中华耳科学杂志
中華耳科學雜誌
중화이과학잡지
CHINESE JOURNAL OF OTOLOGY
2010年
1期
51-56
,共6页
Stathmin%CX26%相互作用蛋白质%酵母双杂交%免疫共沉淀
Stathmin%CX26%相互作用蛋白質%酵母雙雜交%免疫共沉澱
Stathmin%CX26%상호작용단백질%효모쌍잡교%면역공침정
Stathmin%CX26%Protein-protein interaction%Yeast two hybrid technique%Co-immunoprecipitation
目的 筛选和鉴定连接蛋白26(connexin 26,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.
目的 篩選和鑒定連接蛋白26(connexin 26,CX26)的相互作用蛋白質,分析其在小鼠內耳的錶達,探討與聽覺的關繫.方法 酵母雙雜交技術篩選CX26的相互作用蛋白質.在體外真覈細胞錶達繫統HEK293細胞內過錶達人CX26和相互作用蛋白質,觀察它們的定位,併用免疫共沉澱實驗觀察在體外真覈細胞中是否存在相互作用.進一步在小鼠腦組織中用免疫共沉澱方法驗證CX26和相互作用蛋白質在真覈生物體內的相互作用.逆轉錄聚閤酶鏈反應(Reverse transcription polymerase chain reaction,RT-PCR)和蛋白質印跡法(Western blot)分析相互作用蛋白質在小鼠耳蝸的錶達.結果 酵母雙雜交實驗篩選和鑒定到stathmin與CX26相互作用,兩者在體外過錶達繫統HEK293真覈細胞中有共定位,免疫共沉澱實驗證實在體外真覈細胞和真覈生物體內存在相互作用,而且stathmin在小鼠耳蝸錶達.結論 stathmin與CX26相互作用,併且在小鼠耳蝸錶達.
목적 사선화감정련접단백26(connexin 26,CX26)적상호작용단백질,분석기재소서내이적표체,탐토여은각적관계.방법 효모쌍잡교기술사선CX26적상호작용단백질.재체외진핵세포표체계통HEK293세포내과표체인CX26화상호작용단백질,관찰타문적정위,병용면역공침정실험관찰재체외진핵세포중시부존재상호작용.진일보재소서뇌조직중용면역공침정방법험증CX26화상호작용단백질재진핵생물체내적상호작용.역전록취합매련반응(Reverse transcription polymerase chain reaction,RT-PCR)화단백질인적법(Western blot)분석상호작용단백질재소서이와적표체.결과 효모쌍잡교실험사선화감정도stathmin여CX26상호작용,량자재체외과표체계통HEK293진핵세포중유공정위,면역공침정실험증실재체외진핵세포화진핵생물체내존재상호작용,이차stathmin재소서이와표체.결론 stathmin여CX26상호작용,병차재소서이와표체.
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 im-munofluorescence 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.
