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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: The experiment was an early part of gene therapy for degenerative disc disease. The aim was to construct recombinant plasmid pAAV-hSOX9-IRES-tdTomato containing immunofluorescence and packaging with adeno-associated virus, which laid the foundation for later in vivo and in vitro experiments. OBJECTIVE: To construct a human SOX9 overexpression adeno-associated virus packaging pAAV-hSOX9-IRES-tdTomato. METHODS: Plasmid pAAV-IRES-tdTomato and plasmid pUC57-hSOX9 were ligated into pAAV-hSOX9-IRES-tdTomato by enzyme digestion. The adeno-associated virus was packaged by plasmid co-transfection and infected 293AAV cells. The adeno-associated virus Titers were measured titer. RESULTS AND CONCLUSION: After sequencing BLAST analysis, pAAV-hSOX9-IRES-tdTomato was completely consistent with hSOX9 and the titer was 1 × 107TU / mL. The results showed that human SOX9 gene overexpression adeno-associated virus pAAV-hSOX9-IRES-tdTomato packaging was successful.