A kind of African swine fever virus recombinant antigen and its application

A technology of African swine fever virus and recombinant antigen, which is applied in the field of genetic engineering, can solve problems such as difficult to meet the needs of the grassroots, and achieve the effect of improving specificity, good diagnostic sensitivity and high specificity

Active Publication Date: 2021-07-16
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the laboratory diagnosis of African swine fever includes animal inoculation, virus isolation, virus nucleic acid DNA detection and specific antibody detection, among which animal inoculation, virus isolation and virus nucleic acid detection need to be conducted by professionals in laboratories above biosafety level 3 The operation is in progress, it is difficult to meet the needs of the grassroots

Method used

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  • A kind of African swine fever virus recombinant antigen and its application
  • A kind of African swine fever virus recombinant antigen and its application
  • A kind of African swine fever virus recombinant antigen and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Preparation of m35 protein

[0041] The amino acid sequences of p30 and p54 proteins of African swine fever virus were obtained from GenBank, and their antigenic epitopes were predicted by online software ABCpredPrediction, Scratch, NetCTL and IEDB, and the restriction endonucleases BamH I and EcoR I were selected to introduce flexible cleavage sites. Chain connection sequence, optimized to obtain SEQID NO: 2;

[0042] Send the gene sequence to GenScript to construct plasmid pET-28a(+)-m35;

[0043] 1. Inducible expression of m35 recombinant protein

[0044] The pET-28a(+)-m35 vector was transformed into E. coli BL21, spread on a plate, and cultured at 37°C overnight; a single colony was picked, added to 5 mL of LB liquid medium containing Amp, and shaken at 37°C overnight; according to 1: The cultured bacterial liquid was inoculated into 5 mL of LB liquid medium containing Amp resistance at a ratio of 100, and shaken at 37 °C to the bacterial liquid OD. 60...

Embodiment 2

[0060] The establishment of embodiment 2ELISA optimal reaction method

[0061] 1. Determination of optimal antigen coating concentration and optimal antibody dilution

[0062] (1) Dilute the antigen (purified m35) with PBS buffer, use Costar microtiter plate (Costar 42582USA) to coat and detect the diluted antigen, and select the concentration of 1, 0.5, 0.25, 0.125, 0.0625 μg / ml The diluted antigen was kept overnight at 4°C, blocked with 1% BSA at 37°C for 2 hours on the second day, and the liquid was discarded and patted dry.

[0063] (2) Adding samples: Take the ELISA plate coated with African swine fever specific recombinant antigen m35 in step (1), and add 1 / 50, 1 / 100, and 1 / 200 dilutions to the detection well and its duplicate well, respectively. Standard positive serum (serum infected with African swine fever is a positive control) and non-infected negative serum (serum not infected with African swine fever is a negative control), react at 37°C for 60 minutes in the da...

Embodiment 3

[0091] Embodiment 3ELISA kit

[0092] The kit includes: an enzyme-labeled plate coated with antigen standard solution, an HRP-labeled enzyme-labeled secondary antibody and other matching reagents; the enzyme-labeled detection antibody is anti-pig IgG (Sigma SAB3700434-2MG) with HRP-labeled enzyme-labeled secondary antibody );

[0093] The kit also includes sample diluent, washing solution and stop solution;

[0094] The diluent is PBS buffer solution with a pH of 7.4;

[0095] The washing solution is a phosphate buffer containing 0.05% Tween-20;

[0096] The stop solution is 2M sulfuric acid solution;

[0097] Enzyme substrate solution chromogenic solution TMB (Surmodics TMBW-1000-01);

[0098] The positive control is the positive serum of pigs artificially infected with African swine fever in the laboratory;

[0099] The negative control was the serum of pig control group not infected with African swine fever in the laboratory.

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Abstract

The invention relates to an African swine fever virus recombinant antigen and its application, belonging to the technical field of genetic engineering. The m35 recombinant antigen is formed by cascading epitopes selected from the African swine fever virus protein p30 and p54 antigen proteins. The sequence is shown in SEQ ID NO:1, and the nucleotide sequence is shown in SEQ ID NO:2. The recombinant antigen m35 has immune reaction with positive pig serum, can be used as an immune recognition antigen, can be used for the diagnosis of African swine fever virus disease in pigs, and has good diagnostic sensitivity, high specificity and high reliability.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, in particular to an African swine fever virus recombinant antigen and its application. Background technique [0002] African swine fever is an acute, severe and highly lethal infectious disease of pigs caused by African swine fever virus. The World Organization for Animal Health (OIE) lists it as a notifiable animal disease, and my country lists it as a Class I animal disease. Since there is no commercial vaccine available so far, the breeding process mainly relies on the purification and prevention of pig farms. Therefore, the establishment of a low-cost, high-sensitivity, and specific rapid test is particularly important for the effective prevention and control of ASF. [0003] At present, the laboratory diagnosis of African swine fever includes animal inoculation, virus isolation, viral nucleic acid DNA detection and specific antibody detection, among which animal inoculation, vi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/01C12N15/34G01N33/569
CPCC07K14/005C12N2710/12022G01N33/56983G01N2333/01
Inventor 常惠芸高瞻邵军军常艳燕
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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