri)	Survival embryo's sum (individual)	Survival embryo's average (individual/cornua uteri) X ± SD	Survival embryo's suppression ratio (%)	Remarks
							Matched group (training liquid)	30	50 μ l/ only	131	4.4±2.3	0	(1) survival embryo mean is that embryo number (2) the survival embryo inhibiting rate at angle, injection side palace is Yu control group ratio, establish control group and be 0% (3) a Yu control group than P＜0.05 (4) b Yu corresponding dosage Sense Zu than P＜0.05 (5) Zhu enter the antisensenucleic acids time for afternoon 2:00 to 3:00
The transfection agents group	12	15 μ l/ only	87	4.3±2.4	0
						EMO-2 antisensenucleic acids (SEQ ID NO:3)	13 8 17	10 25 33	32 0 19	2.5±2.5 a 0 ab 1.1±1.1 ab	46.8 100.0 75.0
Contrast nucleic acid (SEQ ID NO:4)	8 21	25 33	30 14	3.8±1.7 3.8±2.1	15.8 14.0

Embodiment 5EMO-2's is recombinant expressed

In this embodiment, be template with extractive mice blastocyst mRNA, with the PCR oligonucleotide primers RT-PCR that increases of 5 ' and 3 ' following end of sequence, obtain EMO-2DNA as inserting fragment.

5 ' the end oligonucleotide primers sequence of using in the PCR reaction is:

5’-aaaCATATGatgagcttcatagattgagc-3’(SEQ ID NO：5)

This primer contains the restriction enzyme site of NdeI restricted enzyme, is the part coded sequence that is begun by start codon after this restriction enzyme site;

3 ' end primer sequence is:

5’-tatGGATCCcaggaaacacaggctctagcct-3’(SEQ ID NO：6)，

This primer contains the part coded sequence of restriction enzyme site, translation termination and the EMO-2 of BamHI restricted enzyme.

EMO-2 cDNA PCR product purification after the NdeI/BamHI enzyme action recombinate according to a conventional method with plasmid pUC18 again and be converted into the competence bacillus coli DH 5 alpha, the picking positive colony is identified back purification and order-checking (377 type sequenators of ABI company, BigDye Terminator test kit, PE company).The EMO-2 albumen cDNA NdeI/BamHI endonuclease bamhi of correct sequence is cloned into expression vector pGEX-2T, and (Pharmacia, Piscataway NJ), form carrier pGEX-2T-EMO-2, transform people DH5 α then.Positive colony is identified direction of insertion, the capable 2% agarose gel electrophoresis analysis of enzyme action product with the NdeI/BamHI enzyme action.Confirm through order-checking, inserted complete EMO-2 coded sequence.

Choosing the positive DH5 α clone who expresses EMO-2 is inoculated in 100ml 2 * YTA culture medium, 37 ℃ of 300rpm shaken cultivation 12-15hr, 2 * YTA the culture medium that is diluted in preheating at 1: 10 continues shaken cultivation 1.5hr, add behind the 100mMIPTG to 0.1mM 30 ℃ and induce 2-6hr, 5,4 ℃ of centrifugal 10min of 000g remove supernatant, put and use 50ml 1 * PBS (0.14M NaCl on ice, 2.7mM KCl, 10.1mM Na ₂HPO ₄, 1.8mM KH ₂PO ₄PH7.3) resuspended, add 20%Triton X-100 to 1% jog 30min again after ultrasonic (B.Braun Labsonic U) fragmentation, then 12,4 ℃ of centrifugal 10min of 000g, supernatant with 0.8 μ m membrane filtration after, cross 1ml 50% glutathione S epharose 4B chromatographic column, behind 1 * PBS thorough washing, add 500ul glutathion elution buffer (10mM glutathion, 50mM Tris-HCl, pH8.0) room temperature leaves standstill after 30 minutes and collects eluent, repeat eluting 2-3 time, obtain EMO-2 albumen, the molecular weight size conforms to predicted value.

Embodiment 6 anti-EMO-2 production of antibodies

1. polypeptide is synthetic

Carry out the synthetic of polypeptide in the mode of synthetic multiple antigenic peptide (MAP), the core of MAP is a kind of immunogenic Lys (lysine) core that do not have, and the C end of purpose antigen polypeptide is coupled on two aminoacid of Lys.Can form the multiple antigenic peptide of 8 repetitive sequences.The antigen fragment of two kinds of synthetic polypeptide that obtain is as follows:

Antigen polypeptide EMO-2N:Ala-Glu-Ser-Ser-Arg-Thr-Phe-Pro-Glu-His-Cys-Ala-P ro-Ser-Arg-Leu-Ala (SEQ ID NO:7)

Antigen polypeptide EMO-2C:Thr-Val-Pro-Arg-Lys-Arg-Arg-Ala-Arg-Gly-Lys-Val-L ys-Leu-Ala-Gly (SEQ ID NO:8)

2. generation antibody:

With as above synthetic 2 peptide species respectively as immunogen, each subcutaneous with 0.4mg/Kg multi-point injection rabbit, antigen be dissolved in normal saline and and the emulsifying of isopyknic Freund ' s Freund's complete adjuvant, every carrying out booster immunization two weeks one time, use the solid-phase enzyme immunoassay method, carry out the mirror of antiserum titre and survey.Treat sero-fast tiring when reaching requirement, can get whole blood from the carotid artery of rabbit, and isolate serum, obtain the antiserum (anti-EMO-2N antiserum and anti-EMO-2C antiserum) of two kinds of anti-EMO-2.-20 ℃ freezing stand-by.

Carry out inhibition test in the body of blastocyst implantation by the method for embodiment 4, difference is to replace antisensenucleic acids with antiserum (without transfection agents).The result is as shown in table 2.

Table 2 EMO-2 antiserum is to the influence of mice implantation

Group	Number of animals	Dosage (a μ g/ cornua uteri)	Survival embryo's sum (individual)	Survival embryo average/animal X ± SD	Implantation suppression ratio (%)	Remarks
							The EMO-2C antiserum	10	50	14	1.4	79.3	(1) survival embryo mean is that embryo number (2) the survival embryo inhibiting rate at angle, injection side palace is Yu control group ratio, and establishing control group and be 0% (3) Zhu, to enter the sero-fast time be 6:00 in afternoon
The EMO-2N antiserum	10	50	27	2.7	60.3
						The normal serum matched group	10	50	68	6.6	0

* administration time is that gestation 6:00 in the 4th day afternoon is to 6:30 in afternoon

The antagonist of embodiment 7 screening EMO-2

Press embodiment 4 described methods, following two groups of materials (replacing antisensenucleic acids and transfection agents) are applied to pregnant mouse, observe then inhibitory action in the body of blastocyst implantation: (a) candidate substances+EMO-2 factor; (b) the EMO-2 factor.

If the blastocyst implantation rate of group (a) significantly is lower than the blastocyst implantation rate of group (b) statistically, show that then this candidate substances is the antagonist of EMO-2.

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〉Shanghai Family Planning Science and Research Institute.

＜120〉blastocyst implantation associated protein and uses thereof

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<223>

<400>1

catatttggc agctactggg tggaatagag ggtctaacag taggcccttg gcactttatt 60

ctcttgaagc tgttaaa atg agc ttc ata gat ttg agc aac aag atg cca 110

Met Ser Phe Ile Asp Leu Ser Asn Lys Met Pro

1 5 10

tca ctg ctg ttt ggt acg gaa aac gtc cca ttt tcc ttt cat gtg aaa 158

Ser Leu Leu Phe Gly Thr Glu Asn Val Pro Phe Ser Phe His Val Lys

15 20 25

tca gtg tcc agc tgc atg gct gag tcc tcc agg act ttt cct gag cac 206

Ser Val Ser Ser Cys Met Ala Glu Ser Ser Arg Thr Phe Pro Glu His

30 35 40

tgc gct cca tcg agg ctt gcc tta aca gag gcc tcc cag tgc tca gct 254

Cys Ala Pro Ser Arg Leu Ala Leu Thr Glu Ala Ser Gln Cys Ser Ala

45 50 55

cca gct ccc aag tgg acc ttc tct ttt ttc ttg tcc cat ggt tgc cct 302

Pro Ala Pro Lys Trp Thr Phe Ser Phe Phe Leu Ser His Gly Cys Pro

60 65 70 75

ggg atg gcc aca ttc agg gaa gac act ggc ctc agt agt cag aca cac 350

Gly Met Ala Thr Phe Arg Glu Asp Thr Gly Leu Ser Ser Gln Thr His

80 85 90

act cag gct cct ctc caa gaa tat gga ggc act gcc ata gtt cag acc 398

Thr Gln Ala Pro Leu Gln Glu Tyr Gly Gly Thr Ala Ile Val Gln Thr

95 100 105

agg gca gac cgc tct ggc ctt ggc ctt cac aca ctt cta gca ctt tgt 446

Arg Ala Asp Arg Ser Gly Leu Gly Leu His Thr Leu Leu Ala Leu Cys

110 115 120

tca ccc gga tgt tac cga atc tgg aca aaa aga cgg aac ttc tcc agt 494

Ser Pro Gly Cys Tyr Arg Ile Trp Thr Lys Arg Arg Asn Phe Ser Ser

125 130 135

aat atg cct atc atg cag agg ctc ttc ctg acc cag ttt aca cag ggc 542

Asn Met Pro Ile Met Gln Arg Leu Phe Leu Thr Gln Phe Thr Gln Gly

140 145 150 155

cta aaa ggg tta agg tct cca gcc tct ata gca gat aag gtc ttc tgt 590

Leu Lys Gly Leu Arg Ser Pro Ala Ser Ile Ala Asp Lys Val Phe Cys

160 165 170

tct ctg ccc tac tcg gtg ggc cgg gtg ttg tcc ctt tgg agc cag cac 638

Ser Leu Pro Tyr Ser Val Gly Arg Val Leu Ser Leu Trp Ser Gln His

175 180 185

ggg ccc tct tgt acc ttc aaa ttg cca gct ctt cat tcc acc cct agc 686

Gly Pro Ser Cys Thr Phe Lys Leu Pro Ala Leu His Ser Thr Pro Ser

190 195 200

aag cag cag ggg agc ctg aac gcc ctg agc agc cac acc acc ata cca 734

Lys Gln Gln Gly Ser Leu Asn Ala Leu Ser Ser His Thr Thr Ile Pro

205 210 215

aat gtg cct ctt cca ggc atg gga gct gcc cac acc acc aac agc agt 782

Asn Val Pro Leu Pro Gly Met Gly Ala Ala His Thr Thr Asn Ser Ser

220 225 230 235

cac ctg agg cta gag cct gtg ttt cct gcc ttg gtg cca aag tct tgc 830

His Leu Arg Leu Glu Pro Val Phe Pro Ala Leu Val Pro Lys Ser Cys

240 245 250

ttg gta aca gaa gcc gct gtc agc aca ctg ctg ctc tct gcc tct gag 878

Leu Val Thr Glu Ala Ala Val Ser Thr Leu Leu Leu Ser Ala Ser Glu

255 260 265

ctc gca gtt cct ggc tgt gat gag ctg gat ggt gtg tca gca gcc tgc 926

Leu Ala Val Pro Gly Cys Asp Glu Leu Asp Gly Val Ser Ala Ala Cys

270 275 280

ccc cga cca cag agc agc cct gca cag cag aaa gag gct gag cca gag 974

Pro Arg Pro Gln Ser Ser Pro Ala Gln Gln Lys Glu Ala Glu Pro Glu

285 290 295

aag aga cca aag aaa gtc tca cag atc cgc atc cgg aaa acc att cct 1022

Lys Arg Pro Lys Lys Val Ser Gln Ile Arg Ile Arg Lys Thr Ile Pro

300 305 310 315

aaa cca gat ccc aac ctt acc cca atg ggc ctg cct cgg ccc aaa agg 1070

Lys Pro Asp Pro Asn Leu Thr Pro Met Gly Leu Pro Arg Pro Lys Arg

320 325 330

tta aag aag aag gag ttt agt tta gag gaa atc tat aca aac aag aat 1118

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

335 340 345

tac aag tct cct cct gcg agc agg tgt tta gag acc atc ttt gag gaa 1166

Tyr Lys Ser Pro Pro Ala Ser Arg Cys Leu Glu Thr Ile Phe Glu Glu

350 355 360

ccc aaa gaa cga aat ggt acc ctg atc tct atc agc cag cag aaa agg 1214

Pro Lys Glu Arg Asn Gly Thr Leu Ile Ser Ile Ser Gln Gln Lys Arg

365 370 375

aag cgt gtt ctg gaa ttc cag gat ttt acc gtc cct cga aag aga cga 1262

Lys Arg Val Leu Glu Phe Gln Asp Phe Thr Val Pro Arg Lys Arg Arg

380 385 390 395

gct cga ggt aag gtg aag ctg gcc ggc agc ttc acc agg gcc cag aag 1310

Ala Arg Gly Lys Val Lys Leu Ala Gly Ser Phe Thr Arg Ala Gln Lys

400 405 410

gcg gct ctg cag act cag gag ctg gac gct ctt ctg att cag aag ctg 1358

Ala Ala Leu Gln Thr Gln Glu Leu Asp Ala Leu Leu Ile Gln Lys Leu

415 420 425

atg gaa ctg gag acg ttc ttt gcc aag gag ggg gag cag gag cat cag 1406

Met Glu Leu Glu Thr Phe Phe Ala Lys Glu Gly Glu Gln Glu His Gln

430 435 440

cca gct gct gag aac agt tcg ggg tct cca ctt tgaacttttg cagacctccc 1459

Pro Ala Ala Glu Asn Ser Ser Gly Ser Pro Leu

445 450

tggatgaggt tccttgattt tagccctgtg cccaaaccac tcacaatgcc cagcccatga 1519

atttttactt atttcaaggt gcagcctttt tagaagctgg tgaagaaggg ccctctaatt 1579

ctactgctga gtctagagcc tcaggaatgc tccctttctc ctggggatgg tcctgagtga 1639

ccaggccacc tccttcccgt ctcccatcac tggtggaaaa gccacacatc ttaagcaaca 1699

ctaggaatct aaggcctcgt tgtctgtgtg tccacacgaa agctcctctc cattcatggt 1759

gctgctcgcc caagatgact tagaaacctg gcctggggag gacctggctg cctggtctgt 1819

gtgttctcac cagcttgtta tggtgactga gaaagatgaa ctggtgtgtt tcttgactct 1879

gattctgtct gcccaggacc tttattctga gagaatgtgt gtttgtgtgt gtttgtggct 1939

tttccccatc ttttctccca tctgtgactg cgggtcacta gtgccagagg agccgtccag 1999

gccccagcgt aagttgcact ttttaatgtt atggtgttga ttattttcat ttttcttctt 2059

cttttgtttt tgtattattg ctgcatgttt ggggtctcta aatgtgattt taaaccattt 2119

tgtaagacat taaggagaaa gacaatttac gtcttaggag aatatgtgac aggcattgat 2179

cagcacatgg aggggcagtg ttcccaacca tgtgtttcag gcagccctcc cccaccccca 2239

gcttttcatg tagttcacta gaggaagcac ttttcacctc cttctcagtc ctgcccactg 2299

aatacacata ttcatttagt gacgtgcata aaagcagatt tctctcgtaa ccacttaaaa 2359

actgttaagg tggagtgtgg gtttctctga aatgttggta tatgatgaag ttagcagtgg 2419

tgtcagaggc tgaccaagag tgttttcagc tgctagagtg cctggttctg tcaagggcca 2479

gagccagtgc tctctgagag tgcctcgtgg actctgctcc ctcgtcaaac ctactggaaa 2539

ggtttgcatg ctacaagtag cacagttcct aggtttgcgc ccagaggagt ctgcaggcgg 2599

taggctgccg tctgtcgtct gagcagcagg ccctcagagc tcaccctccc cagtgtcctt 2659

actgtgctcc ttcctctcct gtgtgttctc atcccaccct ctctcccatt cattcttcac 2719

acctaccgct cagaggtcag aagttcaaac tgcttggctg atggctaaga tgctcaagct 2779

agctgctgaa caatgctcat ttcttcccat tctcctggct tggtccaggg acatttctct 2839

gttcgtctgt agagaagagt gagcagtgct gtcctggaat gttggccaac gcttctacga 2899

ttctttcatt ccccatttgt cagtggctaa ggagaagggg caccaccagt tcagggtctt 2959

tatgtgcctt tcctttgcgg gttggtggtc tgggccaccc tgaaacagtt aaactggaca 3019

taagtatggg gaaacgtatc atgtccttca cctcctcagc tgtttaaata agttaacaca 3079

cacctgctgg tgtatggaga cttgaaggtc acagctctat cacagaagag aacaagctag 3139

tttgcacgat ggtcttcagc agtgtatgca gatcctggtt tacacatgat gcccagacca 3199

tacacacgtg acctgtgttc acctgaatct ctctctccct cgttggggca ggagttttag 3259

ttactcagtg cttctgaact ctgtgttgtt acttcaggga ctgaaatgag ttacacattt 3319

tgcccttcta caagtacatc taagacactg tacattagac agcttgcaag ctagtgggca 3379

gcaaacagcc tttcatcagt ttaaccacca tggttactgg ggcatcagat taccccaaaa 3439

aggtgaggtg tccacagctg gggtaccacc agtatacaga cctttagaat ttgtccatga 3499

ctagtgtggc atgtaagagg ctggaaacac tggtctggac tcttagcatc tcggagcaca 3559

ggacttgtcc tgggaatgga agaggaaatc tgccatcttg gtctgccctc cagctcagaa 3619

ctaagagact cagccacacc tgtcttacca tcatgtttca cagtggtcag ccagcctcag 3679

agacacctta gtgtcctcag aaccttggtc tttgacaatg actggaggtg gtggctgctg 3739

gcatgtcacc tgccaaacct ataggagtgg ttctgtaaag gtagaatttc tgaggatggg 3799

gacttgccac tcaagtgatg agaagctgac attaagcaca acatacctag ctgtagctca 3859

cctccagaag tttagtgttt tcatgattat ggggaaagaa gacaaatttc aggttcagcc 3919

agatttcagt cgtttccttg gccacctttc agtctctcgc cagagcagcc tcttccctca 3979

ctagtgcctg tcttcttttc ctcccctgct gtcccatccc gcctctccac ccatctagtt 4039

gtacctcatc acctgttaaa gcagattgtg tctgtcttcc tgctgccctt actataagag 4099

ctgactgaga gaggtgcagt gggaagagat gaagcttttt tgccttactg gagcctgaag 4159

agctcagaaa ctcatgctgt gaatcctaaa ctctgtgctc ctccaagagc tgtgaaaatc 4219

atgacttttt tttttttttt ttccacaatt ttgcctgaaa gggtcttctg tgagggctcc 4279

agagccagcc tgaggtcagt tctagatccc accctctggg catccttccc ctgggtgtgt 4339

cctctgccca ccaggctaag agcagcagca agtctgcagg gctgtggcag caggtccgag 4399

gctgcagtgc agccaagtgg acccagccct ccatctcccc aggctggggc tcagtctctt 4459

ctgtaccatg tgcattaaga tctgtgcaag accaactttt aggttgtgat actttcctcc 4519

tggtacctgg ggggagggga gtatgcagtt ggtgctttct ttaatatcct tggtttgttt 4579

ctatagcttt tcccctggta tcatgaaaag ggccttttta atgaccacat tgtgggtggc 4639

tgtatccaaa gcataaataa ttggccagtg gtatggaatg tcctcacctg cattcctgtc 4699

aggagagggc tactttcctg gactgaacta tatagaactg gctaggaaga tgagttaaat 4759

tttgttcaaa tcacaattaa tattccaatt tagccacact ttgaccagca aaagccatct 4819

tggttgtgaa gctggggccc tcatttagcc gccatccagc ccagcatgct aaaatctgag 4879

gctcattctg tctttataag actgtctggt gttggttgga ttttaattct caacaagagg 4939

cccccttgaa ctaggatctg ggccagtaag ttgctgtccc tagtgttcct gtattatcat 4999

ggctaatagt tcagtgaaat gtgtccatct ttggtaatgc tagtatacat gataccctac 5059

agttcctttt ctgtttgata gtttgtgact ctaaatgccc actgttacat taattaattt 5119

atggaaataa atttttcttg aaaaccattc agataattgt caaggctatt ttgttcttta 5179

gggcttggta gtaggtttga attttcccgg taatattcca attagacatg ctccctccca 5239

ccttaatagg tgtgcttgcc tctcctgtgc cagaacctcc ctcacattcc tgcatggtca 5299

cttccatcac gtcatccaga tctttacatt cccagatcgc tggtcttgaa cgtggtcccc 5359

tcccattcca tatctgctgc ccgcacaccc caaggcattt tgttatggct cctcccccct 5419

gtctgtgcta ataactataa caaaagctct acaaggtcag gacctttcat tttcttagct 5479

ctagaccctt ctggtctctt tggtacctag aacaatgttt tatatttaca gctgtttacc 5539

gcctaaatat cgttttggtc agtgaactgc atattaccat aagatggtaa tggagctgaa 5599

aaattcctgg gacctagcga ggtcacagct gtgatgatgt gttttactca ggtcttggtg 5659

caaacaagcc agtgcattgc cagtcttgta aaaatgtagc a 5700

<210>2

<211>454

<212>PRT

＜213〉mice (Mus musculus)

<400>2

Met Ser Phe Ile Asp Leu Ser Asn Lys Met Pro Ser Leu Leu Phe Gly

1 5 10 15

Thr Glu Asn Val Pro Phe Ser Phe His Val Lys Ser Val Ser Ser Cys

20 25 30

Met Ala Glu Ser Ser Arg Thr Phe Pro Glu His Cys Ala Pro Ser Arg

35 40 45

Leu Ala Leu Thr Glu Ala Ser Gln Cys Ser Ala Pro Ala Pro Lys Trp

50 55 60

Thr Phe Ser Phe Phe Leu Ser His Gly Cys Pro Gly Met Ala Thr Phe

65 70 75 80

Arg Glu Asp Thr Gly Leu Ser Ser Gln Thr His Thr Gln Ala Pro Leu

85 90 95

Gln Glu Tyr Gly Gly Thr Ala Ile Val Gln Thr Arg Ala Asp Arg Ser

100 105 110

Gly Leu Gly Leu His Thr Leu Leu Ala Leu Cys Ser Pro Gly Cys Tyr

115 120 125

Arg Ile Trp Thr Lys Arg Arg Asn Phe Ser Ser Asn Met Pro Ile Met

130 135 140

Gln Arg Leu Phe Leu Thr Gln Phe Thr Gln Gly Leu Lys Gly Leu Arg

145 150 155 160

Ser Pro Ala Ser Ile Ala Asp Lys Val Phe Cys Ser Leu Pro Tyr Ser

165 170 175

Val Gly Arg Val Leu Ser Leu Trp Ser Gln His Gly Pro Ser Cys Thr

180 185 190

Phe Lys Leu Pro Ala Leu His Ser Thr Pro Ser Lys Gln Gln Gly Ser

195 200 205

Leu Asn Ala Leu Ser Ser His Thr Thr Ile Pro Asn Val Pro Leu Pro

210 215 220

Gly Met Gly Ala Ala His Thr Thr Asn Ser Ser His Leu Arg Leu Glu

225 230 235 240

Pro Val Phe Pro Ala Leu Val Pro Lys Ser Cys Leu Val Thr Glu Ala

245 250 255

Ala Val Ser Thr Leu Leu Leu Ser Ala Ser Glu Leu Ala Val Pro Gly

260 265 270

Cys Asp Glu Leu Asp Gly Val Ser Ala Ala Cys Pro Arg Pro Gln Ser

275 280 285

Ser Pro Ala Gln Gln Lys Glu Ala Glu Pro Glu Lys Arg Pro Lys Lys

290 295 300

Val Ser Gln Ile Arg Ile Arg Lys Thr Ile Pro Lys Pro Asp Pro Asn

305 310 315 320

Leu Thr Pro Met Gly Leu Pro Arg Pro Lys Arg Leu Lys Lys Lys Glu

325 330 335

Phe Ser Leu Glu Glu Ile Tyr Thr Asn Lys Asn Tyr Lys Ser Pro Pro

340 345 350

Ala Ser Arg Cys Leu Glu Thr Ile Phe Glu Glu Pro Lys Glu Arg Asn

355 360 365

Gly Thr Leu Ile Ser Ile Ser Gln Gln Lys Arg Lys Arg Val Leu Glu

370 375 380

Phe Gln Asp Phe Thr Val Pro Arg Lys Arg Arg Ala Arg Gly Lys Val

385 390 395 400

Lys Leu Ala Gly Ser Phe Thr Arg Ala Gln Lys Ala Ala Leu Gln Thr

405 410 415

Gln Glu Leu Asp Ala Leu Leu Ile Gln Lys Leu Met Glu Leu Glu Thr

420 425 430

Phe Phe Ala Lys Glu Gly Glu Gln Glu His Gln Pro Ala Ala Glu Asn

435 440 445

Ser Ser Gly Ser Pro Leu

450

<210>3

<211>22

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉oligonucleotide

<400>3

gtcttctgtg ggctgctcct ca 22

<210>4

<211>22

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉oligonucleotide

<400>4

tgaggagcag cccacagaag ac 22

<210>5

<211>29

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉oligonucleotide

<400>5

aaacatatga tgagcttcat agattgagc 29

<210>6

<211>31

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉oligonucleotide

<400>6

tatggatccc aggaaacaca ggctctagcc t 31

<210>7

<211>17

<212>PRT

＜213〉mice (Mus musculus)

<400>7

Ala Glu Ser Ser Arg Thr Phe Pro Glu His Cys Ala Pro Ser Arg Leu

1 5 10 15

Ala

<210>8

<211>16

<212>PRT

＜213〉mice (Mus musculus)

<400>8

Thr Val Pro Arg Lys Arg Arg Ala Arg Gly Lys Val Lys Leu Ala Gly

1 5 10 15

Claims (10)

1. the purposes of an EMO-2 gene is characterized in that, it is used as the promoter that promotes blastocyst implantation.

2. the purposes of an EMO-2 gene is characterized in that, it is used to prepare the medicine that promotes blastocyst implantation.

3. the purposes of an EMO-2 antagonist is characterized in that, it is used as the inhibitor of vitro inhibition blastocyst implantation.

4. the purposes of an EMO-2 antagonist is characterized in that, it is used to prepare the medicine that suppresses blastocyst implantation.

5. purposes as claimed in claim 4 is characterized in that, described medicine is a contraceptive.

6. purposes as claimed in claim 4 is characterized in that, described antagonist is the antisensenucleic acids of EMO-2 or the antibody of anti-EMO-2 polypeptide.

7. the purposes of an EMO-2 agonist is characterized in that, it is used as the promoter of external promotion blastocyst implantation.

8. the purposes of an EMO-2 agonist is characterized in that, it is used to prepare the medicine that promotes blastocyst implantation.

9. whether a definite test compounds is the method for antagonist or the agonist of EMO-2, it is characterized in that it comprises step:

(a) blastocyst of test compounds and EMO-2 polypeptide adding In vitro culture is organized as test, and the blastocyst of EMO-2 polypeptide adding In vitro culture is organized in contrast;

(b) observation test group and matched group blastocyst implantation situation are if the blastocyst implantation quantity of test group, represents then that test compounds is the agonist of EMO-2 greater than matched group; If the blastocyst implantation quantity of test group, represents then that test compounds is the antagonist of EMO-2 less than matched group.

10. a pharmaceutical composition is characterized in that, it contains EMO-2 polypeptide, its agonist or its antagonist and the pharmaceutically acceptable carrier of safe and effective amount.