Chinese Journal of Tissue Engineering Research ›› 2012, Vol. 16 ›› Issue (4): 699-703.doi: 10.3969/j.issn.1673-8225.2012.04.030

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Construction and detection of lentiviral plasmids containing human transforming growth factor beta 3, connective tissue growth factor and tissue inhibitor of metalloproteinases 1 gene 

Jiang Feng, Yue Bin, Ma Xue-xiao, Zhang Guo-qing, Hu You-gu, Chen Bo-hua   

  1. Department of Spine Surgery, Affiliated Hospital of Qingdao University Medical College, Qingdao  266003, Shandong Province, China
  • Received:2011-09-24 Revised:2011-10-28 Online:2012-01-22 Published:2014-04-04
  • Contact: Chen Bo-hua, Chief physician, Department of Spine Surgery, Affiliated Hospital of Qingdao University Medical College, Qingdao 266003, Shandong Province, China 266003 bhchen@hotmail.com
  • About author:Jiang Feng★, Studying for master’s degree, Department of Spine Surgery, Affiliated Hospital of Qingdao University Medical College, Qingdao 266003, Shandong Province, China iamjiangf@163.com
  • Supported by:

    the National Natural Science Foundation of China, No. 81171758*

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Abstract:

BACKGROUND: The former tests showed that multi-gene co-expression system mediated by virus can significantly improve the transgenic curative effect of intervertebral disc degeneration.
OBJECTIVE: To construct the recombinant plasmid of lentivirus containing human transforming growth factor beta 3 (TGFβ3), connective tissue growth factor (CTGF) and tissue inhibitor of metalloproteinases 1 (TIMP1).
METHODS: 2A self-cleaving sequences were used to ligate the cDNA of TGFβ3, CTGF, TIMP1 in a single open reading frame derived from plasmid of lentivirus in order to obtain the recombinant lentiviral plasmid Lenti-TGFβ3-P2A-CTGF-T2A-TIMP1. RT-PCR and Western-blot were used to detect mRNA and protein expressions of TGFβ3, CTGF, TIMP1 in different times after transfection.
RESULTS AND CONCLUSION: RT-PCR and Western-blot detection shows that the mRNA and protein expressions of TGFβ3, CTGF, TIMP1 were detected in transfected 293 cells, the protein expressions reached peak value at 48 hours after transfection, and the protein level of downstream gene was about 80% of upstream gene. The recombinant plasmid, Lenti-TGFβ3-P2A-CTGF-T2A-TIMP1, is constructed successfully, providing a basic model for the packaging of virus and further study on therapy of intervertebral disc degeneration.

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