蛋白磷酸酶2A调节亚基PR55-γ基因克隆及真核细胞表达  

The construction and eukaryotic expression of protein phosphatase 2A regulatory subunit PR55-γ

作　　者：魏波华 梁惠欣[1] 王翠翠[1] 陈连祥[1] 叶静[1] 陆一鸣[1] 

机构地区：[1]上海交通大学医学院附属瑞金医院急诊科,上海200025

出　　处：《内科理论与实践》2014年第3期198-202,共5页Journal of Internal Medicine Concepts & Practice

基　　金：国家自然科学基金项目(项目编号:81270433;81000875;81171846;81372099);上海市科委基础研究重点项目(项目编号:13JC1404001);上海市青年科技启明星计划(项目编号:11QA1404400)

摘　　要：目的:构建蛋白磷酸酶2A(PP2A)调节亚基PR55-γ(PPP2R2C)慢病毒过表达质粒,并在U87细胞株中稳定过表达PPP2R2C,探讨PPP2R2C对PP2A活性的影响。方法:通过反转录(RT)-PCR扩增PPP2R2C的编码序列并克隆至慢病毒载体PWPIR-增强型绿色荧光蛋白(EGFP)中,获得PWPIR-EGFP-PPP2R2C载体,通过酶切鉴定、质粒测序确定质粒克隆成功,用磷酸钙沉淀法将质粒转导至U87细胞中,流式细胞分析术检测U87细胞转导效率,蛋白质印迹法检测目的蛋白表达,通过PP2A蛋白免疫共沉淀法检测PP2A活性。结果:PWPIR-EGFP-PPP2R2C载体构建及U87细胞株稳转过表达PPP2R2C成功,并且细胞转导效率超过90%,外源性PPP2R2C表达率上升近23倍,过表达PPP2R2C能够增强PP2A活性。结论:PWPIR-EGFP-PPP2R2C载体构建成功,并通过慢病毒转导细胞U87细胞株表达PPP2R2C成功,过表达PPP2R2C能够增强PP2A活性,为进一步研究PPP2R2C功能奠定基础。Objective To construct plasmids containing protein phosphatase 2A （PP2A） regulatory subunit PR55-γ （PPP2R2C） gene and transduct it into U87 ceils for over-expressing PPP2R2C, and to explore the effect of PPP2R2C on PP2A activity. Methods PPP2R2C cDNA sequence was obtained by RT-PCR amplification and cloned into slow virus vector PWPIR-enhanced green fluorescent protein （EGFP）; the construction of PWPIR-EGFP-PPP2R2C vector was confinned by restrictive enzyme digestion and DNA sequencing. The plasmid PWPIR-EGFP-PPP2R2C was transducted into U87 cells by calcium phosphate precipitation method. The PPP2R2C expression was detected by Western blotting. Fluo- rescence activated cell sorting was used to detect the transduction efficiency. PP2A activity was detected by PP2A protein coimmunoprecipitation （COIP）. Results PWPIR-EGFP-PPP2R2C plasmid was constructed successfully and expressed in U87 cells, and the transduction efficiency was〉90%. The expression of exogenous PPP2R2C was elevated to about 23 times the endogenous one. Over-expressing PPP2R2C could enhance PP2A activity. Conclusions PPP2R2C was successfully constructed and expressed in U87 cells, and PP2A activity was enhanced with the over-expressing of PPP2R2C, which would provide the foundation for further studies on biological function of PPP2R2C.

关 键 词：蛋白磷酸酶2A 蛋白磷酸酶2A调节亚基PR55-γ 真核表达 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学] Q78[医药卫生—基础医学]