Chinese Journal of Tissue Engineering Research ›› 2010, Vol. 14 ›› Issue (20): 3674-3677.doi: 10.3969/j.issn.1673-8225.2010.20.015

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Construction and identification of a recombinant eukaryotic expression vector containing human heat shock protein 70 gene

Han Shi-wei1, Zhang Yang-de1,2, Wan Xiao-ping1, Chen Yu-xiang2   

  1. 1National Hepatobiliary & Enteric Surgery Research Center, Ministry of Health, Central South University, Changsha  410008, Hunan Province, China;
    2National Key Laboratory of Nanobiological Technology, Ministry of Public Health, Central South University, Changsha  410008, Hunan Province, China
  • Online:2010-05-14 Published:2010-05-14
  • Contact: Zhang Yang-de, Doctor, Professor, Doctoral supervisor, National Hepatobiliary & Enteric Surgery Research Center, Ministry of Health, Central South University, Changsha 410008, Hunan Province, China ZYD@2118.cn
  • About author:Han Shi-wei, Studying for master’s degree, National Hepatobiliary & Enteric Surgery Research Center, Ministry of Health, Central South University, Changsha 410008, Hunan Province, China cco1985@yahoo.com.cn
  • Supported by:

    the 863 Program of State Commission of Science Technology in the Tenth Five-Year Plan of China, No. 2007AA021104*;
    Central South University Innovation Fund for Post-graduate Degree Thesis*

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Abstract:

BACKGROUND: Heat shock protein 70 (Hsp70) has attracted widespread attention by domestic and foreign scholars for the role of tumor immunity in recent years. However, the commonly used methods, such as heat stress and ischemic preconditioning, have disadvantages in inducing exogenous recombinant Hsp70 gene expression. 

OBJECTIVE: To construct a recombinant eukaryotic expression vector which expresses human HSP70 gene.

METHODS: The exogenous Hsp70 gene was connected to eukaryotic expression vector pDC315-EGFP. Then it was transformed to E. coli competent cells. The positive clone was evaluated by gene sequencing and PCR detection and then was transfected to human embryo kidney 293 cells. The exogenous gene expression was tested by fluorescence microscope and Western blot. 

RESULTS AND CONCLUSION:It was confirmed by PCR and sequencing identification analysis that the target gene was cloned correctly to the eukaryotic expression vector. The expression of GFP could be observed by fluorescence microscope and western blot, which indicated that Hsp70 could be efficiently expressed in human embryo kidney 293 cells. Recombinant eukaryotic expression pDC315-EGFP-Hsp70 was constructed successfully

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