一种新颖的棉铃虫单粒包埋核多角体病毒表达系统

A Novel Expression System of Helicoverpa armigera single-nucleocapsid nucleopolydrovirus

将含有低拷贝数的mini Freplicon、一个卡那霉素抗性基因和一个lacZα基因 8.6kb的DNA片段经同源重组置换到棉铃虫核型多角体病毒基因组中的多角体蛋白基因内 ,构建了既能在E .coli内复制又可在昆虫细胞内复制形成完整的病毒粒子棉铃虫核型多角体病毒Bacmid(HaBacmid HZ8)。另外将HaSNPV的多角体蛋白基因和P10启动子序列取代 pFastBacDual质粒上的AcMNPV的多角体启动子序列和P10启动子序列 ,构建插入HaSNPV多角体蛋白基因和P10启动子序列的HapFastBacPhP10供体质粒。利用HapFastBacPhP10供体质粒将eGFP基因转位至HZ8的Tn7附着位点上 ,随后将含有eGFP基因的重组HaBacmidDNA转染至HZAm1细胞内。转染 5d后 ,细胞核内能形成典型的多角体 ,在萤光显微镜下观察到细胞内显示出强烈的绿色萤光。

A 8.5kb fragment containing an \%E.coli\% low copy number mini F replicon, a selectable kanamycin resistance marker and \%lacZ\% gene with attTn7 ( the target site for bacterial transposon Tn7) replaced polyhedrin gene in HaSNPV genome using homologous recombination. HaSNPV genome was cloned and maintained as 132 kb bacterial artificial chromosome (Bacmid) in \%E.coli\%,transfection of the Bacmid(HaBacmid HZ8) into HzAm1 cells led to a productive virus infection . In this paper, the donor plasmid HapFastPhP10 was constructed using the Ha polh gene and the Ha P10 promoter to replace the original Ac P10 promoter and Ac Polh promoter of the pFastBacDual donor plasmid respectively. The eGFP gene was inserted into multiple cloning site(MCS) downsream of the P10 promoter in the pFastBacHaPhpP10 donor plasmid. Recombinant HaBacmid HZ8 was constructed by transposing a mini Tn7 element from a HapFastPhP10 donor plasmid to the mini attTn7 attachment site on the HaBacmid HZ8 when the Tn7 transposition functions are provided in trans by a helper plasmid. Recombinant HaBacmid HZ8 DNA was transfected HzAm1 cells, occlusion bodies and green flusecent were found within HzAm1 cells 5 days after transfection. The results indicated the Habac to Bac expression system based on HaSNPV can effectively express foreign gene.