CAJ | 학술논문

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.
배경:실험위기인치료퇴변추간반실험적전기부분,지재구건함면역형광적pAAV-hSOX9-IRES-tdTomato중조질립병응용선상관병독포장,위후기체내외실험타하기출.목적:구건인SOX9기인과표체선상관병독pAAV- hSOX9-IRES- tdTomato적포장.방법:용매절법장질립pAAV-IRES- tdTomato화질립pUC57- hSOX9련접성pAAV-hSOX9-IRES- tdTomato,용질립공전염방법포장선상관병독,감염293AAV세포,선상관병독순화급용생물학적도측정법진행적도측정.결과여결론:경측서결과BLAST비대분석,pAAV-hSOX9-IRES-tdTomato완전여합성적기인서렬hSOX9상부,적도위1×107TU/mL.결과표명,인SOX9기인과표체선상관병독pAAV-hSOX9-IRES- tdTomato포장성공.
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.