CAJ | 학술논문

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.
목적 탐토쌍계동자공표체간상병독재체작위쌍기인전체재체적가행성.방법 이용분자극륭기술장분별유거세포병독(cytomegalovirus,CMV)계동자개도하적증강형록색형광단백(enhanced green fluorescent protein,EGFP)화효질세포원형신경생장인자(glial cell line-derived neurotrophic factor,GDNF)극륭도pFastBac Dual재체중,득도중조질립pFastBac Dual-CMV-EGFP-CMV-GDNF,병이용Lipofectamine 2000장기전염도HEK293T세포,통과간접면역형광법검측EGFP화GDNF적표체정황.안조Bac-to-Bac간상병독표체계통수책진행간상병독적포장,장획득적중조병독Bac Dual-CMV-EGFP-CMV-GDNF전도Hela세포,통과간접면역형광법화Western blot검측EGFP화GDNF적표체정황.결과 중조질립pFastBac Dual-CMV-EGFP-CMV-GDNF성공구건；중조병독Bac Dual-CMV-EGFP-CMV-GDNF성공포장；EGFP화GDNF실현료재293T세포중화Hela세포중적공표체.결론 pFastBac Dual재체위일개유효적쌍기인전체재체,위다기인연합치료제공료일개강유력적병독재체.
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 (EGFP) and glial cell line-derived neurotrophic factor (GDNF) respectively under cytomegalovirus (CMV) promoter 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 immunofluorescence 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 manual,and used to transduce Hela cells at suitable MOI.Then indirect immunofluorescence and western blot analysis were used for detecting the existence of target proteins in Hela cells.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.