用于高效构建复制缺陷型重组腺病毒载体的两步转化法。

Two-step transformation for highly efficient construction of a replication-defective recombinant adenoviral vector.

作者信息

Yang Wen-yu, Huang Zong-hai, Tang Fu-xiang, Qian Yong, Che Xiao-yan

机构信息

Department of Surgery, Zhujiang Hospital, First Military Medical University, Guangzhou 510282, China.

出版信息

Di Yi Jun Yi Da Xue Xue Bao. 2004 Feb;24(2):164-7.

PMID:14965817

Abstract

OBJECTIVE

To efficiently construct a replication-defective recombinant adenoviral vector using a two-step transformation procedure.

METHODS

Plasmid pAdEasy-1 was linearized and transformed into E.coli BJ5183 to construct BJ5183pAdEasy-1 cells. Cytosine deaminase (CD) gene was obtained from plasmid pBS-CD, and subcloned into the shuttle plasmid to form transfer plamid of pAdtrackCMV-CD, which was then linearized and transformed into BJ5183pAdEasy-1 cells. The recombinant adenovirus plasmid DNA was extracted from the transformed bacteria and digested with Pac I after identification, followed by transfection into 293 packaging cells. PCR was used to detect target gene, and the titer and the infection rate of the recombinant Ad were measured with the aid of green fluorescent protein (GFP) reporter gene. The same recombinant AdCMV-CD was constructed in a one-step transformation method for comparison.

RESULTS

The homologous recombination of the two-step transformation method resulted in a success rate of 76.5% (13/17), while the success rate of the one-step method was only 11.8% (2/17), showing significant difference between the two methods (P=0.000 17 by Fisher's exact test).

CONCLUSION

The two-step transformation procedure is more efficient and convenient than one-step method.

摘要

目的

采用两步转化法高效构建复制缺陷型重组腺病毒载体。

方法

将质粒pAdEasy-1线性化后转化至大肠杆菌BJ5183中，构建BJ5183pAdEasy-1细胞。从质粒pBS-CD中获取胞嘧啶脱氨酶（CD）基因，并亚克隆至穿梭质粒中，形成pAdtrackCMV-CD转移质粒，随后将其线性化并转化至BJ5183pAdEasy-1细胞中。从转化后的细菌中提取重组腺病毒质粒DNA，鉴定后用Pac I酶切，然后转染至293包装细胞中。采用聚合酶链反应（PCR）检测目的基因，并借助绿色荧光蛋白（GFP）报告基因测定重组腺病毒的滴度和感染率。采用一步转化法构建相同的重组腺病毒AdCMV-CD作为对照。