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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 eukaryotic expression vector of non-structural protein NS1 of H7N9 subtype A virus of avian influenza virus and transfect 293T cells to express its encoded protein. Methods: The viral RNA was extracted from Nanjing H7N9 influenza virus [A / Nanjing / 1/2013 (H7N9)]. The full length NS1 gene was amplified by RT-PCR and cloned into pMD18-T vector to construct pMD18-T NS1 plasmid, and the NS1 gene was amplified using the pMD18-T-NS1 plasmid as a template. After double digestion of NS1 gene PCR product and PXJ40-MYC, the eukaryotic expression vector PXJ40-MYC-NS1 was ligated and constructed. The plasmid was transfected into 293T cells after digestion and sequencing, and the expression of NS1 protein was identified by Western blot. Results: After double enzyme digestion and sequencing, the eukaryotic expression vector of NS1 gene was successfully constructed. Western blot showed that the NS1 gene encoding protein was successfully expressed. Conclusion: The eukaryotic expression vector of H7N9 nonstructural protein NS1 was successfully constructed. The construction of this expression vector lays the foundation for the establishment of the cell model and the function of NS1 protein.