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Swine-derived single-chain antibody resistant to porcine epidemic diarrhea viruses and preparing method thereof

A porcine epidemic diarrhea and single-chain antibody technology, applied in the field of genetic engineering, can solve the problems of inactivation of PED and unsatisfactory effects of attenuated vaccines

Active Publication Date: 2016-12-21
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 resistant to porcine epidemic diarrhea viruses and preparing method thereof
  • Swine-derived single-chain antibody resistant to porcine epidemic diarrhea viruses and preparing method thereof
  • Swine-derived single-chain antibody resistant to porcine epidemic diarrhea viruses and preparing method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] The preparation of the single-chain antibody of embodiment 1 porcine-derived anti-epidemic diarrhea virus

[0026] 1: Detection of porcine epidemic diarrhea virus (PEDV) antibody (PEDV strain preserved in this laboratory) on piglets with diarrhea symptoms in the farm, using routine (refer to F.M. ) ELISA method to detect serum antibody titer greater than 1:20 000, collect the pig blood, lysed red blood cells to obtain white blood cells (erythrocyte lysate Shanghai Biyuntian Biotechnology Co., Ltd.), using the Trizol method (TRIZOL Reagent purchased from Invitrogen, USA) 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; AF064687.1; AF064688.1; AF064689.1; AF064690.1) and light chain variable regio...

Embodiment 2

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

[0042] Take the porcine epidemic diarrhea virus (preserved in the laboratory) concentrated by ultracentrifugation, dilute it to 5 μg / mL with antigen coating solution (50mmol / L sodium bicarbonate salt solution, pH=9.6), add to a 96-well plate, 100 μL per well, Coating overnight; add 200 μL of 5% skimmed milk powder solution to each well, block at 37°C for 1 hour, wash 3 times with PBS; mix 50 μL of purified protein supernatant with 50 μL of 4% skimmed milk powder solution, add to the above wells, and incubate at 37°C for 2 hours , washed 3 times with PBST; added 100 μL (1:2000) of E-Tag Mouse mAb (E-tag mouse monoclonal antibody was purchased from RayBiotech Company), reacted at 37°C for 2 h, washed 3 times with PBST; added hydrogen peroxide-labeled sheep Anti-mouse IgG secondary antibody (purchased from Invitrogen, USA) 1000 μL (1:4000), react at 37°C for 1 h, wash 3 times with PBST; OD 450 When the absorb...

Embodiment 3

[0043] Example 3 sequenced the obtained single-chain antibody coding gene, which proved 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 acids The sequence is shown as SEQ ID No.3.

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Abstract

The invention discloses a swine-derived single-chain antibody resistant to porcine epidemic diarrhea viruses and a preparing method thereof. The swine-derived single-chain antibody resistant to the porcine epidemic diarrhea viruses is formed by connecting a heavy chain variable region VH with a light chain variable region VL of an antibody through short connecting peptide Linker. The amino acid sequence of the light chain variable region VL is shown as SEQ ID No.1, and the amino acid sequence of the heavy chain variable region VH is shown as SEQ ID No.2. The molecular weight of the single-chain antibody resistant to the obtained porcine epidemic diarrhea viruses is about 28 kDa, the porcine epidemic diarrhea viruses can be specifically identified, and the single-chain antibody can be used for further blocking infection and invasion of the porcine epidemic diarrhea viruses.

Description

technical field [0001] The invention relates to a pig-derived single-chain antibody against porcine epidemic diarrhea virus and a preparation method thereof, belonging to the technical field of genetic engineering. Background technique [0002] Porcine epidemic diarrhea (porcine epidemic diarrhea, PED) is an acute, highly contagious infectious disease caused by porcine epidemic diarrhea virus (Porcine epidemic diarrhea virus, PEDV). Pigs infected with PEDV often cause acute enteritis and fatal Watery diarrhea until dehydration death, the main symptoms are vomiting, watery diarrhea and dehydration. At present, PED affects a wide range, and cases have been reported all over the world. At the same time, the incidence of PED is high, the infection rate of young pigs is as high as 100%, and the infection mortality rate of piglets under the age of 7 days is as high as 80-100%. The frequent occurrence of PED has caused serious economic losses to the pig industry in my country. V...

Claims

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Application Information

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IPC IPC(8): C07K16/10C12N15/13C12N15/70C12N1/21C12R1/19
CPCC07K16/10C07K2317/20C07K2317/622C12N15/70C12N2800/101
Inventor 朱建国张凡庆王苗苗
Owner SHANGHAI JIAO TONG UNIV
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