医药学论文：简化的两步法细菌内同源重组腺病毒表达载体重组高效制备p53基因

【摘要】 目的 应用细菌内同源重组高效制备p53基因重组腺病毒。方法 设计引物扩增p53基因，在上下游引物分别引入Xhol和HindⅢ酶切位点，将p53基因亚克隆连接到经相同酶切的p shuttle-cmv转移质粒上，转化DH5α筛选卡那霉素抗性菌落构建重组腺病毒转移质粒p shuttle-cmv-p53用Pmel酶切线性化采用两步法进行同源重组：先将腺病毒骨架质料pAdEasy-1转化氯化钙法制备的BJ5183感受态细菌，筛选得到链霉素和氨苄青霉素抗性的AdBJ5183转化菌；再将线性化的p shuttle-cmv-p53转化氯化钙法制备的AdBJ5183感受态细菌，在细菌内进行同源重组，挑选卡那霉素抗性菌落培养并提取质粒，琼脂糖电泳挑选大片断的重组质粒pAdBJ5183-P53分别用限制性内切酶PacI和BstXI及PCR法鉴定。结果 成功构建了p53基因重组腺病毒表达载体。结论 采用简化的两步氯化钙化学转化法可以取代电穿孔法高效构建重组腺病毒表达载体。
【关键词】 腺病毒载体；同源重组；p53基因
【Abstract】 Objective To construct recombinant replication-defective human adenovirus serotype 5 vector carrying wt-p53 gene by simplified two-step homologous recombination protocol in bacteria.Methods wt-p53 gene was firstly subcloned into a transfer vector p shuttle-cmv and the positive recombinant p shuttle-p53 was linearized by PmeI.Then the simplified two-step homologous recombination protocol was employed for the construction of recombinant adenoviral vector.In step one，adenoviral skeletal plasmid pAdeasy-1 was transformed into BJ5183 competent cells by calcium chloride chemical methed to obtain pAdBJ5183;and in step two，linearized psh-p53 plasmid was transformed into pAdBJ5183.The possible positive adenoviral plasmids were selected by agarose gel electrophoresis and indentified by PacI as well as BxtXI cleaved pattern.Results Replication-defective recombinatant adenovirus containing human p53 gene was successfully constructed.Conclusion The results indicated that the calcium chloride chemically simplified two-step homologous recombination protocol can replace the co-transformation by electroperforation and may have broaed utility in system that involve homologous recombination in bacteria.