磷脂酰肌醇3-激酶调节亚基p55PIK表达载体的构建及单克隆株的筛选  

Construction of p55PIK Expression Vector and Screening of Its Stable Cell Line

作　　者：左学良[1] 王桂华[1] 蔡娟[2] 邓豫[1] 董硕[1] 蔡少鑫[1] 李小兰[1] 陶德定[1] 胡俊波[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院分子医学中心,武汉430030 [2]华中科技大学同济医学院附属同济医院肿瘤科,武汉430030

出　　处：《华中科技大学学报（医学版）》2010年第3期366-368,共3页Acta Medicinae Universitatis Scientiae et Technologiae Huazhong

基　　金：国家重点基础研究发展规划项目(No.2009CB521802);国家自然科学基金(No.30872472;30800569);湖北省自然科学基金(No.2008CDB174)资助项目

摘　　要：目的构建表达载体pEGFP-N1-p55PIK,在人胚胎肾细胞株HEK293中筛选其稳定表达的细胞株。方法 PCR扩增p55PIK基因全长,经过Xho1和Xma1酶切、T4DNA连接酶连接、DH5α转化,酶切和测序鉴定以确定构建质粒正确。转染人胚胎肾细胞株HEK293,G418筛选稳定表达p55PIK的单克隆细胞株,应用Western blot方法检测p55PIK蛋白的表达。结果 pEGFP-N1-p55PIK质粒经PCR、酶切、测序鉴定正确,经过G418筛选后获得稳定细胞株,在荧光显微镜下可观察到绿色荧光蛋白在HEK293细胞中的表达,Western blot检测结果显示稳定转染pEGFP-N1-p55PIK的细胞中p55PIK表达水平增高。结论正确构建表达载体pEGFP-N1-p55PIK,并在HEK293细胞中成功筛选出稳定表达p55PIK的细胞株,为进一步探讨p55PIK的功能提供了良好的工具。Objective To construct the recombinant plasmid pEGFP-N1-p55PIK and detect its stable expression in HEK293 cells.Methods The full sequence of p55PIK was got by PCR.After enzyme digestion by Xho1 and Xma1,ligation overnight by T4 DNA ligase and transformation by DH5α,we got the recombinant plasmid of pEGFP-N1-p55PIK,which was identified by enzyme digestion and DNA sequencing.The recombinant plasmid was transfected into HEK293 cells cultured in DMEM containing G418.The p55PIK expression in every stable cell line was detected by using Western blot.Results The recombinant plasmid pEGFP-N1-p55PIK was identified to be correct by enzyme digestion and DNA sequencing.The green fluorescence in HEK293 cells transfected with pEGFP-N1-p55PIK and pEGFP-N1 vector was observed under the fluorescence microscopy.Western blot analysis revealed that the p55PIK expression was significantly increased in HEK293 cells.Conclusion The expression vector pEGFP-N1-p55PIK is successfully constructed,which can stably express p55PIK protein in HEK293 cells.

关 键 词：磷脂酰肌醇3-激酶 p55PIK 基因表达 克隆细胞 

分 类 号：Q7[生物学—分子生物学]