南方医科大学学报2012，Vol.32Issue(3)：317-321,5.DOI:44-1627/R.20120307.1529.010
p38丝裂原激活蛋白激酶基因重组慢病毒载体的构建及其在建立人前列腺癌稳定细胞株中的应用
Construction of a recombinant lentiviral vector of p38 MAPK and establishment of a human prostatic carcinoma cell line stably expressingp38 MAPK
摘要
Abstract
Objective To construct a recombinant lentiviral vector for p38 MAPK and establish a human prostatic carcinoma cell line that stably expresses p38 MAPK. Methods EGFP/p38 fusion gene was subdoned into the lentiviral vector pTYF-EFla-IRES-EGFP. The recombinant lentiviral vector pTYF-EFla-EGFP/p38 was indennfied by restriction enzyme digestion, and packaged in HEK 293T cells using lipofectaminlm2000 with the packaging plasmid psPAX2 and envelope plasmid pMD2.G. The viral titer was tested according to the expression level of GFP. The resulting recombinant lentiviral vector was transduced into human prostatic carcinoma DU145 cells, and stably transduced cells were selected by limiting dilution analysis. The intracellular expression level of total p38 was detected by Western blotting and the cell growth curve was drawn. Results DNA restriction enzyme digestion demonstrated that the recombinant lentiviral vector of the fusion gene EGFP/p38 (pTYF-EFla-EGFP/p38) was constructed successfully. The recombinant lentiviral vector was packaged in 293T with a viral titer of 4.7x106 TU/ml. A stable cell line, EGFP/p38-DU145, was established, which stably expressed exogenous EGFP/p38 MAPK fusion protein as detected by Western blotting and showed a lowered growth rate compared to the control cells. Conclusion We have successfully constructed a recombinant lentiviral vector of the fusion gene EGFP/p38 and established a stable cell line EGFP/p38-DU145. Overexpression of p38 has a significant inhibitory effect on the proliferation of DU145 cells in vitro.关键词
p38丝裂原激活蛋白激酶/稳定细胞株/前列腺癌/慢病毒Key words
p38 MAPK/ stable cell line/ prostatic carcinoma/ lentivirus分类
医药卫生引用本文复制引用
荆玉明,李红卫,罗杰,张艳玲,陈三三,万沛,颜仁和,王宏昌,陈白虹,谭万龙..p38丝裂原激活蛋白激酶基因重组慢病毒载体的构建及其在建立人前列腺癌稳定细胞株中的应用[J].南方医科大学学报,2012,32(3):317-321,5.基金项目
国家自然科学基金(81072113) （81072113）
