非洲猪瘟病毒p30基因的原核表达及间接ELISA抗体检测方法的建立

doi:10.16431/j.cnki.1671-7236.2018.12.029

Abstract

Abstract:

In order to establish an indirect ELISA method for detecting African swine fever virus (ASFV) antibody in serum,p30 gene of ASFV was expressed in prokaryotic system,the expression and immunogenicity of recombinant protein were identified by SDS-PAGE and Western blotting,respectively.Subsequently,the purified recombinant protein was used as a coating antigen,and a method for the detection of ASFV antibodies in serum was established condition optimization,specificity test,sensitivity test and repetitive test.The results showed that the ASFV p30 gene was successfully cloned into the prokaryotic expression vector pET-32a(+),and the recombinant plasmid pET-32a-p30 was obtained.The recombinant plasmid was transformed into E.coli BL21 (DE3) for expression,and the recombinant protein was obtained with 42 ku,it was mainly in the form of inclusion bodies.Western blotting results showed that the purified protein had good immunogenicity.An indirect ELISA method for detecting ASFV antibody was established by using purified P30 recombinant protein as coating antigen.The indirect ELISA method was optimized by square matrix test,and the optimal coating concentration of antigen was determined to be 1.2 μg/mL.The optimal dilution factor of the serum to be tested was 1:100,the optimal blocking solution was 1% BSA,and the optimal dilution of the enzyme-labeled antibody was 1:4 000.The established ASFV indirect ELISA method had a cut-off value of 0.322.The method showed high specificity,only specifically reacted with ASFV-positive serum,and had no cross-reaction with classical swine fever virus,porcine reproductive and respiratory syndrome virus,foot-and-mouth disease virus,pseudorabies virus,porcine circovirus type 2 and porcine epidemic diarrhea virus positive serum.The sensitivity of the method for detecting ASFV positive serum could reach 1:1 600;The intra-assay repeatability and the inter-assay repeatability coefficient of variation were both <10%.The established indirect ELISA method had good specificity,sensitivity and reproducibility,and could be initially applied to the detection of ASFV antibodies.

Key words: African swine fever (ASFV); p30 gene; prokaryotic expression; recombinant protein; indirect ELISA

CLC Number:

S852.65⁺1

WU Jing, WANG Xixi, WU Yingtong, REN Xiao, GUO Xiaoyu. Prokaryotic Expression of p30 Gene of African Swine Fever Virus and Establishment of Indirect ELISA Antibody Detection Method[J]. , 2018, 45(12): 3555-3562.

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Prokaryotic Expression of p30 Gene of African Swine Fever Virus and Establishment of Indirect ELISA Antibody Detection Method

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