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Epitope minimum motif peptide of human zona pellucida protein

A technology of zona pellucida protein and motifs, applied in the fields of bioengineering and immunology, can solve problems such as false positive results

Inactive Publication Date: 2009-10-14
SHANGHAI INST OF PLANNED PARENTHOOD RES +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, detection of anti-self ZP antibodies by porcine ZP antigen ELISA and Western blot is prone to false positive results (Skinner SM, et al. Zonapellucida antigens: Targets for contraceptive vaccines. Am J Reprod Immunol, 35: 163-174, 1996) , and recently we found that there are antibodies that can cross-react with multiple long epitope peptides (8-20 amino acid residues) of the three proteins of huZP in 1:15 diluted serum of normal men and women (unpublished data), that is to say , it is difficult to avoid false positive results even when natural huZP or recombinant huZP3 is used as the detection antigen

Method used

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  • Epitope minimum motif peptide of human zona pellucida protein
  • Epitope minimum motif peptide of human zona pellucida protein

Examples

Experimental program
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Effect test

Embodiment 1

[0019] Example 1: Human egg zona pellucida protein-1 (huZP1) epitope 313-321 Minimal motif identification

[0020] Materials and methods:

[0021] 1. The heat-inducible expression plasmid pXXGST-1 was constructed by the inventor of this patent (patent application number: 200710173305.2). Escherichia coli BL21 (DE3) strain was preserved by the State Key Laboratory of Genetic Engineering, Fudan University.

[0022] 2. Rabbit anti-pig ZP IgGs antibody and rabbit anti-huZP3a and huZP3b antiserum were prepared and preserved by Shanghai Institute of Family Planning Science.

[0023] 3. Restriction enzymes BamH I, Sal I and T4 DNA ligase were purchased from Japan TaKaRa Biotechnology Company, protein low molecular weight standards, horseradish peroxidase-labeled goat anti-rabbit secondary antibody (IgG / HRP), diaminobenzidine (DAB) and nitrocellulose membranes were purchased from Huamei Bioengineering Company.

[0024] 4. QIAprep spin miniprep Kit plasmid extraction kit, QIAquick ...

Embodiment 2

[0033] Example 2: Human egg zona pellucida protein-3 (huZP3) epitope 93-110 Minimal motif identification

[0034] Materials and methods: In addition to rabbit anti-huZP3a and huZP3b antiserum (prepared by the patent applicant. Song Liwen et al. Immunogenicity of human egg zona pellucida protein ZP3a and ZP3b peptides and their antisera inhibit human sperm-Zona translucency binding in vitro. Physiological Acta Sinica, 57:682-688, 2005) and horseradish peroxidase-labeled goat anti-human secondary antibody (IgG / HRP, purchased from PTGLAB Company), others see the corresponding part of Example 1.

[0035] wxya 93-110 The specific steps of epitope peptide minimal motif identification are as follows:

[0036]1. Based on the epitope identification and gene sequence public information (Paterson M, et al. Evaluation of the contraceptive potential recombinant human ZP3 and human ZP3 peptides in a primate model: their safety and efficacy. Am J Reprod Immunol, 40: 198-209, 1998; Chamber...

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Abstract

The invention belongs to the biological engineering and immunology technology fields and specifically relates to a linear epitope minimum motif peptide of human zona pellucida (huZP) protein huZP1 and huZP3, and ZP chimeric peptides. In particular, the invention provides the minimum epitope amino acid sequence on the huZP1 and the huZP3 proteins for detecting ZP antibodies (Leading to the sterility or premature ovarian failure of women) and the 8-peptide sequence containing the epitope minimum motif and of truncated GST188 carrier protein fusion expression. The epitope minimum motif peptide of the huZP1 with immunogenicity can be identified by a rabbit anti-pig ZP antiserum and the 5-epitope minimum peptide motif of huZP3 can be identified by a rabbit antibody human ZP3 antiserum, to prove the value of the epitope minimum motif peptide when used as a single-epitope or multi-epitope peptide contraception antigen or when used as a single-epitope or multi-epitope fusion expression antigen for detecting the ZP antibodies of humans.

Description

technical field [0001] The invention belongs to the technical field of bioengineering and immunology, and in particular relates to the linear epitope minimal motif peptides of human egg zona pellucida (huZP) proteins huZP1 and huZP3. In particular, the present invention provides the minimal epitope amino acid sequences on the huZP1 and huZP3 proteins that can be used to detect auto ZP antibodies (causing female infertility or premature ovarian failure), as well as the minimal epitope containing the epitope expressed in fusion with the truncated GST188 carrier protein. 7 / 8 peptide sequence of the motif. Background technique [0002] Mammalian egg zona pellucida (ZP) is composed of 3 to 4 glycoproteins, and it has been regarded as a potential target antigen for the development of anti-fertilization contraceptive vaccines for more than 30 years (Skinner SM, et al.Zona pellucidaantigens: Targets for contraceptive vaccines. Am J Reprod Immunol, 35: 163-174, 1996; AitkenRJ, et al...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/06C07K5/093C07K7/08
Inventor 徐万祥顾少华何亚萍季朝能谢毅王健唐海平
Owner SHANGHAI INST OF PLANNED PARENTHOOD RES
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