A kind of African swine fever virus cd2v extracellular domain recombinant protein and its application

A technology of African swine fever virus and recombinant protein, which is applied in the field of ELISA detection kit for detection of African swine fever virus and CD2v ectodomain recombinant protein of African swine fever virus, which can solve the problem of different maintenance time, cumbersome operation and limited application and other issues to achieve the effect of setting the critical value accurately and scientifically and reducing the false positive rate

Active Publication Date: 2022-07-15
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, Chinese patent CN110927390A reports a double-antibody sandwich ELISA based on eukaryotically expressed CD2v full-length protein. This method utilizes recombinant pIRESpuro2 eukaryotic expression vector to transfect into CHO-K1 cells to obtain CD2v full-length protein, although the obtained The protein has protein modification, but the yield is low, the operation is cumbersome, the purity is low, and the application is limited
Moreover, the test results of clinical samples show that the ELISA method established by coating different African swine fever virus proteins to detect the same serum sample will give inconsistent results, which may be due to the different production periods and duration of different proteins No, the existing research has not been able to well understand and grasp the law of the ebb and flow of different protein antibodies, so this phenomenon occurs

Method used

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  • A kind of African swine fever virus cd2v extracellular domain recombinant protein and its application
  • A kind of African swine fever virus cd2v extracellular domain recombinant protein and its application
  • A kind of African swine fever virus cd2v extracellular domain recombinant protein and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1 Amplification of CD2v ectodomain gene and construction of recombinant plasmid

[0059] Using the CD2v gene of the ASFV SY-18 genome published in GenBank as a template, the CD2v ectodomain gene was synthesized by Beijing Qingke Biotechnology Co., Ltd. as a PCR amplification template, and the CD2v ectodomain amplification primers were designed as follows, used to amplify the CD2v ectodomain Gene:

[0060] Upstream primer:

[0061] 5'-TAAGAAGGAGATATACCATGGATTATTGGGTTAGTTTTAATAAAACAATAATTTTAGATAG-3';

[0062] Downstream primers:

[0063] 5'-GTGGTGGTGGTGGTGCTCGAGTTAAGTATAAAAATAGTTAGATGACAATG-3'.

[0064] PCR amplification was performed using Platinum SuperFi II Green PCRmaster mix from Thermo Fisher Scientific. The amplification system includes:

[0065] 2×Platinum SuperFi II Green buffer 12.5μL;

[0066] 5×GC buffer 5μL;

[0067] 1 μL each of upstream primer and downstream primer;

[0068] Template 2.5μL;

[0069] ddH 2 O 3 μL;

[0070] The total reactio...

Embodiment 2CD2

[0074] Example 2 Induced expression, purification and identification of CD2v ectodomain protein

[0075] Induce the expression of CD2v extracellular domain protein by the positive clones with correct sequencing, and wait for the OD of the bacterial solution. 600nm Between 0.6 and 0.8, add isopropyl thiogalactoside (IPTG) with a final concentration of 500 μM, induce 8 h at 37 °C with a shaker at 180 rpm, collect the cells, add sterile PBS to resuspend the cells, and perform ultrasonic disruption. Ultrasonic parameters are: power 30W; launch 5s, intermittent 5s. After sonication, centrifuge at 12,000 rpm and 4°C for 20 min, collect supernatant and precipitate, add 5× protein loading buffer, respectively, for identification by SDS-PAGE protein electrophoresis and western blotting (Western Blot, WB). The correctly identified protein band was developed by 0.3M KCl for 5 minutes, and then the gel was cut, and the recovered gel was purified by electrophoresis in a 3500D dialysis bag...

Embodiment 3

[0077] Example 3 Condition optimization of indirect ELISA coated with CD2v ectodomain protein

[0078] The purified CD2v ectodomain protein in Example 2 was coated by ELISA, and the optimal antigen coating concentration and the best clinical sample to be tested were carried out in a chessboard method (in this example, the antibody positive for African swine fever virus was used. Standard porcine serum instead of clinical serum samples) determination of dilution.

[0079] The antigen concentration gradients were controlled to be 1 μg / mL, 2 μg / mL, 4 μg / mL, and 8 μg / mL, respectively; the dilution gradients of clinical serum samples were 1:50, 1:100, 1:200, and 1:400. Take positive sample OD 450nm Value vs. Negative Sample OD 450nm The antigen coating concentration and clinical serum sample dilution when the value ratio is the largest and the OD value of the positive sample is close to 1 are the optimal antigen coating concentration and the optimal clinical serum sample dilution...

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Abstract

The invention belongs to the field of biotechnology, in particular to an African swine fever virus CD2v extracellular domain recombinant protein, and further discloses its application for constructing ELISA, and the prepared ELISA detection kit for detecting African swine fever virus. The scheme of the present invention constructs a prokaryotic-expressed CD2v extracellular domain recombinant protein based on the characteristics of the virus strains currently prevalent in China. The recombinant protein has high yield, high purity and good reactogenicity, and can induce the body to produce neutralizing antibodies. An ELISA kit for detecting African swine fever virus can be further constructed based on the recombinant protein. The kit has high specificity, sensitivity and reproducibility, and can be used as a reliable detection method for monitoring African swine fever virus antibodies or virus identification.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to an African swine fever virus CD2v extracellular domain recombinant protein, and further discloses its application for constructing ELISA, and the prepared ELISA detection kit for detecting African swine fever virus. Background technique [0002] African swine fever (ASF) is a highly contagious, multi-organ hemorrhagic infectious disease caused by African swine fever virus (ASFV) infection. Domestic pigs and wild boars of all ages are susceptible to , morbidity and mortality can be as high as 100%. African swine fever virus is an arbovirus that can infect a variety of soft ticks and spread the virus through the bite of soft ticks. It has become one of the important pathogens that seriously affects the development of the pig industry, and is listed by the World Organization for Animal Health (OIE). Notifiable animal diseases. African swine fever virus was first discovered in Kenya, A...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/70C07K14/01G01N33/543G01N33/569G01N33/68C12R1/19
CPCC12N15/70C07K14/005G01N33/6854G01N33/56983G01N33/543C12N2710/12022C12N2710/12051G01N2333/01G01N2469/20
Inventor 盖新娜周信荣戴云周磊韩军张永宁张桂红杨汉春
Owner CHINA AGRI UNIV
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