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African swine fever virus synthetic peptide ELISA antibody detection kit

A technology of African swine fever virus and kit, which is applied in the field of African swine fever virus synthetic peptide ELISA antibody detection kit, can solve the problems of unsuccessfully developing ASF, and achieve less antigen consumption, high sensitivity, and high purity Effect

Active Publication Date: 2021-07-06
CHINA ANIMAL HUSBANDRY IND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is currently no vaccine available against ASF, and in fact an effective commercial ASF vaccine has never been successfully developed

Method used

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  • African swine fever virus synthetic peptide ELISA antibody detection kit
  • African swine fever virus synthetic peptide ELISA antibody detection kit
  • African swine fever virus synthetic peptide ELISA antibody detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1, Preparation of Antibody Detection Kit for African Swine Fever Virus Synthetic Peptide ELISA Coating Antigen This test uses bioinformatics methods to analyze the p30, p54 and p72 of the Pig / HLJ / 2018 strain sequence (MK333180.1) published by GenBank The main antigenic epitopes of the protein were accurately analyzed, and suitable peptides were screened out, and three peptides were synthesized with a fully automatic peptide synthesizer. 80% of the coated antigen can cover the main neutralizing epitope of African swine fever virus, increasing the detection rate of antibody positive. The polypeptide synthesis method can be a conventional method. The following method is used in the present invention to synthesize the three polypeptides of the present invention as the coating agent of the kit of the present invention.

[0043] The coated antigen of the present invention can be prepared using an Applied Biosystem automatic polypeptide synthesizer (model 433A). Using...

Embodiment 2

[0072] Embodiment 2, the preparation of the positive control serum of African swine fever virus synthetic peptide ELISA antibody detection kit

[0073] 1. Construction of prokaryotic expression vector According to the Pig / HLJ / 2018 strain sequence (MK333180.1) published by GenBank, it was synthesized by Shanghai Jierui Bioengineering Co., Ltd. to express p30 (sequence 4), p54 (sequence 5) and The recombinant expression vectors of the p72 (SEQ ID NO: 6) gene are named pET-28a-p30, pET-28a-p54 and pET-28a-p72, respectively.

[0074] 2. Transform the above pET-28a-p30, pET-28a-p54 and pET-28a-p72 recombinant expression vectors into Escherichia coli BL21DE3 competent cells to obtain the corresponding expression strains, and use the Escherichia coli expression system to express ASFVp30, p54 and p72 protein, the expression method is: culture the recombinant expression bacteria in LB medium to OD 600 When =0.6~0.8, add the inducer isopropyl-β-D-thiogalactoside (IPTG) with a final con...

Embodiment 3

[0076] Embodiment 3, the preparation of African swine fever virus synthetic peptide ELISA antibody detection kit

[0077] African swine fever virus synthetic peptide ELISA antibody detection kit includes:

[0078] (1) A 96-well detachable polystyrene enzyme-linked reaction plate coated with a synthetic peptide antigen of African swine fever virus; 2×96 wells.

[0079] (2) Positive control serum: the hyperimmune serum prepared by immunizing the main antigens of African swine fever virus p30, p54 and p72 recombinant protein was diluted with sample diluent and used as the positive control serum of the kit (1 tube, 1.5ml / tube ).

[0080] (3) Negative control serum: specific pathogen-free (SPF) pig serum, used as the negative control serum of the kit (1 tube, 1.5ml / tube).

[0081] (4) Enzyme-labeled secondary antibody: prepared by diluting 1:30000 with horseradish peroxidase-labeled rabbit anti-pig IgG (purchased from sigma company, product number A5670) as the stock solution, 2 bo...

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Abstract

The invention discloses an African swine fever virus synthetic peptide ELISA antibody detection kit. The kit includes enzyme-labeled plate, positive control serum, negative control serum, enzyme-labeled secondary antibody, sample diluent, 20 times concentrated washing solution, substrate solution A, substrate solution B, stop solution, wherein the enzyme-labeled plate The polypeptide composition coated with African swine fever virus antigen epitope. The antigen epitope polypeptide composition is one or any combination of the polypeptide shown in sequence 1 in the sequence listing, the polypeptide shown in sequence 2 in the sequence listing, and the polypeptide shown in sequence 3 in the sequence listing . This kit uses chemically synthesized antigen peptides to coat the microtiter plate, with less antigen consumption, high sensitivity and specificity, and can efficiently detect whether there are antibodies infected with African swine fever virus. The kit of the invention has high sensitivity, good specificity, convenient operation and good market prospect.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and more specifically, the invention relates to an African swine fever virus synthetic peptide ELISA antibody detection kit. Background technique [0002] The causative agent of African swine fever (ASF) is African swine fever virus (ASFV), which belongs to the genus Asfivirus of the African swine fever virus family (Asfarviridae). ASFV is an enveloped virus with an average particle diameter of about 200nm; the virus genome is a double-stranded DNA molecule with a length of 170-193kb, encoding more than 150 polypeptides, at least 50 of which constitute different structural domains of the virus particle. The 80nm viral core contains the viral genome and nucleoproteins p10 and pA104R, and the viral core is wrapped by a viral capsid composed of p35, p15, p150, p37, p34 and p14 proteins; surrounding the viral capsid are two layers of lipid molecules, the inner The outer envelope consist...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/01G01N33/569
CPCC07K14/005C12N2710/12022G01N33/56983G01N2333/01
Inventor 张蕾董春娜吴冬荀李静肖进李玲王飞向王震李鹏宇张欣齐鹏
Owner CHINA ANIMAL HUSBANDRY IND
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