双表达H9N2禽流感病毒HA基因的假型重组杆状病毒系统的构建

Construction of A Pseudotype Baculovirus Dual Expression System-Based Avian Influenza H9N2 Virus HA Gene

以含有H9N2禽流感病毒HA基因的重组质粒pMD18-H9HA为模板,扩增HA基因,将其亚克隆入杆状病毒表面展示质粒pBACsurf-1中,再次将含有H9HA及gp64的基因片段克隆到质粒pcDNA3.1(+),获得重组质粒pcDNA-H9-HA.然后将含有CMV启动子、H9HA及gp64的基因片段克隆到杆状病毒转移质粒pFastBac-G,得到重组质粒pFastBac-G-H9-HA.将该重组质粒转化到DH10Bac感受态细胞中,得到重组穿梭载体Bacm id-G-H9-HA.利用脂质体转染Sf9昆虫细胞,获得重组杆状病毒BV-Dual-H9-HA.间接免疫荧光实验表明,该重组杆状病毒可以转导哺乳动物细胞并表达HA蛋白.免疫电镜观察结果表明,HA蛋白可以在该重组杆状病毒表面展示.

HA gene of avian influenza H9N2 virus amplified by PCR from the recombinant plasmid pMD18-HA was sub-cloned into pBACsurf-1.The fusion gene containing HA and gp64 was inserted into pcDNA3.1（＋）to construct the recombinant plasmid pcDNA-H9-HA.A fragment of the CMV promoter-H9HA-gp64 fusion gene cassette was excised from pcDNA-H9-HA and inserted into the baculovirus transfer vector pFastBac-G to construct the recombinant plasmid pFastBac-G-H9-HA.The plasmid was transformed into Escherichia coli DH10Bac competent cells to obtain recombinant shuttle vector Bacmid-G-H9-HA.The recombinant baculovirus BV-Dual-H9-HA was generated in Sf9 cells by cotransfection of the recombinant shuttle vector Bacmid-G-H9-HA with LipofectamineTM2000.Indirect immunofluoresent assay demonstrated that BV-Dual-H9-HA efficiently transducted the mammalian cells in vitro and HA gene was successfully expressed.Immunoelectron microscopy showed that HA protein was surface-displayed on the BV-Dual-H9-HA virus.