中国组织工程研究

中国组织工程研究 ›› 2012, Vol. 16 ›› Issue (41): 7728-7732.doi: 10.3969/j.issn.2095-4344.2012.41.026

• 干细胞基础实验 basic experiments of stem cells • 上一篇    下一篇

人转化生长因子β1基因重组腺病毒的构建与鉴定

曾昭勋，尹承慧，邱俊钦，陈宗雄   

  1. 解放军南京军区福州总医院骨科研究所，福建省福州市 350025
  • 收稿日期:2012-01-05 修回日期:2012-02-01 出版日期:2012-10-07 发布日期:2012-10-07
  • 通讯作者: 尹承慧，博士，副主任医师，解放军南京军区福州总医院骨科研究所，福建省福州市 350025 chenghuiyin@hotmail.com
  • 作者简介:曾昭勋★，男，1984年生，江西省泰和县人，汉族，福建医科大学福州总医院临床学院骨科在读硕士，主要从事脊柱伤病与四肢创伤研究。

Construction and identification of recombinant adenovirus vectors encoding human transforming growth factor-beta 1 gene

Zeng Zhao-xun, Yin Cheng-hui, Qiu Jun-qin, Chen Zong-xiong   

  1. Institute of Orthopedics, Fuzhou General Hospital of Nanjing Military Command, Fuzhou 350025, Fujian Province, China
  • Received:2012-01-05 Revised:2012-02-01 Online:2012-10-07 Published:2012-10-07
  • Contact: Yin Cheng-hui, M.D., Associate chief professor, Institute of Orthopedics, Fuzhou General Hospital of Nanjing Military Command, Fuzhou 350025, Fujian Province, China chenghuiyin@hotmail.com
  • About author:Zeng Zhao-xun★, Studying for master’s degree, Institute of Orthopedics, Fuzhou General Hospital of Nanjing Military Command, Fuzhou 350025, Fujian Province, China

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摘要：

背景：转化生长因子β1能促进多种细胞的生长增殖，是调控骨髓间充质干细胞向成骨方向定向分化的主要生长因子。
目的：利用AdMax系统构建人转化生长因子β1(hTGF-β1)基因的重组腺病毒表达载体。
方法：采用PCR方法克隆人转化生长因子β1基因cDNA后，插入线性化表达载体pAV-MCMV-EGFP-3FLAG中，转化E.coli DH5α感受态细胞，构建pAV-MCMV-hTGF-β1重组质粒。
结果与结论：含目的基因的重组腺病毒质粒共转染293细胞进行病毒包装、扩增后, 经PCR、Western Blot检测得到转化生长因子β1重组腺病毒，其滴度约为1.25×1010 pfu/mL。表明利用AdMax系统成功可构建人转化生长因子β1腺病毒载体，并可满足进一步的转化生长因子β1成骨作用的研究。

关键词: 转化生长因子β1, 腺病毒载体, 基因, 转染, AdMax系统

Abstract:

BACKGROUND: Transforming growth factor-beta1 (TGF-β1) can promote the growth and proliferation of many cells and is one of main growth factors that regulate osteoblast differentiation of bone marrow mesenchymal stem cells.
OBJECTIVE: To construct a recombinant adenoviral vector carrying the human TGF-β1 gene using the AdMax system which may be applicable in gene therapy of bone defect.
METHODS: The hTGF-β1 gene sequence was amplified by PCR from cDNA template and then was inserted into shuttle plasmid pAV-MCMV-EGFP-3FLAG. The recombinant shuttle plasmid was constructed and transformed into E.coli DH5α.The recombinant pAV-MCMV-hTGF-β1 was obtained.
RESULTS AND CONCLUSION: The recombinant pAV-MCMV-hTGF-β1 was packaged and amplified in 293 cells. Then the recombinant TGF-β1 adenovirus was constructed successfully. After measuring the titre of virus (1.25×1010 pfu/mL), the target gene was evaluated by PCR and Western Blot. These findings suggest that construction of hTGF-β1 adenoviral vector was successfully achieved using the AdMax system and may be available in the future study of TGF-β1 gene therapy.

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