CN102659923B - Epitope minimal motif peptides of human zona pellucida protein-4 and extended short peptides and application thereof - Google Patents
Epitope minimal motif peptides of human zona pellucida protein-4 and extended short peptides and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biomedicine and biological detection, in particular to two linear epitope minimal motif peptides capable of being identified by human zona pellucida protein-4 (ZP4) monoclonal antibodies MA-1662 and MA-1671 on human ZP4 and extended eight peptides and application thereof. The invention provides two candidate epitopes which can be used for developing human immune birth control recombinant multi-epitope vaccines, and the amino acid sequences of the candidate epitopes are shown as SEQ ID No. 1 and marked as huZP4147-151 or shown as SEQ ID No.2 and marked as huZP4256-260; and the invention also provides candidate epitopes which can be used for developing novel high-specificity high-sensitivity recombinant multi-epitope detection antigens for diagnosing serum ZP antibodies of ZP autoimmune infertile women, and the amino acid sequences of the candidate epitopes are shown as SEQ ID No.3-SEQ ID No.6.
Description
Technical field
The invention belongs to biotechnology and immunological technique field, be specifically related to epitope minimum motif peptide and expansion small peptide and the application that can be identified by special monoclonal antibody human oocyte zona pellucida protein-4.
Background technology
Except mouse, the Mammals egg vitellary membrane (zona pellcida, ZP) including the mankind is all made up of four glycoprotein (being named as respectively ZP1, ZP2, ZP3 and ZP4).As one of antifertility link, thereby blocking-up people essence ovum causes infertile the most desirable approach that beyond doubt can be not controversial in conjunction with fertilization.Therefore, over more than 40 year, the research of ZP pregnancy vaccine is being carried out in many laboratories always in the world.Although the many abundant validity of antifertility that studies confirm that ZP or single ZP3 proteantigen active immunity taking mouse and rabbit etc. as animal model, ovaritis, ovarian follicle and ovarian atrophy all occur animal subject or the pathological phenomenons such as decay occur ovum.For the mankind, the immune side effect of ZP vaccine this respect is obviously unacceptable.For this reason, this major defect that how to overcome ZP vaccine is the focus that related researcher pays close attention to all the time, has also obtained some impressive progresses.For example; first the Dean laboratory of America NI H uses a monoclonal antibody (hereinafter to be referred as monoclonal antibody) to identify a linear B cell epitope (hereinafter to be referred as epi-position) of mouse sperm acceptor ZP3 protein carboxyl terminal; and incomplete antifertility effect (Millar SE, et al. Vaccination with a synthetic zona pellucida peptide produces long-term contraception in female mice. Science 1989 are obtained using its epi-position chemical synthesising peptide as immunogen; 246:935-938).Find that subsequently this epi-position synthetic peptide still occurs ovaritis in some other inbred mouse strain; and prove that it is to be caused by the special pathogenic T cell epitope of a ZP in its long epitope peptide; reject the synthetic peptide vaccine of this t cell epitope and can eliminate mouse ZP immunity ovaritis (Lou YH, et al. A zona pellucida 3 peptide vaccine induces antibodies and reversible infertility without ovarian pathology. J Immunol 1995; 155:2715-2720).Known t cell epitope is made up of 9～30 amino-acid residues conventionally; B cell epitope is made up of 3～8 residues; obviously be tested and appraised B cell epitope minimum motif and just can get rid of t cell epitope (Bagavant H, the et al. Immunogenicity and contraceptive potential of a human zona pellucida 3 peptide vaccine. Biol Reprod 1997 that in a long epitope peptide, may exist; 56:764-770).
Chemosynthesis peptide vaccine was once posted hope can become the 3rd milestone in Vaccine Development history.But, owing to being subject to the restriction of existing linear B cell epitope authentication method, on each target protein, certified epi-position is very limited, thereby cause the chemosynthesis peptide vaccine of development to be all difficult to reach effective virus infection or disease prevention or result for the treatment of, so that do not produce and can be applied to the clinical synthetic peptide vaccine of the mankind so far.Many ZP synthetic peptide vaccine research papers are also all mentioned and need to be developed multi-epitope peptide vaccine, to realize the abundant validity of ZP synthetic peptide vaccine antifertility effect.And to realize such target, obviously depend on the epi-position that can identify more ZP albumen, particularly their minimum motif.Know now that people ZP4 albumen also can be induced people's sperm acrosome reaction and with it in conjunction with (Chiu PC, et al. Effects of native human zona pellucida glycoprotein-3 and-4 on acrosome reaction and zona pellucida binding of human spermatozoa. Biol Reprod 2008; 79:869-877).In addition; also prepared monoclonal antibody (the Bukovsky A of anti-recombinant human ZP4; Gupta SK, et al. Production of monoclonal antibodies against recombinant human zona pellucida glycoproteins:utility in immunolocalization of respective zona proteins in ovarian follicles. J Reprod Immunol 2008; 78:102-114), wherein MA-1662((IgG2a) and MA-1671(IgG1) have and the reactivity of people's ovum and suppress the characteristic (Xu WX, in et al. Mapping of epitopes relevant for induction of acrosome reaction on human zona pellucida glycoprotein-4 using monoclonal antibodies. paper contribution) of ZP4 Mediated Human sperm acrosome reaction.Therefore, use pregnancy vaccine immunogen in view of developing from now on restructuring ZP multi-epitope peptide detectable antigens and/or people, and illustrate people ZP4 albumen and have the needs of biological significance functional domain, the present invention with above two Identification of monoclonals they discernible fine meter amino acids sequences.Technical background of the present invention or main foundation are to have set up more convenient more economical linear epitope; comprise improvement biosynthesizing peptide method (Xu WX, the et al. Minimal motif mapping of a known epitope on human zona pellucida protein-4 using a peptide biosynthesis strategy. J Reprod Immunol 2009 of minimum motif qualification; 81:9-16; Xu WX, et al. Mapping of minimal motifs of B-cell epitopes on human zona pellucida glycoprotein-3. Clin Dev Immunol doi:10:1155/2012/831010).
Existing this respect research report aspect development recombinant multi-epitope peptide vaccine; as: 11 epitope peptide vaccines (Shi YP, the et al. Immunogenicity and in vitro protective efficacy of a recombinant multistage Plasmodium falciparum candidate vaccine. Proc Natl Acad Sci USA 1999 of 7 specific antigens of combination plasmodium; 96:1615-1620) and human chorionic short sexual gland vaccine research (Xu WX, et al. Immunogenic comparison for two different recombinant chimeric peptides (CP12 and CP22) containing one or two copies of three linear B cell epitopes from-hCG subunit. J Biothech 2011 of three epi-positions of combination; 151:15-21) etc.But with regard to single target antigen, above research is subject to the restriction of the number of linear epitope qualification in the past, because combination table limited bits is all also difficult to reach synthetic peptide vaccine prevention or result for the treatment of fully at linear antibody horizontal.
Summary of the invention
The object of the present invention is to provide with two monoclonal antibody human oocyte zona pellucida protein-4(huZP4) minimum motif peptide of two linear epitopes of monoclonal antibody (MA-1662 and MA-1671) qualification, and provide available chemosynthesis or with the expansion small peptide (8 peptide) containing described minimum motif peptide of GST carrier proteins amalgamation and expression or be longer than the antigen of this small peptide (8 peptide); The expansion small peptide (8 peptide) of described minimum motif peptide, minimum motif peptide is also provided or is longer than the application of the antigen of this small peptide (8 peptide).
Human oocyte zona pellucida protein-4(huZP4 provided by the invention) the minimum motif peptide of two linear epitopes, its aminoacid sequence is respectively shown in SEQ.ID No.l and SEQ.ID No.2, the former is designated as huZP4
147-151, aminoacid sequence write a Chinese character in simplified form in one word is PARDR(SEQ.ID No.l); The latter is designated as huZP4
256-260, aminoacid sequence write a Chinese character in simplified form in one word is ENELV(SEQ.ID No.2).
Short skin containing above-mentioned minimum motif skin provided by the invention, its aminoacid sequence is respectively shown in SEQ.ID No.3 and SEQ.ID No.4.Be designated as huZP4-1, the people ZP4 of its expansion
145-152or people ZP4
146-153the aminoacid sequence that poly-peptide one word of epi-position 8 is write a Chinese character in simplified form is: SIPARDRL(SEQ.ID No.3) or IPARDRLP(SEQ.ID No.4).In the occasion of chemosynthesis or this 8 peptide fusion protein of amalgamation and expression, the residue part of expansion can additions and deletions or alternative with other residues.
Short skin containing above-mentioned minimum motif skin provided by the invention, its aminoacid sequence also can be shown in SEQ.ID No.5 and SEQ.ID No.6.Be designated as huZP4-2, the people ZP4 of its expansion
254-261or people ZP4
255-262the aminoacid sequence that poly-peptide one word of epi-position 8 is write a Chinese character in simplified form is: VYENELVA(SEQ.ID No.5) or YENELVAT(SEQ.ID No.6).In the occasion of chemosynthesis or this 8 peptide fusion protein of amalgamation and expression, the residue part of expansion can additions and deletions or alternative with other residues.
The minimum motif peptide that the present invention also provides above-mentioned linear epitope development and design people with applying in ZP multi-epitope peptide pregnancy vaccine; Further also providing containing small peptide (epi-position 8 peptides) or its fusion rotein of above-mentioned minimum motif peptide causes the serum of the woman infertility cause of disease to identify the application of epi-position mark as clinical diagnosis by ZP autoimmunization alone or in combination.
Content of the present invention is more specifically described as follows:
1,people ZP4 albumen (once by priority called after ZPB and ZP1) encoding gene clone [Harrist JD; et al. Cloning and characterization of zona pellucida genes and cDNAs from a variety of mammalian species:the ZPA, ZPB and ZPC gene families. DNA Seq 1994; 4:361-393; Skinner SM, et al. Mapping of dominant B cell epitopes of a human zona pellucida protein (ZP1). Biol Reprod 1999; 61:1373-1380] provided by the U.S.'s state-run commune hospital cell and developmental biology laboratory.From huZP4 cDNA clone, amplify coding huZP4 maturation protein N end large fragment by conventional PCR method and (remove the amino-acid residue 58-234 of N end signal peptide, referred to as huZP4N
58-234) and C holds large fragment, and (the residue 227-463 in removal C end span film district, referred to as huZP4C
227-463) cDNAs, then respectively restructuring insert pSY621 expression plasmid [Shen Yun, Ying Kang, Xu Wanxiang, Xie Yi: the structure of escherichia coli high-level expression carrier pSY621.
fudan Journal(
natural science edition) 2000; 39 (3): 313 – 317], carry out thermal induction expression after transforming respectively intestinal bacteria, result is MA-1671 monoclonal antibody identification restructuring huZP4N albumen in western blot test, and MA-1662 monoclonal antibody identification restructuring huZP4C albumen (result does not show).
,previously contriver had built biosynthetic pXXGST-1 fusion expression vector (the Xu WX of small peptide that is specifically designed to epitope scanning mapping, et al. Minimal motif mapping of a known epitope on human zona pellucida protein-4 using a peptide biosynthesis strategy. J Reprod Immunol, 81:9-16,2009).Therefore,, before above-mentioned two monoclonal antibodies identification of qualification epi-position aminoacid sequence, build first respectively two groups of poly-small peptides of series 18 of crossing over respectively huZP4N and overlapped 10 residues of huZP4C overall length (taking GST188 albumen as fusion expression vector.Concrete operation step: send the poly-peptide of synthetic designed series 18 the positive minus strand fragment of coding DNA containing two ends BamH I and Sal I sticky end outside; After the positive minus strand fragment pairing of DNA annealing, expression plasmid is inserted in restructuring; Transform e. coli bl21 (DE3) Host Strains; Select recombinant clone sequence verification Insert Fragment DNA sequence dna; And object GST188-18 peptide fusion protein is expressed in thermal induction).And then the Western blot of implementing the first round identification 18 poly-peptides of two monoclonal antibodies is identified, be about to carry out SDS-PAGE containing 21 the 18 poly-peptides (numbering P1～P21) of huZP4N with containing 28 the 18 each induction bacterium total proteins that gather peptides (numbering P22～P49) fusion rotein of huZP4C respectively, then electrotransfer, to nitrocellulose filter, finally completes trace qualification with MA-1662 and MA-1671 monoclonal antibody.Result: MA-1671 monoclonal antibody identification P11 and P12 small peptide (fusion rotein), and MA-1662 monoclonal antibody identification P25 small peptide (fusion rotein) (figure does not show).
,express step by aforementioned serial 18 poly-small peptide fusion protein constructions, further expressing GST188 and be carrier crosses over P11 and P12 and merges the poly-peptide fusion protein of series 8 of length and overlapped 7 residues of P25 length (the former numbers P50～P62, the latter numbers P63～P70), then implement second and take turns the meticulous motif immunoblotting qualification (step is with poly-epitope peptide qualification of the first round 18) of MA-1662 and MA-1671 monoclonal antibody identification epi-position.Result: the minimum motif of MA-1671 monoclonal antibody identification is defined as PARDR pentapeptide (Fig. 1) according to consensus sequence in the poly-peptide of four immunoreactivities 8, is defined as ENELV pentapeptide (Fig. 2) and the minimum motif of MA-1662 monoclonal antibody identification is same according to four reactivity 8 peptide consensus sequences.
More than use the qualification of two functional epitope minimum motifs on the people ZP4 albumen of MA-1662 and two special monoclonal antibodies of MA-1671, for Future Design development provides two important candidate's epi-positions without ZP specific T-cells epi-position (causing the crucial cause of disease of ZP immunity ovarian dysfunction) people with ZP multi-epitope peptide vaccine.In addition, these two meticulous epi-positions and/or can expand the chemical synthesising peptide of 8 peptides or with GST188 amalgamation and expression small peptide, also can be used as candidate's epi-position or the epitope peptide of the infertile cause of disease of exploitation diagnosis women ZP autoimmunization multi-epitope peptide detectable antigens, or separately and/or combination as the epi-position mark of diagnosis ZP autoimmune disease.
Brief description of the drawings
Fig. 1. immunoblotting qualification (A) and the list consensus sequence of MA-1671 monoclonal antibody identification epitope minimum motif are analyzed (B).
Note: show the P50-P62 small peptide fusion rotein that Lanes 50-62 representative is expressed on blotting membrane.Figure A note arrow is indicated respectively the poly-peptide (P53-P56) of trace reactive 8.Consensus amino acid sequences (in 10 residue overlaps between P11 and P12 small peptide) in the poly-peptide of the yellow shade albumen test 8 of figure B.
Fig. 2. immunoblotting qualification (A) and the list consensus sequence of MA-1662 monoclonal antibody identification epitope minimum motif are analyzed (B).
Note: show the P63-P70 small peptide fusion rotein that Lanes 63-70 representative is expressed on blotting membrane.Figure A note arrow is indicated respectively the poly-peptide (P64-P67) of trace reactive 8.Consensus amino acid sequences in the poly-peptide of the yellow shade albumen test 8 of figure B.
Embodiment
The present invention can further describe by following example.Illustrative example is not meaned restriction scope involved in the present invention, and this scope is sufficiently clarified in description before.The experimental technique of unreceipted actual conditions in the following example, all write the third edition " molecular cloning: the laboratory manual " (Science Press of the translations such as Huang Peitang according to normal condition and [U.S.] J. Pehanorm Brooker and D.W. Russell, 2002) and [U.S.] E. breathe out Lip river and D. Lay grace and write " antibody technique experiment guide " (Science Presses of the translations such as Shen Guanxin, 2002) step described in, or carry out according to the condition of production and sales business suggestion.
Embodiment 1: the meticulous epi-position qualification of mouse-anti people ZP4 monoclonal antibody MA-1671
Materials and methods:
1. people ZP4 gene clone is provided by the U.S.'s state-run commune hospital cell and developmental biology laboratory.Thermal induction expression plasmid pSY621 and pXXGST-1 by inventor herein build [Shen Yun, Ying Kang, Xu Wanxiang, Xie Yi: the structure of escherichia coli high-level expression carrier pSY621.
fudan Journal(
natural science edition) 2000; 39 (3): 313 – 317; Xu WX, et al. Minimal motif mapping of a known epitope on human zona pellucida protein-4 using a peptide biosynthesis strategy. J Reprod Immunol 2009; 81:9-16].E. coli bl21 (DE3) bacterial strain is by the preservation of genetic engineering National Key Laboratory of Fudan University.
2. mouse-anti huZP4 monoclonal antibody MA-1662 and MA-1671 are by contriver's preparation (Bukovsky A; Gupta SK, et al. Production of monoclonal antibodies against recombinant human zona pellucida glycoproteins:utility in immunolocalization of respective zona proteins in ovarian follicles. J Reprod Immunol 2008; 78:102-114).
3. restriction enzyme EcoR I, BamH I, Sal I, Taq enzyme and T4 DNA ligase, purchased from Japanese TaKaRa Biotechnology company, dye sheep anti mouse two anti-(IgG/HRP), diaminobenzidine (DAB) and the 0.2 μ m nitrocellulose filter of molecular weight of albumen standard, horseradish peroxidase mark in advance purchased from Shanghai Sheng Gong biotechnology service company.Immunoblotting chemoluminescence ECL detection kit is purchased from GE company.
4. QIAprep spin miniprep Kit plasmid extraction test kit, QIAquick PCR product purification test kit and quick gel reclaim test kit purchased from German QIAGEN company.
5. two ends are respectively BamH I and Sal I sticky end, and the middle positive minus-strand dna fragment that adds TAA terminator codon for each 8-18 peptide DNA sequences encoding is synthesized by Shanghai Jierui Biology Engineering Co., Ltd.
6. serial 8/18 peptide DNA sequences encoding is according to people ZP4 protein coding gene and protein amino acid sequence public information (Harrist JD; et al. Cloning and characterization of zona pellucida genes and cDNAs from a variety of mammalian species:the ZPA, ZPB and ZPC gene families. DNA Seq 1994; 4:361-393).Preparation process summary is as follows: from huZP4 gene clone plasmid, going out except the two ends in signal peptide and cross-film district by pcr amplification is huZP4N (amino-acid residue 58-234) and two encoding gene fragments of huZP4C (residue 227-463) of EcoR I and Sal I restriction enzyme sites; After double digestion, pSY621 thermal induction plasmid is inserted in restructuring, and recombinant plasmid transforms e. coli host bacteria after DNA sequencing checking; Abduction delivering huZP4N and huZP4C albumen are for MA-1662 and MA-1671 Identification of monoclonal epitope peptide.
with the human oocyte zona pellucida protein-4(huZP4 of MA-1671 monoclonal antibody) concrete steps of meticulous epi-position qualification are as follows:
1. according to huZP4 (once called after ZPB and ZP1) gene order public information, pcr amplification goes out huZP4N and two large fragment protein coding genes of huZP4C, pSY621 thermal induction prokaryotic expression plasmid is inserted in restructuring, after abduction delivering, carry out SDS-PAGE analysis and complete immunoblotting qualification with MA-1662 and MA-1671 monoclonal antibody, determine that two monoclonal antibodies can identify huZP4N and huZP4C section.
2. (normal chain 5 '-end adds 5 '-gatcc to series 18 peptides (numbering P1-P21) the positive minus strand fragment of coding DNA of overlapped 10 amino-acid residues of huZP4N overall length sequence that design leap can be identified by MA-1671, and 3 '-end adds taag-3 '; Minus strand 5 '-end adds 5 '-tcgactta, and 3 '-end adds g-3 '), send DNA chemosynthesis outside.
3. by positive the complementation of 1 or 2 OD minus strand fragment ddH
2o is dissolved into 20 μ mol/ μ l storage liquid (according to the synthetic report data of DNA); Respectively get 10 μ l storage liquid and 20 ddH
2o is in 1.5 ml Eppendorf pipes, 94 DEG C of heating in water bath 5 min, naturally be down to after room temperature, in 15 μ l reaction volumes, suck 2 μ l annealing fragments, 1 μ l approximately 200 ng/ μ l pXXGST-1 plasmid, 1 μ l T4 DNA ligase and its 1.5 μ l damping fluid through BamH I and Sal I double digestion, connection is spent the night; Connecting fluid transformed competence colibacillus BL21(DE3) Host Strains, coating is containing on the LB flat board of Amp, in 37 DEG C of overnight incubation; The mono-clonal growing on picking ammonia benzyl LB flat board for the second day 3 ml LB nutrient solution abduction deliverings of transferring, taking the GST188 albumen of being expressed by pXXGST-2 as contrast, each GST188-18 peptide (P1-P21) fusion rotein of expressing is through 15%SDS-PAGE analysis confirmation (differing approximately 2 kDa with reference protein electrophoretic mobility), and the each recombinant clone of picking is sent outside and carried out DNA sequencing checking.
4. clone's inoculation correct Insert Fragment sequencing result is added in the 3 ml LB nutrient solutions of Amp, spend the night in 30 DEG C of shaking culture, morning on next day is fresh in the LB nutrient solution of Amp in 1/50 ratio switching, 30 DEG C of shaking culture 2～3 h reach after 0.6～0.8 OD to cell concentration, heighten temperature to 42 DEG C thermal induction 4 h, centrifugal collection thalline, adding loading lysate, to boil 5 min for subsequent use.
5. the total bacterial protein sample of induction is walked to 15%SDS-PAGE simultaneously, after electrophoresis finishes, one clotting glue coomassie brilliant blue staining, two clotting glue carry out nitrocellulose filter electricity (100 mA) transferase 12 h, dye and turn blotting membrane 2 min with ponceau, at object small peptide fusion rotein band place, with the syringe needle mark that punches, water rinses out ponceau dyeing.
6. blotting membrane PBS damping fluid repetitive scrubbing four times, seal and spend the night with 5% skim-milk, PBS damping fluid washing four times, in 8～10 ml reaction solutions, add respectively 1 μ l primary antibodie MA-1671 monoclonal antibody, room temperature reaction 2 h, add the anti-sheep anti-mouse igg of 5 μ l bis-/HRP(1:2000 dilution after the washing of PBS damping fluid), room temperature reaction 1 h, with carrying out ECL chemoluminescence colour developing after the washing of PBS damping fluid, be reactive 18 poly-peptides (fusion rotein) according to immunoblotting results verification P11 and P12.
7. (11 of the P11 that design covering can be identified by MA-1671 and series 8 peptides of two overlapping 18 poly-overlapped 7 amino-acid residues of peptide residue sequence total length of P12, numbering P50-P62) (normal chain 5 '-end adds 5 '-gatcc to the positive minus strand fragment of coding DNA, and 3 '-end adds taag-3 '; Minus strand 5 '-end adds 5 '-tcgactta, and 3 '-end adds g-3 '), send DNA chemosynthesis outside.
8. as aforementioned 3-5 operation steps, complete construction expression and the qualification of epitope minimum motif immunoblotting thereof of 13 serial small peptide fusion roteins, confirm that the P53-P56 being identified by MA-1671 monoclonal antibody is for the poly-peptide fusion protein (Figure 1A) of four reactivities 8.
9. gather according to above-mentioned four reactivities 8 the total total number of atnino acid that in peptide (fusion rotein), yellow shade shows, the final epitope minimum motif of determining mouse-anti huZP4 monoclonal antibody MA-1671 identification is PARDR, and its pentapeptide residue sequence is positioned at 10 residue overlaps of 18 poly-small peptide P11 and P12.
In addition, the meticulous epi-position qualification of MA-1662 monoclonal antibody identification huZP4 albumen, also adopts above-mentioned same method and step.Be summarized as follows: first series 18 peptides (numbering P22-P49) the positive minus strand fragment of coding DNA of overlapped 10 amino-acid residues of huZP4C overall length sequence that can be identified by MA-1662 is crossed in design, recombinant expressed serial 18 poly-small peptide fusion roteins, carry out poly-pepscan mapping of the first round reactive 18 with MA-1662 monoclonal antibody and identify; Then express poly-peptide (P63-P70) fusion rotein of series 8 that covers P25 reactive 18 poly-overlapped 7 residues of peptide, carry out second and take turns meticulous epi-position qualification, finally, according to the total residue in tetra-small peptides of MA-1662 monoclonal antibody identification P64-P67, determine that its epitope minimum motif is ENELV.
Although the invention describes human oocyte zona pellucida protein-4(huZP4) can be by two linear epitope minimum motifs of monoclonal antibody MA-1662 and MA-1671 identification and 8 peptides of their expansions, they both can separately and/or combine as development safety, effective and reversible people uses ZP recombinant multi-epitope vaccine candidate epi-position, also can or be used as alone or in combination the candidate's epitope antigen or the epi-position mark (detect patients serum and combine ZP antibody) that are caused woman infertility etiological diagnosis by ZP autoimmunization by chemosynthesis or amalgamation and expression, but having is a bit apparent for association area investigative technique personnel, without departing from the spirit and scope of the present invention, can make various changes change to 8 peptides or 8 peptide fusion proteins of minimum epitope peptide of the present invention and its expansion.Therefore, claims have covered all these variations within the scope of the present invention.
Fudan University of <110> Shanghai Family Planning Science and Research Institute.
The epitope minimum motif peptide of <120> human oocyte zona pellucida protein-4 and expansion small peptide and application
<130> 001
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<170> PatentIn version 3.3
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Claims (2)
1. an epitope minimum motif peptide for human oocyte zona pellucida protein-4, is characterized in that aminoacid sequence is shown in SEQ.ID No.l, is designated as huZP4
147-151; Or aminoacid sequence is shown in SEQ.ID No.2, is designated as huZP4
256-260.
2. epitope minimum motif peptide as claimed in claim 1 is caused the application of the serum identification epi-position mark of the woman infertility cause of disease by ZP autoimmunization as clinical diagnosis in preparation.
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| CN102659925A (en) * | 2012-05-23 | 2012-09-12 | 上海市计划生育科学研究所 | Human oocyte zona pellucida (ZP)-4 epitope minimum motif peptide identified by anti-recombinant human ZP4N and ZP4C polyclonal antibody |
| CN103524601B (en) * | 2013-09-27 | 2016-01-20 | 上海市计划生育科学研究所 | Epitope peptide in human oocyte zona pellucida protein 4 and application thereof |
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| CN105859849A (en) * | 2016-05-30 | 2016-08-17 | 复旦大学 | Linear epitope minimum motif peptide Asp f 4<136-141> of aspergillus fumigatus Asp f 4 and expanded short peptides thereof |
| CN105859852A (en) * | 2016-05-30 | 2016-08-17 | 复旦大学 | Aspergillus fumigatus Asp f 4 linear antigenic epitope minimum base sequence peptide Asp f 456-61 and extended oligopeptides thereof |
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