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

为建立检测血清中非洲猪瘟病毒（African swine fever virus,ASFV）抗体的间接ELISA方法,本试验将ASFV p30基因进行原核表达,采用SDS-PAGE和Western blotting方法对重组蛋白进行表达鉴定和免疫原性分析,随后以纯化的重组蛋白为包被抗原,经条件优化、特异性试验、敏感性试验和重复性试验,建立一种血清中ASFV抗体的检测方法。结果显示,ASFV p30基因成功克隆到原核表达载体pET-32a （+）中,获得pET-32a-p30重组质粒;转化大肠杆菌BL21（DE3）感受态细胞进行诱导表达,得到P30重组蛋白,重组蛋白大小约为42 ku,主要以包涵体形式存在;Western blotting结果显示,纯化后的蛋白具有良好的免疫原性;以纯化的P30重组蛋白为包被抗原,建立了检测ASFV抗体的间接ELISA方法,通过方阵试验对间接ELISA方法进行优化,最终确定了抗原最佳包被浓度为1.2 μg/mL,待检血清最佳稀释倍数为1：100,最佳封闭液为1% BSA,酶标抗体最佳稀释度为1：4 000,以此建立的ASFV间接ELISA方法临界值为0.322。本方法仅与ASFV阳性血清发生特异性反应,与猪瘟病毒、猪繁殖与呼吸综合征病毒、口蹄疫病毒、伪狂犬病病毒、猪圆环病毒2型及猪流行性腹泻病毒阳性血清均无交叉反应,具有较强的特异性。该方法检测ASFV阳性血清灵敏度可达到1：1 600;批内重复性和批间重复性变异系数均<10%。本试验建立的间接ELISA方法具有良好的特异性、灵敏度和重复性,可初步应用于ASFV抗体的检测。

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

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.