中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (37): 6922-6926.doi: 10.3969/j.issn.1673-8225.2010.37.019

• 组织构建实验造模 experimental modeling in tissue construction • 上一篇    下一篇

构建人热休克蛋白70基因重组腺病毒载体及鉴定

韩世伟，张忠涛，王  宇   

  1. 首都医科大学附属北京友谊医院 北京市  100050
  • 出版日期:2010-09-10 发布日期:2010-09-10
  • 通讯作者: 张忠涛，博士，教授，博士生导师，主任医师，首都医科大学附属北京友谊医院普外科，北京市 100050 zhangzht@medmail.com.cn
  • 作者简介:韩世伟☆，男，1985年生，内蒙古锡林郭勒盟人，蒙古族，首都医科大学附属北京友谊医院在读博士，主要从事肝胆胃肠疾病及肝移植的基础与临床研究。 cco1985@yahoo.com.cn

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

Han Shi-wei, Zhang Zhong-tao, Wang Yu   

  1. Department of General Surgery, Beijing Friendship Hospital, Capital University of Medical Sciences, Beijing  100050, China
  • Online:2010-09-10 Published:2010-09-10
  • Contact: Zhang Zhong-tao, Doctor, Professor, Doctoral supervisor, Chief physician, Department of General Surgery, Beijing Friendship Hospital, Capital University of Medical Sciences, Beijing 100050, China zhangzht@medmail.com.cn
  • About author:Han Shi-wei☆, Studying for doctorate, Department of General Surgery, Beijing Friendship Hospital, Capital University of Medical Sciences, Beijing 100050, China cco1985@yahoo.com.cn

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被引次数

2

摘要：

背景：腺病毒载体作为低毒高效的基因载体已被广泛应用，但是人热休克蛋白70基因腺病毒载体较为少见。
目的：构建重组人热休克蛋白70基因的腺病毒载体，鉴定外源基因在真核细胞中的良好表达。
方法：采用AdMax腺病毒系统将外源基因人热休克蛋白70基因重组入腺病毒载体中，转染人胚肾293细胞并重组包装出毒，检测外源基因的表达和病毒滴度。
结果与结论：观察转染后的人胚肾293细胞出现明显细胞病变效应后，收获并纯化重组病毒；荧光显微镜观察绿色荧光蛋白表达情况良好，Western blot检测人热休克蛋白70蛋白表达良好，收获病毒的滴度为1×1011 efu/mL，证明实验已成功构建携带人热休克蛋白70基因的重组腺病毒载体。

关键词: 热休克蛋白70, 腺病毒载体, 基因载体, 基因重组, 基因治疗

Abstract:

BACKGROUND: Adenovirus vectors have been widely used as a low high effectiveness and low toxicity gene vector, however, little is known about a recombinant adenovirus vector containing human heat shock protein 70 (Hsp70) gene.
OBJECTIVE: To construct a recombinant adenovirus vector containing human Hsp70 gene and to express the gene efficiently in eukaryotic cells.
METHODS: The recombinant adenovirus vector carrying Hsp70 gene was constructed with AdMax system and then transferred human embryonic kidney 293 (HEK293) cells to produce recombinant adenovirus. The exogenous gene expression was detected by Western blot and the viral titer was tested.
RESULTS AND CONCLUSION: The significant cytopathic effects were observed in transfected HEK293 cells, and then the recombinant virus was harvested and purified. The expression of green fluorescence protein could be observed by fluorescence microscope and the expression of Hsp70 could be detected by Western blot. The viral titer was 1×1011 efu/mL. Recombinant adenovirus vector was constructed successfully and packed in HEK293 cells.

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