甲型流感病毒HA基因核酸疫苗表达载体的构建及表达

Construction and Expression of Recombinant Plasmid Containing Influenza A Virus HA Gene

目的构建含有甲型流感病毒HA基因的核酸疫苗表达载体,转染COS-7细胞,并检测目的蛋白的表达。方法将甲型流感病毒New Caledonia/20/99(H1N1)接种鸡胚后,收获尿囊液,提取流感病毒总RNA,RT-PCR法扩增HA基因。经亚克隆后获得质粒pMD-T/HA,酶切鉴定后测序,将其与质粒pVAX1分别双酶切后连接,构建甲型流感病毒表达载体pVAHA。脂质体法转染COS-7细胞,并检测目的蛋白的表达。结果扩增出1700 bp片段,与预期的HA基因大小相符,测序结果与GenBank报道一致,酶切鉴定表明甲型流感病毒表达载体pVAHA构建正确,Western blot检测证明了流感病毒HA蛋白的表达。结论已成功构建了甲型流感病毒HA基因核酸疫苗表达载体pVAHA。

Objective To construct a recombinant plasmid containing influenza A virus HA gene, transfect COS-7 cells and test for the expression of target protein. Methods Inoculate New Caledonia/20/99（HIHI ）strain of influenza A virus to chick embryo, harvest the allantoic fluid and extract the total RNA of influenza A virus for amplification of HA gene by RT-PCR. Subclone the amplified HA gene to pMD18-T vector and identify the constructed recombinant plasmid pMD-T/HA by restriction analysis and sequencing. Digest plasmids pMD-T/HA and pVAX1 with Kpn I/BamH I separately, hnk the recovered HA gene and skeleton of pVAX1 ,and transform to E. coli XL 1-Blue to construct expression vector pVAHA. Transfect COS-7 cells with pVAHA in mediation of liposome and identify the expressed product by Western blot. Resuits The HA gene fragment at a length of 1700 bp was amplified, which was consistent with that expected. The sequence of amplified HA gene was identical to that reported in GenBank. Restriction analysis showed that expression vector pVAHA was constructed correctly. Western blot proved the expression of influenza A virus HA protein. Conclusion A recombinant plasmid as DNA vaccine, containing influenza A virus HA gene, was successfully constructed.