利用双色荧光观察分析重组家蚕杆状病毒对昆虫细胞及蚕体组织的感染

Infection Analysis of Recombinant Bombyx mori Nucleopolyhedrovirus to Insect Cells and Bombyx mori Tissues by Observation of Red and Green Fluorescence

尝试将红色荧光蛋白(RFP)作为昆虫表达体系的分子标签,克隆了rfp基因的读码框,通过Bac-to-Bac杆状病毒表达系统,将该基因插入家蚕核型多角体病毒(BmNPV)中,获得重组杆状病毒BmNPV-rfp。对家蚕细胞的感染表明,rfp适于在昆虫细胞中表达,在显微镜下红色荧光很明显,说明RFP可以作为杆状病毒表达的分子标签。BmNPV-rfp和另一种插入绿色荧光蛋白基因gfp的家蚕重组杆状病毒BmNPV-gfp混合感染家蚕细胞和幼虫的实验表明,二者的荧光很少在同一细胞中发生重叠,说明昆虫细胞大多只感受1次同一来源的病毒。BmNPV-rfp、BmN-PV-gfp与AcNPV混合感染Sf21细胞的结果也证明了该推断,并且还显示AcNPV可以协助BmNPV来源的病毒基因在Sf21细胞中表达以及协助病毒增殖或产生杂交病毒,但尚未明确其机制。

In order to know whether the red fluorescent protein（RFP） could be used as a molecular marker in baculovirus expression system,the open reading frame of rfp gene was cloned and inserted into Bombyx mori nucleopolyhedrovirus（BmNPV） genome.The recombinant virus BmNPV-rfp was obtained.The results of infection in BmN cells demonstrated that the rfp gene was highly expressed and was suitable for application as a fusing marker.When the mixed viruses of BmNPV-rfp and BmNPV-gfp infected BmN cells and Bombyx mori larvae,separated fluorescent light（red or green） was observed in the infected cells,meaning that most insect cells are only sensitive by one time to the virions that derived from the same type of baculovirus.Infection of Sf21 cells by mixed viruses of BmNPV-rfp,BmNPV-gfp and AcNPV（Autographa californica NPV） also proved the above conclusion.Furthermore,it showed that AcNPV could help gene expression and viral amplification of the viruses derived from BmNPV or to form hybrid viruses inSf21 cells.Yet the mechanism remains for further study.