Preparation method of recombinant porcine epidemic diarrhea virus antibody

A porcine epidemic diarrhea and antibody technology, which is applied in the field of preparation of recombinant porcine epidemic diarrhea virus antibody, can solve the problems of long preparation period, activity in the antibody and the like

Inactive Publication Date: 2016-11-23
HEILONGJIANG BAYI AGRICULTURAL UNIVERSITY
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  • Abstract
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  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to provide a preparation method of recombinant porcine epidemic diarrhea virus antibody, which is used to solve the problem that traditional mouse PEDV antibody will produce immune response, long preparation period, and The problem of low neutralization activity

Method used

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  • Preparation method of recombinant porcine epidemic diarrhea virus antibody
  • Preparation method of recombinant porcine epidemic diarrhea virus antibody
  • Preparation method of recombinant porcine epidemic diarrhea virus antibody

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preparation example Construction

[0043] The preparation method of this recombinant porcine epidemic diarrhea virus antibody: choose the way of co-expression of antigen and antibody, co-express PEDV antigen gene S1 and anti-PEDV ScFv gene in the periplasmic cavity of Escherichia coli, and use flow cytometry for three rounds panning. After expressing the ScFv gene in Escherichia coli, the PEDV antibody was purified, and the specificity of the PEDV ScFv antibody was detected by ELISA method, which was named PEDV-ScFv.

[0044] The specific preparation method of the above-mentioned PEDV antibody:

[0045] 1. Construction of anti-PEDV ScFv library

[0046] 1. Detection of animal immunity and serum antibody titer

[0047] Piglets aged 3-6 days were first immunized with attenuated PEDV vaccine, and then challenged with moderately virulent strains, a total of 3 times before and after. Blood was collected one day before each immunization or challenge and one week after the last immunization, and the serum antibody ...

Embodiment 1

[0060] Firstly, a moderately virulent virus strain was selected to challenge the piglets to detect the antibody titer of the piglets, and then the RNA extracted from the spleen of the piglets was reverse-transcribed into cDNA to construct an antibody library. Before preparing the antigen, bioinformatics analysis was performed on the PEDV S gene, and it was found that there were many glycosylation sites, the expression was difficult, and the yield was low. Therefore, the co-expression of antigen and antibody was selected in the later experiment, and flow cytometry was used to Three rounds of screening of the ScFv antibody library were performed. The plasmid was then extracted, amplified by PCR, cloned into a T vector, and sent to Shanghai Sangon for sequencing. Use Primer5.0 and DNAMAN software for sequence analysis, compare the sequence of the plasmid with the porcine antibody on the Internet, use the primer design software Primer Premier 5.0 to analyze and design PCR primers ...

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Abstract

The invention relates to a preparation method of a recombinant porcine epidemic diarrhea virus antibody, comprising: selecting an antigen antibody co-expression mode, co-expressing PEDV (porcine epidemic diarrhea virus) antigen gene and anti-PEDV ScFv gene in Escherichia coli periplasmic cavity, and performing triple sorting by using flow cytometry; using Escherichia coli to express the ScFv gene, then purifying the PEDV antibody, detecting specificity of the PEDV ScFv antibody by using ELISA (enzyme-linked immuno sorbent assay), and naming the antibody as PEDV-ScFv; wherein two specific steps are performed: constructing an anti-PEDV ScFv library and preparing the anti-PEDV antibody. The porcine-derived PEDV antibody provided herein never causes immune response, has high specificity, enables easier screening of an antibody with high neutralizing activity, and has short preparation cycle and high preparation speed.

Description

1. Technical field [0001] The invention relates to the genetic engineering preparation of porcine epidemic diarrhea virus (PEDV) antibody, in particular to a preparation method of recombinant porcine epidemic diarrhea virus antibody. 2. Background technology [0002] Antibodies are the body's natural endogenous proteins, with low toxicity and high specificity, and can directly act on the target. They are widely used in the treatment of diseases and have become an important class of biotechnological drugs for clinical application. As of 2010, the U.S. FDA has approved 25 antibodies for disease treatment, and more than 240 antibodies have been used clinically. The diseases treated include autoimmune diseases and inflammation, cancer, organ transplantation, cardiovascular diseases, infectious diseases and ophthalmic diseases, etc. . The successful clinical use of antibody drugs has generated huge commercial profits. In 2007, the sales volume exceeded 27 billion US dollars, acc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N15/10C40B50/06C12N15/70C12P21/02
Inventor 张华曹宏伟邹德华
Owner HEILONGJIANG BAYI AGRICULTURAL UNIVERSITY
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