一株在HA831中复制并能提高绿色荧光蛋白表达水平的AcMNPV突变体的初步研究

Preliminary Studies on an AcMNPV Mutant with Increased Replication and Reporter Gene Expression in the Non-sensitive Cell Line HA831

棉铃虫卵巢细胞系HA831是一株对AcMNPV不敏感的细胞系.用AcMNPV突变体库中分离到的病毒突变体感染HA831细胞时,得到了一株绿色荧光蛋白基因表达水平明显提高的AcMNPV突变体.分析该突变体基因组,发现其orf18基因被Tn5转座子插入.利用同源重组重新构建了一株在同样位置带有同样插入突变的突变体,命名为AcGFP-i18.AcGFP-i18在Sf9细胞中能正常复制,其感染HA831细胞后,细胞中绿色荧光蛋白的表达水平明显高于对照病毒AcGFP,同时有一定水平的子代病毒产生.这一结果提示orf18位点的插入突变可以一定程度上扩展AcMNPV的宿主细胞范围.

HA831 is a cell line established from cotton bollworm Heliocoverpa armigera that is not susceptible to Autographa californica multiple nucleopolyhedrovirus（AcMNPV） infection.In the current work,an AcMNPV mutant was identified from the mutant library to have improved the expression level of green fluorescence protein gene in HA831.It was found to have Tn5 transposon inserted in the coding region of orf18.A mutant with the same insertion was constructed by homologous recombination and named AcGFP-i18.AcGFP-i18 replicated normally in Sf9 cells,indicating that orf18 is not essential for AcMNPV replication,which is consistent with previous reports.When AcGFP-i18 was used to infect HA831 cells,gfp expression driven by viral polyhedrin promoter was significantly increased,with the fluorescence intensity being 2.1-fold of that of AcGFP-infected cells.The production of progeny virus was seen in HA831 infected by AcGFP-i18,but not that by AcGFP.The result implies that the insertion mutation in orf18 locus extends the host range of AcMNPV.