甲型H1N1流感病毒非结构蛋白NS1真核表达载体的构建与表达

Construction and expression of eukaryotic expression vector of NS1 protein of influenza A (H1N1)

目的:构建甲型H1N1流感病毒非结构蛋白NS1真核表达载体并表达其编码蛋白(转染293T细胞)。方法:从江苏首例甲型H1N1流感病毒毒株(A/Nan jing/1/2009(H1N1))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 Vector中构建pMD18-T-NS1质粒,双酶切pMD18-T-NS1与PXJ40-HA后,构建真核表达载体PXJ40-HA-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。West-ern blot法可见NS1基因编码蛋白的成功表达。结论:成功克隆NS1全长基因,并构建了其真核表达载体,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蛋白功能研究提供了材料。

AIM： To insert the full-length NS1 gene of influenza A（H1N1） into an eukaryotic expression vector PXJ40-HA,and to evaluate the expression of NS1 gene in transfected 293T cells.METHODS： The NS1 gene of influenza A（H1N1） was amplified by RT-PCR and cloned into pMD18-T vector to construct a plasmid,named pMD18-T-NS1.The pMD18-T-NS1 and the PXJ40-HA were both digested using the same restrict enzymes and ligated,yielding the recombinant eukaryotic expression vector PXJ40-HA-NS1.The expression of the NS1 gene in transfected 293T cells was tested by Western blot.RESULTS： The recombinant eukaryotic expression vector PXJ40-HA-NS1 was successfully constructed.The NS1 protein was observed to be expressed in 293T cells.CONCLUSION： The full-length NS1 gene is obtained and its recombinant eukaryotic expression plasmid is successfully constructed.This study is of help to further understanding the biological function of NS1 protein and the mechanism of diseases induced by influenza A virus.