家蚕核型多角体病毒orf90基因在Bac-to-Bac家蚕杆状病毒系统中快速表达

Rapid Expression Bombyx mori Nucleopolyhedrovirus orf90 Gene in Bac-to-Bac/BmNPV Baculovirus Expression System

【目的】利用BmNPV的Bac-to-Bac系统快速表达目的基因orf90,为深入研究该基因打下基础。【方法】将目的基因BmNPVorf90克隆到转移载体pFasBacHTb上,并将报告基因egfp插入到orf90的3'末端,形成重组转移载体pFasBacHTb-egfp-90。在将其转化到含穿梭载体bacmid的感受态细胞DH10Bac中,通过转座作用,经白斑筛选得到重组穿梭载体。纯化DNA,用脂质体介导转染家蚕BmN细胞,得到重组病毒bacmid-egfp-90。【结果】转染细胞72h后在荧光显微镜下可以观察到强烈的绿色荧光。重组病毒穿刺接种家蚕蛹,5d后观察到很强的荧光。结果表明构建的Bac-to-Bac系统能在家蚕细胞和蛹体中正确的快速表达外源基因。【结论】证明BmNPV的Bac-to-Bac是一个快速表达系统,适合在家蚕上广泛应用。

[ Objective ] Present studies describe the rapid expression Bombyx rnori nucleopolyhedrovirus orf90 in Bac-to-Bac/BmNPV baculovirus expression system, which will provide basic information for deeply studying this gene. [Method] The interest gene of BmNPV orf90 was cloned into a transfer vector pFasBacHTb and the report gene of egfp was inserted into downstream of orf90 to form the recombinant donor plasmid pFasBacHTb-egfp-90. The transfer vector was transformed into DH10Bac competent cell which contain the BmNPV bacmid. A recombinant shuttle vector was constructed by site-specific transposition and the colonies containing recombinant bacmid were collected by white selection. The cultured BmN cells were transfected with recombinant bacmid DNA mediating with Lipofectin and the pure recombinant baculovirus bacmid-egfp-90 was obtained. [Result] The intensive green fluorescence was observed both in transfected BmN cells after 72 h and injected silkworm pupae after 5 d. The results indicated that the Bac-to-Bac/ BmNPV expression system could effectively express foreign gene. [Conclusion] This method showed that Bac-to-Bac/BmNPV could be a rapid expression system, which would be wildly used in silkworm.