miR-181a真核表达载体的构建及鉴定

目的: 构建miR-181a真核表达载体，获得食管癌细胞TE11在其中稳定表达的细胞系，为研究miR-181a的功能以及其在食管癌细胞TE11中的作用机理奠定基础。方法: 根据miR-181a成熟序列在基因组中的位置及其上下游250个碱基序列，以293T细胞基因组DNA为模板设计PCR引物，扩增包含miR-181a前体的序列，连接到线性化的pMD18-T Simple载体中，经BamHⅠ和EcoRⅠ双酶切后亚克隆到pcDNA3. 1 ( + )中，并对重组质粒进行双酶切和测序分析；鉴定完全正确的重组质粒转染食管癌细胞TE11，用G418 ( 350 mg/L )筛选4周获得miR-181a稳定表达细胞系TE11-miR-181a，提取TE11-miR-181a细胞总RNA，用real-time PCR鉴定pcDNA3. 1 ( + )-miR-181a可在真核细胞中稳定过表达。结果: 成功构建了miR-181a的真核表达载体，并获得了在TE11细胞中稳定表达重组质粒的细胞系TE11-miR-181a。结论: miR-181a真核表达载体在食管癌细胞TE11中稳定高表达，为进一步研究miR-181a在食管癌细胞TE11中的功能及基因调控机制奠定了基础。

construct and establishan of eukaryotic expression vector of microRNA-21

Objective To construct an eukaryotic expression vector of microRNA-21 and to establish it’s stable expression cell lines for the study of miR-181a function，to provide a technical platform and make a foundation for the mechanism study of microRNA-21 in esophageal cancer cell TE11. Methods According to mature miRNA-21 with its upstream and dowmstream 250bp sequences in the genome，PCR primers were designed and miR-181a precursor sequence was amplified from 293T cell genomic DNA . PCR product was coloned to the linearized pMD18-T Simple vector and then was subcloned into pCDNA3.1(+) by BamHⅠand EcoRⅠdouble-ligated， the recombinant plasmid was analyzed through restriction enzyme digestion and sequencing analysis；The identified plasmid was transfected into the cell line TE11 with lipofectmine 2000，miR-181a stable expression cell line was screened out by G418，Total RNAs were isolated and miR-181a gene expressed in transfected cells were testified by real-time PCR. Results The miR-181a recombinant eukaryotic expression vector has been constructed successfully and effectively expressed in esophageal carcinoma cell TE11. Conclusion The miR-21 recombinant eukaryotic expression vector can stably express in TE11 and may provide an experimental basis for research of microRNAs.