稳定表达CDC50A的卵巢癌细胞株的建立及鉴定

doi:10.16352/j.issn.1001-6325.2022.06.003

基础医学与临床 ›› 2022, Vol. 42 ›› Issue (6): 896-901.doi: 10.16352/j.issn.1001-6325.2022.06.003

稳定表达CDC50A的卵巢癌细胞株的建立及鉴定

韩甜甜, 尹婕, 曾靖, 金滢, 李艳, 潘凌亚^*

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

收稿日期:2022-03-07 修回日期:2022-04-23 出版日期:2022-06-05 发布日期:2022-06-02
通讯作者: * panly@pumch.cn
基金资助:
国家自然科学基金 (81572564)

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

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

Received:2022-03-07 Revised:2022-04-23 Online:2022-06-05 Published:2022-06-02
Contact: * panly@pumch.cn

摘要/Abstract

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。

关键词: CDC50A, 过表达, 卵巢癌, 稳定表达

Abstract: 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.

Key words: CDC50A, over-express, ovarian cancer, stable expression

中图分类号:

Q291

韩甜甜, 尹婕, 曾靖, 金滢, 李艳, 潘凌亚. 稳定表达CDC50A的卵巢癌细胞株的建立及鉴定[J]. 基础医学与临床, 2022, 42(6): 896-901.

HAN Tian-tian, YIN Jie, ZENG Jing, JIN Ying, LI Yan, PAN Ling-ya. Establishment　and identification of an ovarian cancer cell strain stably expressing CDC50A[J]. Basic & Clinical Medicine, 2022, 42(6): 896-901.

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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

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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

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