摘要
目的筛选和鉴定连接蛋白26(connexin26,CX26)的相互作用蛋白质,分析其在小鼠内耳的表达,探讨与听觉的关系。方法酵母双杂交技术筛选CX26的相互作用蛋白质。在体外真核细胞表达系统HEK293细胞内过表达人CX26和相互作用蛋白质,观察它们的定位,并用免疫共沉淀实验观察在体外真核细胞中是否存在相互作用。进一步在小鼠脑组织中用免疫共沉淀方法验证CX26和相互作用蛋白质在真核生物体内的相互作用。逆转录聚合酶链反应(Reverse transcription polymerase chain reaction,RT-PCR)和蛋白质印迹法(Western blot)分析相互作用蛋白质在小鼠耳蜗的表达。结果酵母双杂交实验筛选和鉴定到stathmin与CX26相互作用,两者在体外过表达系统HEK293真核细胞中有共定位,免疫共沉淀实验证实在体外真核细胞和真核生物体内存在相互作用,而且stathmin在小鼠耳蜗表达。结论stathmin与CX26相互作用,并且在小鼠耳蜗表达。
Objective To screen and identify proteins interacting with CX26 and to study their expressions in the murine cochlea.Methods Proteins potentially capable of interacting with CX26 were screened and identified by the yeast two hybrid technique.The co-localization of CX26 and one candidate protein （stathmin） produced from the yeast two hybrid screening in exogenous expression system HEK293 cells were tested using confocal microscopy and immunofluorescence techniques.The in vitro interaction in exogenous expression system HEK293 cells and in vivo interaction in murine brain tissue were examined using co-immunoprecipitation techniques.The expression of the interacting protein in murine cochlea was confimed with RT-PCR and Western blot methods.Results Stathmin interaction with CX26 was demonstrated both in HEK293 cells and in murine brain tissue.Both molecules co-localized in cytoplasma of HEK293 cells.Stathmin also expressed in mice cochlea.Conclusion The data indicate that Stathmin interacts with CX26 and is expressed in murine cochlea.
出处
《中华耳科学杂志》
CSCD
2010年第1期51-56,共6页
Chinese Journal of Otology
基金
国家自然科学基金资助课题(编号:30000094
30572021)
中南大学博士后基金(编号:56890)
