中国组织工程研究

中国组织工程研究 ›› 2011, Vol. 15 ›› Issue (28): 5299-5302.doi: 10.3969/j.issn.1673-8225.2011.28.042

• 组织构建细胞学实验 cytology experiments in tissue construction • 上一篇    下一篇

表达胰高血糖素样肽1重组腺伴随病毒载体的构建

娄明武1，梁  冰1，张明东1，杨广笑2，王全颖2   

  1. 1深圳市龙岗中心医院介入影像科，广东省深圳市  518116
    2西安市华广生物工程有限公司，陕西省西安市  710025
  • 收稿日期:2011-03-02 修回日期:2011-05-25 出版日期:2011-07-09 发布日期:2011-07-09
  • 作者简介:娄明武★，男，1950年生，吉林省长春市人，汉族，1992年吉林医学院毕业，硕士，主任医师，主要从事分子影像学与微创治疗的研究。 mingwulou@163.com
  • 基金资助:

    广东省科学技术厅资助项目(2005B36001082)；深圳市科技和信息局资助项目(JH200505260306A)；深圳市龙岗区委组织部2008年专家提升计划资助项目。

Construction of a recombinant adeno-associated virus plasmid expressing glucagon-like peptide-1

Lou Ming-wu1, Liang Bing1, Zhang Ming-dong1, Yang Guang-xiao2, Wang Quan-ying2   

  1. 1Department of Interventional Imaging, Shenzhen Longgang Central Hospital, Shenzhen   518116, Guangdong Province, China
    2Xi’an Huaguang Bioengineering Co., Ltd., Xi’an   710025, Shaanxi Province, China
  • Received:2011-03-02 Revised:2011-05-25 Online:2011-07-09 Published:2011-07-09
  • About author:Lou Ming-wu★, Master, Chief physician, Department of Interventional Imaging, Shenzhen Longgang Central Hospital, Shenzhen 518116, Guangdong Province, China mingwulou@163.com
  • Supported by:

    a grant from Science and Technology Bureau of Guangdong Province, No. 2005B36001082*; a grant from Bureau of Technology & Information of Shenzhen city, No. JH200505260306A*; Expert Training Plan Program of Organization Department of Longgang District, Shenzhen in 2008*

PDF

300

被引次数

2

摘要：

背景：胰高血糖素样肽1(glucagon-like peptide-1, GLP-1)在人体内半衰期过短限制了其应用。
目的：构建可表达GLP-1的重组腺伴随病毒。
方法：将NT4-GLP-1融合基因插入腺伴随病毒包装质粒pSSHG-CMV中，构建pSSHG/NT4-GLP-1重组腺伴随病毒包装质粒。采用磷酸钙共沉淀法将辅助质粒pAAV/Ad、腺病毒质粒pFG140及pSSHG/NT4-GLP-1转染至293细胞系，用其感染Hela细胞。
结果与结论：重组质粒pSSHG/NT4-GLP-1经限制性内切酶EcoRⅠ酶切鉴定可见342 bp的目的片段，说明NT4-GLP-1融合基因已经成功重组于腺伴随病毒包装质粒pSSHG-CMV内。免疫细胞化学结果显示，转染pSSHG/NT4-GLP-1重组腺伴随病毒包装质粒的Hela细胞内有大量棕黄色颗粒，阳性率达到70%以上，说明NT4-GLP-1重组腺伴随病毒在细胞中可以表达GLP-1。

关键词: 胰高血糖素样肽1, 腺伴随病毒, 重组质粒, 构建, 组织构建

Abstract:

BACKGROUND: Glucagon-like peptide-1 (GLP-1) is limited by half-life in human body.
OBJECTIVE: To construct a recombinant adeno-associated virus vector expressing GLP-1.
METHODS: The fusion gene NT4-GLP-1 was inserted into the adeno-associated virus packaging plasmid pSSHG-CMV to generate the adeno-associated virus packaging plasmid pSSHG/NT4-GLP-1. 80% of fused 293 cells were co-transfected by using the calcium phosphate precipitation method, with aid packaging plasmids pAAV/Ad, pFG140 and the recombinant adeno-associated virus packaging plasmid pSSHG/NT4-GLP-1. Virus effects were confirmed by immunohistochemistry at the same time.
RESULTS AND CONCLUSION: Restriction enzyme EcoRⅠidentification showed that the recombinant plasmid pSSHG/NT4-GLP-1 contained a 342 bp target segment, indicating that the fusion gene NT4-GLP-1 has been successfully transfected into recombinant adeno-associated virus packaging plasmid pSSHG/NT4-GLP-1. Immunocytochemical staining results showed that Hela cells transfected by plasmid pSSHG/NT4-GLP-1 contained a large number of brown yellow particles, with the rate of positive cells more than 70%. These findings suggest that adeno-associated virus packaging plasmid pSSHG/NT4-GLP-1 expressed GLP-1.

中图分类号: