AFP与CEA基因的启动子克隆及肿瘤细胞特异性表达的研究  

Cloning of AFP and CEA Gene Promoters and Analysis of Their Tumor Specific Activity

作　　者：雷文[1] 王世兵[1] 马步云[1] 叶静[1] 夏玉龙[1] 王毅刚[1] 

机构地区：[1]浙江理工大学生命科学学院新元医学与生物技术研究所,杭州310018

出　　处：《浙江理工大学学报（自然科学版）》2012年第1期119-124,共6页Journal of Zhejiang Sci-Tech University(Natural Sciences)

基　　金：国家自然科学基金资助项目(30800093);浙江省自然科学基金资助项目(Y2090935);浙江理工大学科研启动基金(1016845-Y;1016834-Y);浙江省大学生科技创新计划资助课题(2010R406036)

摘　　要：为了研究AFP和CEA基因启动子的转录活性与肿瘤特异性,本实验利用PCR技术扩增AFP和CEA基因上游序列,并将其克隆到pGL3-Basic载体中构建pGL3-AFP和pGL3-CEA荧光报告质粒。将构建的pGL3-AFP和pGL3-CEA质粒分别与pRL-TK共转染人结肠癌细胞株SW620、肝癌细胞株Huh-7、肺癌细胞株A549、宫颈癌细胞株Hela、人肝正常细胞株QSG-7701、人肺正常细胞株Beas-2b,用双荧光报告系统检测这两种质粒在不同细胞里的荧光活性表达。酶切和测序结果证实成功构建了双荧光素酶报告质粒pGL3-AFP和pGL3-CEA,双荧光素酶报告基因检测结果证明AFP和CEA均具有一定的肿瘤特异性。这些结果表明AFP和CEA均具有一定的肿瘤特异性,为进一步研究AFP和CEA基因的表达调控机制和探讨靶向基因-病毒治疗提供依据。Clone the AFP and CEA promoters and construct their luciferase report vector of AFP and CEA gene in order to study their transcriptional targeting activity and tumor specific properties. The AFP and CEA gene promoter fragment are generated by PCR （polymerase chain reaction） and then subcolned into the MCS of luciferase report gene vector pGL3-basic to generate the recombined plasmid pGL3-AFP and pGL3-CEA, pGL3-basic, pGL3-AFP, pGL3-CEA as well as pGL3-control are cotransfected with pRL- TK into SW620 cell, Huh-7 cells, A549 cells, Hela cells, QSG-7701 cells and Beas-2b cells respectively. The luciferase activities are measured by dual luciferase reporter （DLR） system. Sequencing and restricted digestive results show that AFP and CEA gene promoters are successfully cloned into plasmid pGL3-basic and constructed their luciferase report plasmids pGL3-AFP and pGL3-CEA respectively. DLR results indicate their high transcriptional activitity and tumor specific properties.

关 键 词：AFP CEA双荧光素酶报告基因 转录靶向性 

分 类 号：Q789[生物学—分子生物学]