Human oocyte zona pellucida protein multi-epitope chimeric peptide antigen and preparation method thereof

A zona pellucida and multi-epitope technology, applied in the fields of immunology and bioengineering, can solve problems such as unrealistic, high cost, and difficult purification

Inactive Publication Date: 2010-04-21
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, due to their difficulty in purification and high cost, it is obviously unrealistic to use natural huZP or recombinant huZP3 proteins as routine detection antigens

Method used

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  • Human oocyte zona pellucida protein multi-epitope chimeric peptide antigen and preparation method thereof
  • Human oocyte zona pellucida protein multi-epitope chimeric peptide antigen and preparation method thereof
  • Human oocyte zona pellucida protein multi-epitope chimeric peptide antigen and preparation method thereof

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Experimental program
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Effect test

Embodiment 1

[0038] The specific operation steps of human ZPCP bioengineering preparation are as follows:

[0039] 1. Molecular design of human ZPCP: based on the public information on the BCE identification of the three human ZP proteins, and our analysis of the deduced huZP3 protein BCE (huZP3 22-30 and huZP3 301-320 ) identification and confirmation, the target ZPCP selects 9 BCE or BCE peptides of human ZP protein (the latter is more than 8 amino acid residues), and the increase or decrease of individual residues at both ends of their BCE or BCE peptides is based on our analysis of the 5 BCE peptides. Minimal motif analysis results (where huZP3 301-320 The epitope peptide contains 2 minimal motifs, suggesting the existence of two BCEs), and the molecular design target based on the target ZPCP hydrophilicity and isoelectric point (pI) should not be too high or too low. At the same time, as an ideal immunogen, HBsAG was selected based on public information 19-33 、TT 580-599 , bRNase ...

Embodiment 2

[0046] The specific operation steps of rabbit immunization with ZPCP and its immunogenicity analysis are as follows:

[0047] 1. Active immunization with purified ZPCP protein: immunize New Zealand male white rabbits according to the conventional method. For the first immunization, 1 mg ZPCP immunogen emulsified with Freund's adjuvant was used, and after two weeks, it was used with incomplete Freund's adjuvant at intervals of two weeks. A dose of emulsified 0.5mg ZPCP immunogen was used to boost the immunization twice, and the rabbit antiserum was collected one week later for future use. Negative control rabbits were only injected with Freund's adjuvant.

[0048]2. ELISA analysis of rabbit anti-ZPCP antibody: on a 96-well ELISA plate, add 100 μl of purified recombinant ZPCP protein (100 ng / well) as an embedded antigen, and place it overnight at 4 ° C; the next day, use 0.05% Tween- Wash unbound ZPCP protein with 20 pBS (PBST), then dilute rabbit anti-ZPCP antiserum 1:50 with ...

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Abstract

The invention belongs to the technical field of immunology and bioengineering and particularly relates to human oocyte zona pellucida protein multi-epitope chimeric peptide (ZPCP) and a preparation method and an application thereof. A ZPCP molecule designed by the invention is combined with three proteins of huZP (huZP1, huZP2 and huZP3), nine linear B cell epitopes (BCE) and four wide-adaptability helper T cell epitopes (TCE) and comprises a section of pig ZP4 (pZP4), N terminal 25 peptide and a beta turn forming peptide (GPSL). The ZPCP molecule has the advantages that an amino acid residue sequence of the ZPCP molecule or a DNA coding sequence thereof can be highly expressed in a escherichia coli; a targeting expression protein with the electrophoresis uniformity higher than 95 percent can be conveniently obtained; the ZPCP molecule can generate a high level anti ZPCP antibody in an induction way in a rabbit body as an immunogen, including anti each embedded BCE antibody which can be detected in serum.

Description

technical field [0001] The invention belongs to the technical field of immunology and bioengineering, in particular to a human zonapellucida (huZP) multi-epitope chimeric peptide (chimeric peptide, CP) and a preparation method thereof, and also relates to the multi-epitope chimeric peptide Peptide application. 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.Am J Reprod Immunol, 35:163-174, 1996; Aitken RJ, et al. Am J Reprod Immunol, 35:175-180, 1996). Since the use of allogeneic or heterogeneous recombinant or deglycosylated ZP as an immunogen interferes with normal ovarian function, since the first case of peptide vaccine based on ZP antigenic epitope (B-cell epitope, BCE) was reported in 1989, (Millar SE, et al. Science, 246:935-938, 1989), the identification...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C12N15/62C12N15/70C12P21/02A61K39/00A61P15/18G01N33/543C12R1/19
Inventor 徐万祥顾少华何亚萍季朝能王健谢毅唐海平
Owner SHANGHAI INST OF PLANNED PARENTHOOD RES
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