人pEGFP-PKARIIβ重组质粒的构建及其在BGC-823胃癌细胞中的表达  

Construction of a eukaryotic expression plasmid encoding the human PKAR IIβ gene and its expression in human gastric cancer BGC-823 cells

作　　者：王桂玲[1] 姜佩佳[1] 王孝会[1] 陈薇[1] 

机构地区：[1]中国医科大学基础医学院细胞生物学教研室卫生部细胞生物学重点实验室,辽宁省沈阳市110001

出　　处：《世界华人消化杂志》2011年第14期1446-1450,共5页World Chinese Journal of Digestology

基　　金：国家自然科学基金资助项目;No.30871294~~

摘　　要：目的:研究人的蛋白激酶A调节亚基RIIβ(PKARIIβ)基因的绿色荧光蛋白(GFP)真核表达载体融合蛋白在胃癌细胞BGC-823内的表达和定位,初步探讨其在肿瘤细胞中的分子作用机制.方法:以pRSETB-PKARIIβ质粒为模板,PCR扩增出全长PKARIIβ编码序列,用XhoⅠ和EcoRⅠ双酶切后,亚克隆至含有GFP标签的融合表达载体pEGFP-C1中.测序正确后,将重组质粒转染到胃癌细胞BGC-823中,提取细胞蛋白进行Westernbolt检测.利用共聚焦激光扫描显微镜观察pEGFP-PKARIIβ在人胃癌细胞BGC-823内的定位.结果:将人全长PKARIIβ编码序列克隆到了真核表达载体pEGFP-C1中,酶切鉴定片段为1.2kb,在预期的位置产生条带.Western blot检测到了融合蛋白正确表达,分子量约为72000Da.并观察到GFP-PKARIIβ在HEK293和BGC-823细胞内都有表达,且定位以细胞质为主,在细胞核内少量表达.结论:成功构建了人全长PKARIIβ编码序列的pEGFP-PKARIIβ真核表达载体,证实GFP-PKARIIβ蛋白在BGC-823细胞质内特异表达,为进一步研究PKARIIβ的在胃癌中的功能提供了重要的实验材料.AIM：To construct a eukaryotic expression plasmid encoding the human protein kinase A regulatory subunit II beta（PKAR IIβ） gene and to examine its expression and localization in BGC-823 gastric cells using green ？ uorescent protein as a reporter.METHODS：The coding sequence of the PKAR IIβ gene was amplified from the plasmid pRSETB-PKARIIβ by PCR and subcloned into pEGFP-C1 vector after digestion with Xho I and EcoR I.After the identity of recombinant plasmid was verified by direct sequencing,the plasmid was transfected into BGC-823 cells.The expression of the recombinant plasmid in BGC-823 cells was detected by Western blot.The localization of GFP-PKARIIβ in BGC-823 cells was observed by laser scanning confocal microscopy.RESULTS：The coding sequence of the PKARIIβ gene was inserted into the pEGFP-C1 vector successfully.Restriction enzymes digestion showed that the length of the insert was 1.2 kb,matching the expected size.The expression of GFP-PKARIIβ fusion protein,which had a molecular weight of 72 000 Da,was detected in BGC-823 cells by Western blot.The GFP-PKARIIβ protein was localized predominantly to the cytoplasm but sparsely to the nucleus of HEK293 and BGC-823 cells.CONCLUSION：A recombinant plasmid expressing the PKARIIβ gene has been success-fully constructed and provides a tool for future investigation of PKARIIβ functions.The GFP-PKARIIβ fusion protein was expressed mainly in the cytoplasm of HEK293 and BGC7901 cells.

关 键 词：PKARIIβ 真核表达 绿色荧光蛋白 基因转染 胃癌细胞 

分 类 号：R735.2[医药卫生—肿瘤]