pAAV-hSOX9-IRES-tdTomato重组质粒构建腺相关病毒包装(英文)  

pAAV-hSOX9-IRES-tdTomato recombinant plasmid constructs adeno-related virus package

作　　者：刁泽正[1] 燕国清[1] 张志伟[1] 房晶[1] 徐鹏[1] 西永明[1] 任山[1] 刘勇君[1] 隋爱华[1] 

机构地区：[1]青岛大学医学院附属医院脊柱外科,山东省青岛市266003

出　　处：《中国组织工程研究》2012年第20期3759-3762,共4页Chinese Journal of Tissue Engineering Research

摘　　要：背景:实验为基因治疗退变椎间盘实验的前期部分,旨在构建含免疫荧光的pAAV-hSOX9-IRES-tdTomato重组质粒并应用腺相关病毒包装,为后期体内外实验打下基础。目的:构建人SOX9基因过表达腺相关病毒pAAV-hSOX9-IRES-tdTomato的包装。方法:用酶切法将质粒pAAV-IRES-tdTomato和质粒pUC57-hSOX9连接成pAAV-hSOX9-IRES-tdTomato,用质粒共转染方法包装腺相关病毒,感染293AAV细胞,腺相关病毒纯化及用生物学滴度测定法进行滴度测定。结果与结论:经测序结果BLAST比对分析,pAAV-hSOX9-IRES-tdTomato完全与合成的基因序列hSOX9相符,滴度为1×107TU/mL。结果表明,人SOX9基因过表达腺相关病毒pAAV-hSOX9-IRES-tdTomato包装成功。BACKGROUND： As the preliminary experiment for gene therapy in intervertebral disc degeneration, this study aims to construct a recombinant plasmid containing fluorescent pAAV-hSOX9-IRES-tdTomato for adeno-associated virus packaging, in a broader attempt to lay the foundation for late experiments in vitro and in vivo. OBJECTIVE： To construct human SOX9 gene overexpressing adeno-associated virus, pAAV-hSOX9-IRES-tdTomato, packaging. METHODS： The plasmid pAAV-IRES-tdTomato and plasmid pUC57-hSOX9 were connected into pAAV-hSOX9-IRES-tdTomato by enzyme digestion method. The adeno-associated virus was packaged with plasmid co-transfections method. The recombinant pAAV-hSOX9-IRES-tdTomato was transfected into 293AAV cell by calcium phosphate transfection. The purification and drop of adeno-associated virus was tested by determination of biological titer. RESULTS AND CONCLUSION： The results of BLAST sequence comparison analysis showed that, pAAV-hSOX9-IRES-tdTomato exactly matched the synthetic gene sequence hSOX9. The titer is 1×107 TU/mL. Human gene SOX9 recombinant adenoviruses, pAAV-hSOX9-IRES-tdTomato, have been constructed successfully.

关 键 词：腺相关病毒 SOX9 转染 椎间 盘细胞 病毒包装 

分 类 号：R392.7[医药卫生—免疫学]