Objective Three kinds of different internal promoters were inserted into the lentiviral vector to drive the expression of CD19-specifc chimeric antigen receptor (CAR)-redirected T lymphocytes with the green fluorescent protein(GFP) and the GFP expression efficiency of transduced cells driven by lentivirus were compared.Methods Three GFP reporter lentivirus vectors carrying different promoters and anti-CD19-CAR,including CMV,EF1α,and CMV-EF1α were selected.Three different vectors' names abbreviate to pHAGE-CMV-EFlα-EGFP-CD19,pHAGE-CMV-EGFP-CD19,pHAGE-EF1α-EGFP-CD19.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 after transfecting 293T cells,and virus supernatant was collected after 72 h and centrifuged.Nucleic acid copy number in RNA and the expression of EGFP and CD3 zeta were identified through fluorescence quantitative PCR,flow cytometry and western blotting,respectively.The titers of the lentiviral vectors were determinde by scoring GFP expression following swerial dilutions of the viral supernatant on 293T cells.T lymphocytes was infected with lentivims produced from these reporter vectors.Then,fluorescence microscope,wesyern blotting were used to detect the GFP expression strength.Results Results of fluorescence microscope maintained that different internal promotors driving the expressing effect is different.293T as targeted cell,the transfected 293T cells were found containing strong expression of GFP.The amount of the 293T cells expressing GFP driven by CMV-EF1α promoter was the largest,while the EF1α promoter was the smallest.Flow cytometry results show that the rate of transduction is 72.4％,20.6％ and 14.5％,respectively.T lymphocytes as targeted cell,Western blot showed that CMV-EF1α was the largest among all promoters,while the CMV was the smallest.Conclusion In the study of gene expression mediated by lenuvirus,proper cell lines and promoters should be selected to obtain high efficiency.%目的 高效包装含绿色荧光蛋白(GFP)的嵌合性抗原受体CD19重组慢病毒载体,对比不同启动子在T细胞中绿色荧光蛋白的表达效率.方法 构建3种不同启动子及CD19-CAR的质粒pHAGE-CMV-EF1α-GFP-CD19、pHAGE-CMV-GFP-CD19、pHAGE-EF1α-GFP-CD19,转染293T细胞,镜下观察转染的细胞状态,72h后收集上清,PCR定量病毒载体RNA核酸拷贝数,通过流式细胞术以及Western blot法测定单、双启动子启动GFP及CD3ζ的表达情况;慢病毒液分别转染T淋巴细胞后,通过荧光显微镜检测慢病毒转染效果,Western blot法测定蛋白的表达水平.结果 荧光显微镜观察:以293T细胞为靶细胞时,启动子由强到弱依次为CMV-EF1α＞CMV ＞EF1α,流式结果显示,在293T细胞中的转导率分别为72.4％、20.6％、14.5％;Western blot法检测结果显示,在T细胞中启动子表达强弱为CMV-EF1α ＞EF1o＞CMV.结论 在以重组慢病毒为载体的基因研究过程中,为了提高病毒在感染细胞的转染率,需要正确选择启动子以及相应的靶细胞.
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