人miR-205真核表达载体的构建及鉴定  

Construction and identification of eukaryotic expression vector of human miR-205

作　　者：吴义高[1] 肖戈[1] 徐文清[1] 黄福[1] 胡卫列[2] 王尉[2] 

机构地区：[1]南方医科大学研究生学院,广州510515 [2]广州军区广州总医院泌尿外科研究所

出　　处：《山东医药》2012年第44期20-22,F0002,共4页Shandong Medical Journal

基　　金：国家自然科学基金项目(81172421);国家自然科学基金青年科学基金资助项目(81001142)

摘　　要：目的构建真核表达载体pcDNA3.1(+)-miR-205,并使其在肾上腺皮质癌细胞SW-13中稳定表达。方法依据miRbase数据库中pre-miR-205序列设计引物,PCR扩增pre-miR-205基因并将其克隆至线性化的pcD-NA3.1(+)质粒中,获得重组表达载体pcDNA3.1(+)-miR-205,经双酶切及测序分析后,将其及对照空载体转染肾上腺皮质癌SW-13细胞,采用实时定量RCR法鉴定miR-205在SW-13细胞中表达。结果酶切和测序结果均证实pcDNA3.1(+)-205重组质粒构建成功,经实时定量RCR检测表明转染pcDNA3.1(+)-205的SW-13细胞中miR-205阳性高表达。结论真核表达载体pcDNA3.1(+)-205在SW-13中稳定转染,为进一步研究miR-205在肾上腺皮质癌细胞SW-13中的功能及基因调控机制奠定了实验基础。Objective To construct a eukaryotic expression vector of pcDNA3.1 （ ＋ ）-miR-205 and express it stably in adrenal cortical carcinoma SW-13 cell line. Methods PCR primers were designed and amplificated according to the pre-miR-205 sequence in miRbase database, and then the PCR product was cloned into linearized plasmid pcDNA3. 1 （ ＋ ）, finally, a new plasmid pcDNA3.1 （ ＋ ） -205 was constructed and identificated by the restriction endonuclease digestion and DNA sequencing, pcDNA3.1 （ ＋ ） -205 was transfected into SW-13 cells and the miR-205 expression was detected by real-time quantitative PCR. Results Sequencing and restriction endonuclease digestion showed the pcDNA3.1 （ ＋ ） - 205 was correctly constructed. The result of real-time quantitative PCR showed that the expression of miR-205 was higher in cells transfected with pcDNA3.1 （ ＋ ）-205 than the control cells. Conclusion The eukaryotic expression vector of miR- 205 is stably expressed in adrenal cortical carcinoma SW-13 cells, which provides miR-205 an experimental basis for further study of function and gene regulation mechanisms in tumors.

关 键 词：肾上腺皮质肿瘤 微小RNA 真核表达载体 miR-205 

分 类 号：R736.6[医药卫生—肿瘤]