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目的 构建表达人尿激酶原的重组腺病毒载体,为临床基因治疗提供实验依据。方法 采用亚克隆获得的pCA14-pro-UK重组质粒与质粒pJM17共转染培养的293细胞,经同源重组,得到含有pro-UKcDNA的重组腺病毒。扩增纯化后,局部转染经球囊损伤后的兔股动脉。7d后,分别收集转染的血管段和对照血管段,以Western印迹和免疫组化检测表达。结果 (1)经PCR扩增及体外感染A549细胞结果表明,获得了具有生物活性的含人pro-UKcDNA的重组腺病毒;(2)重组腺病毒的滴度为1×109噬菌斑形成单位/ml(PFU/ml);(3)免疫组化及Western印迹分析显示:重组腺病毒能在损伤的兔股动脉内膜表达尿激酶原。结论 构建得到的携带人尿激酶原cDNA的重组腺病毒能在损伤血管部位表达。
Objective To construct a recombinant adenovirus vector expressing human pro-urokinase and provide experimental evidence for clinical gene therapy. Methods The subcloned pCA14-pro-UK recombinant plasmid and plasmid pJM17 were co-transfected into 293 cells and the recombinant adenovirus containing pro-UK cDNA was obtained by homologous recombination. After amplification and purification, the rabbit femoral artery after local injury was transfection. After 7 days, the transfected and control blood vessels were collected and detected by Western blot and immunohistochemistry. Results (1) The results of PCR amplification and in vitro infection of A549 cells showed that the biologically active recombinant adenovirus containing human pro-UK cDNA was obtained. (2) The titer of the recombinant adenovirus was 1 × 109 plaque formation units / ml (PFU / ml); (3) Immunohistochemistry and Western blot analysis showed that the recombinant adenovirus could express urokinase in injured rabbit femoral artery intima. Conclusions The constructed recombinant adenovirus carrying human pro-urokinase cDNA can express in damaged blood vessels.