中国组织工程研究

中国组织工程研究 ›› 2012, Vol. 16 ›› Issue (24): 4505-4508.doi: 10.3969/j.issn.1673-8225.2012.24.028

• 组织构建细胞学实验 cytology experiments in tissue construction • 上一篇    下一篇

重组pcDNA4 His/Max人骨形态发生蛋白9真核表达载体的构建与鉴定

江 维1，胡侦明2，郝 杰2，甘 强1   

  1. 1重庆医科大学，重庆市 400016；
    2重庆医科大学附属第一医院骨科，重庆市 400016
  • 收稿日期:2011-10-20 修回日期:2011-11-30 出版日期:2012-06-10 发布日期:2013-11-05
  • 通讯作者: 胡侦明，博士，教授，重庆医科大学附属第一医院骨科，重庆市 400016 zhenminghu62@yahoo.com.cn
  • 作者简介:江维★，男，1985年生，重庆市人，汉族，重庆医科大学在读硕士，主要从事骨质疏松、骨折修复的研究。

Construction and identification of a recombinant pcDNA4 His/Max human bone morphogenetic protein eukaryotic expression vector

Jiang Wei1, Hu Zhen-ming2, Hao Jie2, Gan Qiang1   

  1. 1Chongqing Medical University, Chongqing 400016, China;
    2Department of Orthopedics, First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China
  • Received:2011-10-20 Revised:2011-11-30 Online:2012-06-10 Published:2013-11-05
  • Contact: Hu Zhen-ming, Doctor, Professor, Department of Orthopedics, First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China zhenminghu62@yahoo.com.cn
  • About author:Jiang Wei★, Studying for master’s degree, Chongqing Medical University, Chongqing 400016, China
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摘要：

背景：腺病毒临床应用存在安全隐患，利用真核表达载体表达蛋白为解决安全性问题提供了一种方法。
目的：将人骨形态发生蛋白9的cDNA构建于pcDNA4 His/Max，得到重组真核表达载体pcDNA4 His/Max人骨形态发生蛋白9。
方法：将已有的Padtrack-cmv-人骨形态发生蛋白9进行PCR扩增，电泳回收人骨形态发生蛋白9片段，将pcDNA4 His/Max用Not Ⅰ和KpnⅠ双酶切，得到pcDNA4 His/Max酶切片段，连接人骨形态发生蛋白9和pcDNA4 His/Max片段得到重组质粒pcDNA4 His/Max人骨形态发生蛋白9，将该载体用DH5α转化，阳性克隆扩增及纯化、序列分析鉴定。
结果与结论：通过PCR及序列分析证明pcDNA4 His/Max人骨形态发生蛋白9真核表达载体的人骨形态发生蛋白9基因长度为1.3 kb，与报道的人骨形态发生蛋白9全长序列一致，无突变。结果证实，实验成功构建了pcDNA4 His/Max人骨形态发生蛋白9真核表达载体。

关键词: 人骨形态发生蛋白9, 真核表达载体, 基因, 腺病毒, 阳性克隆

Abstract:

BACKGROUND: Adenovirus mediated method has been used numerously in human bone morphogenetic protein 9 (hBMP-9) induced osteogenesis researches, but the security is the most important problem. Eukaryotic expressing vector is a way to solve this question.
OBJECTIVE: To reconstruct a recombinant DNA pcDNA4 His/Max hBMP-9 eukaryotic expressing vector by amplifying hBMP9 gene and then cloning into pcDNA4 His/Max.
METHODS: Padtrack-cmv-hBMP-9 was amplified by PCR, and hBMP-9 gene was retrieved by electrophoresis. pcDNA4 His/Max was digested by NotⅠ, Kpn Ⅰand then the hBMP-9 gene was cloned into pcDNA4 His/Max. The recombinant plasmid was transformed by DH5α, clonal expansion, and purification, and then verified by sequencing.
RESULTS AND CONCLUSION: The cloned hBMP-9 gene was 1.3 kb long, having the same length and sequence of the gene that human possessed. Eukaryotic expressing vector of pcDNA4 His/Max hBMP-9 has been constructed successfully.

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