猪瘟病毒Erns和E2蛋白重组表达及抗体间接ELISA检测方法的建立

本研究将CSFV Erns和E2基因的主要抗原区进行克隆,构建pET32a-Erns和pET32aEK-E2重组质粒,将其转化大肠杆菌BL21(DE3)进行诱导表达和纯化,以纯化的rErns、rE2重组蛋白为包被抗原,经条件优化,分别建立CSFV rErns和rE2抗体间接ELISA检测方法。SDS-PAGE结果显示,分别获得约为47 kDa和42 kDa的重组蛋白,rErns重组蛋白主要在上清液中存在,rE2重组蛋白主要以包涵体形式存在。Western blot结果显示,纯化后的重组蛋白具有良好的免疫原性。通过方阵试验进行了ELISA反应条件优化,确定了Erns蛋白最佳包被量为2.5μg/mL,rE2蛋白最佳包被量为0.625μg/mL,待检血清最佳稀释倍数为1∶100,最佳封闭液为0.25%PVA溶液,HRP标记的兔抗猪IgG二抗最佳稀释度为1∶2000,临界值分别为0.24和0.33。上述方法仅与CSFV阳性血清发生特异性反应,检测敏感性可达到1∶1600,批内重复性和批间重复性变异系数均小于10%。本研究建立的间接ELISA方法可初步应用于CSFV Erns和E2抗体的检测,鉴别E2亚单位疫苗免疫抗体和野毒感染抗体,有利于猪瘟净化工作的实施。

Expression of Recombinant Erns and E2 Proteins of CSFV and Development of Indirect ELISA for Detection of Antibodies

In this study,the Erns and E2 genes representing the main antigenic regions of Classical swine fever virus(CSFV)were cloned and pET32a-Erns and pET32aEK-E2 recombinant plasmids were constructed and transformed into E.coli BL21(DE3)for protein expression.The rErns and rE2 proteins were purifi ed and used as coating antigens for development of indirect ELISA assay.The molecular masses of rErns and rE2 proteins were about 47 kDa and 42 kDa representatively as visualized in SDS-PAGE.rErns protein was mainly present in the supernatant and rE2 protein mainly existed in the form of inclusion bodies.Western blot results showed that the purifi ed recombinant protein had good immunogenicity.The ELISA reaction conditions were developed and optimized by square matrix test.The optimum coating amounts were determined to be 2.5μg/mL for rErns protein and 0.625μg/mL for rE2 protein.Other optimized factors included serum dilution at 1:100,0.25%PVA blocking solution,HRP-labeled rabbit anti-porcine IgG dilution at 1:2000 and the critical positive values at 0.24 for rErns protein and 0.33 for rE2 protein.Both ELISA methods reacted specifi cally with CSFV-positive serum and the detection sensitivity reached to 1:1600.In addition,the intra-assay repeatability and inter-assay repeatability coeffi cients of variation were<10%for both methods.The indirect ELISA methods developed in this study could be used for the detection of CSFV Erns and E2 antibodies and differentiation of E2 subunit vaccination antibodies and wild-type infected antibodies,which might be benefi cial to the implementation of eradication program.