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African swine fever virus CD2v extracellular domain recombinant protein and application thereof

A technology of African swine fever virus and recombinant protein, applied in the field of African swine fever virus CD2v ectodomain recombinant protein, ELISA detection kit for detection of African swine fever virus, can solve the problems of cumbersome operation, limited application, inconsistency, etc.

Active Publication Date: 2021-06-01
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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  • African swine fever virus CD2v extracellular domain recombinant protein and application thereof
  • African swine fever virus CD2v extracellular domain recombinant protein and application thereof
  • African swine fever virus CD2v extracellular domain recombinant protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1 Amplification of CD2v Extracellular Domain Gene and Construction of Recombinant Plasmid

[0059] Using the ASFV SY-18 genome CD2v gene 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 to amplify the CD2v ectodomain Gene:

[0060] Upstream primers:

[0061] 5'-TAAGAAGGAGATATACCATGGATTATTGGGTTAGTTTTTAATAAACAATAATTTTAGATAG-3';

[0062] Downstream primers:

[0063] 5'-GTGGTGGTGGTGGTGCTCGAGTTAAGTATAAAAATAGTTAGATGACAATG-3'.

[0064] Platinum SuperFi II Green PCRmaster mix from Thermo Fisher Scientific was used for PCR amplification, and the amplification system included:

[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 2.5 μL;

[0069] wxya 2 O 3 μL;

[0070] The total volume of ...

Embodiment 2CD2

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

[0075] Induce the expression of CD2v ectodomain protein on the positive clones with correct sequencing, and wait for the bacterial solution OD 600nm Add isopropylthiogalactopyranoside (IPTG) with a final concentration of 500 μM between 0.6 and 0.8, induce 8 hours on a shaker at 37°C at 180 rpm, collect the bacteria, add sterile PBS to resuspend the bacteria, and perform ultrasonic disruption. Ultrasonic parameters are: power 30W; emission 5s, intermittent 5s. After the sonication, centrifuge at 12000rpm and 4°C for 20min, collect the supernatant and precipitate, add 5× protein loading buffer respectively for SDS-PAGE protein electrophoresis and western blot (Western Blot, WB) identification. The correctly identified protein bands were developed with 0.3M KCl for 5 minutes, and then the gel was cut. The recovered gel was purified by electrophoresis in a 3500D dialysis ba...

Embodiment 3

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

[0078] The CD2v ectodomain protein purified in Example 2 was coated with ELISA, and the optimal antigen coating concentration and the optimal clinical test sample were carried out in the mode of checkerboard method (in this embodiment, the antibody-positive African swine fever virus Determination of dilution of standard pig serum instead of clinical serum samples).

[0079] The antigen concentration gradients were controlled at 1 μg / mL, 2 μg / mL, 4 μg / mL, and 8 μg / mL; the dilution gradients of clinical serum samples were 1:50, 1:100, 1:200, and 1:400. Positive sample OD 450nm Value and negative sample OD 450nm The antigen coating concentration and clinical serum sample dilution when the value ratio is the largest and the positive sample OD value is close to 1 are the best antigen coating concentration and the best clinical serum sample dilution. The results are shown in T...

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Abstract

The invention belongs to the technical field of biology, particularly relates to an African swine fever virus CD2v extracellular domain recombinant protein, and further discloses the application of the African swine fever virus CD2v extracellular domain recombinant protein to construction of ELISA (enzyme-linked immuno sorbent assay) and an ELISA detection kit prepared from the African swine fever virus CD2v extracellular domain recombinant protein and used for detecting the African swine fever virus. According to the scheme, prokaryotic expression CD2v extracellular domain recombinant protein is constructed based on the characteristics of strains popular in China at present, the recombinant protein has high acquisition amount, high purity and good reactogenicity and can induce an organism to generate a neutralizing antibody, and an ELISA kit for detecting the African swine fever virus can be further constructed based on the recombinant protein. The kit has high specificity, sensitivity and repeatability, and can be used as a reliable detection method for monitoring the African swine fever virus antibody or recognizing the virus.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to an African swine fever virus CD2v ectodomain recombinant protein, and further discloses its application for constructing an ELISA, and the prepared ELISA detection kit for detecting the 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), domestic pigs and wild boars of all ages are susceptible , the morbidity and mortality can be as high as 100%. African swine fever virus is an arbovirus, which can infect a variety of soft ticks and transmit the virus through the bites of soft ticks. It has become one of the important pathogens that seriously affect the development of the pig industry. The World Organization for Animal Health (OIE) listed it as Notifiable animal diseases. African swine fever virus was first discovered in Kenya, Afri...

Claims

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Application Information

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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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