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 × 扫一扫
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Di CHEN1,†(), Le-hong ZHANG2, Wei-qiang PENG1
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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.
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。
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。
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。
成功构建ERβ1基因及其剪切变异体ERβ△真核表达载体。
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△).
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.
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.
This study successfully constructs the eukaryotic cell express cloning IRES2-EGFP-ERβ1 and IRES2-EGFP-ERβ△.