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目的构建p38丝裂原激活蛋白激酶基因(MAPK)重组慢病毒载体并建立表达外源性p38基因的人前列腺癌稳定细胞株。方法采用限制性内切酶酶切、T4 DNA连接酶连接等方法,将EGFP/p38融合基因插入慢病毒载体pTYF-EF1α-IRES-EGFP中,构建启动子EF1α调控的EGFP/p38共表达慢病毒载体pTYF-EF1α-EGFP/p38,经酶切鉴定后,利用慢病毒三质粒系统,通过脂质体法转染包装细胞HEK 293T细胞,收集病毒上清后转导人前列腺癌DU145细胞株,经有限稀释法筛选重组EGFP/p38稳定细胞株,用Western blotting检测细胞总p38的表达,用计数法绘制细胞的生长曲线。结果经酶切鉴定,成功构建EGFP/p38重组慢病毒载体,并包装慢病毒,检测病毒悬液的滴度为4.7×106 TU/ml,利用此病毒悬液感染DU145细胞,成功筛选到EGFP/p38稳定表达细胞株,Western blotting显示该细胞株能稳定表达外源性EGFP/p38融合蛋白,这种过表达p38的细胞株的生长速度明显低于对照组。结论成功构建p38 MAPK重组慢病毒载体并建立了表达外源性p38 MAPK基因的稳定细胞株EGFP/p38-DU145,细胞内p38过表达能够抑制DU145细胞的增殖。
Objective To construct p38 mitogen - activated protein kinase gene (MAPK) recombinant lentiviral vector and construct a human prostate cancer cell line expressing exogenous p38 gene. Methods The EGFP / p38 fusion gene was inserted into the lentiviral vector pTYF-EF1α-IRES-EGFP by restriction endonuclease digestion and T4 DNA ligase, and the lentiviral vector EGFP / p38 co-expressing EF1α was constructed The vector pTYF-EF1α-EGFP / p38 was identified by restriction enzyme digestion and then transfected into HEK 293T cells by lipofection method using the lentivirus three plasmid system. The supernatant of the transfected human prostate cancer DU145 cell line was obtained. The recombinant EGFP / p38 stable cell line was screened by limited dilution method. The expression of total p38 was detected by Western blotting. The cell growth curve was drawn by counting method. Results Recombinant EGFP / p38 lentiviral vector was successfully constructed and packaged with lentivirus. The titer of the virus suspension was 4.7 × 106 TU / ml. The virus suspension was used to infect DU145 cells and EGFP / p38 stably expressing cell line. Western blotting showed that the cell line stably expressed exogenous EGFP / p38 fusion protein. The growth rate of this over-expressing p38 cell line was significantly lower than that of the control group. Conclusion The recombinant lentiviral vector p38 MAPK was successfully constructed and a stable cell line EGFP / p38-DU145 expressing exogenous p38 MAPK gene was constructed. Overexpression of p38 could inhibit the proliferation of DU145 cells.