| | | 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
| | 西医学 评职论文下载邹昌勇，全家妩Δ，田生和，杨京生，吴季南 【摘要】 目的西医学 评职论文下载 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西医学 评职论文下载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.
复制缺陷型腺病毒可以高效介导基因的体内外转移；感染细胞范围广，不但可以感染分裂期细胞，也可感染静止期细胞；感染细胞时其DNA不整合到宿主细胞染色体中，安全可靠。因此成为基因治疗中常用的载体。重组腺病毒载体的构建方法有真核细胞内质粒重组法、酵母人工染色体克隆系统、Cre/loxp定点重组系统、末端蛋白-DNA复合物共转染法等［１～３］。以上方法牵涉到的步骤和环节较多、工作量大、实验周期长。1998年He等［4］建立了细菌内质粒共转化同源重组方法，该法简便快速、实验周期短，但是共转化需要电穿孔仪，且同源重组成功率不到20%。笔者在实际工作中对该法进行了改进，采用简化的两步氯化钙化学转化法代替电穿孔共转化法构建了p53重组腺病毒表达载体。
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