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

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

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(PPT)元件、泛醌启动子(PUB)和绿色荧光蛋白基因(GFP)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(包括包装质粒△NRF、转移质粒pTK153和包膜蛋白质粒VSV-G)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(FACS)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(63.04±7.24)%,病毒滴度测定为(3.09±0.61)×106U/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(37.98±6.26)%。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。

This study was purposed to constmcte the three-plasmid system of 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, ubiqttinone promoter （PUB） and GFP were ligated to plasmid pLO134 using subcloning technology to construct plasrnid pTK153. Human kideny 293T cells were co-transfected with the three-plasmid system containing packaging plasmid ANRF, plasmid pTK153 and envelope plasmid VSV-G by using 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 （ m. o. i. ） of 3. The expression of GFP in mouse T lymphocytes was observed by fluorescence microscopy and fluorescence-activated cell sorting （FACS）. The results showed that the transfection efficacy was 63.04 ±7.24% in 293T cells analysed by FACS and the viral titer was （ 3.09± 0.61 ）×10^6 U/ml. The expression of GFP was also evident in mouse T lymphocytes and the transduction efficacy was （ 37.98 ±6.26 ） %. It is concluded that the three-plasmid system of lentiviral vector containing GFP gene is successfully constructed and the transduction efficacy is high in mouse T lymphocytes.