pMAL-p2X系统表达番鸭呼肠孤病毒MW9710株σC蛋白

Expression ofσC gene of muscovy duck reovirus,MW9710 strain,by using pMAL-p2X system

根据Genbank中MDRV 89026毒株S4基因序列设计引物,通过RT-PCR获得番鸭呼肠孤病毒MW9710株σC蛋白基因,将此σC蛋白基因插入原核表达载体pMAL-p2X中,得到重组表达质粒(pMAL-p2X-σC)并转化大肠杆菌BL21(DE3)中,经IPTG诱导后能高效表达,SDS-PAGE和Western-blot检测结果发现表达的σC蛋白分子质量约为72 kDa,并以可溶性蛋白和包涵体2种形式同时存在,且具有良好的反应原性。原核表达的可溶性σC蛋白为进一步开展抗原表位和诊断试剂盒研究奠定了基础。

To obtain recombinant Muscovy duck reovirus （MDRV） σC protein by prokaryotic expression for further study on its functions, the encoding sequence of the σC gene from the MDRV MW9710 strain was amplified with RT - PCR and inserted into pMAL - p2X vector to establish the prokaryotic expression plasmid. After transforming the plasmid, pMAL - p2X - σC, into E. coil BL21 （DE3）, the bacteria were induced by IPTG. The protein was expressed efficiently in both soluble and insoluble forms, and was positively recognized by the reference serum. The SDS - PAGE and western - blot analysis showed the recombinant pMAL - p2X - σC produced had an apparent molecular weight of 72KDa. The soluble σC protein was purified and obtained using MBP - affinity chromatography. The result provided a foundation for the gene＇s epitope identification and development of a clinical diagnostic kit in the future.