通用腺伴随病毒载体的构建及表达报告基因GFP的实验研究.DOCVIP

临床医学论文-通用腺伴随病毒载体的构建及表达报告基因GFP的实验研究 ???????????? 作者：郑国玺，韦俊荣，祝康，侯瑾，施典羽，朱宏亮，杨广笑，王全颖 【关键词】? 重组腺伴随病毒；载体；报告基因；基因治疗；NIH3T3 　　摘要：目的? 构建基因治疗通用型腺伴随病毒(AAV)载体并检测其转染外源基因作用。方法? 使用限制性内切酶切除pSSV9int质粒中的AAV病毒Rep和Cap基因元件后，插入了重组腺病毒专用穿梭质粒pACCMVpLpA的含有CMV启动子、多克隆位点(MCS)和多聚腺苷酸信号(PolyA)的表达盒，构建了重组AAV通用载体质粒pSSHGCMV。在该质粒MCS插入绿色荧光蛋白(GFP)基因后，使用pSSHGCMVGFP、GFP140和pAAV/Ad三种质粒共转染293包装细胞，制备GFP重组AAV。应用斑点杂交实验检测重组病毒滴度，并将该病毒感染新生大鼠NIH3T3细胞，荧光显微镜观察GFP表达。结果? 重组AAV的滴度在浓缩前可达2×1011cfu/mL，浓缩后可达2×1013cfu/mL，表明成功地构建了重组AAV载体。插入外源基因GFP后，在包装病毒和辅助质粒的联合作用下，能产生具有感染性的重组AAV。感染了重组GFPAAV的大鼠NIH3T3细胞表达了明显的GFP荧光。结论? 构建的重组AAV通用载体pSSHGCMV可转染外源基因，这为进一步的基因治疗奠定了基础。 　　关键词：重组腺伴随病毒；载体；报告基因；基因治疗；NIH3T3 　　Construction of a gengeral adenoassociated virus vector and GFP expression in rat NIH3T3 transduced by the rAAV vector 　　ABSTRACT: Objective?To construct a universal adenoassociated virus (AAV) vector and to detect the ability of the rAAV vector to transfer gene into the target cell. Methods?By using recombinant technique, the gene elements of AAV Rep and Cap genes between ITRs of pSSV9int- plasmid were replaced by expressing cassette in pACCMVpLpA plasmid, a recombinant adenovirus shuttle vector. The expressing cassette included a CMV promoter, a multicloning sites (MCS) and a polyA signal. The new plasmid constructed was named pSSVHGCMV, which was a universal adenoassociated virus (AAV) vector. After GFP report gene was inserted into MCS of the plasmid, the rAAV vector expressing GFP, pSSVHGCMVGFP plasmid, have been constructed and then it introduced into 293 cell by method of Ca3(PO4)2 using three plasmids of pSSVHGCMVGFP, pGF140 and pAAV/Ad. After the cell was transfected for 72h, the rAAV was harvestd and the titrations of rAAV stocks and rAAV concentrated were detected by dotblot test using digGFP probe. The GFP expression in rat NIH3T3 was observed by fluorescence microscop after the cell was infected for 24, 48 and 72h. Results?Titration of rAAV stock produced using new rAAV vector and procedure ranged between 1?011 and 2?0