人ob基因克隆及其亚细胞定位的研究  

Cloning and Sub-cellular Localization of Human Obese Gene

作　　者：陈香凝[1] 阴彦辉[1] 施青青[1] 孙敏[1] 傅德智[1] 高波[1] 李碧春[1] 

机构地区：[1]扬州大学动物科学与技术学院,江苏扬州225009

出　　处：《生物技术》2011年第2期7-10,共4页Biotechnology

基　　金：国家"863"高技术研究发展计划项目(2007AA100504);江苏省高校自然科学重大基础研究项目(08KJA230001)资助

摘　　要：目的:旨在克隆人肥胖(obese,ob)基因的全长cDNA序列,与EGFP重组构建融合蛋白表达载体,并分析其亚细胞水平的定位。方法:提取人脂肪细胞总RNA,采用RT-PCR方法扩增出人ob基因cDNA,并克隆至真核表达载体pEGFP-C1,重组质粒转染NIH-3T3细胞,荧光显微镜分析EGFP-ob融合蛋白的亚细胞定位。结果:克隆的ob基因cDNA为501bp,共编码167个氨基酸,与GenBank公布的人ob基因序列一致,荧光显微镜分析表明,重组的EGFP-ob融合蛋白主要分布于NIT-3T3的细胞质中。结论:成功克隆了人ob基因的cDNA序列,构建人ob基因的真核表达载体pEGFP-C1-ob,融合蛋白EGFP-ob定位于NIH-3T3细胞质中。Objective：The aim of this study is to clone full-length cDNA of obese（ob） gene in humans and analysis its sub-cellular localization through EGFP fusion protein.Method：cDNA of ob gene was cloned by RT-PCR from total RNA extracted from human fat tissue.The RT-PCR product was sub cloned into pMD19-T vector.The sequence analysis was then performed.The cDNA of ob gene was inserted into upstream of an expression vector pEGFP-C1,which contains the enhanced green fluorescent protein（EGFP）,to construct fusion expression vector named pEGFP-C1-ob.The recombinant plasmid,named pEGFP-C1-ob,was identified by PCR and restriction analysis.pEGFP-C1-ob was transfected into the eukaryotic cells of NIH-3T3 by electroporation,followed by the observation of transfected NIH-3T3 cells under inverted microscope after 24h.Result：The cloned full-length cDNA sequence of ob gene,501 bp in size,was determined to encode 167 amino acids and show 100% homology with the published sequence in the GenBank.The eukaryotic expression vector pEGFP-C1-ob was constructed and expressed in NIH-3T3 cells successfully.EGFP-ob fusion protein was identified to locate at the cytoplasm of NIH-3T3 cells.Conclusion：cDNA of ob gene was cloned successfully,and recombinant plasmid pEGFP-C1-ob was constructed and expressed in the cytoplasm of NIH-3T3 cells.

关 键 词：人 肥胖基因 EGFP蛋白 亚细胞定位 

分 类 号：Q786[生物学—分子生物学]