人pre-mir181a腺病毒载体的构建与鉴定

Construction and identification of human pre-mir181a adenovirus

目的探讨构建携带人pre-mir181a基因的重组腺病毒载体,为基因治疗研究提供一种新的方法,并观察其在神经胶质瘤细胞U251细胞中的表达。方法以从Genesil公司购买的含有mir181a的质粒为模板扩增pre-mir181a基因,将回收的PCR产物片段克隆入pGenesil载体,获得重组质粒pGenesil-pre-mir-181a。从pGenesil-pre-mir181a上通过LR体外同源重组将mir181a microRNA表达框转移至pGSadeno腺病毒表达载体上。在体外用不同感染复数(M·O·I)值rAd5-mir181ab分别转染神经胶质瘤细胞U251细胞。采用流式细胞仪、RT-PCR方法检测目的基因的转染效率和表达。结果PCR、酶切鉴定以及序列测定与比对分析表明,重组腺病毒rAd5-mir181a构建正确。神经胶质瘤细胞U251细胞转染rAd5-mir181a的最高效率可达99%,转染后的神经胶质瘤细胞U251细胞表达相应的mir181a。结论本实验成功构建了含mir181a基因的重组腺病毒rAd5-mir181a载体,在体外能高效转染神经胶质瘤细胞U251细胞。

Objective human pre-mirl 81 a gene for To investigate the construction of an adenovirus based vector carrying a new approach of gene therapy, and observe their expression in human glioma cell line U251 cell. Methods The coding sequence of pre-mir181a was amplified by PCR from the plasmid containing the human mirl 81 a purchased from Genesil company. The retrieved PCR fragment was cloned into vector pGenesil and acquired recombinant plasmid pGenesil-pre-mir-181a. After that mirl 81 a microRNA expression frame was transfer to pGSadeno adenovirus vector via LR in vitro homologous recombination from pGenesil-pre-mir181 a. Different multiplicity of infection （M·O·I） of rAd5-mir181 ab was differently tranfected to glioma cell line U251 cell. Then the gene transfer efficiency and expression was detected via Flow cytometry and RT-PCR assay. The recombinant adenovirus rAd5-mirlSla was identified by PCR and digest with restriction enzyme. The best transfection showed an efficiency of 99 % in glioma cell line U251 cell. Conclusions The recombinant rAd5-mirl 81 a vector carrying mirl 81 a gene is successfully constructed and efficiently expressing in glioma cell line U251 cell.