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African swine fever virus p72 recombinant protein and colloidal gold immunochromatography test paper constructed by same

A technology of African swine fever virus and immunochromatographic test paper, which is applied in the field of virus epidemic diagnosis and animal quarantine

Pending Publication Date: 2021-08-31
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are fewer reports and commercial products on colloidal gold test strips for ASFV diagnosis

Method used

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  • African swine fever virus p72 recombinant protein and colloidal gold immunochromatography test paper constructed by same
  • African swine fever virus p72 recombinant protein and colloidal gold immunochromatography test paper constructed by same
  • African swine fever virus p72 recombinant protein and colloidal gold immunochromatography test paper constructed by same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1p72

[0050] Expression of embodiment 1p72 recombinant protein

[0051] Using the African swine fever virus ASFV-SY18 sequence published in GenBank (GenBank accession number: MH766894) as a template, the p72 gene fragment was synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0052] The following expression primers were designed to amplify the p72 gene:

[0053] Upstream primer: 5'-GAGCTCGGTACCCTCGAGTTAGGTACTGTAACGCAG-3';

[0054] Downstream primer: 5'-CTGCAGGTCGACAAGCTTTCAATGGCATCAGGAGGAGCT-3'.

[0055] PCR amplification system (total system 25 μL) includes:

[0056]

[0057] The specific PCR amplification program includes: 98°C for 5min; 98°C for 40s, 55°C for 40s, 68°C for 3min, 35 cycles; 68°C for 10min.

[0058] After the amplified product was subjected to 1% agarose gel electrophoresis, the Omega gel recovery kit was used for gel recovery, and the gel recovery product was used directly or stored at -20°C for future use.

[0059] Select XhoI and HindIII restriction ...

Embodiment 2

[0067] Embodiment 2 recombinant protein purification

[0068] The p72 recombinant protein expressed in the supernatant in Example 1 was affinity-purified on a nickel column prepacked column and then further purified by molecular sieves. The main operation steps included: taking 10 mL of the protein supernatant and adding it to 800 μL of nickel column, mixing it thoroughly, and placing it at 4°C After incubation for 12 h, centrifuge at 3000 rpm and 4°C for 10 min, discard the supernatant, and wash with 10 mM, 20 mM, 50 mM, and 100 mM imidazole solutions at pH = 7.4, respectively. The amount of each gradient washing solution is 30 mL. After washing, 5 mL of 250 mM imidazole solution with pH=7.4 was used for elution, and the collected eluate was passed through a molecular sieve of a protein purifier for further protein purification.

Embodiment 3

[0069] Example 3 colloidal gold particle labeling purified p72 recombinant protein

[0070] Take 0.01% HAuCl 4 Put 100mL of aqueous solution in a beaker, heat to boil fully, add 1mL of 1% trisodium citrate aqueous solution, continue to heat and boil for 5min, until the solution is orange-red, and the prepared colloidal gold particles are about 30nm, and stored at 4°C.

[0071] Take 500 μL of the above colloidal gold solution, add 1 μL, 2 μL, 4 μL, 8 μL and 16 μL of K at a concentration of 0.2 mol / L, respectively. 2 CO 3 The solution was mixed well, and then 0.4 μg, 0.8 μg, 1.6 μg, 3.2 μg and 6.4 μg of the above-mentioned purified p72 recombinant protein were added respectively, mixed well and then allowed to stand at room temperature for 20 minutes. Continue to add 50 μL of BSA with a concentration of 10%, mix well, block at room temperature for 10 minutes, centrifuge at 4°C, 1000rpm for 10min to remove excess colloidal gold, absorb the supernatant, and centrifuge at 4°C, 75...

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Abstract

The invention belongs to the field of virus and epidemic disease diagnosis and animal quarantine, particularly relates to an African swine fever virus p72 recombinant protein, and further discloses colloidal gold immunochromatography test paper constructed based on the recombinant protein and a method for detecting an African swine fever virus antibody. According to the colloidal gold test paper based on African swine fever virus p72 protein double-antigen sandwich, a recombinant antigen of ASFV strong immunogenicity main structural protein p72 is marked by colloidal gold, and ASFV antibody in porcine serum is detected in a double-antigen sandwich mode. According to the double-antigen sandwich colloidal gold immunochromatography test paper, after a sample is collected, the sample is mixed with a sample diluent according to a certain proportion and then dropwise added into a sample hole, a detection result can be obtained within 5 minutes, the result is accurate, the double-antigen sandwich colloidal gold immunochromatography test paper can be directly used for detecting a virus antibody in suspicious porcine serum, and the double-antigen sandwich colloidal gold immunochromatography test paper is especially suitable for on-site ASFV serological diagnosis, epidemiological investigation and swine international trade quarantine inspection.

Description

technical field [0001] The invention belongs to the field of virus epidemic diagnosis and animal quarantine, and specifically relates to a recombinant protein of African swine fever virus p72, and further discloses a colloidal gold immunochromatographic test paper constructed based on the recombinant protein and a method for detecting antibodies to African swine fever virus. Background technique [0002] African swine fever (ASF) is a highly contagious and fatal infectious disease that infects domestic pigs and various wild boars caused by the African swine fever virus (ASFV). Its morbidity and mortality can be as high as 100%. Organization (OIE) listed as a legally notifiable animal disease, and even listed as a first-class animal disease in my country. The main clinical symptoms of the disease are high fever, extensive hemorrhage of internal organs, respiratory system and nervous system dysfunction. ASFV was first discovered in Kenya in 1921, and then spread to neighboring...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/34G01N33/569G01N33/68G01N33/558G01N33/58G01N33/532
CPCC12N15/70C07K14/005G01N33/56983G01N33/6854G01N33/558G01N33/587G01N33/532C12N2710/12022C12N2710/12051G01N2333/01G01N2469/10
Inventor 杨汉春周信荣赵全红章健李明伟周磊盖新娜韩军张桂红
Owner CHINA AGRI UNIV
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