[1]张炜炜,王涛,艾国平,等.小鼠微小RNA miR-22真核表达载体的构建及功能初步研究[J].第三军医大学学报,2007,29(09):803-805.
 ZHANG Wei-wei,WANG Tao,AI Guo-ping,et al.Construction of eukaryotic expression vector of mouse microRNAs miR-22[J].J Third Mil Med Univ,2007,29(09):803-805.
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小鼠微小RNA miR-22真核表达载体的构建及功能初步研究
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《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

卷:
29卷
期数:
2007年第09期
页码:
803-805
栏目:
论著
出版日期:
2007-05-15

文章信息/Info

Title:
Construction of eukaryotic expression vector of mouse microRNAs miR-22
作者:
张炜炜王涛艾国平粟永萍王军平邹仲敏邓均董世武
第三军医大学军事预防医学院防原医学教研室,全军复合伤研究所,创伤、烧伤与复合伤国家重点实验室
Author(s):
ZHANG Wei-wei WANG Tao AI Guo-ping SU Yong-ping WANG Jun-ping ZOU Zhong-min DENG Jun DONG Shi-wu
State Key Laboratory of Trauma, Burns and Combined Injury, Institute of Combined Injury, College of Preventive Medicine, Third Military Medical University, Chongqing 400038, China
关键词:
微小RNA真核表达载体miR-22基因组片段HelaNorthern  blot
Keywords:
microRNAs eukaryotic expression vector miR-22 genomic sequence Hela Northern blotting
分类号:
R394-33; R394.3
文献标志码:
A
摘要:
目的      从基因组DNA扩增miR-22前体对应的基因组片段并克隆到pIRES2-EGFP,构建miR-22的真核表达载体。      方法      利用PCR技术从基因组DNA扩增miR-22前体对应的基因组片段,克隆到质粒pUCm-T,测序正确后构建到真核表达载体pIRES2-EGFP中,经BamHⅠ和EcoRⅠ双酶切后1.5%琼脂糖凝胶电泳鉴定,并进行序列测定。脂质体Lipofectamin 2000法转染Hela细胞后G418筛选获得稳定转染克隆,提取总RNA,通过RT-PCR方法对neo片段进行鉴定,采用Northern blot技术检测miR-22的表达。      结果      PCR扩增得到的334 bp片段与预期的miR-22前体对应的基因组片段序列一致;成功构建了真核表达载体pIRES2-EGFP/miR-22,测序结果正确。重组质粒转染Hela细胞后,经G418筛选成功获得阳性克隆。Northern blot分析显示miR-22在Hela细胞内高效表达。      结论      本实验成功克隆了miR-22的前体对应的基因组片段,构建其真核表达载体,并在Hela细胞内获得高表达,为深入研究微小RNA miR-22的功能奠定了基础。
Abstract:
Objective      To clone microRNA22 fragment from human genomic DNA and construct it into the eukaryotic expression vector.        Methods      MicroRNA22 genomic sequence was amplified from mouse genomic DNA by PCR and cloned into pUCm-T plasmid, then sequenced. The sequence was cloned into eukaryotic expression vector pIRES2-EGFP plasmid, then identified with BamHⅠ and EcoRⅠ in 1.5% agarose gel electrophoresis and sequenced. Hela cells were tansfected with the identified pIRES2-EGFP/miR-22 and pIRES2-EGFP by Lipofectamin 2000.       Results      The 334-bp genomic fragment amplified by PCR was correct. Eukaryotic expression vector pIRES2-EGFP/miR-22 was successfully constructed and confirmed by sequencing. G418-resistant clones of Hela cells were obtained and confirmed as positive by RT-PCR. Northern blotting showed the expression of miR-22 increased.        Conclusion      The successful cloning of genomic sequence corresponding to pre-miR-22 and construction of its eukaryotic expression vector has laid the foundation for the further study of miR-22 function.

参考文献/References:

张炜炜, 王涛, 艾国平, 等. 小鼠微小RNA miR-22真核表达载体的构建及功能初步研究[J]. 第三军医大学学报, 2007, 29(9):803-805.

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更新日期/Last Update: 2008-10-24