Porcinized single-chain antibody resisting porcine epidemic diarrhea virus and preparation method of porcinized single-chain antibody

A porcine epidemic diarrhea and single-chain antibody technology, applied in biochemical equipment and methods, viruses/bacteriophages, chemical instruments and methods, etc., can solve problems such as uncommercialized products

Active Publication Date: 2016-08-31
HENAN AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

There have been many reports on humanized single-chain antibodies, but porcine or similar expression vectors from other species have not been commercialized, and there are few similar reports

Method used

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  • Porcinized single-chain antibody resisting porcine epidemic diarrhea virus and preparation method of porcinized single-chain antibody
  • Porcinized single-chain antibody resisting porcine epidemic diarrhea virus and preparation method of porcinized single-chain antibody
  • Porcinized single-chain antibody resisting porcine epidemic diarrhea virus and preparation method of porcinized single-chain antibody

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Experimental program
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Embodiment 1

[0032] Anti-porcine epidemic diarrhea virus porcine-derived single-chain antibody, its gene sequence structure is V H -Linker-V L -Fc, the sequence is shown in SEQ ID NO.5, wherein the heavy chain variable region gene sequence V H As shown in SEQ ID NO.1, the connecting peptide linker sequence is shown in SEQ ID NO.4, the light chain variable region gene sequence V L As shown in SEQ ID NO.2, the gene sequence of the Fc region of the porcine antibody constant region is shown in SEQ ID NO.3.

Embodiment 2

[0034]A preparation method for porcine epidemic diarrhea virus porcine-derived single-chain antibody, comprising the following steps:

[0035] 1) Construction of BL21 / pET28a-scFv-Fc

[0036] Contains heavy chain variable region gene sequence V H (as shown in SEQ ID NO.1), connecting peptide linker sequence (as shown in SEQ ID NO.4), light chain variable region gene sequence V L (as shown in SEQ ID NO.2), the Fc segment gene sequence of the porcine antibody constant region (as shown in SEQ ID NO.3), and the scFv of the 5' end restriction site SacI and the 3' end restriction site SalI- The Fc sequence was synthesized by Shanghai Sangon Bioengineering Co., Ltd., and the sequence is shown in SEQ ID NO.6.

[0037] The vector pET28a and the synthetic sequence scFv-Fc were digested by restriction endonucleases SacI and SalI respectively. The stepwise double digestion system was: 1 μL SacI (10 units / μL), 2 μL 10×L Buffer, 1 μg of synthetic sequence scFv-Fc or vector pET28a,dH 2 Ma...

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Abstract

The invention discloses a porcinized single-chain antibody resisting a porcine epidemic diarrhea virus and a preparation method of the porcinized single-chain antibody, and belongs to the field of biotechnology. A gene sequence of a heavy-chain variable region of the single-chain antibody is as shown in SEQ ID NO.1; the gene sequence of a light-chain variable region is as shown in SEQ ID NO.2. The single-chain antibody also comprises the gene sequence of a porcine anti-body constant region Fc segment as shown in SEQ ID NO.3, wherein the gene sequence of the heavy-chain variable region is connected with the gene sequence of the light-chain variable region through a linker sequence as shown in SEQ ID NO.4. The length of scFv-Fc is 1128bp; the molecular weight translated into a polypeptide chain is about 40kDa and is 1.4 that of natural immune globulin; the penetrability between tissues can be improved by reduction of the molecular weight; and the Elisa result shows that PEDV can be identified by the scFv-Fc.

Description

technical field [0001] The invention relates to an anti-porcine epidemic diarrhea virus porcine-derived single-chain antibody, and also relates to a preparation method of the antibody, which belongs to the field of biotechnology. Background technique [0002] Porcine epidemic diarrhea (Porcine epidemic diarrhea, PED) is caused by porcine epidemic diarrhea virus (Porcine epidemic diarrhea virus, PEDV) and is characterized by severe diarrhea, vomiting and dehydration in pigs of different ages. Intestinal infectious disease. Currently, research on PEDV is mainly focused on cloning its spike protein (S), main membrane protein (M) and nucleocapsid protein (N) by means of genetic engineering, and the expression products are then used as protective antigens to immunize animals to generate immune responses. Such as the disclosed classic epidemic strain CV777 strain and attenuated strain ZJ08 strain (CN103725651A), SD10 strain (CN103820399A). For another example, Zheng Fengmei et a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N1/21C12N15/70C12N15/66
CPCC12N15/66C12N15/70C07K16/10C07K2317/21C07K2317/622C12N2800/101
Inventor 杨国庆刘慧敏常洪涛宋晓峰
Owner HENAN AGRICULTURAL UNIVERSITY
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