KDM2B过表达慢病毒载体构建及其在人卵巢癌细胞中的表达  

Construction of lentiviral vector with KDM2B over-expression and its expression in ovarian cancer cells

作　　者：蒙家华 况燕[1] 卢芳芳[1] 易叶叶 徐红[1] 

机构地区：[1]广西医科大学第一附属医院,南宁530021

出　　处：《山东医药》2018年第5期5-8,共4页Shandong Medical Journal

基　　金：国家自然科学基金资助项目(81360389);广西自然科学基金资助项目(2015GXNSFAA139152)

摘　　要：目的构建赖氨酸特异性脱甲基酶2B(KDM2B)过表达慢病毒载体,并探讨其是否能在人卵巢癌细胞A2780及SKOV3中稳定表达。方法根据Gen-Bank数据库提供的KDM2B基因序列,用限制性内切酶获得线性化载体,制备目的基因片段并用GV慢病毒载体改造和包装病毒,对阳性克隆重组产物进行测序及鉴定。用LV-KDM2B过表达慢病毒感染A2780、SKOV3细胞(LV-KDM2B感染组),同时感染LV-CON作为阴性对照(LV-CON感染组),未感染的A2780、SKOV3细胞作为空白对照组。采用RT-PCR和Western blotting法分别检测细胞中KDM2B mRNA及蛋白表达。结果慢病毒阳性克隆载体测序结果与目标序列完全一致。LV-KDM2B感染组A2780、SKOV3细胞中KDM2B mRNA及蛋白表达均较LV-CON感染组、空白对照组高,比较差异均有统计学意义(P均<0.05)。结论 KDM2B过表达慢病毒载体构建成功,且其可在人卵巢癌细胞A2780、SKOV3中稳定表达。Objective To construct the lentiviral vector with KDM2 B over-expression and to explore whether it can be stably expressed in the human ovarian cancer A2780 and SKOV3 cells. Methods According to the KDM2 B gene nucleotide sequence provided by Gen-Bank database,the linearized vector was obtained using restriction endonuclease,and target gene fragment was prepared and packaged with GV lentiviral vector. The positive clone recombinant products were sequenced and identified. A2780 and SKOV3 cells were infected with LV-KDM2 B over-expression lentivirus（ LV-KDM2 B infection group）,while cells infected with LV-CON were taken as the negative controls（ LV-CON infection group）,and the uninfected A2780 and SKOV3 cells served as the blank control group. KDM2 B mRNA and protein expression levels were analyzed by real-time quantitative PCR（ RT-q PCR） and Western blotting,respectively. Results The lentivirus positive clone sequencing was consistent with the target sequence. The mRNA and protein expression levels of KDM2 B in the A2780 and SKOV3 cells of the LV-KDM2 B group were higher than those of the LV-CON infection group and blank control group,and the differences were statistically significant（ all P 0. 05）. Conclusion The lentiviral vector with LVKDM2 B over-expression is successfully constructed,and KDM2 B gene can be stably expressed in the human ovarian cancer A2780 and SKOV3 cells.

关 键 词：卵巢癌 赖氨酸特脱甲基酶2B A2780细胞 SKOV3细胞 

分 类 号：R737.31[医药卫生—肿瘤]