中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (46): 8604-.doi: 10.3969/j.issn.1673-8225.2010.46.013

• 织构建与生物活性因子 • 上一篇    下一篇

携RSKA/hIGF-1杂交基因重组质粒的构建及表达

郑水长，高仕长，倪卫东，梁安霖   

  1. 重庆医科大学附属第一医院骨科，重庆市  400010
  • 出版日期:2010-11-12 发布日期:2010-11-12
  • 通讯作者: 髙仕长，博士，副教授，重庆医科大学附属第一医院骨科，重庆市 400010 gaoshichang2002@yahoo.com.cn
  • 作者简介:郑水长★，男，1978年生，重庆医科大学在读硕士，主要从事周围神经损伤研究。 zsczdd_2000@126.com
  • 基金资助:

    重庆市卫生局科研项目(07-2-088)：skeletalα-actin/hIGF-1真核表达质粒提高神经修复疗效的实验研究。

Construction of recombinant plasmid PBS-RSKA/hIGF-1 and its expression

Zheng Shui-chang, Gao Shi-chang, Ni Wei-dong, Liang An-lin   

  1. Department of Orthopedics, First Affiliated Hospital of Chongqing Medical University, Chongqing  400010, China
  • Online:2010-11-12 Published:2010-11-12
  • Contact: Gao Shi-chang, Doctor, Associate professor, Department of Orthopedics, First Affiliated Hospital of Chongqing Medical University, Chongqing 400010, China gaoshichang2002@yahoo.com.cn
  • About author:Zheng Shui-chang★, Studying for master’s degree, Department of Orthopedics, First Affiliated Hospital of Chongqing Medical University, Chongqing 400010, China zsczdd_2000@126.com
  • Supported by:

    the Science and Technology Program of Health Bureau of Chongqing, No. 07-2-088*

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摘要：

背景：骨骼肌肌萎缩的治疗一直是基础和临床研究的难点和热点之一，近年来基因治疗在该领域得到了广泛的研究和关注。
目的：拟构建携带大鼠骨骼肌α-肌动蛋白/人胰岛素样生长因子1 (rat skeletal α-actin/human insulin-like growth factor-1，RSKA/hIGF-1)杂交基因的重组真核表达质粒，并观察其在C2C12细胞内表达。
方法：登录Genbank数据库，查找RSKA启动子序列、hIGF-1 cDNA序列和人生长激素3′尾端非编码区序列(3′UTR)，把3段基因序列拼接后进行全基因合成，再将合成的全基因融合到PbluescriptⅡ SK(+)[PBS]质粒上。用酶切电泳及测序检查质粒重组后序列的正确性；重组质粒转染C2C12细胞，应用RT-PCR和Western blot法分别鉴定转染细胞中hIGF-1 mRNA和蛋白的表达。
结果与结论：重组质粒PBS-RSKA/hIGF-1酶切图谱与预期相同，测序验证插入片段全序列无改变。转染C2C12细胞后，RT-PCR及Western blot结果显示有特异条带出现，且差异明显。结果证明成功构建携RSKA/hIGF-1杂交基因的重组质粒，并在C2C12细胞中正确表达。

关键词: 胰岛素样生长因子1, &alpha, -肌动蛋白, 生长激素, 基因重组, 转染

Abstract:

BACKGROUND: The treatment for skeletal muscle atrophy is a difficult and hot point both in basic and clinical researches. In recent years, gene therapy has aroused extensive attention. 
OBJECTIVE: To construct a recombinant plasmid for expressing the rat skeletal α-actin/human insulin-like growth factor-1 (RSKA/hIGF-1) gene and to observe its expression in C2C12 cells.
METHODS: The construct consists of rat skeletal a-actin promoter sequence, hIGF-1 coding sequence and human growth hormone 3’UTR region. The construct was initially cloned into a pBluescript Ⅱ SK(+) after synthesized, which contains the plasmid origin of replication and Ampicillin resistance gene. The sequence of synthesized hIGF-1 was confirmed by restriction analysis and DNA sequencing, then transfected into C2C12 cell. HIGF-1 gene was detected by RT-PCR and hIGF-1 was found by Western blot.
RESULTS AND CONCLUSION: The restriction map of PBS-RSKA/hIGF-1 was identical to expectation, and sequencing verified that the inserted fragment was not changed. Specific bands could be seen in transfected C2C12 cells by RT-PCR and Western blot. Recombinant plasmid PBS-RSKA/hIGF-1 constructed successfully and it can be effectively expressed after being transfected into C2C12 cells in vitro.

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