双启动子共表达杆状病毒载体的构建和鉴定

Construction and identification of co-expressing recombinant baculovirus vector with dual promoters

目的 探讨双启动子共表达杆状病毒载体作为双基因传递载体的可行性.方法 利用分子克隆技术将分别由巨细胞病毒（cytomegalovirus,CMV）启动子介导下的增强型绿色荧光蛋白（enhanced green fluorescent protein,EGFP）和胶质细胞源形神经生长因子（glial cell line-derived neurotrophic factor,GDNF）克隆到pFastBac Dual载体中,得到重组质粒pFastBac Dual-CMV-EGFP-CMV-GDNF,并利用Lipofectamine 2000将其转染到HEK293T细胞,通过间接免疫荧光法检测EGFP和GDNF的表达情况.按照Bac-to-Bac杆状病毒表达系统手册进行杆状病毒的包装,将获得的重组病毒Bac Dual-CMV-EGFP-CMV-GDNF转导Hela细胞,通过间接免疫荧光法和Western blot检测EGFP和GDNF的表达情况.结果 重组质粒pFastBac Dual-CMV-EGFP-CMV-GDNF成功构建；重组病毒Bac Dual-CMV-EGFP-CMV-GDNF成功包装；EGFP和GDNF实现了在293T细胞中和Hela细胞中的共表达.结论 pFastBac Dual载体为一个有效的双基因传递载体,为多基因联合治疗提供了一个强有力的病毒载体.

Objective To explore the feasibility of co-expressing recombinant baculovirus vector with dual promoters as dual-gene delivery vector. Methods After enhanced green fluorescent protein （ EG- FP） and glial cell line-derived nFurotrophic factor （GDNF） respectively under cytomegalovirus （CMV） pro- moter were cloned into the pFastBac Dual vector, the recombinant plasmid pFastBac Dual-CMV-EGFP-CMV- GDNF was generated, and then transfected into HEK293T cells with Lipofectamine2000. Indirect immuno- fluorescence was applied to examining the co-expression of EGFP and GDNF in transfected 293T cells. Bac Dual-CMV-EGFP-CMV-GDNF was produced according to Bac-to-Bac Baculovirus Expression System manu- al, and used to transduce Hela cells at suitable MOI. Then indirect immunofluorescence and western blot a- nalysis were used for detecting the existence of target proteins in Hela ceils. Results Plasmid pFastBac DuaI-CMV-EGFP-CMV-GDNF and Bac Dual-CMV-EGFP-CMV-GDNF were successfully generated. EGFP and GDNF were co-expressed in one single 293T cell or Hela cell. Conclusions The pFastBac Dual vector was proved to be an effective dual-gene delivery vector, which supplied a new viral vector for multi-gene therapy.