CENPK基因过表达载体的构建及鉴定  

Construction and Identification of CENPK Gene Overexpressed Vector

作　　者：吴伟刚[1] 刘妙娜[1] 刘利平[1] 刘琢冰 刘威龙[1] 陈心春[1] 周伯平[1] 

机构地区：[1]广东医学院附属深圳市第三人民医院,深圳市肝病研究所,深圳市感染免疫重点实验室,广东深圳518112

出　　处：《中国肿瘤》2015年第6期505-509,共5页China Cancer

基　　金：国家自然科学基金(81372227);深圳市科技计划项目(CXZZ20130515163643)

摘　　要：[目的]构建着丝粒蛋白K(cetromere protein K,CENPK)基因过表达载体,并对其在肝癌FOCUS细胞中的表达进行鉴定。[方法]1从人肝癌组织中扩增出CENPK目的基因并将其克隆到p CMV-3Tag-3载体上,构建真核表达载体p CMV-CENPK;2利用脂质体法将构建好的p CMV-CENPK真核表达载体转染肝癌FOCUS细胞,并用G418筛选出CENPK基因稳定过表达的肝癌FOCUS细胞株;3通过Real-time PCR及Western Blot的方法对筛选出来的细胞进行鉴定。[结果]1经双酶切分析及测序验证,成功构建了真核表达载体p CMV-CENPK。2通过Real-time PCR及Western blot的方法鉴定,CENPK基因在筛选出来的肝癌FOCUS细胞株中成功过表达。[结论]成功构建真核表达载体p CMV-CENPK并使其在肝癌FOCUS细胞中稳定过表达,为进一步研究CENPK基因在肝癌发生发展中的作用奠定了基础。[Purpose ] To construct CENPK gene overexpressed vector and to identify its expression in liver cancer FOCUS cells. [Methods] Amplify the CENPK gene from liver cancer tissues of hu- man,and cloned it into pCMV-3Tag-3 vector,to construct eukaryotic expression vector pCMV- CENPK,then to transfect it into FOCUS cells by lipofection method,and to select it by G418. Cells selected by G418 were confirmed by Real-time PCR and Western Blot. [Results] Being ver- ified with double endonuclease digestion and DNA sequencing,the eukaryotic expression vector pCMV-CENPK was successfully constructed. The FOCUS cells selected by G418 were successfully overexpressing GENPK gene ,which was confirmed by Real-time PCR and Western blot. [Conclu- sion ] The eukaryotic expression vector pCMV-CENPK is successfully constructed and was stably overexpressed on FOCUS cells,which lay the foundation for further study of CENPK gene on the role of carcinogenesis and progress of liver cancer.

关 键 词：CENPK基因 过表达 真核表达载体 肝癌FOCUS细胞 

分 类 号：R735.7[医药卫生—肿瘤]