摘要
目的:应用酵母双杂交(yeast two hybrid,Y2H)和GST-Pull down技术鉴定肝细胞生成素205(hepatopoietin205,HPO205)与细胞色素C(cytochrome c,Cytc)间的相互作用.方法:采用PCR技术扩增HPO205和Cytc编码基因,分别将其构建Y2H质粒pDBLeu和pPC86的重组载体.应用Y2H技术将二者的重组质粒共转染酵母MaV203进行鉴定,同时用GST-Pulldown方法对其验证.结果:成功克隆HPO205和Cytc编码基因至Y2H载体和相应表达载体,包括pDBLeu-GRER、pDBLeu-CYCS、pPC86-GRER、pPC86-CYCS.经过Y2H鉴定发现,pDBLeu-GRER+pPC86-CYCS共转后能激活Ura和His两个报告基因,而pDBLeu-CYCS+pPC86-GRER共转则不能激活任何一个报告基因.GST-Pulldown实验显示,GST-CYCS能将HPO205沉淀下来,而GST空蛋白不能沉淀HPO205,证实HPO205与Cytc存在相互作用.结论:HPO205可能通过Cytc参与电子传递或/和细胞凋亡过程.
AIM： To identify the interaction between hepatopoietin 205 （HPO205） and cytochrome C （Cytc）.METHODS： The coding genes of HPO205 and Cytc, amplified by polymerase chain reaction, were cloned into pDBLeu and pPC86 vector respectively. The interaction was confirmed by cotransformation with the recombinant plasmids into MaV203 of yeast two-hybrid system （Y2H）, and verified by GST-Pull down assay simultaneously. RESULTS： The coding genes of HPO205 and Cytc were successfully cloned into relevant vectors, and the obtained vectors were named as pDBLeu-GRER, pDBLeu-CYCS, pPC86-GFER and pPC86-CYCS. After Y2H identification, we found that co-transformation of pDBLeu-GFER pPC86-CYCS activated reporter genes Ura and His, but co-transformation of pDBLeu-CYCS and pPC86-GFER activated no reporter genes. GST-Pull down assay showed that HPO205 was deposited by GST-CYCS, but not by GST, verifying the interaction between HPO205 and Cytc. CONCLUSION： The interaction between HPO205 and Cytc suggests that HPO205 participates in the biology processes of electron transfer or （and） apoptosis via Cytc.
出处
《世界华人消化杂志》
CAS
北大核心
2008年第12期1281-1286,共6页
World Chinese Journal of Digestology
基金
国家自然科学创新群体基金资助项目
No.30621063
国家高新技术研究发展计划(863计划)资助项目
No2006AA02A310
国家重点基础研究发展规划(973计划)资助项目
No2006CB910802
安徽省人才开发基金资助项目
No.2002Z035
安徽省2006研究实验基地科研带头人培养专项基金资助项目~~
