中国组织工程研究

中国组织工程研究 ›› 2011, Vol. 15 ›› Issue (24): 4489-4492.doi: 10.3969/j.issn.1673-8225.2011.24.029

• 组织构建基础实验 basic experiments in tissue construction • 上一篇    下一篇

分化型胚胎软骨发育基因1特异性小干扰RNA表达质粒的构建和鉴定

魏  炎1，郏雁飞2，郑  燕2，马晓丽2，汪运山1, 2   

  1. 1山东省医学科学院基础医学研究所，山东省济南市 250062
    2山东大学附属济南市中心医院，山东省济南市 250013
  • 收稿日期:2010-12-08 修回日期:2011-01-07 出版日期:2011-06-11 发布日期:2011-06-11
  • 通讯作者: 汪运山，博士，教授，博士生导师，山东省医学科学院基础医学研究所，山东省济南市 250062；山东大学附属济南市中心医院，山东省济南市 250013 sdjnwys@163. com
  • 作者简介:魏炎★，女，1985年出生，山东省邹平县人，汉族，山东省医学科学院在读硕士，主要从事肿瘤免疫研究。 weiyan985@ 126.com
  • 基金资助:

    济南市科技局科技计划项目(200905045-1)，国家自然科学基金(81000869)资助项目。

Construction and identification of small interfering RNA expression plasmid target to differentiated embryo-chondrocyte expressed gene 1  

Wei Yan1, Jia Yan-fei2, Zheng Yan2, Ma Xiao-li2, Wang Yun-shan1, 2   

  1. 1Institute of Basic Medicine, Shandong Academy of Medical Sciences, Jinan  250062, Shandong Province, China
    2Jinan Central Hospital, Shandong University, Jinan  250013, Shandong Province, China
  • Received:2010-12-08 Revised:2011-01-07 Online:2011-06-11 Published:2011-06-11
  • Contact: Wang Yun-shan, Doctor, Professor, Doctoral supervisor, Institute of Basic Medicine, Shandong Academy of Medical Sciences, Jinan 250062, Shandong Province, China; Jinan Central Hospital, Shandong University, Jinan 250013, Shandong Province, China sdjnwys@163.com
  • About author:Wei Yan★, Studying for master’s degree, Institute of Basic Medicine, Shandong Academy of Medical Sciences, Jinan 250062, Shandong Province, China weiyan985@126.com
  • Supported by:

    the Plan of Science and Technology Bureau of Jinan City, No. 200905045-1*; the National Natural Science Foundation of China, No.81000869*

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被引次数

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摘要：

背景：分化型胚胎软骨基因1可调控肿瘤生长、凋亡、衰老相关因子，与肿瘤的发生发展有着重要的联系。
目的：构建针对人分化型胚胎软骨基因1的小干扰RNA表达载体。
方法：从NCBI中查找人分化型胚胎软骨基因1基因全长mRNA序列，利用Katahdin提供的在线小干扰 RNA模板序列设计软件，设计针对分化型胚胎软骨基因1的2条shRNA 的 DNA 模板单链，合成靶向分化型胚胎软骨基因1基因转录可形成茎环结构的寡聚核苷酸，退火后与酶切后的pGreenPuro™ shRNA Cloning and Expression Lentivector质粒连接，在JM-109菌株中扩增，并进行质粒DNA琼脂糖凝胶电泳分析、紫外分光光度计检测、菌落PCR以及测序鉴定。
结果与结论：将含有分化型胚胎软骨基因1目标序列25 bp 的双链 DNA插入片段，连接到pGreenPuro™ shRNA Cloning and Expression Lentivector质粒形成重组质粒。质粒DNA琼脂糖凝胶电泳和紫外分光光度计分析结果确认所提质粒纯度较高，可用于后续实验。菌落PCR结果表明产物大小约为170 bp，与预期相符。测序结果表明pGreenPuro™ shRNA Cloning and Expression Lentivector质粒已经插入人分化型胚胎软骨基因1的干扰合成片段，无碱基突变。成功构建了靶向分化型胚胎软骨基因1-小干扰 RNA表达载体。

关键词: 分化型胚胎软骨基因1, 小干扰 RNA, 构建, 质粒, 基因表达

Abstract:

BACKGROUND: Differentiated embryo-chondrocyte expressed gene 1 (DEC1) is a basic helix-loop-helix transcription factor, which is closely associated with some malignant cancers.
OBJECTIVE: To construct small interfering RNA (siRNA) expression plasmid target to DEC1.  
METHODS: The mRNA sequence of DEC1 gene was searched from NCBI. Utilize of Katahdin siRNA technology, DEC1-siRNA oligonucletides were inserted into pGreenPuro™ shRNA Cloning and Expression Lentivector, after annealing, then transformed into JM-109. The recombinant plasmid was identified by Agarose gel electrophoresis analysis, ultraviolet spectrophotometer analysis, PCR and DNA sequencing.  
RESULTS AND CONCLUSION: The recombinant plasmid pGreenPuro™ shRNA Cloning and Expression Lentivector-DEC1 was obtained by connecting 25 bp segment containing DEC1 sequence to pGreenPuro™ shRNA Cloning and Expression Lentivector. Agarose gel electrophorsis analysis and ultraviolet spectrophotometer analysis confirmed that plasmid DNA had higher purity. DNA sequencing showed Targeting siRNA oligonucleotides were correctly inserted into the eukaryotic expression vector pGreenPuro™ shRNA Cloning and Expression Lentivector without base mutation. The interference vector pGreenPuro™ shRNA cloning and expression lentivector-DEC1 was successfully constructed.

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