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中华普通外科学文献(电子版) ›› 2013, Vol. 07 ›› Issue (04) : 265 -268. doi: 10.3877/cma.j.issn.1674-0793.2013.04.006

所属专题: 文献

论著

ERβ1基因及其剪切变异体真核表达载体的构建与鉴定
陈迪1,(), 章乐虹2, 彭伟强1   
  1. 1. 516001 惠州市中心人民医院乳腺外科
    2. 广州医科大学第二附属医院乳腺外科
  • 收稿日期:2013-03-05 出版日期:2013-08-01
  • 通信作者: 陈迪
  • 基金资助:
    广东省科技计划项目(2010B031600294)

Construction and identification of eukaryotic expression plasmid of human estrogen receptorβ1 and its variant

Di CHEN1,(), Le-hong ZHANG2, Wei-qiang PENG1   

  1. 1. Department of Breast Surgery, Huizhou Municipal Central Hospital, Huizhou 516001, China
  • Received:2013-03-05 Published:2013-08-01
  • Corresponding author: Di CHEN
  • About author:
    Corresponding author: CHEN Di, Email:
引用本文:

陈迪, 章乐虹, 彭伟强. ERβ1基因及其剪切变异体真核表达载体的构建与鉴定[J]. 中华普通外科学文献(电子版), 2013, 07(04): 265-268.

Di CHEN, Le-hong ZHANG, Wei-qiang PENG. Construction and identification of eukaryotic expression plasmid of human estrogen receptorβ1 and its variant[J]. Chinese Archives of General Surgery(Electronic Edition), 2013, 07(04): 265-268.

目的

应用基因克隆技术构建雌激素受体β1(ERβ1)及其5-8外显子缺失变异体ERβ△5-8(文中简称ERβ△),为ERβ功能的研究奠定基础。

方法

组织中提取总RNA,RT-PCR法扩增基因ERβ1及ERβ△,限制性酶切并克隆于以带EGFP绿色荧光基因及Myc报告基因的非融合真核细胞表达质粒IRES2-EGFP,菌群转化扩增后琼脂糖凝胶电泳,限制性酶切后行DNA测序鉴定。

结果

PCR扩增及限制性酶切法证实重组真核表达质粒的构建成功。测序表明ERβ1长度为1593 bp,ERβ△长度为972 bp,与GeneBank上公布的蛋白编码区序列一致。

结论

成功构建ERβ1基因及其剪切变异体ERβ△真核表达载体。

Objective

To construct eukaryotic expression plasmid of human estrogen receptor β1(ERβ1) and the 5th to 8th exon missing variant ERβ△5-8(in this paper referred to as ERβ△).

Methods

Genomic fragment of ERβ1 and ERβ△ was amplified by RT-PCR. The recombinant plasmid was cleared by restriction endonuclease and cloned into eukaryotic expression vector IRES2-EGFP, which carried EGFP gene and Myc report gene and then transformed into the E.coli DH5α. The recombinant plasmid was identified by agarose gel electrophoresis. The genomic fragment of ERβ1 and ERβ△ was determined and analyzed by DNA sequencing.

Results

Eukaryotic expression plasmid was identified by PCR and restriction endonuclease. Sequencing results show that the length of fragments is the same with the CSD of ERβ1 and its variant in the GeneBank.

Conclusion

This study successfully constructs the eukaryotic cell express cloning IRES2-EGFP-ERβ1 and IRES2-EGFP-ERβ△.

图1 PCR扩增片段,可见ERβ1大小约1600 bp,ERβ△约为1000 bp
图2 载体骨架大小为6515 bp,可见目的基因与载体重组成功
图3 重组质粒分别测序,结果拼接后与目的基因一致
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