小鼠miR-214真核表达载体的构建及其鉴定  

Construction and identification of pcDNA3. 1(+/-)-miR-214 recombinant vector in COS-7 cells

作　　者：施冰[1] Les Elzenaar Ralphvan Oort Yigal Pinto 

机构地区：[1]北京军区总医院干部病房一科,北京100700 [2]Heart Failure Research Center,University of Amsterdam

出　　处：《中国临床保健杂志》2013年第6期634-635,共2页Chinese Journal of Clinical Healthcare

基　　金：国家自然科学基金资助项目(81170225)

摘　　要：目的构建小鼠miR-214的pcDNA3.1(+/-)真核表达载体并在COS-7细胞中转染检测其表达。方法小鼠基因组DNA扩增得到miR-214序列,将酶切后的miR-214克隆到真核表达载体pcDNA3.1(+/-),重组pcDNA3.1(+/-)-miR-214经过酶切和测序鉴定后转染COS-7细胞并应用萤光定量PCR法检测miR-214表达水平。结果小鼠miR-214真核表达载体序列分析完全正确;转染COS-7细胞后miR-214的表达水平可增加1.41倍。结论成功构建了pcDNA3.1(+/-)-miR-214真核表达载体并能在真核细胞中进行表达,为进一步进行miR-214的功能研究提供了实验基础。Objective To construct the recombinant eukaryotic expression vector pcDNA3.1(+/-)-miR-214 and investigate its transfection and expression in COS-7 cell.Methods The PCR product of miR-214 was ac-quired from genomic DNA from mice.The miR-214 PCR product was digested with restriction enzyme EcoRI and No-tI,then the digested miR-214 was cloned into vector pcDNA3.1(+/-).The recombinant plasmid pcDNA3.1(+/-)-miR-214 was identified with restriction enzyme digestion and sequence analysis.It was then transfected into COS-7 cells by lipofectamin reagent and the expression of miR-214 was determined by real time PCR.Results Restriction enzyme digestion analysis and DNA sequencing indicated the correct construction of the recombinant plasmid pcD-NA3.1(+/-)-miR-214.The expression of miR-214 in COS-7 cells increased after transfection with pcDNA3.1 (+/-)-miR-214.Conclusion The construction of the eukaryotic plasmid pcDNA3.1(+/-)-miR-214 has been achieved successfully.

关 键 词：微RNAS 遗传载体 真核细胞 COS细胞 小鼠 

分 类 号：R346[医药卫生—基础医学]