利用杆状病毒实现外源基因在哺乳动物细胞内表达

The expression of exogenous genes in mammalian cells by using baculovirus

杆状病毒是一类专职感染无脊椎动物的病毒,目前表达载体和基因转导载体广泛应用于科学研究和生物医药领域。研究在杆状病毒Bac-to-Bac操作系统基础上将杆状病毒载体改造成能在哺乳动物细胞内表达外源基因的载体。首先,将Bac-to-Bac操作系统中pFastBac-Dual质粒中的PH和p10启动子替换成巨细胞病毒(CMV)启动子和真核生物延伸因子(EF1)基因的启动子;然后将egfp基因和mCherry基因分别插入到EFI启动子和CMV启动子下游;通过转座获得表达egfp和mCherry基因的重组杆状病毒基因组,并转染Sf9细胞获得重组杆状病毒。重组杆状病毒可以在昆虫细胞和哺乳动物细胞内产生绿色荧光和红色荧光,表明以CMV启动子和EF1启动子构建的重组杆状病毒既可以在昆虫细胞内表达外源基因,又可以通过转导在哺乳动物细胞内表达外源基因。该载体可以应用于特殊蛋白的真核表达和基因治疗药物的研究开发。

Baculovirus is a kind of virus that infects invertebrates,and they are widely used in scientific research and biomedicine as expression vector and gene transduction vector.Based on“Bac-to-Bac”operating system of baculovirus,we transformed baculovirus vector into a transduction vector with capable of expressing exogenous genes in mammalian cells in this study.First,the PH and p10 promoters of pFastBac-Dual plasmids in the Bac-to-Bac operating system were replaced with the CMV promoter and EF1 promoter.Then,the egfp gene and mCherry gene were inserted into the downstream of EF1 promoter and CMV promoter respectively.The trans⁃position was performed to obtain the recombinant baculovirus genomeexpressing egfp and mCherry genes,and recombinant baculovirus was obtained by transfect the genome to Sf9 cells.The recombinant baculovirus can generate green and red fluorescence in insect cells and mammalian cells,indicating that the recombinant baculovirus constructed with CMV promoter and EF1 promoter can express exog⁃enous genes in insect cells as well as mammalian cells through transduction.This vector can be used for eukaryotic expression of spe⁃cial proteins and research and development of gene therapy drugs.