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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 investigate the interaction between HPO205 and cytochrome C oxidase (COX) 6B1 by yeast two-hybrid (Y2H) and GST-Pull down (GST-Pull down) Function to provide basis. Methods The coding genes of HPO205 and COX6B1 were amplified by polymerase chain reaction (PCR), and the recombinant Y2H plasmid vector was constructed respectively into pDBLeu and pPC86. Then the two recombinant plasmids were cotransfected with yeast MaV203 Y2H identification, and its GST-Pull down technology to verify. Results HPO205 and COX6B1 encoding genes were successfully cloned into Y2H vector and corresponding expression vector. After identified by Y2H, three reporter genes of Ura, LacZ and His were activated after pDBLeu-GRER and pPC86-COX6B1 co-transfection; GST-Pull down experiments showed that GST-COX6B1 can precipitate HPO205. Conclusion HPO205 may affect mitochondrial oxidative phosphorylation through the interaction with COX6B1.