中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (20): 3674-3677.doi: 10.3969/j.issn.1673-8225.2010.20.015

• 组织构建与生物活性因子 tissue construction and bioactive factors • 上一篇    下一篇

重组外源性人热休克蛋白70基因真核表达载体的构建及鉴定

韩世伟1，张阳德1，2，万小平1，陈玉祥2   

  1. 1中南大学卫生部肝胆肠外科研究中心，湖南省长沙市  410008；
    2中南大学卫生部纳米生物技术重点实验室，湖南省长沙市  410008
  • 出版日期:2010-05-14 发布日期:2010-05-14
  • 通讯作者: 张阳德，博士，教授，博士生导师，中南大学卫生部肝胆肠外科研究中心，湖南省长沙市 410008 zyd@2118.cn
  • 作者简介:韩世伟，男，1985年生，内蒙古自治区锡林郭勒盟人，蒙古族，中南大学湘雅医学院在读硕士，主要从事肝胆肠疾病的基础与临床研究。 cco1985@yahoo.com.cn
  • 基金资助:

    国家科技部“十五”863计划项目(2007AA021104)，课题名称：Hsp70、GM-CSF双修饰DU145、PC3激素非依赖性前列腺癌细胞株全细胞疫苗的研制；中南大学研究生学位论文创新基金，课题名称：Hsp70、GM-CSF双修饰DU145、PC3激素非依赖性前列腺癌细胞株全细胞疫苗的研制。

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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431

被引次数

2

摘要：

背景：热休克蛋白70的肿瘤免疫作用近年来受到国内外学者的广泛关注，然而目前常用的诱导内源性热休克蛋白70表达的方法，如热应激、缺血预处理等均存在一些弊端。
目的：拟构建携带并正确表达外源性人热休克蛋白70基因的重组真核表达载体。
方法：外源性热休克蛋白70基因连接入真核表达载体pDC315-EGFP中，转化大肠杆菌感受态细胞，重组真核表达载体外源基因测序及PCR检测鉴定阳性克隆后转染人胚肾293细胞，荧光显微镜及western blot鉴定外源基因在细胞中的表达。
结果与结论：经PCR和测序证实外源性人热休克蛋白70基因正确重组入真核表达载体pDC315-EGFP，转染293细胞后荧光显微镜观察到绿色荧光蛋白GFP的表达，Western blot检测到热休克蛋白70在293细胞中有效表达。

关键词: 热休克蛋白70, 真核表达载体, 基因载体, 基因重组, 基因治疗

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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