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

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

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，将质粒转化入大肠杆菌。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10^7.3TCID50／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 TGF-β1。结论成功构建了能表达TGF-β1 shRNA的重组腺病毒载体rAd TGF-β（A）及对照载体rAd TGF-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。

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- U6sbTGF-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 pDC316- 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 10^7.3 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.