miRNA-7真核表达载体的构建与鉴定  

Construction and identification of eukaryotic expression vector encoding miRNA-7

作　　者：宋永祥[1,2] 李颖[1,3] 秦安东[1,3] 廖珍媛[1,3] 李永菊[1,3] 罗军敏[1,3] 任涛[4] 徐林[1,3] 

机构地区：[1]遵义医学院免疫学教研室,贵州遵义563000 [2]遵义医学院附属医院心胸外科,贵州遵义563000 [3]贵州省免疫学研究生教育创新基地,贵州遵义563000 [4]同济大学附属东方医院,上海200120

出　　处：《西安交通大学学报（医学版）》2013年第4期454-459,共6页Journal of Xi’an Jiaotong University（Medical Sciences）

基　　金：贵州省优秀科技教育人才省长专项资金(No.2009C457);贵州省科技技术厅项目(No.2009C491);遵义医学院博士启动基金项目(No.2008F329)~~

摘　　要：目的构建miR-7真核表达载体并观察其对人肺癌细胞体外生长的影响。方法以人肺癌细胞系95D细胞基因组DNA为模板,PCR法扩增miR-7前体序列,经BamHⅠ和HindⅢ双酶切后将其亚克隆入真核表达载体pcDNA3.1(-),并进行酶切及测序鉴定;将构建成功的pcDNA3.1(-)-pri-miR-7载体(命名为p-miR-7)体外瞬时转染95D细胞,应用Real-time PCR特异探针法检测miR-7成熟体的表达水平,并利用MTT法检测细胞生长的改变。结果酶切和测序验证成功构建了miR-7真核表达载体p-miR-7;表达载体p-miR-7瞬时转染95D细胞后可有效表达miRNA-7成熟体,并显著抑制95D细胞的体外生长(P<0.05)。结论成功构建了miR-7的真核表达载体,为后续深入研究miR-7在肺癌发生中的作用及机制提供了前期实验基础。Objective To construct a eukaryotic expression vector encoding miR-7 and detect its effect on the growth of human lung cancer cells.Methods Pri-miR-7 sequence was amplified from genomic DNA of human 95D cells by PCR and subcloned into eukaryotic expression vector pcDNA3.1（-） using restriction site of BamHⅠ and HindⅢ.The positive recombinant pcDNA3.1（-）-pri-miR-7（p-miR-7） was identified by enzyme cleaving and sequencing.p-miR-7 was transiently transfected into 95D cells and the expression level of miRNA-7 was determined by Real-time PCR using specific probe before and after transfection.The proliferation of 95D cells was also accessed by MTT assay.Results Eukaryotic expression vector encoding miR-7（p-miR-7） was identified as correct by enzyme cleaving and sequencing.The expression level of miR-7 in 95D cells transiently transfected with p-miR-7 was increased significantly（P0.05）.The proliferation of 95D cells in vitro was markedly inhibited（P0.05）.Conclusion The eukaryotic expression vector pcDNA3.1（-）-pri-miR-7 is successfully constructed,which provides the experimental foundation for further studies on the role and mechanism of miR-7 in the pathogenesis of lung cancer.

关 键 词：miR-7 真核表达 肺癌 MTT法 

分 类 号：R392[医药卫生—免疫学]