Liu S, Liu D, Ding J
Molecular Medicine Center, Cardiovascular Institute, CAMS and PUMC, Beijing 100037.
Zhongguo Yi Xue Ke Xue Yuan Xue Bao. 1999 Dec;21(6):425-30.
To construct the recombinant adenovirus which contained human pro-UK cDNA and to provide experimental basis for clinical gene therapy.
The recombinant plasmid pCA14-pro-UK and the plasmid pJM17 were cotransfected into cultured 293 cells and the replication-deficient adenovirus were harvested, identified, propagated and titered. The recombinant virus were then transferred into balloon-injured femoral arteries of rabbits and the pro-UK expression was detected by immunohistochemistry and Western Blot.
PCR amplification and in vitro transfection of A549 cells confirmed that pro-UK had been inserted into adenovirus genome correctly, and immunohistochemistry and Western blot analysis showed that pro-UK expressed evidently in the balloon-injured sites of the rabbit femoral arteries.
The recombinant adenovirus that constructed could express pro-UK cDNA in vivo successfully.
构建含人尿激酶原（pro-UK）cDNA的重组腺病毒，为临床基因治疗提供实验依据。
将重组质粒pCA14-pro-UK与质粒pJM17共转染至培养的293细胞，收获、鉴定、扩增并测定复制缺陷型腺病毒的滴度。然后将重组病毒转染至兔球囊损伤的股动脉，通过免疫组织化学和蛋白质印迹法检测pro-UK的表达。
PCR扩增及A549细胞的体外转染证实pro-UK已正确插入腺病毒基因组，免疫组织化学和蛋白质印迹分析表明pro-UK在兔股动脉球囊损伤部位有明显表达。
构建的重组腺病毒能在体内成功表达pro-UK cDNA。
