猪瘟病毒E2蛋白主要抗原区的原核表达及其间接ELISA检测方法的建立与应用

采用RT-PCR方法扩增猪瘟病毒(CSFV)E2蛋白主要抗原区基因,克隆入原核表达载体pET32a(+),转化大肠埃希菌BL21构建重组表达菌,经SDS-PAGE和Western blot鉴定目的蛋白得到成功表达。利用纯化的重组蛋白作为包被抗原,通过反应条件优化,建立了间接ELISA抗体检测方法。ELISA抗原最适包被浓度为0.5μg/mL(0.05μg/孔),最佳封闭液为5g/L BSA,待检血清最适稀释度为1∶100,作用时间为1h,酶标抗体最适稀释度为1∶2 000,最适作用时间为45min,室温显色10min。用该方法检测牛病毒性腹泻病毒(BVDV)、猪圆环病毒2型(PCV-2)、猪繁殖与呼吸综合征病毒(PRRSV)、伪狂犬病病毒(PRV)阳性血清结果均为阴性;批内和批间重复试验变异系数分别<5%和<8%,表明本方法具有较好的特异性和重复性。应用△E2-ELISA对350份血清样品进行检测,阳性率为77.71%,高于IDEXX试剂盒检测阳性率(67.14%),与IDEXX试剂盒阳性符合率91.06%,阴性符合率50%,总符合率77.43%。表明本试验建立的间接ELISA方法适于临床CSFV血清抗体的检测。

Prokaryotic Expression of the Main Antigenic Region of CSFV E2 and Establishment and Application of an Indirect ELISA for Serum Antibody Detection

The main antigenic region gene of CSFV E2 protein was amplified by RT-PCR,and cloned into pET32a(+).The recombinant bacteria were obtained by transforming BL21 with positive recombinant plasmid.SDS-PAGE and Western blot analysis showed that recombinant protein was expressed successfully.An indirect ELISA was developed for detection of antibodies to CSFV with purified recombinant E2 protein.The optimal concentration of coating antigen was 0.5 μg/mL(0.05 μg/well).The sealing buffer was 5 g/L BSA.The serum sample was diluted by 1∶100 and incubated for 1h at 37 ℃.HRP conjugated rabbit anti-pig IgG was diluted by 1∶2 000 and the reaction time was 45 min at 37 ℃.Colour development was performed at room temperature for 10 min before terminated with stopping solution.Coating antigen had no cross reaction with the antibodies against BVDV,PCV-2,PRRSV and PRV.The intra-batch and inter-batch variation coefficients were lower than 5% and 8%,respectively.350 serum samples were tested with the △E2-ELISA and the positive rate was 77.71%,which was higher than that of IDEXX ELISA kit(67.14%).The positive coincidence rate was 91.06%,the negative coincidence rate was 50%,and the total coincidence rate was 77.43%.It indicated that the △E2-ELISA is suitable for large-scale epidemiological investigation for CSFV infection.