摘要
目的研究双启动子对增强型绿色荧光蛋白(EGFP)表达的影响及双启动子的启动效率。方法分别将H1、U2、U3和U6启动子克隆至pEGFP-N1载体中的MCS位点处,构建CMV与以上各个启动子的双启动子表达载体。将重组载体转染293T细胞,荧光显微镜观察,流式细胞术检测转染效率及EGFP基因的表达效率(以荧光指数表示)。结果双启动子重组表达载体经PCR和酶切证明构建正确。流式细胞术检测表明,pEGFP-N1载体与分别插入H1、U2、U3和U6启动子的双启动子重组表达载体的转染效率分别为26.57%±1.54%、28.57%±0.99%、16.14%±1.69%、22.63%±1.77%和17.89%±1.84%;EGFP基因的表达效率分别为142.79±31.26、103.59±25.90、19.67±0.52、58.16±14.58和15.40±1.92。结论CMV与H1启动子联合驱动EGFP的表达效果与CMV单独驱动相近,而CMV分别与U2、U3和U6启动子联合驱动EGFP的表达效果显著低于CMV单独驱动。
Objective To investigate the effect of dual promoter on expression of enhancement green fluorescent protein（EGFP）as well as its promotion efficacy.Methods Promoters H1,U2,U3 and U6 were cloned to the MCS site of vector pEGFP-N1 containing CMV promoter respectively,and the constructed dual promoter expression vectors were transfected to 293T cells.The transfected cells were observed by fluorescent microscopy,and the transfection efficacy and EGFP gene expression efficacy（expressed as fluorescent index）were determined by flow cytometry.Results Both PCR and restriction analysis proved that the dual promoter expression vectors were constructed correctly.The transfection efficacies of pEGFP-N1 and dual promoter expression vectors inserted with H1,U2,U3 and U6 promoters were 26.57% ± 1.54%,28.57% ± 0.99%,16.14% ± 1.69%,22.63% ± 1.77% and 17.89% ± 1.84%,and the EGFP gene expression efficacies in the cells transfected with the recombinant plasmids were 142.79 ± 31.26,103.59 ± 25.90,19.67 ± 0.52,58.16 ± 14.58 and 15.40 ± 1.92,respectively.Conclusion The expression efficacy driven by CMV promoter combined with H1 promoter were similar to that by CMV promoter alone.However,the expression efficacies driven by CMV promoter combined with U2,U3 and U6 respectively were significantly lower than that by CMV promoter alone.
出处
《中国生物制品学杂志》
CAS
CSCD
2009年第10期957-960,967,共5页
Chinese Journal of Biologicals
关键词
双启动子
增强型绿色荧光蛋白
流式细胞术
Dual promoter
Enhancement green fluorescent protein
Flow cytometry
