携带小鼠RORγt基因慢病毒载体的构建及其在293FT细胞中的表达  

Construction of Lentiviral Vector Carrying Mouse RORγt and Expression of RORγt in 293FT Cells

作　　者：陈翀[1,2] 张焕新[2] 曾令宇[1,2] 张滢[2] 张建军[2] 徐开林[1,2] 

机构地区：[1]徐州医学院附属医院血液科,江苏徐州221002 [2]徐州医学院移植免疫实验室,江苏徐州221002

出　　处：《中国实验血液学杂志》2010年第6期1600-1603,共4页Journal of Experimental Hematology

基　　金：国家自然科学基金资助项目;编号30971281

摘　　要：本研究构建携带小鼠ROFγt和glp基因的重组慢病毒载体,检测RORγt蛋白在293FT细胞中的表达。用RT-PCR扩增小鼠RORγt基因,将其克隆至PCR2.1T载体,酶切制备RORγtDNA片段,插入MigR1质粒后将RORγt-IRES-GFP定向连入慢病毒转移质粒pTK208,生成pXZ9-RORγt。用脂质体介导三质粒共转染法转染293FT细胞,荧光显微镜下观察GFP表达情况,以Western blot鉴定RORγt的表达。结果表明,RT-PCR扩增出RORγt基因并克隆入PCR2.1T载体;经亚克隆构建了慢病毒转移质粒XZ9-RORγt。质粒经脂质体转染293FT细胞获慢病毒颗粒,慢病毒颗粒体外高效感染293FT细胞,感染后Western blot检测到RORγt蛋白表达。结论:成功构建慢病毒载体pXZ9-RORγt,并在293细胞内获得表达。This study was aimed to construct a lentiviral vector carrying mouse RORγt and glp gene, and to detect the expression of RORγt in the 293FT cells. The RORγt fragment was amplified by RT-PCR from mouse thymus and cloned into PCR 2.1 vector. The RORγt DNA fragment was prepared by digestion and inserted into MigR1 plasmid, then the RORγt-IRES-GFP was directionally linked with lentiviral transfer plasmid pTK208 to generate a lentiviral vector pXZ9-RORγt. The recombinant lentivirus were produced by co-transfected three plasmids into 293FT packing cells using lipofectamine 2000. After transfection, the lentiviral supernatant was collected and concentrated via ultracentrifugation. The 293FT cells were infected by the concentrated lentivirus, GFP expression was examined under a fluorescent microscope and the expression of RORγt protein was detected by Western blot. The results showed that the RORγt fragment was amplified from cDNA of mouse thymus and recombinant lentiviral vector pXZ9-RORγt was constructed successfully. High titer lentivirus were prepared after one round ultracentrifugation. RORγt expression could be detected in 293FT cells after virus infection. It is concluded that the lentiviral vector pXZ9-RORγt containing mouse RORγt-IRES-GFP is successfully constructed; RORγt can express in 293FT cells via lentiviral vector transduction, which provides an optional tool for further research on the mechanism of RORγt controlling Th17 cell differentiation.

关 键 词：慢病毒载体 RORΓT 293FT细胞 

分 类 号：R392.4[医药卫生—免疫学] Q789[医药卫生—基础医学]