H7N9亚型禽流感病毒非结构蛋白-1真核表达载体的构建与表达

Cloning and eukaryotic expression of non-structural protein-1 of a human influenza virus subtype H7N9

目的:构建甲型H7N9亚型禽流感病毒非结构蛋白NS1真核表达载体并转染293T细胞以表达其编码的蛋白。方法:从南京分离株H7N9流感病毒[A/Nanjing/1/2013(H7N9)]提取病毒RNA,采用RT-PCR技术扩增NS1全长基因,将其克隆至pMD18-T载体中构建pMD18-T-NS1质粒,以pMD18-T-NS1质粒为模板扩增NS1基因。双酶切NS1基因PCR产物与PXJ40-MYC后,连接构建真核表达载体PXJ40-MYC-NS1,经酶切及测序鉴定后将质粒转染到293T细胞中,通过Western blot鉴定NS1蛋白的表达。结果:经双酶切、测序鉴定证实NS1基因的真核表达载体构建成功。Western blot法可见NS1基因编码蛋白的成功表达。结论:成功构建了H7N9非结构蛋白NS1真核表达载体,该表达载体的构建为后期建立稳定表达NS1蛋白的细胞模型和NS1蛋白功能研究奠定基础。

Objective：To construct the full-length NS1 gene of influenza A （H7N9） into an eukaryotic expression vector PXJ40- MYC ,and study the expression of NS1 gene in transfected 293T cell. Methods：The NS1 gene of influenza A （H7N9） was amplified by RT-PCR and cloned into pMD18-T vector to construct a plasmid,named pMD18-T-NS1.The PCR product of pMD18-T-NS1 plasmid and the PXJ40-MYC were double digested using the same restrict enzymes,the recombinant eukaryotic expression vector PXJ40-MYC-NS1 was subsequently yielded. The expression of the NS1 gene in transfected 293T cells was tested by Western blot. Results：The recombinant eukaryotic expression vector PXJ40-MYC-NS1 was successfully constructed. The NS1 protein was finally expressed in 293T. Conclusion：The full-length NS1 gene was obtained as well as its recombinant eukaryotic expression plasmid was successfully constructed. The construction of eukaryotic expression plasmid of NS1 gene made it possible to further study the function of NSI protein and the mechanism of diseases induced by influenza A virus.