一种快速稳定构建重组杆状病毒的方法

A Rapid and Stable Method for Construction of Recombinant Baculovirus

快速稳定地将外源基因插入杆状病毒基因组获得重组杆状病毒,是提高昆虫杆状病毒表达系统应用效率的关键技术之一。在Ac Bacmid的基础上,通过λRed同源重组技术敲除病毒基因组lef2和orf1629基因的3＇端部分序列获得RDAc Bacmid-DualKo,并用Bsu36Ⅰ线性化之后,经重叠PCR获得5＇端和3＇端各含有50 bp同源臂及以绿色荧光蛋白（GFP）基因作为外源基因的表达盒片段,再将线性化杆状病毒载体片段与表达盒片段共转染sf9细胞,3~4 d即可获得重组杆状病毒rvAcBacmid-GFP。SDS-PAGE和Western blot结果表明,被rvAcBacmid-GFP感染的sf9细胞能高效表达绿色荧光蛋白。该方法通过线性化复制缺陷型杆状病毒载体和携带有同源臂及外源基因的重叠PCR片段,在胞内完成同源重组获得重组杆状病毒,避免了繁琐的病毒空斑纯化和分子克隆操作,获得的重组杆状病毒中无不稳定因子BAC及影响下游应用研究的抗性基因,为快速构建重组杆状病毒高效表达外源基因及相关研究应用提供了新的技术平台。

Inserting foreign genes into baculoviral genome to construct recombinant baculovirus is a key technology for improving the utilization efficiency of baculovirus expression vector system. In this work,RDAc Bacmid-DualK o was constructed through deleting the 3＇ ends of lef2 and orf1629 genes in Ac Bacmid by λRed recombinant system firstly and linearized by Bsu36Ⅰrestriction endonuclease. Then,the expression cassette containing the homologous arms of 50 bp at 3＇ ends of lef2 and orf1629 genes and gfp gene sequence were obtained by overlapping PCR. Finally,recombinant baculovirus rvA-c Bacmid-GFP was successfully obtained within 3- 4 days via cotransfecting sf9 cells with linearized RDAc Bacmid-DualK o and the expression cassette. SDS-PAGE and Western blotting analysis indicated that green fluorescent protein was highly expressed in sf9 cells after infected by rvA c Bacmid-GFP. Through linearization of replication-defective baculovirus vector and overlapping PCR of sequences carrying homologous arms and foreign genes,the recombinant baculovirus was constructed by homologous recombination in cells. There is no need for complicated virus plaque purification and molecular cloning,and the recombinant baculovirus has no unstable factor BAC and antibiotic resistance genes which affect application at the downstream. It provided a new platform to rapidly construct recombinant baculovirus for highly expressing foreign genes and related studies.