表达Cre酶复制缺陷型重组腺病毒载体的构建及鉴定  

Construction and characterization of recombinant replication-defective adenovirus vector expressing Cre recombinase

作　　者：段素素[1] 张云[1] 杨帆[1] 王颖[1] 李文辉[1] 王树惠[1] 刘力[1] 

机构地区：[1]中国医学科学院中国协和医科大学基础医学研究所病原微生物学系,北京100005

出　　处：《基础医学与临床》2006年第6期593-596,共4页Basic and Clinical Medicine

基　　金：北京市自然科学基金(7032035)

摘　　要：目的构建Cre-loxP条件性基因敲除系统中Cre酶的重组腺病毒表达载体,作为体细胞条件性基因敲除的基础,并为传统ES细胞条件性基因敲除提供新的选择。方法用pAd-easy系统在大肠杆菌内经同源重组的方法构建Cre酶的复制缺陷型重组腺病毒载体;W estern b lot方法鉴定Cre蛋白的表达。结果在大肠杆菌内构建出重组腺病毒质粒,在包装细胞系内包装出重组病毒颗粒;测定病毒滴度为106pfu/L;重组病毒在细胞内表达Cre蛋白。结论表达Cre酶的重组腺病毒载体构建成功,阳性重组质粒的鉴定方法得以改进,为进一步的体细胞Cre-loxP条件性基因敲除提供了基础。Objective To construct and to characterize a recombinant adenovirus vector expressing Cre recombinase protein, a major component of Cre-loxP system. Methods To construct recombinant adenovirus vector containing Cre gene by homology recombination in E. coli, and to identify Cre protein expressed by recombinant virus by Westem Blotting. Results We constructed recombinant adenovirus plasmid in E. coli and produced virus particles in its packaging cell line; virus titer was 10^9 pfu/ml; the recombinant virus expressed Cre protein in cell. Conclusion We successfully constructed recombinant adenovirus vector expressing Cre recombinase and improved the method of identifying positive recombinant adenovirus plasmids. Our results laid a technical base for Cre-loxP conditional gene targeting in somatic cells.

关 键 词：CRE重组酶 Cre-loxP条件性基因打靶 腺病毒载体 

分 类 号：Q78[生物学—分子生物学]