中国组织工程研究 ›› 2012, Vol. 16 ›› Issue (4): 699-703.doi: 10.3969/j.issn.1673-8225.2012.04.030

• 骨与关节损伤基础实验 basic experiments of bone and joint injury • 上一篇    下一篇

人转化生长因子β3、结缔组织生长因子和基质金属蛋白酶抑制剂1基因重组慢病毒多表达质粒的构建及活性检测*★

江  峰,岳  斌,马学晓,张国庆,胡有谷,陈伯华   

  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:2011-09-24 修回日期:2011-10-28 出版日期:2012-01-22
  • 通讯作者: 江峰★,男,1984年生,山东省文登市人,汉族,青岛大学医学院附属医院在读硕士,主要从事脊柱外科研究。 iamjiangf@163.com
  • 作者简介:江峰★,男,1984年生,山东省文登市人,汉族,青岛大学医学院附属医院在读硕士,主要从事脊柱外科研究。 iamjiangf@163.com
  • 基金资助:

    国家自然基金项目(81171758)。

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
  • 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*

摘要:

背景:前期试验显示单个病毒载体介导的多基因共表达系统能够显著提高椎间盘退变转基因疗效。
目的:构建人转化生长因子β3、结缔组织生长因子和基质金属蛋白酶抑制剂1基因重组慢病毒多表达质粒。
方法:应用全基因合成技术,以“自剪切多肽2A”串联目的基因,并克隆到慢病毒表达质粒构建重组慢病毒质粒Lenti-TGF-β3-P2A-CTGF-T2A-TIMP1。转染293细胞后,应用RT-PCR和Western-Blot技术分别检测转染后不同时间点目的基因mRNA和蛋白质表达水平。
结果与结论:RT-PCR和Western-blot技术检测结果显示重组质粒成功表达了3种目的基因,并于转染后48 h左右达到峰值,“2A”结构下游基因蛋白质表达量约为上游基因的80%。说明成功构建了携带人转化生长因子β3、结缔组织生长因子和基质金属蛋白酶抑制剂1基因的慢病毒多表达质粒。

关键词: 转化生长因子&beta, 3, 结缔组织生长因子, 基质金属蛋白酶抑制剂1, 慢病毒, 293细胞, 基因治疗

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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