CAJ | 학술논문

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。
목적응용기인극륭기술구건자격소수체β1（ERβ1）급기5-8외현자결실변이체ERβ△5-8（문중간칭ERβ△），위ERβ공능적연구전정기출。방법조직중제취총RNA，RT-PCR법확증기인ERβ1급ERβ△，한제성매절병극륭우이대EGFP록색형광기인급Myc보고기인적비융합진핵세포표체질립IRES2-EGFP，균군전화확증후경지당응효전영，한제성매절후행DNA측서감정。결과 PCR확증급한제성매절법증실중조진핵표체질립적구건성공。측서표명ERβ1장도위1593 bp，ERβ△장도위972 bp，여GeneBank상공포적단백편마구서렬일치。결론성공구건ERβ1기인급기전절변이체ERβ△진핵표체재체。
Objective To construct eukaryotic expression plasmid of human estrogen receptorβ1(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△.