中国组织工程研究

中国组织工程研究 ›› 2011, Vol. 15 ›› Issue (11): 1986-1989.doi: 10.3969/j.issn.1673-8225.2011.11.021

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

携带DREAM基因小分子干扰RNA重组质粒的构建

陈  敏1，项红兵2，田玉科1   

  1. 华中科技大学同济医学院附属同济医院麻醉科，湖北省武汉市 430030
    2广州中医药大学附属第二医院麻醉科，广东省广州市 510120
  • 收稿日期:2010-09-16 修回日期:2010-10-12 出版日期:2011-03-12 发布日期:2011-03-12
  • 通讯作者: 田玉科，教授，博士生导师，华中科技大学同济医学院麻醉科，湖北省武汉市 430030 Yktian@tjh. tjmu.edu.cn
  • 作者简介:陈敏☆，男，1972年生，湖北省十堰市人，汉族，华中科技大学同济医学院在读博士，主治医师，主要从事疼痛机理的研究。现在解放军广州军区武汉总医院麻醉科工作。

Construction of recombinant DREAM-targeting small interfering RNA expressing plasmids

Chen Min1, Xiang Hong-bing2, Tian Yu-ke1   

  1. 1Department of Anesthesiology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan  430030, Hubei Province, China
    2Department of Anesthesiology, Second Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine, Guangzhou  510120, Guangdong Province, China
  • Received:2010-09-16 Revised:2010-10-12 Online:2011-03-12 Published:2011-03-12
  • Contact: Tian Yu-ke, Professor, Doctoral supervisor, Department of Anesthesiology, Second Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine, Guangzhou 510120, Guangdong Province, China Yktian@tjh.tjmu.edu.cn
  • About author:Chen Min☆, Studying for doctorate, Attending Physician, Department of Anesthesiology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China wuhan_chenmin@ sina.com

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摘要：

背景：DREAM是一种多功能蛋白，在细胞中不同位置与不同靶蛋白结合，体外细胞培养和动物实验均证明DREAM参与了许多疾病的发病机制。
目的：构建携带DREAM基因的小分子干扰RNA重组质粒。
方法：设计并合成shRNA对应的两条互补的寡核苷酸链，pDC316-EGFP-U6质粒经BamHⅠ和HindⅢ双酶切与退火后的寡核苷酸连接，转化感受态E.coli DH5α，获得阳性克隆进行PCR和测序鉴定。
结果与结论：经PCR、酶切及测序证实，重组质粒pDC316-EGFP-DREAM-shRNA-U6片段大小为473 bp，其中插入的片断序列和位点与预期完全一致，说明pDC316-EGFP-DREAM-shRNA-U6重组质粒构建成功。

关键词: 转录因子, DREAM, 重组质粒, RNA干扰, 组织工程

Abstract:

BACKGROUND: DREAM is a multi-functional protein, which combine with different target proteins at different sites in cells. In vitro cultivate tests and animal experiments confirmed that DREAM is involving in onset mechanism of many different diseases.
OBJECTIVE: To construct recombinant DREAM-targeting small interfering RNA (siRNA) expressing plasmids. 
METHODS: Oligonucleotide containing the small hairpin of DREAM was designed and synthesized, which was inserted into the pDC316-EGFP-U6 plasmid double digested by BamHⅠ and Hind Ⅲ. The liation product was transformed competence E.coli DH5α. Positive clones were identified by PCR and sequencing. 
RESULTS AND CONCLUSION: The result of PCR and gene sequencing confirmed that the pDC316-EGFP-DREAMshRNA-U6 recombinant plasmid with 473 bp had been constructed successfully.

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