A型流感病毒核蛋白原核表达载体的构建与表达

目的:构建A型流感病毒核蛋白(NP)的原核表达质粒,分析其在大肠杆菌中的表达状况.方法:采用逆转录聚合酶链式反应技术从A型流感病毒基因组中扩增出编码核蛋白的基因片段,克隆至pGEM-T Easy载体,PCR筛选阳性克隆并测序.经酶切后将目的片段亚克隆至表达载体pGEX-4T-1,转化大肠杆菌BL2(CDE3),PCR和酶切鉴定转化菌落.将阳性菌落经异丙基硫代-β-D-半乳糖苷(IPTG)诱导,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和免疫印迹分析目的蛋白的表达.结果:RT-PCR扩增出NP基因序列,将其亚克隆至表达载体pGEX-4T-1构建成重组表达质粒,并在BL2(CDE3)中表达了NP融合蛋白.结论:成功构建A型流感病毒NP的重组表达质粒,并在大肠杆菌中表达了NP融合蛋白.

Construction and expression of influenza A virus NP prokaryotic expression plasmid

Aim:To construct an expression plasmid encoding the sequence of influenza A virus nucleoprotein(NP) and analyze its expression in E.coli BL2(CDE3).Methods:The sequence encoding NP was amplified by RT-PCR from influenza A virus genome RNA and cloned into pGEM-T Easy vector,then identified by colony PCR and confirmed by DNA sequencing.The target gene fragment was subcloned into pGEX-4T-1 vector correctly after digested with restricted endonucleases.The recombinant plasmid pGEX-4T-1-NP was transformed into E.coli BL2(CDE3) and identified by colony PCR.The expression of influenza NP was induced with IPTG,and analyzed by 120 g/L SDS-PAGE and Western blot.Results:The NP fragment was amplified by RT-PCR and subcloned into pGEX-4T-1 properly.The recombinant fusion protein was expressed in BL2(CDE3).Conclusion:The recombinant plasmid pGEX-NP has been successfully constructed and the recombinant protein is expressed in E.coli BL2.