甲型H1N1流感病毒血凝素HA1蛋白的真核表达与纯化

Eukaryotic expression and purification of haemagglutinin HA1 protein of influenza A virus subtype H1N1

目的构建甲型SWH1N1流感病毒血凝素HA1蛋白的杆状病毒真核表达载体,转染sf9细胞表达并纯化其编码蛋白。方法从江苏甲型H1N1流感病毒毒株[A/Jiangsu/1/2009(H1N1)]提取病毒RNA,采用RT-PCR技术扩增HA1基因,将其克隆至pMD18-T Simple Vector中构建pMD18-T/HA1质粒,双酶切pMD18-T/HA1与p-fast后,连接构建真核表达载体p-fast-HA1,转化DH10Bac细胞,转座构建杆状病毒HA1-Bacmid,经酶切及测序鉴定后将HA1-Bacmid转染到sf9细胞中,通过免疫印迹法鉴定HA1蛋白的表达,采用亲和层析法纯化HA1蛋白。结果经双酶切、测序鉴定证实HA1基因的真核表达载体构建成功;免疫印迹法证实HA1基因的表达;纯化获得了高纯度的HA1蛋白。结论成功克隆了HA1基因,并构建了其杆状病毒真核表达载体,该表达载体的构建为后期的流感快速检测以及基因工程疫苗制备奠定了良好的基础。

Objective To construct type A SWH1N1 influenza virus haemagglutinin HA1gene in Baculovirus expression system,and to express and purify its encoded peptide.Methods The HA1 gene of influenza A （SWH1N1） was amplified by RT-PCR from a Jiangsu type A H1N1 influenza virus strain[A/ Jiangsu/1/2009（H1N1） ].After T-A cloning and sequencing validation,HA1 gene was cloned into p-fast vector.Following the transposition of pFastBac-HA1 into the bacmid in DH10Bac E.coli competent cells,the recombinant bacmid （rBacmid-HA1） was verified by PCR analysis.The rBacmid-HA1 was transfected into SF9 cells for expression and the recombinant peptide got purified by Ni-based affinity chromatography.Results The Baculovirus expression vector HA1-Bacmid was successfully constructed.The expression of HA1 gene was detected by Western Blot.The highly homogenous re-HA1 was obtained by purification.Conclusions The cloning and expression strategy employed in this study should lay a foundation for the research which designates hemagglutinin as a potential candidate in diagnosis and vaccine development.