高效转染人T细胞的逆转录病毒载体系统的构建及应用

Construction and application of retroviral vector system useful for high-efficiency transfection into human T cells

目的：构建能高效转染人T细胞并带有快速筛选标签的逆转录病毒载体系统，并与传统的逆转录病毒载体系统做比较。方法：首先在原载体pCMMP-EGFP的基础上，构建携带内部核糖体进入位点（IRES）基因的逆转录病毒载体pCMMP-IRES-GFP。将嵌合T细胞受体基因插入该载体中，与另外两个辅助载体pMD．MLVgag．pol和pHDM．G共同转染包装细胞293T。48h后收培养上清，高速离心病毒。同时将嵌合T细胞受体基因插入传统的逆转录病毒载体pLXSN中，并转染包装细胞PA317，用G418加压筛选出转染的PA317细胞克隆。扩大培养48h后，收细胞培养上清即为病毒液，分别用上述两种方法得到的病，譬液感染NIH333细胞，检测病毒的滴度。取适量病毒液感染川PHA激活后的人原代T细胞，48h后在荧光显微镜下观察绿色荧光，并用流式细胞术（FCM）检测病毒感染的效率。结果：成功地构建逆转录病毒载体pCMMP—IRES-GFP。将目的基因插入该载体中，与pMD．MLVgag．pol和pHDM．G共同转染包装细胞293T。培养48h后，离心收获浓缩的上清中含有滴度为2．15×10^11VP／L的病毒。以其感染用PHA激活后的人原代T细胞48h后，在荧光显微镜下能观察到绿色荧光蛋白（GFP）的表达。FCM检测病毒对T细胞的感染效率为50％～60％，并可用FCM进行分选。而用pLXSN载体转染的PA317细胞，包装得到的病毒滴度为6．43×10^9VP／L，病毒感染T细胞的效率仅为5％～10％。结论：构建了能高效转染T细胞并带有快速筛选标签的逆转录病毒载体系统，为T细胞的基础与临床研究打下了基础。

AIM： To construct a retroviral vector system with high-efficiency transfection into human T cells and carrying a rapid screening label, and compare it with the traditional retroviral vector system. METHODS： Retroviral vector pCMMP-IRES-GFP was first constructed by inserting the internal ribosome entry site-green fluorescent protein （IRESGFP） cDNA into retroviral vector pCMMP, The chimeric TCR gene was inserted into pCMMP-IRES-GFP and then co-transfected into packaging cell line 293T with other two assistant vectors pMD, MLVgag. pol and pHDM. G. After 48 h, the culture supernatant was harvested and condensed by centrifugation. Meanwhile, the chimeric TCR gene was inserted into pLXSN and then transfected into packaging cell line PA317. The transfected PA317 cells were obtained by G418 pressure screening. The cells culture supernatant containing viruses was harvested after being cultured for 48 h. The viral titer was determined by NIH3T3 cells infection. The preactivated primary human T lymphocytes were infected by appropriate volume of viral fluid and detected by fluorescent microscopy or flow cytometry after 48 h. RESULTS： A retroviral vector pCMMP-IRES-GFP was constructed successfullly. Compared with the traditional vector system, the viral titer was 2.15 × 10^11 VP/L vs 6.43 × 10^9VP/L, and efficiency of transfection into preactivated primary T cells was 50% - 60% vs 5% - 10%. Furthermore, the infected cells were also detected by fluorescent microscope and could sorted by fluorescent activated cell sorting. CONCLUSION： A retroviral vector system with high transfection efficiency into T lymphocytes and carrying a rapid screening label has been constructed, which establishes the fundation for basic and clinical studys on T lymphocytes.