人Toll样受体2细胞外区基因重组腺病毒穿梭载体的构建及鉴定  

Construction and identification of the recombinant adenovirus shuttle vector carrying the human Toll-like receptor 2 extracellular domain gene

作　　者：孙守勋[1] 李强[1] 石妍[2] 刘静[1] 蒲晓允[1] 

机构地区：[1]第三军医大学附属新桥医院检验科,重庆400037 [2]解放军第201医院麻醉科,辽宁辽阳111000

出　　处：《医学研究生学报》2009年第2期120-123,I0001,共5页Journal of Medical Postgraduates

基　　金：全军“十一五”面上项目课题(批准号:2006B060);创伤、烧伤与复合伤国家重点实验室开放基金资助项目(批准号:2006B-2)

摘　　要：目的:克隆人Toll样受体2(TLR2)细胞外区基因,并构建含有目的基因的重组腺病毒穿梭质粒pAdTrack-CMV-TLR2。方法:应用RT-PCR方法从人外周血单个核细胞中扩增TLR2细胞外区基因,克隆入pUCm-T载体并转化大肠埃希菌感受态DH5α,提取质粒进行酶切鉴定及DNA测序分析后,用KpnⅠ和HindⅢ双酶切,将目的片段定向克隆至经相同处理的pAdTrack-CMV腺病毒穿梭质粒中,双酶切鉴定并测序验证。结果:RT-PCR扩增得到约1000bp的目的基因片段,TA克隆后测序鉴定与GenBank中的序列一致,经酶切连接成功地将目的基因插入到腺病毒穿梭质粒pAdTrack-CMV中。结论:成功克隆了人TLR2细胞外区基因,并构建了含目的基因的重组腺病毒穿梭载体。Objective： To clone the human TLR2 extracellular domain gene and construct the recombinant adenovirus shuttle plasmid pAdTrack-CMV-TLR2. Methods： The TLR2 extracellular domain gene was amplified from total RNA of human peripheral blood mononuclear cells by RT-PCR and inserted into the pUCm-T vector. The recombinant was transformed into E coli DH5α. After confirmed by enzyme digestion and sequencing, the DNA fragment, digested with Kpn Ⅰ and Hind Ⅲ, was directionally cloned into the adenovirus shuttle plasmid pAdTrack-CMV. The recombinant plasmid was verified by double digestion and DNA sequencing. Results ： The gene fragment of 1 000 bp was obtained by RT-PCR. After TA cloned, its sequence conformed with that reported in the GenBank, and the cloned gene was exactly inserted into the adenovirus shuttle vector. Conclusion： The TLR2 extracellular domain gene was correctly cloned and the recombinant adenovirus shuttle vector successfully constructed.

关 键 词：TOLL样受体2 重组腺病毒 穿梭质粒 单个核细胞 

分 类 号：Q784[生物学—分子生物学]