甲型流感病毒SwH1N1血凝素蛋白HA1真核表达载体的构建与表达

目的构建甲型流感病毒SwH1N1血凝素蛋白(HA1)的真核表达载体,并表达其编码蛋白HA1。方法利用RT-PCR技术扩增HA1基因,克隆至pMD18-T Simple Vector中构建pMD18-T-HA1质粒。双酶切pMD18-T-HA1与PXJ40后回收并连接回收片段,构建PXJ40-HA1真核表达载体,鉴定后转染293T细胞,用免疫印迹法(western-bolt)鉴定重组HA1蛋白的表达。结果实验成功构建HA1基因的真核表达载体,并在真核细胞中表达出分子量为40kD的重组蛋白。结论成功构建的甲型流感病毒SwH1N1HA1基因的真核表达载体,可为后期的流感快速检测及基因工程疫苗的制备奠定良好的基础。

Cloning and eukaryotic expression of haemagglutinin HA1 protein of Influenza A Virus SwH1N1

Objective To construct a eukaryotic expression plasmid for influenza virus A SwH1N1 haemagglutinin HA1 gene and express HA1 gene in 293T cells. Methods The HA1 gene of influenza A SwH1NI was amplified by RT-PCR and cloned into pMD18-T Simple vector by T-A cloning to construct pMD18-T-HA1. Double digestion of pMD18-T-HA1 and PXJ40 and ligation of the gel-purified fragments constructed PXJ40-HA1 plasmid. PXJ40-HA1 plasmid was verified transfect- ed into 293T cells, expression of recombinant HA1 was confirmed by Western Blot analysis. Results Eukaryotic expression plasmid PXJ40-HA1 was successfully constructed. HA1 （a 40 kD protein） was successfully expressed in eukaryotic cells. Con- elusion The successful construction of HA1 eukaryotic expression plasmid should lay a foundation for the research on rapid di- agnosis and vaccine development.