【摘要】 目的在人类免疫缺陷病毒-1(HIV-1)慢病毒载体中引入三种不同的内部启动子来驱动绿色荧光蛋白基因(GFP)的表达,初步比较内部启动子的效率。方法慢病毒载体三质粒系统中,转移质粒的构建采用限制性内切酶酶切、T4DNA连接酶连接方法和内切酶酶切鉴定。磷酸钙沉淀法将三质粒共转染293T包装细胞。病毒滴度的测定采用6孔板培养感染细胞,荧光显微镜计数GFP阳性细胞。通过荧光显微镜观察绿色荧光的表达情况判断启动子的效率。结果构建了含PPT元件和含不同内部启动子和GFP的质粒。转染293T细胞后均观察到较强的绿色荧光。表达荧光的293T细胞数以巨细胞病毒(CMV)启动子最多,肝细胞特异性启动子(LSP)较少,泛醌启动子(PUB)介于两者之间。CMV为内部启动子的慢病毒载体滴度5×106I U/ml,其他二种启动子的滴度(1~2)×105I U/ml。结论在所选择的三种内部启动子中,CMV启动子的效率最高,LSP较强,PUB介于两者之间。更多还原

【Abstract】 Objective Three kinds of different internal promoters were inserted into the lentiviral vector deriving from human immunodeficiency virus-1（HIV-1） to drive the expression of the green fluorescent protein（GFP） and the efficiencies of the internal promoters were compared.Methods The restriction enzemies and T₄ DNA ligase were used to construct the vector plasmids,and the recombinant plasmids were identified by the restriction exzemies.Human embroic kideny 293T cells were cotransfected with the three plasmids by calcium phosphate DNA precipation,and the expression of GFP was observed under fluorescent microscope.The titers of the lentiviral vectors were determinde by scoring GFP expression following swerial dilutions of the viral supernatant on 293T cells.Results The plasmids containing the PPT element and different internal promoters and GFP gene were constructed.The transfected 293T cells were found containing strong expression of GFP.The amount of the 293T cells expressing GFP driven by CMV promoter was largest among all promoters,while the LSP promoter was the smallest.The titer of the lentiviral vector with CMV internal promoter was 5×10⁶IU/ml and others were 1～2×10⁵IU/ml.Conclusion Under our experiment condition,the efficiency of CMV promoter was the strongest,the LSP promoter was the weakest,while the PUB promoter was at somewhere between the CMV and the LSP promoters.更多还原