【摘要】 目的:利用细菌内同源重组法快速构建携带增强型绿色荧光蛋白(EGFP)和人内皮型一氧化氮合酶(he-NOS)cDNA的重组腺病毒质粒和制备表达EGFP和heNOS的重组腺病毒。方法:质粒载体pHCMV SP1A-heNOS用EcoRⅠ酶切,回收heNOS cDNA,亚克隆至腺病毒穿梭质粒中,形成转移质粒pShuttle-heNOS-EGFP;然后用PmeI线性化后的pShuttle-heNOS-EGFP转化含腺病毒骨架质粒pAdEasy-1的感受态BJ5183,使其在细菌内发生同源重组,获得阳性重组质粒pAdEasy-heNOS-EGFP。重组质粒经PCR、酶切和测序鉴定后经PacⅠ酶切,转入AD 293细胞,包装成重组腺病毒Ad-heNOS-EGFP,纯化,鉴定,滴度测定。结果:heNOS cDNA成功地插入到穿梭质粒中,pShuttle-heNOS-EGFP与pAdeasy-1在BJ5183中成功发生了同源重组,得到了重组腺病毒质粒pAdEasy-heNOS-EGFP。重组腺病毒经PCR检测表明携带有目的基因heNOS,滴度约为6.5×1015pfu/L。结论:细菌内同源重组法构建腺病毒载体具有高效、省时、省力的特点,制备出的高滴度重组腺病毒Ad-heNOS-EGFP为勃起功能障碍的基因治疗奠定了基础,携带的EGFP标记基因可以直观地检测靶细胞感染和外源基因的表达情况。更多还原

【Abstract】 Objective To construct recombinant adenoviral plasmid containing human endothelial nitric oxide synthase (heNOS) cDNA using homologous recombination in bacteria and to prepare recombinant adenovirus expressing enhanced green fluorescent protein (EGFP) and heNOS. Methods HeNOS cDNA was digested from plasmid pHCMV SP1A-heNOS with EcoR I and subcloned into the shuttle vector to generate the transfer plasmid pShuttle-heNOS-EGFP. The vector pShuttle-heNOS-EGFP was linearized with Pme I and transformed into ultracompetent BJ5183 bacteria containing pAdEasy-1. The positive clone of homologous recombination (pAdEasy-heNOS-EGFP) was identified by PCR, enzyme digestion, and DNA sequencing. The positive recombinant adenoviral plasmid was digested with Pac I and transfected AD293 cells with Lipofectamine 2000 to package recombinant adenovirus particles. The recombinant adenovirus was purified with density gradient centrifugation of cesium chloride. PCR was used to characterize the recombinant adenovirus expressing heNOS. The titer and purity of the recombinant adenovirus was determined by ultraviolet spectrometry. Results HeNOS cDNA was successfully inserted into the shuttle vector. Homologous recombination occurred between pShuttle-heNOS-EGFP and pAdEasy-1 within BJ5183 to generate pAdEasy-heNOS-EGFP. The recombinant adenovirus was confirmed to be successfully packaged within AD293 cells with PCR. The titer of the purified Ad-heNOS-EGFP was 6.5 ×1015 pfu/L. Conclusion The construction of adenovirus plasmid by homologous recombination in bacteria can be quickly and easily performed. The preparation of recombinant adenovirus Ad-heNOS-EGFP with high titer provides a basis for gene therapy of erectile dysfunction. EGFP expression is a useful tool for directly monitoring the infection of target cells and the expression of gene of interest.更多还原