中国组织工程研究

中国组织工程研究 ›› 2012, Vol. 16 ›› Issue (36): 6753-6757.doi: 10.3969/j.issn.2095-4344.2012.36.018

• 干细胞因子及调控因子 stem cell factors and regulatory factors • 上一篇    下一篇

趋化因子受体4基因重组腺病毒载体的构建及鉴定

潘廷明1，徐 皓2，陈建梅2，宋晨阳3，姚晓东2   

  1. 1福建中医药大学附属第二人民医院骨科，福建省福州市 350003；
    2解放军南京军区福州总医院骨科研究所，福建省福州市 350025；
    3福建医科大学附属协和医院骨科，福建省福州市350000
  • 收稿日期:2012-01-20 修回日期:2012-02-28 出版日期:2012-09-02 发布日期:2012-09-02
  • 通讯作者: 徐 皓，解放军南京军区骨科研究所所长，主任医师，教授，硕士生导师，解放军南京军区福州总医院骨科研究所，福建省福州市 350025 xiuhao@medmail.com.cn
  • 作者简介:潘廷明★，男，1982年生，福建省闽侯市人，汉族，2009年福建医科大学毕业，硕士，医师，主要从事脊髓损伤与修复研究。 3467890@163.com

Construction and identification of recombinant adenovirus vector containing CXC chemokine receptor 4 gene

Pan Ting-ming1, Xu Hao2, Chen Jian-mei2, Song Chen-yang3, Yao Xiao-dong2   

  1. 1Department of Orthopedics, Second People's Hospital of Fujian University of Traditional Chinese Medicine, Fuzhou 350003, Fujian Province, China;
    2Department of Orthopedics, Fuzhou General Hospital of Nanjing Military Region, Fuzhou 350025, Fujian Province, China;
    3Department of Orthopedics, Union Hospital, Fujian Medical University, Fuzhou 350001, Fujian Province, China
  • Received:2012-01-20 Revised:2012-02-28 Online:2012-09-02 Published:2012-09-02
  • Contact: Xu Hao, Chief physician, Professor, Master’s supervisor, Department of Orthopedics, Fuzhou General Hospital of Nanjing Military Region, Fuzhou 350025, Fujian Province, China
  • About author:Pan Ting-ming★, Master, Physician, Department of Orthopedics, Second People's Hospital of Fujian University of Traditional Chinese Medicine, Fuzhou 350003, Fujian Province, China 3467890@163.com

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被引次数

1

摘要：

背景：目前研究发现，基质细胞衍生因子1/趋化因子受体4轴具有介导骨髓间充质干细胞定向迁移的作用。
目的：构建小鼠趋化因子受体4基因重组腺病毒载体。
方法：从C57BL/6小鼠中提取总RNA，以RT-PCR方法获得小鼠趋化因子受体4基因全长1 080 bp的完整编码序列，通过穿梭质粒pAdTrack-CMV，将目的片段克隆入AdEasy-1腺病毒DNA中，获得重组腺病毒DNA，通过脂质体转染293细胞，经包装扩增后，获得重组腺病毒pAdTrack-CMV-CXCR4，并感染293细胞，PCR鉴定、Western blot检测蛋白表达。
结果与结论：含趋化因子受体4基因的重组腺病毒pAdTrack-CMV-CXCR4构建成功，经PCR鉴定鉴定重组病毒中含有趋化因子受体4cDNA全长，经序列测定表明趋化因子受体4cDNA序列正确无丢失和错配现象，病毒滴度高，Western blot检测感染后293细胞趋化因子受体4的蛋白表达较未感染293细胞明显增加。

关键词: 趋化因子受体4, 重组腺病毒, 构建, 鉴定, 293细胞, 载体

Abstract:

BACKGROUND: Recent studies have demonstrated that stromal cell-derived factor-1 (SDF-1)/CXC chemokine receptor 4 (CXCR4) is capable of promoting the migration of bone marrow mesenchymal stem cells (BMSCs).
OBJECTIVE: To construct recombinant adenovirus vector containing CXCR4 gene and provide the basis for further research of CXCR4 gene for application.
METHODS: The total RNA of mouse was extracted and CXCR4 gene which is 1080 bp in size was amplified by RT-PCR. Fragment containing CXCR4 was cloned into the shuttle vector pAdTrack-CMV that carried a green fluorescence protein (GFP) gene to generate a recombinant plasmid pAdTrack-CMV-CXCR4. The recombinant adenovirus pAdEasy-CMV-CXCR4 was transferred into HEK293 cells for packaging and amplification. The viral titer was determined and the insert of CXCR4 gene was verified by PCR method and western blot.
RESULTS AND CONCLUSION: The recombinant adenovirus vector (pAdEasy-CMV-CXCR4) was successfully constructed and the sequence was corrected by PCR and sequencing, which possesses high titers. Western blot showed CXCR4 protein expression in infected HEK293 cells was markedly higher than that in uninfected HEK293 cells.

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