Swine-derived single-chain antibody capable of resisting swine transmissible gastroenteritis viruses and preparation method of swine-derived single-chain antibody

A single-chain antibody, infectious technology, applied in the field of genetic engineering, can solve the problems of PED inactivation and the unsatisfactory effect of attenuated vaccines

Active Publication Date: 2017-05-10
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Vaccination is one of the ways to prevent the occurrence of this disease, but the effect of the developed PED inactivated and attenuated vaccines is not satisfactory

Method used

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  • Swine-derived single-chain antibody capable of resisting swine transmissible gastroenteritis viruses and preparation method of swine-derived single-chain antibody
  • Swine-derived single-chain antibody capable of resisting swine transmissible gastroenteritis viruses and preparation method of swine-derived single-chain antibody
  • Swine-derived single-chain antibody capable of resisting swine transmissible gastroenteritis viruses and preparation method of swine-derived single-chain antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Preparation of porcine-derived single-chain antibody against transmissible gastroenteritis virus

[0026] 1: Porcine transmissible gastroenteritis virus antigen detection (positive antigen is the TGEV strain preserved in our laboratory) is carried out to piglets with diarrhea symptoms in the farm, with routine (referring to F.M. "Guideline") ELISA method to detect serum antibody titer greater than 1:20000, collect the pig blood, lyse red blood cells to obtain white blood cells (erythrocyte lysate Shanghai Biyuntian Biotechnology Co., Ltd.), use Trizol method (TRIZOL Reagent purchased from Invitrogen Corporation of the United States) ) to extract total RNA. Using the extracted total RNA as a template, Oligo primer was used to synthesize the first-strand cDNA according to the product instructions of the reverse transcription kit (cDNA first-strand synthesis kit was purchased from TaKaRa Company).

[0027] 2: The heavy chain variable region sequence (AF064686.1;...

Embodiment 2

[0041] Example 2 Indirect ELISA screening of antigen-specific ScFv

[0042] Take the porcine transmissible gastroenteritis virus (preserved in our laboratory) concentrated by ultracentrifugation, dilute it to 5 μg / mL with antigen coating solution (50mmol / L sodium bicarbonate salt solution, pH=9.6), and add it to a 96-well plate. Well 100 μL, 4 Coated overnight; add 200 μL of 5% skimmed milk powder solution to each well, 37 Block for 1 hour, wash with PBS 3 times; mix 50 μL of purified protein supernatant with 50 μL of 4% skimmed milk powder solution, add to the above wells, incubate at 37°C for 2 hours, wash with PBST 3 times; add E-Tag Mouse mAb (E-tag Tag mouse monoclonal antibody (purchased from RayBiotech Company) 100 μL (1:2000), reacted at 37°C for 2 h, washed 3 times with PBST; 4000), reacted at 37°C for 1 h, washed 3 times with PBST; developed color with TMB for 15 min, terminated the reaction with 2 mol / L sulfuric acid, and read OD 450 When the absorbance value w...

Embodiment 3

[0044] The obtained single-chain antibody coding gene was sequenced to prove that it consists of 747 nucleotides and 249 amino acids deduced accordingly. The nucleotide sequence is shown in SEQ ID No.4, and the amino acid sequence is shown in SEQ ID No.4. ID No.3 shown.

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Abstract

The invention discloses a swine-derived single-chain antibody capable of resisting swine transmissible gastroenteritis viruses and a preparation method of the swine-derived single-chain antibody. The swine-derived single-chain antibody is formed by linking a heavy-chain variable region with a light-chain variable region through a short linker peptide, wherein an amino acid sequence of the light-chain variable region is shown as SEQ ID No.1, and an amino acid sequence of the heavy-chain variable region is shown as SEQ ID No.2. The swine-derived single-chain antibody capable of resisting the swine transmissible gastroenteritis viruses is about 28 kDa in molecular weight and capable of specifically recognizing the swine transmissible gastroenteritis viruses and can be used for further blocking infection and invasion of the swine transmissible gastroenteritis viruses.

Description

technical field [0001] The invention relates to a pig-derived anti-porcine transmissible gastroenteritis virus single-chain antibody and a preparation method thereof, belonging to the technical field of genetic engineering. Background technique [0002] Transmissible gastroenteritis (TGE) is an acute, highly contagious infectious disease caused by porcine transmissible gastroenteritis virus (TGEV) of the Coronaviridae family Coronaviridae. The main clinical symptom of pigs infected with TGEV is vomiting , watery diarrhea and dehydration. TGE was first reported by Dolye and Hutching in the United States in 1946, and then successively reported cases in Europe, America, Asia and other countries, and now it has become a worldwide pig disease. At the same time, the incidence of TGE is high, and pigs of all ages can get the disease, especially piglets within two weeks of age, with a mortality rate as high as 100%. The frequent occurrence of PED has caused serious economic losses...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N15/13C12N15/70A61K39/42A61P31/14C12R1/19
CPCA61K2039/505C07K16/10C07K2317/56C07K2317/622C12N15/70C12N2800/101
Inventor 朱建国张凡庆王苗苗
Owner SHANGHAI JIAO TONG UNIV
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