猪流行性腹泻病毒M和S蛋白的原核表达及多克隆抗体的制备

Prokaryotic expression of the M and S proteins of PEDV and preparation of their polyclonal antibodies

采用PCR方法对猪流行性腹泻病毒(PEDV)M蛋白和S蛋白基因的亲水区进行扩增,扩增片段(命名为Ma和Sa)分别克隆至原核表达载体pET-32a(+)和pGEX-6p-1中,获得的重组质粒pET-32a-Ma和pGEX-6p-1-Sa转化大肠杆菌BL21,经IPTG诱导表达后进行SDS-PAGE及Westernblot鉴定。表达产物经SDS-PAGE后切胶免疫BALB/c小鼠制备多克隆抗体,间接ELISA方法检测小鼠免疫后血清效价,Westernblot和间接免疫荧光方法检测抗体的特异性。结果表明目标蛋白获得了正确表达,且制备的多克隆抗体具有较高的敏感性和特异性。PEDV Ma和Sa蛋白的表达及多克隆抗体的制备将为进一步构建PEDV的病毒样颗粒(VLP)抗原和建立血清学检测方法奠定基础。

Porcine epidemic diarrhea （PED） , an important swine disease causing high morbidity and mortality in neonatal piglets, have caused tremendous economic losses to swine industry in China, so the development of new type vaccines and establishment of serological detection methods are of importance. The hydrophilic gene regions of M and S proteins （ named Ma and Sa） of PEDV were amplified by PCR, then cloned into the prokaryotic expression vector pET-32a （＋） and pGEX-6p-1, respectively. The obtained recombinant plasmid pET- 32a-Ma and pGEX-6p-1-Sa were transformed into E. coli BL-21 strain, induced by IPTG and detected by SDS-PAGE and Western blot. BALB/C mice were immunized with the recombinant proteins cut from SDS-PAGE gel to get polyclonal antibodies. Indirect ELISA, Western blot and indirect immunofluorescence assay （IFA） were used to detect serum titers and specificity of the antibodies. The results showed that the polyclonal antibodies have high sensitivity and specificity. The expression of PEDV Ma and Sa and preparation of their polyclonal antibodies laid the foundation for constructing virus-like particles （VLPs） of PEDV and developing serological detection methods.