【摘要】 用RT-PCR扩增鸭源坦布苏病毒YY5株的E蛋白基因片段,分别插入到表达载体pMBP-c、pET-GST、pET-His、pET-DsbA中,构建了一系列原核表达载体,转化人大肠杆菌E.coli Trans10或DE3(plysS)宿主菌。工程菌株经诱导后进行SDS-PAGE,结果显示MBP-E融合蛋白得到了有效表达并且以可溶性蛋白的形式存在,且能与自然感染康复的鸭源多抗和鼠源单抗反应。以MBP-E免疫小鼠制备的高免血清,在体外可以中和DDTMUV,中和抗体效价达到1:46。重组蛋白MBP-E具有良好的免疫反应原性、免疫原性和体外免疫保护特性,这为进一步研制DTMUV重组亚单位疫苗奠定基础。更多还原

【Abstract】 The envelope protein gene of duck Tembusu virus(DTMUV) was amplified by RT-PCR from DDTMUV YY5-infected DF-1 cells and cloned into several prokaryotic expression vectors pMBP-c,pET-GST,pET-His,pET-DsbA separately to construct recombinant expression plasmids pMBP-E,pET-GST-E,pET—His-E and pET-DsbA-E.Then the constructs were transformed into bacteria E.coli Trans 10 or BL21(DE3)plysS.The bacteria harboring each expression vector were induced by IPTG and protein expression was detected by SDS-PAGE.The results show the MBP-E fusion protein was expressed in soluble form and capable of reacting specifically with anti-DDTMUV antiserum from naturally DDTMUV-infected rehabilitation ducks and mouse anti-DDTMUV monoclonal antibody.The antiserum prepared by immunizing mouse with MBP-E could neutralize DDTMUV in vitro and its titer measured by neutralization test reached 1:46,indicating that the MBP-E fusion protein has good immunogenicity and reactionogenicity which lay a foundation for developing sub-unit vaccines of DDTMUV.更多还原