中国组织工程研究

中国组织工程研究 ›› 2012, Vol. 16 ›› Issue (20): 3759-3762.doi: 10.3969/j.issn.1673-8225.2012.20.034

• 组织构建基础实验 basic experiments in tissue construction • 上一篇    下一篇

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

刁泽正，燕国清，张志伟，房  晶，徐  鹏，西永明，任  山，刘勇君，隋爱华   

  1. 青岛大学医学院附属医院脊柱外科，山东省青岛市   266003
  • 收稿日期:2011-10-09 修回日期:2012-01-11 出版日期:2012-05-13 发布日期:2012-05-13
  • 通讯作者: 西永明，副教授，青岛大学医学院附属医院脊柱外科，山东省青岛市 266003
  • 作者简介:刁泽正★，男，1985年生，山东省淄博市人，汉族，青岛大学医学在读硕士，主要从事脊柱外科研究。

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

Diao Ze-zheng, Yan Guo-qing, Zhang Zhi-wei, Fang Jing, Xu Peng, Xi Yong-ming, Ren Shan,        Liu Yong-jun, Sui Ai-hua   

  1. Department of Spinal Surgery, Affiliated Hospital of Qingdao University Medical College, Qingdao  266003, Shandong Province, China
  • Received:2011-10-09 Revised:2012-01-11 Online:2012-05-13 Published:2012-05-13
  • Contact: Xi Yong-ming, Associate professor, Department of Spinal Surgery, Affiliated Hospital of Qingdao University Medical College, Qingdao 266003, Shandong Province, China wwuomen@yahoo.com.cn
  • About author:Diao Ze-zheng★, Studying for master’s degree, Department of Spinal Surgery, Affiliated Hospital of Qingdao University Medical College, Qingdao 266003, Shandong Province, China diaozezheng@foxmail.com

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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。
 

关键词: 腺相关病毒, SOX9, 转染, 椎间盘细胞, 病毒包装

Abstract:

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.

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