Epitope peptide in human spawn ZP (zona pellucida) protein 4 and application of epitope peptide

A technology of zona pellucida protein and protein, applied in the field of epitope peptides, which can solve the problems of widely used diagnostic methods that affect the accuracy of clinical diagnosis, easy to produce non-specific cross-reactions, false positives, etc.

Inactive Publication Date: 2014-01-22
SHANGHAI INST OF PLANNED PARENTHOOD RES +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the general use of 18 or 20-mer long epitope peptide antigen enzyme-linked immunoassay (ELISA) to diagnose autoimmune diseases and such as influenza virus infection, there is often a false positive rate of about 30%, which affects the accuracy of clinical diagnosis and wide application of the established diagnostic method
[0004] The reason for the high false positive rate in this case is very simple, that is: since it is clear that the linear epitope is composed of 3-8 amino acid residues, synthetic peptides of 18 or 20 polymers or longer (too short and difficult to be used as antigens for ELISA coating) It is obvious that there may be N unknown epitopes in the detection panel), and due to many reasons such as natural infection of viruses and bacteria (such as influenza virus infection, etc.) and autoimmune diseases, there are usually tens of thousands of antibodies in the human body. Antibodies with low titers, so these two factors are likely to produce non-specific cross-reactions when the above patient serum dilution is unlikely to be high (generally 1:20-1:100), resulting in high false positive rate detection result

Method used

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  • Epitope peptide in human spawn ZP (zona pellucida) protein 4 and application of epitope peptide
  • Epitope peptide in human spawn ZP (zona pellucida) protein 4 and application of epitope peptide
  • Epitope peptide in human spawn ZP (zona pellucida) protein 4 and application of epitope peptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0102] Embodiment 1: Preparation of rabbit anti-human ZP4C antiserum

[0103] Materials and methods:

[0104] 1. Human ZP4 gene clone was donated by Dr. Jurrien Dean, Laboratory of Cell and Developmental Biology, National Institutes of Health. For the construction of heat-inducible expression plasmids pSY621 and pXXGST-1, see Shen Yun, Ying Kang, Xu Wanxiang, Xie Yi: Construction of high-efficiency expression vector pSY621 in Escherichia coli. 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. Escherichia coli BL21 (DE3) strain was preserved by the State Key Laboratory of Genetic Engineering, Fudan University.

[0105] 2. For the preparation of mouse anti-huZP4 monoclonal antibody MA-1662, see Bukovsky A, Gupta SK, et al. Production of monoclonal antibodies against recombinant human zona pellucida glycoprot...

Embodiment 2

[0113]Example 2 The first round of epitope scanning mapping with the target protein of 18 peptides

[0114] Material:

[0115] 1. For the relevant experimental materials, see the part 1 of the embodiment

[0116] 2. The two ends are BamH I and Sal I cohesive ends respectively, and the positive and negative strand DNA fragments with 18 peptide coding DNA sequences plus TAA stop codon in the middle are synthesized by Shanghai Jierui Bioengineering Co., Ltd.

[0117] 3. The series of 18 peptide-encoding DNA sequences are based on the public information of the human ZP4 protein-encoding gene and protein amino acid sequence (Harris 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).

[0118] Method (identification of antigenic (reactive) 18 peptide (fusion protein)):

[0119] 1. After using the rabbit anti-recombinant human ZP4C antiserum to verify the immunobl...

Embodiment 3

[0125] Example 3 Identification of Two Fine Epitope Peptides in Human ZP4C

[0126] Material

[0127] 1. For the relevant experimental materials, see the part 1 of the embodiment

[0128] 2. The two ends are BamH I and Sal I cohesive ends respectively, and the positive and negative strand DNA fragments in the middle are each 8-peptide coding DNA sequence plus TAA stop codon, which were synthesized by Shanghai Jierui Bioengineering Co., Ltd.

[0129] method

[0130] 1. According to the two adjacent reactive 18 peptides of P30-P31 and P33-P34 in Example 2, design a series of 8 peptides (numbering P110-P122 and P134-P146) covering their full-length sequence overlapping 7 amino acid residues , image 3 Only P134-P141 is shown in B) encoding DNA positive and negative strand fragments (positive strand 5'-end plus 5'-gatcc, 3'-end plus taag-3'; negative strand 5'-end plus 5'-tcgactta, 3 '-end plus g-3'), send DNA chemical synthesis;

[0131] 2-5. The identification steps from se...

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Abstract

The invention discloses an epitope peptide in human spawn ZP protein 4 and an application of the epitope peptide, and particularly relates to a short peptide separated from human spawn ZP protein-4 (a human ZP protein 4 227-463 bit). The formula I of the short peptide is shown as follows: X1-X-X2 (the formula I), and the following characteristics are met: a), the X in the formula I is 6-7 amino acid core fragments; b), the number sum of the X1 and the X2 in the formula I is smaller than or equal to 2; c), the sequences of the core fragments are selected from SEQ ID NO.: 1-6; and d), the short peptide is provided with activity combined with an anti-human ZP 4C antibody, wherein the '-' expresses a peptide link or a peptide joint. The epitope peptide can be combined and applied to detection of the existence or content of various antibodies of anti-human ZP 4 protein in a sample, so that women's sterility caused by ZP autoimmunity can be diagnosed at high specificity and high sensitivity; and besides, the epitope peptide can also be used for preparing recombination human ZP multi-epitope-peptide contraceptive vaccine with safe and effective advantages.

Description

technical field [0001] The invention belongs to the technical fields of bioengineering and immunology, and particularly relates to a group of epitope peptides in human egg zona pellucida protein 4 and applications thereof. Background technique [0002] ZP protein (human egg zona pellucida protein), including ZP4 protein, one of the constituent proteins, also plays an important role in the fertilization process of sperm eggs, and is the second sperm receptor. Anti-ZP antibodies in normal women's serum are generally negative, and the existence of anti-ZP antibodies will bind to ZP protein, which will reduce the motility of sperm, affect sperm passing through cervical mucus and ascending, and affect the combination of sperm and ZP protein, thereby preventing Sperm and egg combine and cause infertility. In infertile women with ZP autoimmunity, there are anti-ZP protein antibodies in the serum, but due to the unavailability of ZP protein antigens, the existing general detection ...

Claims

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Application Information

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IPC IPC(8): C07K7/06G01N33/68A61K39/00A61P15/18
Inventor 徐万祥顾少华赛提旭·古普塔王健唐海平季朝能何亚萍谢毅
Owner SHANGHAI INST OF PLANNED PARENTHOOD RES
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