Chinese Journal of Tissue Engineering Research ›› 2012, Vol. 16 ›› Issue (20): 3759-3762.doi: 10.3969/j.issn.1673-8225.2012.20.034

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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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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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