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Epitope antigen polypeptide of African swine fever virus and application of epitope antigen polypeptide of African swine fever virus

A technology of African swine fever virus and antigen epitope, 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 low antigen consumption, high sensitivity and high purity Effect

Active Publication Date: 2019-12-27
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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  • Epitope antigen polypeptide of African swine fever virus and application of epitope antigen polypeptide of African swine fever virus
  • Epitope antigen polypeptide of African swine fever virus and application of epitope antigen polypeptide of African swine fever virus
  • Epitope antigen polypeptide of African swine fever virus and application of epitope antigen polypeptide of African swine fever virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1, Preparation of African swine fever virus synthetic peptide ELISA antibody detection kit coated antigen

[0045] In this experiment, bioinformatics methods were used to accurately analyze the main epitopes of the p30, p54 and p72 proteins of the Pig / HLJ / 2018 strain sequence (MK333180.1) published by GenBank, and to screen out suitable peptides. Three peptides were synthesized by the peptide synthesizer, and the sequences were shown in sequence 1, sequence 2, and sequence 3 in the sequence table, and the coated antigen with a purity of about 80% was produced, which can cover the main neutralizing epitope of African swine fever virus , improve 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.

[0046] The coated antigen of the present in...

Embodiment 2

[0075] Embodiment 2, the preparation of the positive control serum of African swine fever virus synthetic peptide ELISA antibody detection kit

[0076] 1. Construction of prokaryotic expression vector According to the Pig / HLJ / 2018 strain sequence (MK333180.1) published by GenBank, p30 (sequence 4), p54 (sequence 5) and p72 were synthesized and expressed by Shanghai Jierui Bioengineering Co., Ltd. The recombinant expression vectors of the gene (SEQ ID NO: 6) are named pET-28a-p30, pET-28a-p54 and pET-28a-p72 respectively;

[0077] 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

[0079] Embodiment 3, the preparation of African swine fever virus synthetic peptide ELISA antibody detection kit

[0080]African swine fever virus synthetic peptide ELISA antibody detection kit includes:

[0081] (1) A 96-well detachable polystyrene enzyme-linked reaction plate coated with a synthetic peptide antigen of African swine fever virus; 2×96 wells.

[0082] (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 ).

[0083] (3) Negative control serum: specific pathogen-free (SPF) pig serum, used as the negative control serum of the kit (1 tube, 1.5ml / tube).

[0084] (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 b...

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Abstract

The invention discloses an epitope antigen polypeptide of an African swine fever virus and an application of the epitope antigen polypeptide of the African swine fever virus. The epitope antigen polypeptide of the African swine fever virus is a polypeptide as shown in a sequence 2 or a polypeptide as shown in a sequence 3 in a sequence table. A composition of the epitope antigen polypeptide is thecomposition formed by the polypeptide as shown in the sequence 2 in the sequence table and the polypeptide as shown in the sequence 3 in the sequence table. According to the epitope antigen polypeptide or the composition thereof disclosed by the invention, a detection kit is prepared by coating an elisa plate with a chemically synthesized antigen peptide, so that the epitope antigen polypeptide is small in amount of antigen, high in sensitivity and high in specificity; whether an African swine fever virus antibody exists or not can be efficiently detected; and the kit has the advantages of being high in sensitivity, good in specificity and convenient to operate, and has a 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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IPC IPC(8): G01N33/68G01N33/569
CPCG01N33/6854G01N33/56983G01N2333/01G01N2469/20
Inventor 董春娜张蕾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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