含人β1转化生长因子短发夹RNA重组腺病毒载体的构建和鉴定

目的 构建含人β1转化生长因子(TGF-β1)短发夹RNA(short hairpin RNA,shRNA)的复制缺陷型腺病毒载体.方法 通过聚合酶链式反应(PCR)法将带有TGF-β1 shRNA 序列构建至U6启动子(U6sh TGF-β1)下游,与T载体连接,将质粒转化人大肠杆菌.将U6sh TGF-β1连到穿梭质粒pDC316上,与腺病毒基因组质粒pBHGlox△E1、3Cre共转染293T细胞,得到重组腺病毒载体(rAd TGF-β1).经PCR鉴定正确后,进行扩增、纯化、滴度测定及感染性鉴定.结果 PCR法得到目的 片段U6sh TGF-β1,经测序,序列与设计方案完全一致.rAd TGF-β1具有良好的感染性,腺病毒滴度可达107.3TCID50/ml.双引物PCR法鉴定Ad TGF-β1可扩增出腺病毒E2b区片段及特异性片段U6sh TGFβ1.结论 成功构建了能表达TGF-β1 shRNA 的重组腺病毒载体rAd TGF-β1(A)及对照载体rAdTGF-β1(B),可能为临床应用RNA干涉--新的基因技术防治瘢痕疙瘩提供高效的方法.

Construction and identification of replication recombinent adenovirus vector of human TGF-β1 shRNA

Objective To construct the recombinant adenovirus vector with human gene TGF-β1 (shRNA,short hairpin RNA)by Cre/lox P system.Methods Human U6 promoters following TGF-β1 shRNA(U6shTGF-β1)were obtained with PCR.Then the promoters were ligated to T-vector.After plasmids pT-U6shTGF-β1 being formed.these plasmids were transformed into E.coli JM109.The target gene fragment was subcloned into a shuttle plasmid PDC316 to construct recombinant shuttle plasmid PDC3 16-TGF-β1.Then the combinant shuttle plasmid and adenovirus genomic plasmid pBHGlox△E1 and 3Cre were cotransfected into 293T cell to construct recombinant adenovirus AdTGF-β1,which was then identified by infection test and PCR amplification.Results U6shTGF-β1 was obtained.The sequence of cloned target fragment was completely consistent with designed sequence.After purification and concentration,the titer of AdTGF-β1 reached 107.3 TCID50/ml.Ad TGF-β1 could transfect 293T cell.Both adenovirus and TGF-β1 target gene fragment could be amplified from rAdTGF-β1 by PCR.Conclusions The recombinant adenovirus expression vector of TGF-β1 and control gene are successfully constructed.This study establishes a foundation for further study on TGF-β1 RNAi vaccines and new gene therapy for human keloid and hypertrophic scar.