摘要
目的构建可调控性大鼠小肠TGF-β1表达的重组腺病毒载体。方法按RNA提取试剂（TRIzol Reagent）说明书步骤从大鼠小肠组织抽提总RNA，克隆TGF-β1基因；经穿梭载体与可调控性腺病毒载体骨架连接，鉴定后在HEK293细胞包装。获得高滴度病毒后。取上清检测绿色荧光蛋白（GFP）的表达，观察目的基因的表达情况。结果扩增出与预期大小一致的cDNA片段，其中TGF-β1大小为1173bp；其基因序列与GenBank登录的大鼠TGF-β1基因序列完全一致。重组TGF-β1腺病毒获得的滴度约为10nPFU／ml；病毒包装后绿色荧光蛋白表达的检测结果显示，绿色荧光蛋白随着时间的延长，表达量逐渐增高；与预期结果一致。结论构建的可调控性大鼠小肠TGF-β1重组腺病毒载体得到了正确表达，为进一步研究TGF-β1干扰树突状细胞分化成熟诱导小肠移植免疫耐受提供了依据。
Objective To construct a recombinant adenovirus vector which regulates the expression of rat transforming growth factor beta （TGF-β1）. Methods Total RNA was extracted from F344/N rat small intestine pre-treated with Con A to clone TGF-β1. pTRE-shutfle vector was used as mediator to ligate TGF-β1 gene and backbone of replication-incompetent adenoviral vector. The constructed recombinant adenovirus contained tetracycline-responsive element which could regulate the expression of inserted genes. After identification, the desired recombinant adenovirus was packaged in HEK 293 cells. Superuatant of high titer adenovirus was collected to detect the TGF-β1 gene expression by green fluorescent protein（GFP）. Results The constructed recombinant adenovirus was identified by restriction endonucleases cutting, sequencing, PCR and GFP examination. Condusion Rat TGF-β1 recombinant adenovirus is established successfully, which provides material and evidence for further research of dendritic cell （DC） modified by TGF-β1 to induce immune tolerance in rat heterotopic small bowel transplantation.
出处
《中华胃肠外科杂志》
CAS
2008年第1期76-79,共4页
Chinese Journal of Gastrointestinal Surgery
基金
国家自然科学基金资助项目（30571803）
关键词
转化生长因子Β
腺病毒载体
小肠
树突细胞
大鼠
Transforming growth factor beta
Adenovirus vectors
Intestine, small
Dendritic cells
Rats
