【摘要】 [目的]构建大麦黄矮病毒运动蛋白(BYDV-MP)基因的原核表达载体,并在E.coli-BL21中进行高效表达。[方法]根据GenBank中收录的大麦黄矮病毒PAV6株系的基因序列设计合成1对引物,应用RT-PCR技术从感染大麦黄矮病毒的叶片中扩增得到大麦黄矮病毒(BYDV)的运动蛋白(MP)基因,利用基因重组技术构建pGEX-4T-1-MP,将其转化至大肠杆菌表达菌株E.coli-BL21(DE3)中,用IPTG诱导重组质粒pGEX-4T-1-MP在E.coli-BL21中进行表达。[结果]对重组质粒进行酶切分析及序列测定,鉴定正确后,与GeneBank中收录的大麦黄矮病毒PAV6株系的MP基因序列比对分析,同源性达100%。在IPTG诱导下,高效表达出相对分子质量约43 kD的蛋白,蛋白质电泳(SDS-PAGE)结果表明,表达的蛋白条带与预期的GST-MP融合蛋白相符。[结论]成功构建了pGEX-4T-1-MP的原核表达载体,在E.coli-BL21中高效表达了MP,为进一步研究该蛋白的结构和功能奠定基础。更多还原

【Abstract】 [Objective] To construct the prokaryotic expression vector of the movement protein gene of barley yellow dwarf virus(BYDV-MP) and efficiently express the movement protein in E.coli-BL21.[Method] According to movement protein gene sequence of the Barley Yellow Dwarf Virus(BYDV) PAV6 in Genebank,a pair of primers were designed and synthesized,BYEDV-MP gene was amplified from infected wheat leaves by RT-PCR,pGEX-4T-1-MP was constructed by using recombinant DNA technology and then expressed in E.coli-BL21(DE3).[Result] The construct of pGEX-4T-1-MP was verified by restriction endonuclease and sequenced.Compared with the sequence of movement protein genes of The Barley Yellow Dwarf Virus(BYDV) in the GeneBank,their homology was 100%.The expression of the GST-MP fusion protein was induced with IPTG.Its molecular weight was about 43 KDa,the results was identified by electrophoreses of SDS-PAGE,the expression of protein bands was consistent with the expected size.[Conclusion] The recombinant prokaryotic expression vector in pGEX-4T-1-MP was constructed successfully and highly expressed in E.coli-BL21,which laid a foundation for the further study on function and structure of MP.更多还原