甲型流感病毒H1N1HA 蛋白在果蝇S2细胞中的表达及免疫原性研究

摘要

In order to obtain immunogenic hemagglutinin (HA) of influenza virus A (HIND , the HA was expressed in drosophila S2 cells and its immunogenicity was analyzed in the BALB/c mice. The full-length of optimized gene of H1N1 HA plus a specific T4-foldon sequence at the N-terminal end was synthesized and cloned into the plasmid pMT/Bip/V5-His A; the recombinant plasmid pMT-HA was co-transfected with the plasmid pCoHygro into the S2 cells, and the stable cell lines were selected by hygromycin B; HA was induced by copper sulfate in the serum-free medium, and identified by KLISA, SDS-PAGK electrophoresis and western blot. After purification with Ni2 -NTA, the purified rHA was immunized into the BALB/c mice with the alum, and the Ab titers were evaluated by ELISA. As a result, the recombinant plasmid pMT-HA for expression of the H1N1 HA in the S2 cells was successfully constructed, and the stable S2 cell lines expressing HA was obtained; ELISA, SDS-PAGE and WB analysis showed that the recombinant HA was expressed in the secretive and trimer form; the recombinant HA could elicit effective Ab immune response in the BALB/c mice. These results suggest that the purified rHA in the trimer form has good immunogenicity, and is a promising immunogen for future vaccine development.%目的 在果蝇S2细胞表达甲型流感H1N1 HA并分析其免疫原性.方法 根据GenBank发表的甲型流感病毒(H1N1)HA基因序列,经过密码子优化,全基因合成编码HA的基因,并于其N端引入特定的空间折叠序列,定向连接至表达载体pMT/ Bip/ V5-His A,构建重组表达载体pMT-HA.酶切鉴定正确后,用脂质体转染法与辅助质粒pCoHygro 共转染果蝇S2细胞,潮霉素加压筛选稳定细胞系,在无血清培养基中以硫酸铜溶液诱导表达,SDS-PAGE电泳及Western blot鉴定,经镍柱纯化后免疫小鼠,ELISA检测其诱导的特异性抗体水平.结果 获得了稳定表达HA的S2细胞株,目的 蛋白以分泌形式表达于上清,并形成三聚体,可以被H1N1 HA 抗体识别;免疫小鼠后可诱导机体产生抗HA特异性抗体.结论 获得了纯化的三聚体形式表达的HA蛋白,并具有较好的免疫原性,具有很好的疫苗应用前景.