高效化学转化菌内同源重组法构建重组腺病毒  

Construction of Recombinant Adenovirus by An Efficient Method of Chemical Transformation and Homologous Recombination in Bacteria

作　　者：尹冰楠[1] 李冬田[1] 李秋香[1] 佟惠春[1] 李光明[1] 

机构地区：[1]天津医科大学医学微生物学教研室,300070

出　　处：《天津医药》2005年第7期437-440,共4页Tianjin Medical Journal

基　　金：天津市卫生局科技基金资助项目(项目编号:99KY047)

摘　　要：目的:对细菌内同源重组法构建重组腺病毒AdEasy系统加以改进,为制备具有更高抗肿瘤疗效的生物治疗制剂奠定基础。方法:将自杀基因CD和HSV-TK插入穿梭质粒pAdTrack-CMV中,PmeⅠ酶切后,用酚∶氯仿抽提、乙醇沉淀或切胶回收纯化;分别采用电穿孔转化法和化学转化法将线性穿梭质粒(pAdTrackCMV-CDglyTK)与腺病毒骨架质粒pAdEasy-1在细菌内实现同源重组;将重组腺病毒质粒rpAdEasyGFP-CDglyTK以脂质体介导至293细胞中进行包装、扩增。结果:采用切胶回收化学转化法获得阳性重组率最高(100%)。经PCR、酶切以及测序等方法鉴定,证实融合自杀基因已成功插入腺病毒中。结论:对细菌内同源重组法构建重组腺病毒的方法进行了改进,在降低实验成本的同时获得了较原法更高的阳性重组率,并成功构建了复制缺陷性重组腺病毒rAd-CDglyTK。Objective The AdEasy system to construct recombinant adenoviruses by homologous recombination in bacteria was modified to establish the basis for preparing a biological preparation with more anti-tumor efficiency. Methods The suicide genes of CD and HSV-TK were inserted into the shuttle plasmid pAdTrack-CMV and the resultant plasmid was digested by Pme I and then purified by phenol chloroform extraction and ethanol precipitation or gelextraction. The linearized shuttle plasmid pAdTrackCMV-CDglyTK and the backbone plasmid pAdeasy-1 were co-tranformed into bacteria by electro-transformation or chemical transformation to attain homologous recombination. The recombinant plasmid rpAdEasyGFP-CDglyTK was tranformed into 293 cells by liposome to package and proliferate adenovirused. Results The chemical transformation with gel extraction got the highest positive rate of recombination 100%. The fusion suicide genes were successfully inserted into adenoviruses identified by PCR amplification digestion and sequencin. Conclusion The method of homologous recombination in bacteria to consturct recombinant adenoviruses was improved to decrease the cost of experiments as well as to obtain the highest positive recombinant rate. The replication-deficient recombinant adenovirus rAd-CDglyTK was successfully constructed.

关 键 词：重组腺病毒 细菌内同源重组法 AdEasy系统 化学转化法 高效 生物治疗制剂 融合自杀基因 穿梭质粒 293细胞 脂质体介导 肿瘤疗效 基因CD 氯仿抽提 乙醇沉淀 骨架质粒 实验成本 CMV HSV PME 电穿孔 PCR 插入 酶切 回收 阳性 

分 类 号：R734.2[医药卫生—肿瘤] R730.5[医药卫生—临床医学]