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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。
Objective: To construct the eukaryotic expression vector of non-structural protein H1N1 of influenza A (H1N1) virus and express its encoded protein (transfected 293T cells). Methods: The viral RNA was extracted from the first influenza A (H1N1) virus strain in Jiangsu Province (A / Nan jing / 1/2009 (H1N1)). The full length NS1 gene was amplified by RT-PCR and cloned into pMD18-T vector After construction of pMD18-T-NS1 plasmid and double digestion of pMD18-T-NS1 and PXJ40-HA, the eukaryotic expression vector PXJ40-HA-NS1 was constructed and transfected into 293T cells by enzyme digestion and sequencing. NS1 protein expression was identified by Western blot. Results: After double enzyme digestion and sequencing, the eukaryotic expression vector of NS1 gene was successfully constructed. Western-ern blot showed that the NS1 gene encoding protein was successfully expressed. CONCLUSION: The full-length NS1 gene was successfully cloned and its eukaryotic expression vector was constructed. The construction of this expression vector will provide the material for establishing the cell model of stable expression of NS1 protein and the function of NS1 protein in the later stage.