Single-chain antibody for porcine epidemic diarrhea resistant virus and application thereof

A single-chain antibody, light chain technology, applied in antiviral agents, antiviral immunoglobulins, applications, etc., can solve the problems of easy production of anti-antibodies, affecting therapeutic effects, and large molecular weight of antibodies

Active Publication Date: 2018-10-16
青岛博隆基因工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there are few porcine-derived phage antibody display platforms, the molecular weight of the antibody is relatively large, and the antigen-binding region is easily blocked, and the folding ability of the single-chain antibody is limited, and the traditional monoclonal antibody is basically a mouse-derived antibody, which is prone to produce anti-bacterial antibodies during use. Antibodies, affecting its therapeutic effect
The existing porcine epidemic diarrhea vaccines cannot fully prevent and control the outbreak of the disease, which has brought huge economic losses to the pig industry

Method used

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  • Single-chain antibody for porcine epidemic diarrhea resistant virus and application thereof
  • Single-chain antibody for porcine epidemic diarrhea resistant virus and application thereof
  • Single-chain antibody for porcine epidemic diarrhea resistant virus and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1, Preparation of Phage Antibody Display Library

[0042] 1. Primer design and synthesis

[0043] Primers were designed respectively according to the sequences of pig IgG heavy chain (GenBank number: AM177137, light chain Kappa chain (GenBank number: AY518084) and light chain lambda chain (GenBank number: AF345512). The nucleotide sequence and length of the amplified fragment of each primer See Table 1.

[0044] Table 1

[0045]

[0046] Note: The part in italics indicates the linker, and the underline indicates the recognition site for restriction endonuclease SfiI. The name containing "for" means upstream, the name containing "back" means downstream, VH means heavy chain, VL (Kappa) means light chain Kappa chain, VL (Lambda) means light chain lambda chain, S means C or G , Y is C or Y, K is G or T, R is A or G, W is A or T.

[0047] The respective primers shown in Table 1 were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.

[0048] 2. Extrac...

Embodiment 2

[0101] Example 2, Detection of the binding activity of the phage antibody display library

[0102] The binding activity of the phage antibody display library was detected by ELISA method.

[0103] Take porcine epidemic diarrhea virus S protein (i.e. antigen) and dilute it with pH9.6, 0.1M sodium carbonate-sodium bicarbonate buffer solution to obtain antigen dilution solution with a concentration of 100 μg / μL.

[0104] 1. Take a 96-well plate, add 100 μL of antigen diluent to each well, and leave overnight at 4°C.

[0105] 2. After completing step 1, take the 96-well plate, add 100 μL blocking solution (obtained by dissolving 0.05 g BSA in 100 mL pH7.4, 10 mM PBS buffer) to each well, and let stand at 37° C. for 2 hours.

[0106] 3. After completing step 2, take the 96-well plate, add 100 μL of phage antibody display library to each well, and let stand at 37° C. for 2 hours.

[0107] 4. After completing step 3, take the 96-well plate, add 100 μL of HRP-labeled anti-M13 antibo...

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Abstract

The invention discloses a single-chain antibody for a porcine epidemic diarrhea resistant virus and application thereof. The single-chain antibody provided by the invention is a polypeptide composed of a light chain variable region, a connecting peptide, and a heavy chain variable region, wherein the connecting peptide is positioned between the light chain variable region and the heavy chain variable region; the light chain variable region is shown as amino acid residues at 1-107 sites from N terminal in a sequence 1 of a sequence table; the heavy chain variable region is shown as amino acid residues at 123-240 sites from N terminal in the sequence 1 of the sequence table. The single-chain antibody provided by the invention is capable of neutralizing the porcine epidemic diarrhea virus, and has an important application value in prevention and / or treatment of porcine epidemic diarrhea.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a single-chain antibody against porcine epidemic diarrhea virus and its application. Background technique [0002] Porcine Epidemic Diarrhea (PED) is an intestinal infectious disease caused by Porcine Epidemic Diarrhea Virus (PEDV). Diarrhea, vomiting, emaciation, dehydration, and eventually death. Although the application of vaccines has played a certain role in the control of the disease, the disease still occurs and spreads to varying degrees in some areas. [0003] Phage antibody display technology provides a more efficient way for efficient screening and directional transformation of genetically engineered antibodies. With the development of animal immunization technology, the space will become more and more narrow, and the use of in vitro methods to screen antibodies will attract more and more attention. The phage display technology is different from the traditio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N15/13A61K39/42A61P31/14A61P1/12
CPCA61K2039/505A61P1/12A61P31/14C07K16/10C07K2317/622
Inventor 尹燕博孙举王冬冬刘宏
Owner 青岛博隆基因工程有限公司
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