靶向大鼠诱导型一氧化氮合酶基因的shRNA重组腺病毒载体的构建及鉴定

目的 构建携带增强型绿色荧光蛋白基因(EGFP)标志针对大鼠诱导型一氧化氮合酶(iNOS)基因的shRNA重组腺病毒载体并在人胚肾-293细胞中扩增制备重组病毒.方法 利用AdMax包装系统,用先期构建的带有EGFP标记基因的穿梭质粒pDC316-iNOS-shRNA-EGFP,与骨架质粒pBHGlox_El,3Cre共转染293包装细胞,同源重组产生复制缺陷型重组腺病毒载体Ad5-inosshRNA-EGFP,反复感染293细胞扩增病毒后,离子交换法纯化病毒,并测定病毒颗粒数及滴度.结果 经聚合酶链反应(PCR)检测和EGFP表达证明已成功构建了携带iNOS-shRNA-EGFP的重组腺病毒载体;扩增纯化后,测得重组腺病毒颗粒数为3.6×1011 VP/ml,A260/A280值约为1.25,病毒活性为7.94×109IU/ml.结论 已成功构建重组腺病毒载体Ad5-inos-shRNA-EGFP,为利用基因激活技术治疗勃起功能障碍的研究奠定实验基础.

Abstract：

Objective To construct an adenovirus vector expressing short hairpin RNA (shRNA)of rat inducible oxide synthase (iNOS) carrying enhanced green fluorescent protein (EGFP) and amplify the adenovirus vector in HEK-293 cells. Methods The shuttle plasmid pDC316-inos-shRNA-EGFP that was constructed at an earlier date, was cotransfected with the adenovirus skeleton plasmid pBHGlox_E1,3Cre into 293 cells to obtain the produced replication defective recombinant adenovirus vector Ad5-inosshRNA-EGFP. The recombinant adenovirus was propagated by repeat infection of 293 cells and purified by ion exchange method, then the virus particles were counted and the purity and titer were determined.Results Recombinant adenoviral vector Ad-ACE-shRNA was constructed successfully, which was confirmed by polymerase chain reaction (PCR) and GFP expression. After amplification and purification, the virus particle count, A260/A280 and titer of recombinant adenovirus were 3.6 × 1011 VP/ml, 1.25 and 7.94 ×109 IU/ml,respectively. Conclusion Recombinant adenovirus vector Ad5-inos-shRNA-EGFP is successfully constructed, which laid a foundation for gene activation in erectile dysfunction treatment.

Construction and identification of recombinant adenovirus expressing short hairpin RNA of rat inducible nitric oxide synthase

Objective To construct an adenovirus vector expressing short hairpin RNA (shRNA)of rat inducible oxide synthase (iNOS) carrying enhanced green fluorescent protein (EGFP) and amplify the adenovirus vector in HEK-293 cells. Methods The shuttle plasmid pDC316-inos-shRNA-EGFP that was constructed at an earlier date, was cotransfected with the adenovirus skeleton plasmid pBHGlox_E1,3Cre into 293 cells to obtain the produced replication defective recombinant adenovirus vector Ad5-inosshRNA-EGFP. The recombinant adenovirus was propagated by repeat infection of 293 cells and purified by ion exchange method, then the virus particles were counted and the purity and titer were determined.Results Recombinant adenoviral vector Ad-ACE-shRNA was constructed successfully, which was confirmed by polymerase chain reaction (PCR) and GFP expression. After amplification and purification, the virus particle count, A260/A280 and titer of recombinant adenovirus were 3.6 × 1011 VP/ml, 1.25 and 7.94 ×109 IU/ml,respectively. Conclusion Recombinant adenovirus vector Ad5-inos-shRNA-EGFP is successfully constructed, which laid a foundation for gene activation in erectile dysfunction treatment.