猪流行性腹泻病毒流行毒株S1蛋白的原核表达与鉴定

Prokaryotic expression and identification of S1 protein of prevalent strain of Porcine epidemic diarrhea virus

为了获得具有良好活性的猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)流行毒株重组蛋白,试验采用PCR扩增、克隆、诱导表达、Western-blot检测、间接ELISA等方法对PEDV流行毒株S1蛋白的主要抗原区域进行了原核表达、鉴定与初步应用研究。结果表明:经PCR扩增得到大小为1 095 bp的S1蛋白主要抗原区域基因;将目的片段定向克隆至pET-30a原核表达载体中,经IPTG诱导获得大小为48. 0 ku的重组蛋白;Western-blot检测显示,重组蛋白可与PEDV阳性血清发生特异性反应;用以重组蛋白为包被抗原初步建立的PEDV抗体间接ELISA方法检测PEDV感染猪场,其阳性感染率达98. 52%,无PEDV免疫史和感染史猪场的阳性感染率为0。说明试验获得了具有良好生物学活性的PEDV流行毒株重组蛋白。

In order to obtain the recombinant protein of porcine diarrhea virus(PEDV)prevalent strain with good activity,those methods were used in this experiment including PCR amplification,cloning,inducing expression,Western-blot and indirect ELISA,for the research on the prokaryotic expression,identification and preliminary application of S1 protein major antigen region of PEDV prevalent strain.The results showed that the major antigen region of S1 protein with a size of 1 095 bp were amplified by PCR.The target fragments were cloned into pET-30 a prokaryotic expression vector and the recombinant protein with a size of 48.0 ku was obtained by IPTG induction.Western-blot detection showed that the recombinant protein had a specific reaction with PEDV positive sera.The indirect ELISA method established using the recombinant protein as the coating antigen,was applied in PEDV-infected pig farms for detecting PEDV antibodies.The positive rate of PEDV antibodies was98.52%,and that of pig farms with no history of PEDV immunization or infection was 0.The results suggested that S1 recombinant protein of PEDV prevalent strain with good biological activity was obtained in this experiment.