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[目的]构建甲型流感病毒H1N1亚型NS1蛋白真核表达载体,并在293T细胞中表达。[方法]采用RT-PCR技术,从甲型流感病毒H1N1毒株提取的病毒总RNA中,扩增NS1全长基因,将其克隆至pMD18-TVector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,通过免疫印迹法鉴定NS1蛋白的表达。[结果]酶切、测序证明重组真核表达载体PXJ40-HA-NS1构建成功,免疫印迹法可见NS1基因编码蛋白表达。[结论]成功构建甲型流感病毒H1N1亚型NS1蛋白真核表达载体PXJ40-HA-NS1,并在293T细胞中传染表达,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 H1N1 subtype NS1 protein of influenza A virus and express it in 293T cells. [Method] The full-length NS1 gene was amplified from the total RNA extracted from influenza A virus H1N1 by RT-PCR and cloned into pMD18-TVector to construct pMD18-T-NS1 plasmid. After digested with pMD18-T-NS1 and PXJ40-HA, the eukaryotic expression vector PXJ40-HA-NS1 was constructed and transfected into 293T cells. The expression of NS1 protein was identified by Western blotting. [Results] Enzyme digestion and sequencing proved that the recombinant eukaryotic expression vector PXJ40-HA-NS1 was constructed successfully. The expression of NS1 gene was found by immunoblotting. [Conclusion] The eukaryotic expression vector PXJ40-HA-NS1 of the H1N1 subtype NS1 protein of influenza A virus was successfully constructed and transfected into 293T cells. The construction of this expression vector will establish a cell model and a NS1 protein stable expression NS1 protein Functional studies provide the material.