稳定表达CDC50A的卵巢癌细胞株的建立及鉴定  

Establishment and identification of an ovarian cancer cell strain stably expressing CDC50A

作　　者：韩甜甜[1] 尹婕[1] 曾靖[1] 金滢[1] 李艳[1] 潘凌亚[1] HAN Tian-tian;YIN Jie;ZENG Jing;JIN Ying;LI Yan;PAN Ling-ya(Department of Obstetrics and Gynecology,National Clinical Research Center for Obstetrics&Gynecologic Disease,Peking Union Medical College Hospital,CAMS&PUMC,Beijing 100730,China)

机构地区：[1]中国医学科学院,北京协和医学院,北京协和医院妇产科,国家妇产疾病临床研究中心,北京100730

出　　处：《基础医学与临床》2022年第6期896-901,共6页Basic and Clinical Medicine

基　　金：国家自然科学基金(81572564)。

摘　　要：目的以分子克隆技术建立稳定表达人细胞周期调控蛋白50A(CDC50A)的SKOV3细胞株。方法通过分子克隆技术将CDC50A定向连接到表达载体pLVX-IRES-GFP上。pLVX-CDC50A-GFP表达质粒经慢病毒包装,稳定转染SKOV3,建立稳定表达CDC50A的SKOV3细胞株,通过流式细胞测量术、免疫印迹、免疫荧光分析其Flag标签蛋白表达。结果通过电泳及酶切鉴定证实了pLVX-CDC50A-GFP表达载体的构建,荧光显微镜定性观察病毒感染效率,流式细胞测量术检测感染效率为86.5%,免疫印迹法只检测到pLVX-CDC50A-GFP表达质粒的SKOV3细胞株有Flag标签蛋白表达,阴性对照组无蛋白表达。结论成功构建了CDC50A的表达载体,并通过慢病毒感染建立了过表达CDC50A的SKOV3细胞株,为开展CDC50A在卵巢癌中的机制研究奠定了基础。Objective To establish a stable-expressing human cell cycle regulatory protein 50A(CDC50A)SKOV3 cell strain expressing vector containing CDC50A by molecular cloning.Methods CDC50A was introduced into the expression vector pLVX-IRES-GFP by molecular cloning method.The pLVX-CDC50A-GFP expression plasmid was packaged by lentivirus and stably transfected into SKOV3.SKOV3 cell strain with high-expressing CDC50A was established.The expression of Flag protein was analyzed by flow cytometry,Western blot and immunofluorescence.Results The construction of pLVX-CDC50A-GFP expressing vector was confirmed by gel electrophoresis and endonuclease digestion.The efficiency of infection was observed qualitatively under fluorescence microscope and detected quantitatively by flow cytometry as 86.5%.Flag-tag protein was detected by Western blot to present only in the positive group.Conclusions The SKOV3 cell strain was established by infecting with lentivirus of pLVX-CDC50A-GFP,laying a foundation for the mechanism study of CDC50A in ovarian cancer.

关 键 词：CDC50A 过表达 卵巢癌 稳定表达 

分 类 号：Q291[生物学—细胞生物学]