慢病毒载体介导绿色荧光蛋白基因在小鼠T淋巴细胞中的表达

慢病毒载体介导绿色荧光蛋白基因在小鼠T淋巴细胞中的表达

【摘要】本研究构建含绿色荧光蛋白基因的慢病毒载体三质粒系统，并观察其在小鼠T淋巴细胞中的表达情况。应用亚克隆技术将多聚嘌呤通道元件、泛醌启动子和绿色荧光蛋白基因连接至pLO134载体，构建成pTK153载体。之后应用磷酸钙沉淀法将慢病毒载体三质粒系统共转染293T细胞，12小时后在荧光显微镜下观察绿色荧光蛋白表达情况，72小时收集病毒上清并感染小鼠T淋巴细胞，在荧光显微镜下和应用流式细胞仪观察感染情况。结果表明：慢病毒载体的三质粒系统转染293T细胞12小时后在荧光显微镜下观察到绿色荧光蛋白表达，FACS分析转染效率为%，病毒滴度测定为

×106 U/ml。感染小鼠T淋巴细胞后，荧光显微镜下观察到GFP的表达，FACS分析转导效率为%。结论：成功构建了含绿色荧光蛋白基因的慢病毒载体，对小鼠T淋巴细胞有较高的感染效率。

【关键词】慢病毒载体 T淋巴细胞绿色荧光蛋白基因

Expression of Green Fluorescent Protein Gene in Mouse T Lymphocytes Mediated by Lentiviral Vector

Abstract This study was purposed to

the lentiviral vector carrying the green fluorescent protein (GFP) gene and to investigate the expression of GFP in T lymphocytes of the mouse. The polypurine tract (PPT) element, ubiquinone promoter (PUB) and GFP were ligated to plasmid pLO134 using subcloning technology to construct plasmid pTK153. Human kideny 293T cells were

system containing packaging plasmid

△NRF, plasmid pTK153 and envelope

calcium phosphate DNA precipation and the expression of GFP was observed under

fluorescence microscope after 12 hours. The viral particles were collected after transfection 72 hours, were frozen at -80℃ and were used to infect mouse T lymphocytes at multiplicity of infection ( .) of 3. The expression of GFP in mouse T lymphocytes was observed by fluorescence microscopy and

(FACS). The results showed that the transfection efficacy was ±% in 293T cells analysed by FACS and the viral titer was (±)×106 U/ml. The expression of GFP was also evident in mouse T lymphocytes and the transduction efficacy was (±)%. It is concluded that

vector containing GFP gene is successfully constructed and the transduction efficacy is high in mouse T lymphocytes.

Key words lentiviral vector; mouse T

lymphocytes; green fluorescent protein gene

J Exp Hematol 2007; 15(1):125-128

逆转录病毒载体系统已被广泛应用于临床前及临床的基因转移研究，虽然对其复制以及转导外源目的基因至细胞的相关机理研

究得较为深入，但是逆转录病毒载体不能有效转导非分裂期细胞，因而其应用于临床基因治疗受到了一定的限制［1］。围绕这一问题，近年来人们把目光投向了来源于包括人类免疫缺陷病毒在内的慢病毒载体。LV 最大的特点是可以感染非分裂期细胞如神

经细胞、肝细胞、造血干细胞、胰岛细胞、肌肉细胞等［2］。我们构建了以为基础的LV三质粒系统，并以绿色荧光蛋白基因作为标志基因，观察其在小鼠T淋巴细胞中的表达情况。

LV三质粒系统的构建及鉴定

用限制性内切酶BamHⅠ和BglⅡ分别从pTK131、pTK145及pTK54切下PPT元件、PUB 启动子及GFP基因，用T4DNA连接酶依次将PPT元件、PUB启动子及GFP基因连接至由

BamHⅠ酶切并经牛小肠碱性磷酸酶去除

的载体pLO134上，连接产物转化感受态细胞DH5α，同时设立空白对照。37℃过夜培养，次日从连接板挑取6-8个克隆进行扩增培养，用Promega公司质粒小量提取试剂盒提取质粒后，分别用限制性内切酶EcoRⅠ、SacⅡ和AgeⅠ+XbaⅠ鉴定连接产物的正反向。包装质粒△NRF、包膜蛋白质粒、pTK131、pTK145及pTK54均为本科徐开林教授构建。

慢病毒载体包装细胞系的建立

应用Ivitrogen 公司无内毒素的质粒中量提取试剂盒提取pTK153、△NRF及

用于转染。转染前24小时选择生长状态良好的293T细胞按1∶3传代，24小时后细胞生长达60%-70%汇合时采用磷酸钙沉淀法进行转染。将转移质粒pTK153 15 μg、ΔNRF 10 μg、μg、CaCl2 125 μl，补加消毒的双蒸水至500 μl，在涡旋混合器上边振荡边加入500 μl 2×BES，室温放置30分钟。将转染液加到培养板中，轻轻混匀，3% CO2、37℃培养箱中培养3小时后，