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Easy-to-segment human-mouse zona pellucida fusion protein as well as preparation method and application thereof

A technology of zona pellucida protein and fusion protein, applied in anti-animal/human immunoglobulins, fusion peptides, chemical instruments and methods, etc., can solve the problems of high cost, time-consuming and laborious

Pending Publication Date: 2020-12-29
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In vitro expression of the N-terminal peptide of human ZP3. Journal of Jinan University (Medical Science) 2013, 34(6): 583-587); not only time-consuming and laborious, but also costly

Method used

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  • Easy-to-segment human-mouse zona pellucida fusion protein as well as preparation method and application thereof
  • Easy-to-segment human-mouse zona pellucida fusion protein as well as preparation method and application thereof
  • Easy-to-segment human-mouse zona pellucida fusion protein as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Preparation of easily split hZP3 and mZP2 fusion protein

[0033] 1. Construction of gene expression vectors with hZP3 and mZP2 chimeric peptides linked by enterokinase restriction sites

[0034] (1) Preparation of pET-mZP2 plasmid

[0035] This plasmid is the expression vector of pET transformed mZP2 (see reference 1: Nie Chuncheng, Yu Yanhong, Xie Yan, Cao Zuowu. Gene cloning of mouse ZP2 peptide and construction of prokaryotic expression vector. Journal of Jinan University (Medical Edition) 2011,32(4):374~378).

[0036]The mZP2 peptide is the 604-1545 fragment of the mouse ZP2-encoded DNA, inserted between the EcoRI and XhoI sites of the multi-cloning site, and can encode the 192-505 peptide in the middle of mZP2, followed by the DNA sequence of the 6X His tag peptide , with a BamHI site upstream.

[0037] ----BamH I-EcoR

[0038] The DNA sequence is as follows:

[0039] ATGGGTCGGGATCCGAATTCACCCTTGAAGGATGCCATAGTACAAGGATTTAATCTTCTGATTGACAGCCAGAAAGTAAC...

Embodiment 2

[0064] Example 2 hZP3 and mZP2 fusion protein preparation of antiserum

[0065] 1. Preparation of antiserum

[0066] Human ZP3 protein and mouse ZP2 protein separated by enzyme digestion were mixed with Freund's adjuvant to immunize white rabbits for a total of 5 times, blood was collected to separate serum, and antisera of mZP2 and hZP3 peptides were prepared respectively.

[0067] Antiserum was used to react with human egg zona pellucida and mouse zona pellucida respectively to test its immunospecificity.

[0068] 2. Identification of immune specificity of antiserum

[0069] (1) Immunohistochemical experiments were carried out with mZP2 peptide antiserum and mouse ovary slices to test the specific reaction between the antiserum and the mouse egg zona pellucida. Immunofluorescence showed that fluorescent signals were found in the egg zona pellucida of the ovarian slices, and the results were as follows Figure 4 , indicating that the mZP2 peptide antiserum contains anti-mou...

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Abstract

The invention provides an easy-to-segment human-mouse zona pellucida fusion protein as well as a preparation method and application thereof. The easy-to-segment human-mouse zona pellucida fusion protein is formed by connecting a human zona pellucida peptide fragment and a mouse zona pellucida peptide fragment through a connecting peptide with a cleavage site, the zona pellucida protein with two species is obtained, the fusion protein capable of generating the human zona pellucida and the mouse zona pellucida can be expressed at a time, the fusion protein can be used for simultaneously preparing antibodies for resisting human zona pellucida and mouse zona pellucida, and can also be used for separating the mouse zona pellucida peptide fragment and the human zona pellucida peptide fragment bycutting the connecting peptide of the fusion protein through specific protease to respectively prepare pure human zona pellucida protein or pure mouse zona pellucida protein.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, and more particularly relates to an easily split human-mouse egg zona pellucida fusion protein and its preparation method and application. Background technique [0002] The zona pellucida is a layer of cell shell outside the egg cell. It plays an important role in egg development and fertilization. It is an important target molecule in fertilization research and a candidate antigen for the development of contraceptive vaccines. Anti-Zona pellucida antibodies are therefore an important tool in fertilization and development studies. However, due to the limited natural sources, the traditional method of preparing anti-zona pellucida antibodies is greatly restricted. Now the preparation of antibodies is mainly to obtain the zona pellucida antigen through gene expression. Due to the particularity of the molecular structure of the zona pellucida, it brings some difficulties Practical diffi...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/70C07K16/18C07K16/06
CPCC07K14/47C12N15/70C07K16/18C07K16/06C07K2319/50C07K2319/40C07K2319/21
Inventor 曹佐武
Owner JINAN UNIVERSITY
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