Chen Chong, Zhang Huan-Xin, Zeng Lin-Yu, Zhang Yin, Zhang Jian-Jun, Xu Kai-Lin
Department of Hematology, Xuzhou Medical College Affiliated Hospital, Xuzhou 221002, Jiangsu Province, China.
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2010 Dec;18(6):1600-3.
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.
本研究旨在构建携带小鼠RORγt和glp基因的慢病毒载体，并检测RORγt在293FT细胞中的表达。通过RT-PCR从小鼠胸腺中扩增RORγt片段，并克隆到PCR 2.1载体中。通过酶切制备RORγt DNA片段并插入MigR1质粒，然后将RORγt-IRES-GFP与慢病毒转移质粒pTK208定向连接，生成慢病毒载体pXZ9-RORγt。使用脂质体2000将三种质粒共转染到293FT包装细胞中，产生重组慢病毒。转染后，收集慢病毒上清液并通过超速离心进行浓缩。用浓缩的慢病毒感染293FT细胞，在荧光显微镜下检查GFP表达，并通过蛋白质免疫印迹法检测RORγt蛋白的表达。结果显示，从小鼠胸腺cDNA中扩增出RORγt片段，成功构建了重组慢病毒载体pXZ9-RORγt。经过一轮超速离心制备出高滴度慢病毒。病毒感染后可在293FT细胞中检测到RORγt表达。结论是成功构建了含有小鼠RORγt-IRES-GFP的慢病毒载体pXZ9-RORγt；RORγt可通过慢病毒载体转导在293FT细胞中表达，为进一步研究RORγt调控Th17细胞分化的机制提供了一种可选工具。
