猪源札幌病毒衣壳蛋白的原核表达及免疫原性检测

Prokaryotic expression and immunogenicity detection of capsid protein of sapporo virus from swine

为获得猪源札幌病毒(SaV)衣壳蛋白(VP1),以SaV CH430株的RNA为模板,采用RT-PCR扩增VP1基因;将其克隆到原核表达载体pET-30a中,并将成功构建的原核表达质粒转化到大肠杆菌BL21(DE3)中进行诱导表达。结果显示,获得的VP1基因全长为1 635bp,编码545个氨基酸。SDS-PAGE结果显示,目的条带大小为63ku,与预期结果相符。重组菌在37℃、IPTG终浓度为0.8mmol/L、诱导表达6h时重组蛋白的表达量达到最大值,表达的目的蛋白主要以包涵体的形式存在。将纯化的VP1重组蛋白免疫家兔,得到了超免疫血清。Western-blot分析表明,该重组蛋白与超免疫血清具有良好的反应原性。上述试验结果为深入研究VP1蛋白的功能和研制ELISA诊断试剂盒奠定了基础。

The present study aims to express the capsid protein(VP1)of sapporo virus(SaV)from swine.Using SaV(CH430)RNA as a template,RT-PCR was utilized to amplify the full-length coding sequence of VP1 gene.The VP1 gene was cloned into pMD19-T vector,and then was subcloned into prokaryotic expression vector pET-30 ato construct the recombinant plasmid pET-30a-SaV-VP1.The recombinant plasmid was transformed into the competent E.coli BL21(DE3)cells for expression.The VP1 gene was 1635 bp in size,encoding 545 amino acids.Induced with IPTG and detected by SDS-PAGE,a 63 ku band was identified,which was as expected.At the same time,the best conditions for expression were studied,and the results indicated that recombinant proteins reached a maximum level under the conditions of 37 ℃,0.8mmol/L IPTG and 6hinduction time.Further tests showed that the target protein in the form of inclusion bodies,which was used to get hyper-immune serum.It turned out that the VP1 gene of SaV capsid protein was stably and efficiently expressed.Western-blot shows that expression products had good reactogenicity with hyper-immune serum.The above-mentioned results paved the way for further studies on functions of VP1 and the development of ELISA kits for detection of sapporo virus.