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Porcine single-chain antibody for resisting hog cholera viruses and preparation method thereof

A single-chain antibody and swine fever virus technology, applied in the field of genetic engineering, can solve problems such as the impact of results, expensive reagents and instruments, and complicated operations, and achieve high specificity, easy transformation, and strong penetrating power

Active Publication Date: 2019-02-05
SHANGHAI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] However, the reported single cell expansion method is complicated in the first step, such as a large number of experimental steps, a single B cell needs to be cultured individually, and it is easy to cross-culture, which affects the results; at the same time, reagents and instruments are expensive, which are urgently needed improved question

Method used

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  • Porcine single-chain antibody for resisting hog cholera viruses and preparation method thereof
  • Porcine single-chain antibody for resisting hog cholera viruses and preparation method thereof
  • Porcine single-chain antibody for resisting hog cholera viruses and preparation method thereof

Examples

Experimental program
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Effect test

Embodiment

[0045] Embodiment A kind of obtaining method of pig-derived anti-single-chain antibody of classical swine fever virus

[0046] 1. Using live classical swine fever vaccine as the cell source for passage, immunize two 30-day-old piglets by injection, one portion, and carry out secondary immunization at intervals of two weeks; for the immunized piglets, refer to Osper et al. Guidelines" method to detect serum antibody titers; details are as follows:

[0047] (1) Dilute CSFV (1:100) with bicarbonate coating solution to coat a 96-well plate;

[0048] (2) 8% skimmed milk closed;

[0049] (3) The collected pig serum was diluted 1:100, 100 μl / well;

[0050] (4) HRP-labeled goat anti-pig IgG1: 250 dilutions, 100 μl / well;

[0051] (5) Chromogenic solution A:B=1:1 50μl / well;

[0052] (6) Stop solution 2M H 2 SO 4 50μl / well;

[0053]Results: The OD value of pig serum after the second immunization: 0.987.

[0054] 2. Labeling E2 protein with FITC

[0055] (1) E2 protein (concentr...

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Abstract

The invention relates to a porcine single-chain antibody for resisting hog cholera viruses and a preparation method thereof. The single-chain antibody comprises a heavy-chain variable area and a light-chain variable area, wherein the heavy-chain variable area and the light-chain variable area are connected by virtue of a connecting peptide; the amino acid sequence of the heavy-chain variable areais as shown by SEQ ID No.1, and the amino acid sequence of the light-chain variable area is as shown by SEQ ID No.2; and the amino acid sequence of the connecting peptide is GGGGSGGGGSGGGGS. The single-chain antibody of the invention can be specifically combined with the hog cholera viruses, the in-vivo expression can be carried out directly by virtue of a recombinant vaccine, the neutralizing antibody can be rapidly and efficiently produced in an induction manner without depending on the immune system of the body, and the porcine single-chain antibody is used for blocking the infection of thehog cholera viruses.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a swine-derived single-chain antibody against swine fever virus and a preparation method thereof. Background technique [0002] Swine fever vaccine immunization often leads to immunization failure because it cannot induce effective neutralizing antibodies. In vaccine research, by screening monoclonal neutralizing antibodies, recombinant expression vectors are used to construct recombinant bacterial strains expressing CSF neutralizing antibody proteins, and then antibody vaccines are developed. [0003] Since the 1970s, mouse hybridoma cell technology and phage display technology have become common technical means for screening and preparing monoclonal antibodies. Most of the antibody drugs currently on the market at home and abroad are also obtained by using these two technologies . [0004] These two technical methods have many advantages, such as stron...

Claims

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Application Information

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IPC IPC(8): C07K16/10C12N15/13C12N15/70
CPCC07K16/1081C07K2317/622C12N15/70
Inventor 刘惠莉李本强陶洁程靖华石迎
Owner SHANGHAI ACAD OF AGRI SCI
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