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#alpha#_(1A)-肾上腺素受体与骨形成蛋白-1片段在人胚肾293细胞中的结合

徐琦, 张坦, 韩启德, 张幼怡

北京大学第三医院血管医学研究所,分子心血管学教育部重点实验室.北京 100083

摘要

用酵母双杂交方法发现骨形成蛋白-1(BMP-1)的片段可以与#alpha#_(1A)-肾上腺素受体(AR)的细胞内游离C末端结合。进一步探讨了二者在哺乳动物表达系统人胚肾293细胞(HEK293)中的相互作用。采用PCR方法构建含BMP-1片段cDNA的真核表达质粒PCP3HA,将其与含全长人#alpha#_(1A)-AR cDNA的质粒PDT-#alpha#_(1A)分别或共同转染HEK293细胞,用免疫印迹法检测到#alpha#_(1A)-AR和BMP-1在HEK293细胞有相应的蛋白表达。用酶联免疫吸附实验对免疫印迹鉴定过的细胞裂解液进行检测,观察到空白对照组,单独转染PDT-#alpha#_(1A)和单独转染PCP3HA的细胞,OD_(490)值分别为0.034±0.027、0.042±0.019、0.030±0.0096,三者之间无显著性差异。共转染PDT-#alpha#_(1A)和PCP3HA的细胞,OD_(490)值为0.57±0.12,较其它三组具有显著性差异(均P<0.001)。免疫共沉淀结果显示,单独转染PDT-#alpha#_(1A)或PCP3HA的细胞,免疫沉淀产物中均不能检测到BMP-1片段,只有共转染PDT-#alpha#_(1A)和PCP3HA的细胞,在其免疫沉淀产物中能检测到BMP-1片段。ELISA和免疫沉淀结果均表明#alpha#_(1A)-AR与BMP-1的片段在HEK293细胞中存在蛋白水平的相互作用。

关键词： α1A-肾上腺素受体; 骨形成蛋白-1; 酶联免疫吸附实验

Binding between #alpha#_(1A)-adrenergic receptor and segment of bone morphogenetic protein-1 in human embryonic cell 293

Xu Qi, Zhang Tan, Han Qide, Zhang Youyi

Institute of Vascular Medicine,Peking University Third Hospital and Key Laboratory of Molecular Cardiology,Education Ministry.Beijing 100083

Abstract

In co-immunoprecipitation experiments, HEK293 cells expressing #alphla#_(1A)-AR or/and segment of BMP-1 were lysed and incubated with anti-FLAG M2 antibody, then the immunoprecipitation pellet was immunoblotted with either the HRP conjugated anti-FLAG antibody or the anti-HA antibody, which recognizes the HA-tagged segment of BMP-1. Segment of BMP-1 was present in the pellet immunoprecipitation of PDT-#alpha#_(1A) and PCP3HA co-transfected group. In conclusion, the results indicate that #alpha#_(1A)-AR and the segment of BMP-1 are present in the same complex in HEK293 cells.

Key words: Receptor adrenergic α1A;Bone morphogenetic proteins enzyme-linked;Immunosorbent assays

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引用本文：

徐琦, 张坦, 韩启德, 张幼怡. #alpha#_(1A)-肾上腺素受体与骨形成蛋白-1片段在人胚肾293细胞中的结合[J]. 生理学报 2003; 55 (6): .

Xu Qi, Zhang Tan, Han Qide, Zhang Youyi. Binding between #alpha#_(1A)-adrenergic receptor and segment of bone morphogenetic protein-1 in human embryonic cell 293. Acta Physiol Sin 2003; 55 (6): (in Chinese with English abstract).