中国组织工程研究

中国组织工程研究 ›› 2011, Vol. 15 ›› Issue (18): 3357-3360.doi: 10.3969/j.issn.1673-8225.2011.18.031

• 器官移植基础实验 basic experiments of organ transplantation • 上一篇    下一篇

大鼠热休克蛋白72基因真核表达载体构建及在COS7细胞中的表达

晏春根，黄丹文，任光圆，朱冬芳   

  1. 绍兴文理学院附属医院内科，浙江省绍兴市 312000
  • 收稿日期:2010-10-22 修回日期:2011-03-21 出版日期:2011-04-30 发布日期:2011-04-30
  • 作者简介:晏春根★，男，1970年生，江西省丰城市人，汉族，2003年上海第二医科大学毕业，硕士，教授，主要从事消化内科（肝脏疾病）方面的研究。 ycg28@163.com
  • 基金资助:

    浙江省自然科学基金(Y2100961)；浙江省科技计划项目(2009C33129)。

Construction of eukaryotic expression vector carrying rat heat shock protein 72 gene and its expression in COS7 cells

Yan Chun-gen, Huang Dan-wen, Ren Guang-yuan, Zhu Dong-fang   

  1. Department of Internal Medicine, the Affiliated Hospital of Shaoxing University, Shaoxing  312000，Zhejiang Province, China
  • Received:2010-10-22 Revised:2011-03-21 Online:2011-04-30 Published:2011-04-30
  • About author:Yan Chun-gen★, Master, Professor, Department of Internal Medicine, the Affiliated Hospital of Shaoxing University, Shaoxing 312000，Zhejiang Province, China ycg28@163.com
  • Supported by:

    the Natural Science Foundation of Zhejiang Province, No. Y2100961*; Science and Technology Plan of Zhejiang Province, No. 2009C33129*

PDF

425

被引次数

7

摘要：

背景：体外克隆、表达热休克蛋白，尤其正常时少量或不表达，而应激时大量表达的热休克蛋白72对研究其缺血再灌注损伤中的作用尤为重要。
目的：构建热休克蛋白72基因真核表达载体并于COS7细胞内表达，为HSP72蛋白免疫调节功能的研究奠定基础。
方法：采用RT-PCR技术从BABL/C大鼠肝细胞中扩增出热休克蛋白72 cDNA，经限制性核酸内切酶消化后，插入真核表达载体pcDNA3.1(+)的相应酶切位点，并将重组质粒转染COS7细胞，进行基因表达鉴定。
结果与结论：重组质粒插入基因序列检测证实为热休克蛋白72 cDNA，并能在COS7细胞稳定表达。成功构建热休克蛋白72真核表达载体，并于COS7细胞中成功转录与表达。

关键词: 热休克蛋白72, 基因表达, COS7细胞, 转录, 鉴定

Abstract:

BACKGROUND: In vitro cloning and expression of heat shock proteins (HSP), especially HSP72, is important in the study of ischemia-reperfusion injuries.
OBJECTIVE: To construct eukaryotic expression vector carrying HSP72 and study its transient expression in COS7 cells, and to establish the foundation for further research of the immune function of HSP72.
METHODS: HSP72 cDNA was amplified from hepatocytes of BABL/C mice by RT-PCR, and correctly inserted into corresponding sites of eukaryotic expression vector pcDNA3.1(+)after restriction endonuclease digestion, and the recombinant plasmid was transfected into COS7 cells and gene expression was determined by RT-PCR.
RESULTS AND CONCLUSION: The gene fragment inserted into the vector pcDNA3.1(+)was confirmed by nucleotide sequencing, and the recombinant plasmid mRNA was successfully expressed in COS7 cells.

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