人LGR5基因克隆及其基因转染细胞株的构建  

Cloning of Human LGR5 Gene and Construction of Its Gene Transfected Cell Line

作　　者：邓云[1,2] 陆婷[1] 葛彦[1] 居颂文[3,4] 居颂光[1] 

机构地区：[1]苏州大学医学部基础医学与生物科学学院免疫学系,江苏苏州215123 [2]解放军第一00医院妇产科,江苏苏州215007 [3]南京医科大学附属苏州医院,江苏苏州215008 [4]苏州市立医院北区消化疾病与营养研究中心,江苏苏州215008

出　　处：《中国血液流变学杂志》2013年第1期1-3,72,共4页Chinese Journal of Hemorheology

基　　金：国家自然科学基金青年科学基金资助项目（81000912）;江苏省自然科学基金资助项目（BK2012621）;“青蓝工程”资助（2010）

摘　　要：目的克隆人LGR5分子并构建其稳定表达的基因转染细胞株.方法提取人宫颈癌细胞株HeLa细胞总RNA,通过RT-PCR克隆获得人LGR5全长cDNA,将人LGR5全长cDNA重组入逆转录病毒载体pEGZ-term,通过293T细胞的包装获得具有感染力的完整重组病毒载体,收集培养上清感染L929细胞,筛选构建稳定表达人LGR5分子的基因转染细胞株.结果成功克隆人LGR5全长cDNA和构建pEGZ/LGR5逆转录病毒表达载体,成功获得稳定表达人LGR5的基因转染细胞株L/LGR5.结论成功克隆了人LGR5基因并构建了稳定表达LGR5分子的基因转染细胞株,为进一步研究LGR5分子的生物学功能奠定了基础.Objective To clone human LGR5 full length cDNA and construct its gene transfected cell line that stably expressed the human LGR5.Methods Human LGR5 full length cDNA was cloned from human cervical cancer cell line HeLa by RT-PCR and subcloned into retroviral expressing vector pEGZ-term.The recombinant plasmid together with its helper virus vector was cotransfected into the package cell 293T with Lipofectamine 2000.The L929 cells were infected with the supernatant of the transfected 293T cells,and then were selected with Zeocin.The Zeocin resistant cells were harvested for screening their GFP and LGR5 expression by fluorescence microscope and flow-cytometry.Results Human LGR5 gene is cloned and subcloned into retroviral expressing vector pEGZ-term successfully.Gene transfected line L929/LGR5 that stably expressed the human LGR5 was constructed.Conclusion Human full length LGR5 cDNA is cloned and its retroviral expressing vector is constructed successfully.Gene transfected cell line which stably expressed the human LGR5 is established successfully.It provides a valuable tool to study the biological function of human LGR5 molecule.

关 键 词：LGR5 克隆 基因转染细胞株 

分 类 号：R730.3[医药卫生—肿瘤]