摘要
目的以酵母双杂交(Y2H)和GST融合蛋白沉降技术(GST-Pull down)来探讨肝细胞生成素205(HPO205)与细胞色素C氧化酶(COX)6B1间存在的相互作用,为研究HPO205的功能提供依据。方法聚合酶链式反应(PCR)扩增HPO205和COX6B1的编码基因,分别构建至pDBLeu和pPC86的重组Y2H质粒载体。然后将二者的重组质粒共转染酵母MaV203进行Y2H鉴定,并用GST-Pull down技术对其验证。结果成功克隆HPO205和COX6B1编码基因至Y2H载体和相应表达载体。经过Y2H鉴定发现,pDBLeu-GRER和pPC86-COX6B1共转后能激活Ura、LacZ、His 3个报告基因;GST-Pull down实验显示,GST-COX6B1能沉淀HPO205。结论 HPO205可能通过与COX6B1相互作用影响线粒体氧化磷酸化过程。
Objective To identify the interaction of proteins hepatopoietin 205 （HPO205） and COX6B1, which may provide a clue for further study on the functions of HPO205. Methods The coding genes of HPO205 and COX6B1 ,after having been amplified by polymerase chain reaction （PCR）, were cloned into pDBLeu and pPC86 respectively. The interaction was identified by co-transformation with the recombinant plasmids into MaV203 of yeast two-hybrid system （Y2H） and verified by GST-Pull down. Results The recombinant plasmids inclusive of the coding genes of HPO205 and COX6B1 were successfully constructed. Y2H identification implied the co-trans- formation of pDBLeu-GFER with pPC86-COX6B1 could activate reporter genes LacZ, Ura and His in MaV203. HPO205 was also precipitated by GST-COX6B1. Conclusion HPO205 may affect mitochondrial oxidative phos- phorylation through the interaction with COX6B1.
出处
《安徽医科大学学报》
CAS
北大核心
2012年第11期1278-1282,共5页
Acta Universitatis Medicinalis Anhui
基金
安徽省高等学校省级优秀青年人才基金重点项目(编号:2011SQRL064ZD)
安徽医科大学校基金(编号:2010xkj015)
关键词
肝细胞生成素205
COX6B1
蛋白质相互作用
hepatopoietin 205
cytochrome c oxidase subunit Vib polypeptide 1
protein-protein interaction
