Eukaryotic expression single-chain antibody of porcine epidemic diarrhea virus N protein, and preparation method and application thereof

A porcine epidemic diarrhea and single-chain antibody technology, applied in the field of genetic engineering, can solve the problems of rapid mutation, economic loss, and high incidence of PEDV

Active Publication Date: 2021-11-23
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

PED is highly contagious and has a high incidence rate. The mortality rate of newborn piglets after infection is even as high as 100%. The disease has broken out in many countries around the world, causing huge economic losses and affecting the development of the pig industry all over the world.
Vaccination is an effective way to prevent viral diseases, but due to the rapid mutation of PEDV, commercial vaccines are not ideal for the prevention of mutant strains

Method used

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  • Eukaryotic expression single-chain antibody of porcine epidemic diarrhea virus N protein, and preparation method and application thereof
  • Eukaryotic expression single-chain antibody of porcine epidemic diarrhea virus N protein, and preparation method and application thereof
  • Eukaryotic expression single-chain antibody of porcine epidemic diarrhea virus N protein, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Construction of swine-derived anti-porcine epidemic diarrhea virus N protein single-chain antibody library, including:

[0034] Step 1. Immunize pigs with recombinantly expressed porcine epidemic diarrhea virus N protein. When the serum antibody titer reaches 1:64000 detected by indirect ELISA, the spleen tissue of the pig is taken and the total RNA is extracted by Trizol method (TRIzol reagent is purchased from Invitrogen Company). . Using the extracted total RNA as a template, using Oligo primer, according to the operation steps of the product instructions of the reverse transcription kit (reverse transcription cDNA synthesis kit purchased from TaKaRa Company), cDNA was synthesized.

[0035] Step 2. Analyze the variable region sequence of the porcine antibody coding gene in the published literature, and design primers for amplifying the variable region of the heavy chain and light chain of the antibody according to the sequence, as shown in Table 1), where VH-B and VH...

Embodiment 2

[0044] Indirect ELISA screening of porcine-derived single-chain antibody against porcine epidemic diarrhea virus N protein, specifically: coating the purified recombinantly expressed porcine epidemic diarrhea virus N protein in a 96-well plate overnight at 4°C; Blocked with 4% BSA-containing blocking solution at 37°C for 2h; washed 3 times with PBST buffer, added the periplasmic luminal protein of the extracted colonies to each well of the 96-well plate, reacted at 37°C for 2h; washed 3 times with PBST buffer, Add anti-Myctag mouse monoclonal antibody 100 μL (1:3000 dilution), react at 37°C for 2 h; wash 3 times with PBST buffer, add HRP-labeled goat anti-mouse IgG (H+L) 100 μL (1:6000 dilution), React at 37°C for 1 h; wash with PBST buffer for 3 times, add TMB chromogenic solution, and protect from light for 15 min; add 2 mol / L sulfuric acid to stop the reaction, and read the OD450 value with a microplate reader. The periplasmic luminal protein extracted from the empty plasmi...

Embodiment 3

[0046] Sequence analysis of the recombinant scFv, specifically: performing DNA sequencing on the obtained positive cloned single-chain antibody encoding gene to prove that it consists of 723 nucleotides and 241 amino acids deduced accordingly, and the nucleotide sequence is as shown in SEQ ID As shown in No.4, the amino acid sequence is shown in SEQ ID No.3.

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Abstract

The invention provides a eukaryotic expression single-chain antibody of porcine epidemic diarrhea virus N protein, and a preparation method and application thereof. The eukaryotic expression single-chain antibody comprises a porcine single-chain antibody fragment VH-Linker-VL, wherein the amino acid sequence of a porcine antibody heavy chain variable region VH is shown as SEQ ID No. 1, the amino acid sequence of a porcine antibody light chain variable region VL is shown as SEQ ID No. 2, and Linker is intermediate connecting peptide. The eukaryotic expression single-chain antibody can be specifically combined with the porcine epidemic diarrhea virus N protein, can inhibit the replication of porcine epidemic diarrhea virus in host cells, and can be used for the research of preparing a medicine for preventing and treating porcine epidemic diarrhea.

Description

technical field [0001] The invention relates to a technology in the field of genetic engineering, in particular to a porcine-derived anti-porcine epidemic diarrhea virus N protein eukaryotic expression single-chain antibody and a preparation method and application thereof. Background technique [0002] Porcine epidemic diarrhea (PED) is an intestinal disease caused by porcine epidemic diarrhea virus (PEDV) infection in pigs with clinical manifestations of diarrhea, vomiting, dehydration and even death. PED is highly contagious and has a high morbidity rate. The mortality rate of infected newborn piglets is even as high as 100%. The disease has broken out in many countries around the world, causing huge economic losses and affecting the development of the pig industry around the world. Vaccination is an effective method to prevent viral diseases, but due to the rapid mutation rate of PEDV, the preventive effect of commercial vaccines on mutant strains is not ideal. SUMMARY ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N15/13C12N15/85A61K39/42A61P31/14
CPCC07K16/10C12N15/85A61P31/14C07K2317/56C07K2317/622C12N2800/107A61K2039/505Y02A50/30
Inventor 朱建国王凤青张蕾程曼玲
Owner SHANGHAI JIAO TONG UNIV
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