摘要
目的 利用细菌内同源重组法构建含 1 2密码子点突变的k ras基因重组腺病毒。方法 用限制性内切酶kpnⅠ +xhoⅠ从载体pcDNA3 k ras1 2 (Val)中切出 1 2密码子点突变的k ras基因片断 ,亚克隆至经同样酶切的腺病毒穿梭质粒pAdTrack CMV中 ,形成转移质粒pAdTrack CMV/k ras 1 2 (Val) ,将之PmeⅠ酶切线性化后与腺病毒基因重组质粒pAdEasy 1共转化大肠杆菌BJ51 83 ,抽提经鉴定含目的基因的重组体质粒 ,PacⅠ酶切后用脂质体转染 2 93细胞 ,包装成重组体腺病毒Ad k ras1 2 (Val)。采用PCR方法对重组体腺病毒进行鉴定 ,利用穿梭质粒pAdTrack CMV中带有GFP报告基因 ,对病毒滴度和感染效率进行监测。结果 利用CaCl2 法由pAdTrack CMV/k ras 1 2 (Val)和pAdEasy 1共转化大肠杆菌BJ51 83 ,可获得 2 5%左右的阳性重组体细菌克隆。PCR检测表明重组腺病毒已含有目的基因 ,滴度为 1 .2× 1 0 1 2 pfu/ml。结论 细菌内同源重组法构建腺病毒相比于传统的细胞内同源重组法 ,具有成功率高、方法简便、快捷、实验周期短的优点 。
Objective To construct the recombinant adenovirus of mutant k ras by using the method of homogenous recombination in bacteria. Methods Mutant k ras gene was liberated from the vector of pcDNA3 k ras 12(Val) via KpnⅠ+XhoⅠ digestion, and subcloned into shuttle vector of pAdTrack CMV, forming transfer vector of pAdTrack CMV/k ras 12(Val). Then it was linearized with PmeⅠ and cotransformed into BJ5183 cells with adenovirual geonomis plasmid of pAdEasy 1. The DNA of identified recombinant plasmid was digested with PacⅠ and transfected to 293 cells to package adenovirus. The PCR technique was used to detect target gene. The titre and its infection rate of the Ad k ras 12(Val) was measured with the aid of GFP expression. Results There were over 25% positive recombinant bacterial clones after co transformation of BJ5183 bacterial cells with pAdTrack CMV/k ras 12 (Val) and pAdEasy 1 by method of CaCl 2. PCR test indicated each the recombinant adenovirus contained the insert of k ras 12(Val). The titre of purified recombinant adenovirus was 1.2×10 12 ?pfu/ml. Conclusion The method of homologous recombination in bacteria is convenient and efficient, which compared with that of in cell and the pepared Ad k ras 12(Val) paves a sound foundation for further study.
出处
《中国肺癌杂志》
CAS
2002年第1期14-17,共4页
Chinese Journal of Lung Cancer
基金
国家自然科学基金 ( 30 0 0 0 2 0 6 )
教育部博士点基金 ( 2 0 0 0 55)资助
