摘要
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-TEasy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。
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.
出处
《郑州大学学报（医学版）》
CAS
北大核心
2007年第5期887-890,共4页
Journal of Zhengzhou University(Medical Sciences)
关键词
A型流感病毒
核蛋白
克隆
表达
influenza A virus
nucleoprotein
clone
expression
