慢病毒介导的稳定过表达人谷氨酰半胱氨酸连接酶催化亚基基因的WRL68细胞株构建

Construction and identification of stable WRL68 cell line with overexpression of GCLC gene mediated by lentiviral vector

目的利用慢病毒载体构建稳定过表达人谷氨酰半胱氨酸连接酶催化亚基(GCLC)的WRL68细胞株。方法采用PCR方法合成GCLC基因全长,经Not I、Nsi I酶切后克隆到慢病毒载体LV6上;采用PCR、酶切、测序鉴定重组质粒LV6-GCLC;将重组质粒转染293T细胞,收集培养上清并感染WRL68细胞,经嘌呤霉素筛选出稳定过表达细胞株。采用增强型绿色荧光蛋白检测转染情况,采用Real-time PCR及Western blotting鉴定所构建的细胞株;MTT法检测细胞活性;采用酶联免疫吸附(ELISA)法检测细胞谷胱甘肽(GSH)含量。结果 PCR、酶切及测序结果表明重组慢病毒载体构建成功;重组慢病毒转染293T细胞后可观察到荧光及蛋白表达,包装过表达慢病毒并检测其浓缩滴度为1.0×109 TU/ml;用1μg/ml嘌呤霉素成功筛选出稳定过表达细胞株;GCLC过表达组中GCLC的m RNA和蛋白的表达量高于空载体转染组及对照组(P<0.05);GCLC过表达组、空载体转染组及对照组的细胞活性间比较,差异无统计学意义(P>0.05);GCLC过表达组GSH含量明显高于空载体转染组及对照组(P<0.05)。结论成功构建了稳定过表达人GCLC的WRL68细胞株。

Objective To over express glutamate-cysteine ligase catalytic subunit（GCLC） in WRL68 cells by the lentiviral vector. Methods The full length GCLC gene was synthesized by PCR method and cloned to lentiviral vector,recombinant plasmid was identified by PCR,restriction enzymes and DNA sequence analysis. The recombinant plasmid was transfected into293 T cell,the lentivirus was added to WRL68 and selected with puromycin for the stable transfected cells. Green fluorescent protein（GFP） was observed under fluorescence microscope to determine efficiency of infection,real-time PCR and Western blotting were used to test the expression of GCLC. Cell viability was tested by MTT,and the content of GSH was analyzed by enzyme linked immunosorbent assays（ELISA）. Results PCR,restriction enzymes and DNA sequence analysis showed that GCLC was successfully bound to the lentiviral vector. The expression of GFP could be observed after recombinant lentiviruses were transfected into 293 T cell. The viruses with overexpression of GCLC were obtained and the titer of concentrated viruses was 1.0×10^9TU/ml. Real-time PCR and Western blotting showed that GCLC was successfully overexpressed in WRL68 cells.The result of MTT showed the cell viability had no difference in control cells and GCLC overexpression cells. The content of GSH in GCLC overexpression cells were highly expressed compared with others. Conclusion The lentiviral vector-mediated GCLC gene overexpression WRL68 cell strain is successfully constructed in the present study.