CN102659923A - 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, being specifically related to human oocyte zona pellucida protein-4 can be by the epitope minimum motif peptide of special monoclonal antibody identification and expansion small peptide and application.
Background technology
Except mouse, comprise that (zona pellcida ZP) is made up of four gp (being named as ZP1 respectively, ZP2, ZP3 and ZP4) human Mammals egg vitellary membrane.As one of antifertility link, thereby the smart ovum of blocking-up people combines fertilization to cause infertile the most desirable approach that beyond doubt can be not controversial.Therefore, the research of ZP pregnancy vaccine is being carried out in many in the world laboratories always over more than 40 year.Though with mouse and rabbit etc. is the abundant validity of antifertility that many researchs of animal model have confirmed ZP or single ZP3 proteantigen active immunity, ovaritis, ovarian follicle and ovarian atrophy all take place animal subject or pathological phenomenons such as decay take place ovum.As far as human, the immune spinoff of ZP vaccine this respect obviously is unacceptable.For this reason, this major defect that how to overcome the ZP vaccine is the focus that the correlative study personnel pay close attention to all the time, has also obtained some impressive progresses.For example; The Dean laboratory of America NI H at first 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 have been obtained as immunogen with its epi-position chemical synthesising peptide; 246:935-938).Find that subsequently the synthetic peptide of this epi-position ovaritis still occurs in some other inbred mouse strain; And prove that it is to be caused by the special pathogenic T cell epitope of the ZP in its long epitope peptide; Reject the synthetic peptide vaccine of this t cell epitope and then 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 usually; The B cell epitope then 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, possibly exist; 56:764-770).
The chemosynthesis peptide vaccine was once posted hope can become the 3rd milestone on the vaccine development history.But; Owing to receive the restriction of existing linear B cell epitope authentication method; Certified epi-position is very limited on each target protein; Thereby the chemosynthesis peptide vaccine that causes developing all is difficult to reach effective virus infection or disease prevention or result of treatment, so that produces so far and can be applied to human clinical synthetic peptide vaccine.Many ZP synthetic peptide vaccine RPs are also all mentioned needs development multi-epitope peptide vaccine, to realize the abundant validity of ZP synthetic peptide vaccine antifertility effect.And to realize such target, obviously depend on and can identify the proteic epi-position of ZP more, particularly their minimum motif.Known that now people ZP4 albumen also can induce people's sperm acrosome reaction and combine (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 with it; 79:869-877).In addition; Monoclonal antibody (the Bukovsky A that has also prepared 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 ((IgG2a) and MA-1671 (IgG1) have the reactivity and the characteristic that suppresses ZP4 Mediated Human sperm acrosome reaction (Xu WX is in the submission of et al. Mapping of epitopes relevant for induction of acrosome reaction on human zona pellucida glycoprotein-4 using monoclonal antibodies. paper) with people's ovum to MA-1662.Therefore; In view of the ZP of development reorganization from now on multi-epitope peptide detects antigen and/or human pregnancy vaccine immunogen; And illustrating the needs that people ZP4 albumen has the biological significance domain, the present invention has identified their discernible meticulous epi-position aminoacid sequences with above two monoclonal antibodies.Technical background of the present invention or main basis are to have set up more convenient more economical linear epitope; Comprise improvement biosynthesizing peptide method (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 that minimum motif is identified; 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) the 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, more than research receives the restriction of the number of linear epitope evaluation in the past, because of the combination table limited bits all also is difficult to reach synthetic peptide vaccine prevention or result of treatment fully at linear antibody horizontal.
Summary of the invention
The object of the present invention is to provide the minimum motif peptide of two linear epitopes identifying with two monoclonal antibody human oocyte zona pellucida proteins-4 (huZP4) monoclonal antibody (MA-1662 and MA-1671), and available chemosynthesis is provided or with the expansion small peptide that contains said minimum motif peptide (8 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 said minimum motif peptide, minimum motif peptide also is provided or is longer than the antigenic application of this small peptide (8 peptide).
The minimum motif peptide of two linear epitopes of human oocyte zona pellucida protein provided by the invention-4 (huZP4), its aminoacid sequence are respectively shown in SEQ.ID No.l and the SEQ.ID No.2, and the former is designated as huZP4
147-151, aminoacid sequence write a Chinese character in simplified form in the one of which 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 the one of which word is ENELV (SEQ.ID No.2).
The short skin that contains above-mentioned minimum motif skin provided by the invention, its aminoacid sequence is respectively shown in SEQ.ID No.3 and the SEQ.ID No.4.Be designated as huZP4-1, the people ZP4 of its expansion
145-152Or people ZP4
146-153Epi-position 8 is gathered the aminoacid sequence that peptide one word writes a Chinese character in simplified form: 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, but the residue of expansion part additions and deletions or alternative with other residues.
The short skin that contains above-mentioned minimum motif skin provided by the invention, its aminoacid sequence also can be shown in SEQ.ID No.5 and the SEQ.ID No.6.Be designated as huZP4-2, the people ZP4 of its expansion
254-261Or people ZP4
255-262Epi-position 8 is gathered the aminoacid sequence that peptide one word writes a Chinese character in simplified form: 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, but the residue of expansion part additions and deletions or alternative with other residues.
The present invention also provides the minimum motif peptide of above-mentioned linear epitope in development and design human ZP multi-epitope peptide pregnancy vaccine, to use; Further also provide small peptide (epi-position 8 peptides) or its fusion rotein of containing above-mentioned minimum motif peptide to be used as clinical diagnosis is caused the serum Identification Lists bit flag thing of the woman infertility cause of disease by ZP autoimmunization application alone or in combination.
Content of the present invention further specifically describes as follows:
1,People ZP4 albumen (once by priority called after ZPB and ZP1) encoding sox 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] provide by the U.S.'s state-run commune hospital cell and developmental biology laboratory.From huZP4 cDNA clone, amplifying coding huZP4 maturation protein N with conventional PCR method holds big fragment (to remove the amino-acid residue 58-234 of N end signal peptide, abbreviate huZP4N as
58-234) and C holds big fragment, and (the residue 227-463 in removal C end span film district abbreviates huZP4C as
227-463) cDNAs, then respectively reorganization insert the 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 respectively behind the transformed into escherichia coli and express, the result is MA-1671 monoclonal antibody identification reorganization huZP4N albumen in western blot test, and MA-1662 monoclonal antibody identification reorganization huZP4C albumen (result does not show).
,Previous contriver has made up 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 identifying above-mentioned two monoclonal antibody Identification Lists amino acids sequences, make up two groups of series 18 of crossing over huZP4N and overlapped 10 residues of huZP4C overall length respectively at first respectively and gather small peptide (with GST188 albumen is fusion expression vector.Concrete operations step: send synthetic institute design of series 18 outside and gather peptide and contain two ends BamH I and the positive minus strand fragment of coding DNA of Sal I sticky end; Expression plasmid is inserted in the positive minus strand fragment pairing of DNA annealing back reorganization; Transformed into escherichia coli BL21 (DE3) host bacterium; Select the recombinant clone sequence verification and insert the fragment dna sequence dna; And purpose GST188-18 peptide fusion protein is expressed in thermal induction).And then the first round identification 18 of implementing two monoclonal antibodies gathers the Western blot evaluation of peptide; Soon containing 21 18 of huZP4N respectively gathers peptide (numbering P1～P21) gathers peptide (P22～P49) the bacterium total protein of respectively inducing of fusion rotein carries out SDS-PAGE to numbering with 28 18 that contain huZP4C; Then electrotransfer is accomplished trace with MA-1662 and MA-1671 monoclonal antibody at last and is identified to nitrocellulose filter.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).
,Gather small peptide fusion rotein construction expression step by aforementioned serial 18; Further expressing GST188 and be carrier crosses over P11 and gathers peptide fusion protein with the series 8 of P12 merging length and overlapped 7 residues of P25 length (the former numbers P50～P62; The latter numbers P63～P70), implement then second take turns MA-1662 and MA-1671 monoclonal antibody identification epi-position meticulous motif immunoblotting identify (step is gathered epitope peptide with the first round 18 and identified).The result: the minimum motif of MA-1671 monoclonal antibody identification gathers in the peptide consensus sequence according to four immunoreactivities 8 and confirms as PARDR pentapeptide (Fig. 1), confirms 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 with the evaluation of two functional epitope minimum motifs on the people ZP4 albumen of MA-1662 and two special monoclonal antibodies of MA-1671, two important candidate's epi-positions are not provided for development and design from now on has ZP specific T-cells epi-position (the crucial cause of disease that causes ZP immunity ovarian dysfunction) human ZP multi-epitope peptide vaccine.In addition; These two meticulous epi-positions and/or can expand 8 peptides chemical synthesising peptide or with GST188 amalgamation and expression small peptide; Also can be used as the infertile cause of disease of exploitation diagnosis women's ZP autoimmunization and detect antigenic candidate's epi-position or epitope peptide with multi-epitope peptide, or separately and/or combination as the epi-position mark of diagnosis ZP autoimmune disease.
Description of drawings
Fig. 1. the immunoblotting of MA-1671 monoclonal antibody identification epitope minimum motif is identified (A) and tabulation consensus sequence analysis (B).
Annotate: show the P50-P62 small peptide fusion rotein that Lanes 50-62 representative is expressed on the blotting membrane.Figure A annotates arrow and indicates trace reactive 8 to gather peptide (P53-P56) respectively.The yellow shade albumen test of figure B property 8 is gathered consensus amino acid sequences in the peptide (in 10 residue overlaps between P11 and P12 small peptide).
Fig. 2. the immunoblotting of MA-1662 monoclonal antibody identification epitope minimum motif is identified (A) and tabulation consensus sequence analysis (B).
Annotate: show the P63-P70 small peptide fusion rotein that Lanes 63-70 representative is expressed on the blotting membrane.Figure A annotates arrow and indicates trace reactive 8 to gather peptide (P64-P67) respectively.The yellow shade albumen test of figure B property 8 is gathered consensus amino acid sequences in the peptide.
Embodiment
The present invention can further pass through following case description.Illustrative example is not meaned restriction scope involved in the present invention, this scope before description in made a thorough statement on and illustrated.The experimental technique of unreceipted actual conditions in the following example; All write the third edition " molecular cloning: the laboratory manual " (Science Press of translations such as Huang Peitang according to normal condition and [U.S.A] J. Sa nurse Brooker and D.W. Russell; 2002) and [U.S.A] E. breathe out Lip river and D. Lay grace and write " antibody technique experiment guide " (Science Press that Shen Guanxin etc. translates; 2002) step described in, or carry out according to the condition of production and sales business suggestion.
Embodiment 1: the meticulous epi-position of mouse-anti people ZP4 monoclonal antibody MA-1671 is identified
Material and method:
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 the inventor herein make up [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 be available from Japanese TaKaRa Biotechnology company, and the sheep anti mouse that dyes molecular weight of albumen standard, HRPO mark in advance is two anti-, and (IgG/HRP), diaminobenzidine (DAB) and 0.2 μ m nitrocellulose filter are available from Shanghai life worker biotechnology service company.Immunoblotting chemoluminescence ECL detection kit is available from GE company.
4. QIAprep spin miniprep Kit plasmid extraction test kit, QIAquick PCR product purification test kit and quick gel reclaim test kit available 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 the 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 the huZP4 gene clone plasmid through pcr amplification go out except that signal peptide and the two ends of striding the film district be huZP4N (amino-acid residue 58-234) and two encoding sox fragments of huZP4C (residue 227-463) of EcoR I and Sal I restriction enzyme sites; PSY621 thermal induction plasmid is inserted in reorganization behind the double digestion, and recombinant plasmid is transformed into escherichia coli host bacterium after the dna sequencing checking; Abduction delivering huZP4N and huZP4C albumen supply MA-1662 and MA-1671 monoclonal antibody to identify epitope peptide usefulness.
The concrete steps of identifying with the meticulous epi-position of the human oocyte zona pellucida protein-4 (huZP4) of MA-1671 monoclonal antibody are following:
1. according to huZP4 (once called after ZPB and ZP1) gene order public information; Pcr amplification goes out huZP4N and two big fragment protein coding genes of huZP4C; PSY621 thermal induction prokaryotic expression plasmid is inserted in reorganization; Carry out behind the abduction delivering that SDS-PAGE analyzes and with MA-1662 and the evaluation of MA-1671 monoclonal antibody completion immunoblotting, confirm that two monoclonal antibodies can discern huZP4N and huZP4C section.
2. the design leap can (normal chain 5 '-end adds 5 '-gatcc, and 3 '-end adds taag-3 ' by series 18 peptides of overlapped 10 amino-acid residues of huZP4N overall length sequence of MA-1671 identification (numbering P1-P21) the positive minus strand fragment of coding DNA; Minus strand 5 '-end adds 5 '-tcgactta, and 3 '-end adds g-3 '), send the DNA chemosynthesis outside.
3. the complementary positive minus strand fragment of 1 or 2 OD is used 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 pipe; 94 ℃ of heating in water bath 5 min; Naturally after reducing to room temperature; In 15 μ l reaction volumes, suck 2 μ l annealing fragment, the about 200 ng/ μ l of 1 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; Connect liquid transformed competence colibacillus BL21 (DE3) host bacterium, coating contains on the LB flat board of Amp, in 37 ℃ of overnight cultures; The mono-clonal that grew on the picking ammonia benzyl LB flat board in the second day 3 ml LB nutrient solution abduction deliverings of transferring; GST188 albumen to be expressed by pXXGST-2 is contrast; Each GST188-18 peptide (P1-P21) fusion rotein of expressing is through 15%SDS-PAGE analysis confirmation (differing about 2 kDa with the reference protein electrophoretic mobility), and each recombinant clone of picking is sent outside and carried out the dna sequencing checking.
4. will insert the correct clone of sequencing fragment result inoculates in the 3 ml LB nutrient solutions that add Amp; Spend the night in 30 ℃ of shaking culture; After 30 ℃ of shaking culture 2～3 h reach 0.6～0.8 OD to cell concentration, heighten temperature to 42 ℃ thermal induction 4 h in the fresh LB nutrient solution that contains Amp of 1/50 ratio switching morning on next day; Centrifugal collection thalline, it is subsequent use to add that a kind lysate boils 5 min.
5. inductive total bacterial protein sample is walked 15%SDS-PAGE simultaneously; After electrophoresis finishes; One clotting glue is used coomassie brilliant blue staining, and two clotting glue carry out nitrocellulose filter electricity (100 mA) transferase 12 h, changes blotting membrane 2 min with ponceau dyeing; With the syringe needle mark that punches, water rinses out ponceau dyeing at purpose small peptide fusion rotein band place.
6. blotting membrane spends the night with the sealing of 5% skim-milk with PBS damping fluid repetitive scrubbing four times, PBS damping fluid washing four times; In 8～10 ml reaction solutions, add 1 μ l, one anti-MA-1671 monoclonal antibody respectively; Room temperature reaction 2 h, PBS damping fluid washing back adds 5 μ l, two anti-sheep anti-mouse igg/HRP (1:2000 dilution), room temperature reaction 1 h; With carrying out ECL chemoluminescence colour developing after the washing of PBS damping fluid, gather peptide (fusion rotein) for reactive 18 according to immunoblotting results verification P11 and P12.
7. design covers (11 of series 8 peptides that can be gathered overlapped 7 amino-acid residues of peptide residue sequence total length by the P11 of MA-1671 identification and two overlapping 18 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 the DNA chemosynthesis outside.
8. like aforementioned 3-5 operation steps, accomplish the construction expression and the epitope minimum motif immunoblotting thereof of 13 serial small peptide fusion roteins and identify, confirm to be gathered peptide fusion protein (Figure 1A) by four reactivities 8 of P53-P56 confession of MA-1671 monoclonal antibody identification.
9. gather the total total number of atnino acid that yellow shade shows in the peptide (fusion rotein) according to above-mentioned four reactivities 8; Final confirm that the epitope minimum motif of mouse-anti huZP4 monoclonal antibody MA-1671 identification is PARDR, its pentapeptide residue sequence be positioned at 18 gather small peptide P11 and P12 10 residue overlaps.
In addition, the proteic meticulous epi-position of MA-1662 monoclonal antibody identification huZP4 is identified, also adopts above-mentioned same method and step.Be summarized as follows: at first the design leap can be by series 18 peptides of overlapped 10 amino-acid residues of huZP4C overall length sequence of MA-1662 identification (numbering P22-P49) the positive minus strand fragment of coding DNA; Recombinant expressed serial 18 gather the small peptide fusion rotein, carry out the first round reactive 18 with the MA-1662 monoclonal antibody and gather pepscan mapping evaluation; Then express to cover the series 8 that P25 reactive 18 gathers overlapped 7 residues of peptide and gather peptide (P63-P70) fusion rotein; Carry out second and take turns meticulous epi-position evaluation; Finally, confirm that its epitope minimum motif is ENELV according to the total residue in four small peptides of MA-1662 monoclonal antibody identification P64-P67.
Can be although the invention describes human oocyte zona pellucida protein-4 (huZP4) 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 make up as development safety, effective and reversible human ZP recombinant multi-epitope vaccine candidate epi-position; Also can or be used as candidate's epitope antigen or the epi-position mark (detect patient's serologic group and close ZP antibody) that causes the woman infertility etiological diagnosis by ZP autoimmunization alone or in combination through chemosynthesis or amalgamation and expression; But have is conspicuous for association area investigative technique personnel a bit; Promptly under the situation that does not break away from the spirit and scope of the present invention, can do various variations changes to 8 peptides or 8 peptide fusion proteins of minimum epitope peptide of the present invention and its expansion.Therefore, accompanying claims has covered all these changes within the scope of the present invention.
< 110>Fudan University of Shanghai Family Planning Science and Research Institute.
< 120>epitope minimum motif peptide of human oocyte zona pellucida protein-4 and expansion small peptide and application
<130> 001
<160> 6
<170> PatentIn?version?3.3
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Pro?Ala?Arg?Asp?Arg
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<400> 2
Glu?Asn?Glu?Leu?Val
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<210> 3
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Ser?Ile?Pro?Ala?Arg?Asp?Arg?Leu
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Ile?Pro?Ala?Arg?Asp?Arg?Leu?Pro
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Claims (4)
1. the epitope minimum motif peptide of a human oocyte zona pellucida protein-4 is characterized in that aminoacid sequence is shown in the SEQ.ID No.l, is designated as huZP4
147-151Perhaps aminoacid sequence is shown in the SEQ.ID No.2, is designated as huZP4
256-260
2. expansion small peptide that contains epitope minimum motif peptide as claimed in claim 1; It is characterized in that its aminoacid sequence is shown in SEQ.ID No.3 and the SEQ.ID No.4; In the occasion of chemosynthesis or this 8 peptide fusion protein of amalgamation and expression, but the residue of expansion part additions and deletions or alternative with other residues;
Perhaps its aminoacid sequence is shown in SEQ.ID No.5 and the SEQ.ID No.6, in the occasion of chemosynthesis or this 8 peptide fusion protein of amalgamation and expression, but the residue of expansion part additions and deletions or alternative with other residues.
3. epitope minimum motif peptide as claimed in claim 1 is used in preparation human ZP multi-epitope peptide pregnancy vaccine.
4. the expansion small peptide of epitope minimum motif peptide as claimed in claim 2 prepares the application that is caused the serum Identification Lists bit flag thing of the woman infertility cause of disease as clinical diagnosis by ZP autoimmunization alone or in combination.
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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 |
| CN103524601A (en) * | 2013-09-27 | 2014-01-22 | 上海市计划生育科学研究所 | Epitope peptide in human spawn ZP (zona pellucida) protein 4 and application of epitope peptide |
| CN105859847A (en) * | 2016-05-30 | 2016-08-17 | 复旦大学 | Linear epitope minimum motif peptide Asp f 4<226-232> of aspergillus fumigatus Asp f 4 and expanded short peptides thereof |
| CN105859854A (en) * | 2016-05-30 | 2016-08-17 | 复旦大学 | Linear epitope minimum motif peptide Asp f 3<72-78> of aspergillus fumigatus Asp f 3 and expanded short peptides thereof |
| CN105859850A (en) * | 2016-05-30 | 2016-08-17 | 复旦大学 | Linear epitope minimum motif peptide Asp f 4<130-134> of aspergillus fumigatus Asp f 4 and expanded short peptides thereof |
| CN105866435A (en) * | 2016-05-30 | 2016-08-17 | 复旦大学 | Minimum motif peptide Asp f 3 of linear epitope of aspergillus fumigatus Asp f 3<123-127> and expanded oligopeptides of minimum motif peptide Asp f 3 |
| CN105859853A (en) * | 2016-05-30 | 2016-08-17 | 复旦大学 | Aspergillus fumigatus Asp f 3 linear antigenic epitope minimum base sequence peptide Asp f 3141-148 and extended oligopeptides thereof |
| CN105859855A (en) * | 2016-05-30 | 2016-08-17 | 复旦大学 | Aspergillus fumigatus Asp f 3 protein linear antigenic epitope minimum base sequence peptide Asp f 35-9 and extended oligopeptides thereof |
| CN105859851A (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 4104-109 and extended oligopeptides thereof |
| CN105859848A (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 4219-226 and extended oligopeptides 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 |
| CN105859856A (en) * | 2016-05-30 | 2016-08-17 | 复旦大学 | Linear epitope minimal motif peptide Asp f 3<35-41> of aspergillus fumigatus Asp f 3 and extended short peptide thereof |
| CN105859857A (en) * | 2016-05-30 | 2016-08-17 | 复旦大学 | Linear epitope minimum motif peptide Asp f 3<16-23> of aspergillus fumigatus Asp f 3 and expanded short peptides thereof |
| 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 |
| CN105924509A (en) * | 2016-05-30 | 2016-09-07 | 复旦大学 | Minimal sequence motif peptide Asp f 3<12-17> of linear epitope of aspergillus fumigatus Asp f 3 and extended short peptide containing minimal sequence motif peptide of linear epitope of aspergillus fumigatus Asp f 3 |
| CN105924508A (en) * | 2016-05-30 | 2016-09-07 | 复旦大学 | Minimal sequence motif peptide Asp f 3<67-72> of linear epitope of aspergillus fumigatus Asp f 3 and extended short peptide containing minimal sequence motif peptide of linear epitope of aspergillus fumigatus Asp f 3 |
| CN109486842A (en) * | 2017-09-12 | 2019-03-19 | 上海市计划生育科学研究所 | The carrier of small peptide recombinant clone building screening and its application |
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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 |
| CN103524601A (en) * | 2013-09-27 | 2014-01-22 | 上海市计划生育科学研究所 | Epitope peptide in human spawn ZP (zona pellucida) protein 4 and application of epitope peptide |
| 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 |
| CN105924509A (en) * | 2016-05-30 | 2016-09-07 | 复旦大学 | Minimal sequence motif peptide Asp f 3<12-17> of linear epitope of aspergillus fumigatus Asp f 3 and extended short peptide containing minimal sequence motif peptide of linear epitope of aspergillus fumigatus Asp f 3 |
| CN105924508A (en) * | 2016-05-30 | 2016-09-07 | 复旦大学 | Minimal sequence motif peptide Asp f 3<67-72> of linear epitope of aspergillus fumigatus Asp f 3 and extended short peptide containing minimal sequence motif peptide of linear epitope of aspergillus fumigatus Asp f 3 |
| CN105866435B (en) * | 2016-05-30 | 2018-07-13 | 复旦大学 | The linear epitope minimum motif peptide Asp f 3 of aspergillus fumigatus Asp f 3123-127And its extension small peptide |
| CN109486842A (en) * | 2017-09-12 | 2019-03-19 | 上海市计划生育科学研究所 | The carrier of small peptide recombinant clone building screening and its application |
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