通用腺相关病毒载体构建及表达报告基因GFP的研究

Construction of a General Adeno-associated Virus Vector and GFP Expressing in Rat Astrocytes Transduced by the rAAV Vector

目的 构建基因治疗通用型 AAV载体并检测它转导外源基因作用。方法 使用限制性内切酶切出p SSV9int-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 -p ACCMVp L p A的含有CMV启动子、多克隆位点 (MCS)和多聚腺苷酸信号 (Poly A)的表达盒 ,构建了重组 AAV通用载体质粒 p SSHG-CMV。在该质粒 MCS插入 GFP基因后 ,我们使用 p SSHG-CMV-GFP、p GF14 0和 p AAV/Ad 3种质粒共转染 2 93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度 ,并将该病毒感染新生大鼠星形胶质细胞 ,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× 10 11,浓缩后可达 2× 10 13 ,表明成功的构建了重组 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 GFP-AAV的大鼠星形胶质细胞表达了明显的 GFP荧光。结论 本文构建的重组 AAV通用载体 p SSHG-CMV,可转导外源基因 。

ObjectiveTo construct an universal adeno-associ ated virus(AAV) vector and to detect the ability of the rAAV vector to transfer gene into target cells. MethodsBy using recombinant techniques ,gene elements of AAV Rep and Cap genes between ITRs of pSSV9inte-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 to be pSSVHG-CMV,which was an universal adeno-associated virus (AAV) vector. After GFP report gene was inserted into MCS of the plasmid,the rAAV vector expressing GFP,pSSVHG-CMV-GFP plasmid,have been constructed and then it was introduced into 293 cells by Ca 3(PO 4) 2 me thod using three plasmids of pSSHG-CMV-GFP?pGF140 and pAAV/Ad. When the cell transfected after 72h,the rAAV was harvested and the titrations of rAAV stocks and rAAV concentrated was detected by dot-blot test using dig-GFP probe. The GFP expressing in rat astrocytes was observed by fluorescence microscope when cells infected after 24,48 and 72h. ResultsTi tration of rAAV stock produced using new rAAV vector and procedure ranged betwee n 1×10 11 and 2×10 12 total particles/ml and it was increased 100 ti mes in rAAV concentrated than it did in rAAV stock. The astrocytes infected rAAV-GFP expressed significantly GFP fluorescence,that it showed that rAAV-GFP is an infectious virus. ConclusionsThe rAAV vector constructed in this paper,pSSHG-CMV plasmid,can transfer exterior gene into target cell usi ng proceeding of recombinant AAV and it may be used on the research of genes therapy.