猪流行性腹泻病毒COE基因的原核表达及其表达产物的生物活性分析

Prokaryotic expression of porcine epidemic diarrhea virus COE gene and biological activity analysis of the expressed protein

为了获得猪流行性腹泻病毒(PEDV)COE基因在原核表达载体pET-32a(+)中的表达产物,并检测表达产物的生物活性,根据猪流行性腹泻病毒COE基因的全序列设计引物,以pCMV-PEDVCOE质粒作为模板,通过PCR扩增得到COE基因编码区的序列,将其克隆至含有6×His tag的原核表达载体pET-32a(+)中进行诱导表达。表达产物经SDS-PAGE分析和Western-blot鉴定,并用Ni-NTA亲和层析柱纯化,以此纯化产物为包被抗原建立了ELISA检测方法,并用该方法对39份感染PEDV猪血清进行了检测。结果显示,COE基因在大肠杆菌中经IPTG诱导表达,获得与预期分子质量相符的融合蛋白条带。经Western-blot检测,该蛋白能与Anti-PEDV单克隆抗体发生特异性反应。该融合蛋白纯化、复性后的质量浓度为0.7mg/mL。采用以融合COE蛋白为包被抗原建立的ELISA对39份PEDV感染猪血清的检测结果都为阳性,说明该融合蛋白具备作为包被抗原建立ELISA抗体检测方法的潜力。

In order to obtain the prokaryotic expression products and detect biological activity of the expressed protein,COE gene of porcine epidemic diarrhea virus(PEDV)was amplified by PCR method from plasmid pCMV-PEDVCOE,cloned into pET-32a(+)vector and expressed with IPTG induction.The fusion COE protein was detected by SDS-PAGE and Western-blot and purified by Ni-NTA affinity chromatography.ELISA detection method was established using the purified product as coating antigen.Thirtynine PEDV-infected pig sera were detected.SDS-PAGE and Western-blot showed that the COE gene had been successfully expressed in Escherichia coli with IPTG induction and the expressed protein could specifically react with anti-PEDV monoclonal antibody.The mass concentration of fusion protein was 0.7mg/mL after purification and dialysis.ELISA results showed that 39PEDV-infected swine sera were positive,indicating that the recombinant protein could be used as coating antigen to establish ELISA antibody detection method.