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α_(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 被引量:2

Binding between α1A -adrenergic receptor and segment of bone morphogenetic protein-1 in human embryonic cell 293
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摘要 用酵母双杂交方法发现骨形成蛋白-1(bone morphogenetic protein-1,BMP-1)的片段可以与α1A-肾上腺素受体(adrenergic receptor,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(human embryonic cell 293,HEK293)中的相互作用。采用PCR方法构建含BMP-1片段eDNA的真核表达质粒PCP3HA,将其与含全长人α1A-AR eDNA的质粒PDT-α1A分别或共同转染HEK293细胞,用免疫印迹法检测到α1A-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PDTα1A-和单独转染PCP3HA的细胞,OD490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-α1A和PCP3HA的细胞,OD490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-α1A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-α1A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α1A-AR与BMP-1的片段在HEK293细胞中存在蛋白水平的相互作用。 Using matchmaker yeast two-hybrid system, it has been demonstrated that there exists an interaction between the cellular C terminal of α1A-adrenergic receptor (α1A-AR) and a segment of bone mor-phogenetic protein-1 ( BMP-1 ). In the present study binding between the two proteins was further determined in human embryonic cell 293 ( HEK293 ) , a mammalian expression system. Mammalian expression vector PCP3HA was constructed by PCR and consisted of segments of BMP-1 cDNA, and vector PDT-α1A consisted of the full-length cDNA of human α1A-AR. They were transfected to HEK293 cells and examined by Western blot. α1A-AR and the segment of BMP-1 could be detected in the lysis of transfected cells. Then binding betweenα1A-AR and the segment of BMP-1 in HEK293 cell was determined by enzyme-linked im-munosorbent assays ( ELISA) and co-immunoprecipitation. In ELISA experiment, the ELISA microwell plate was first coated with anti-FLAG M2 antibody, which recognizes the FLAG-tagged α1A-AR, then the cell lysis, anti-HA rabbit polyclonal antibody and HRP conjugated anti-rabbit antibody were added in turn. The OD490 values among the control group, PDT-α1A transfection group and PCP3HA transfection group, ex- hibited no significant difference (0. 034 ±0. 027 , 0. 042 ±0. 019, 0. 030 ±0. 0096) , but the OD490 values of PDT-α1A and PCP3HA co-transfection group (0. 57±0. 12) were significantly higher than those of the other groups (P<0. 001, respectively). In co-immunoprecipitation experiments, HEK293 cells expressing α1A-AR or/and segment of BMP-1 were lysed and incubated with anti-FLAG M2 antibody, then the immu-noprecipitation pellet was Jmmunoblotted 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-α1A and PCP3HA co-transfected group. In conclusion, the results indicate that α1A-AR and the segment of BMP-1 are present in the same complex in HEK293 cells.
出处 《生理学报》 CAS CSCD 北大核心 2003年第6期692-698,共7页 Acta Physiologica Sinica
基金 This work was supported by the National Basic Research Priorities Programme of China (No. G2000056906) the National Natural Science Foundation of China (No. 30270540 30171083).
关键词 α1A-肾上腺素受体 骨形成蛋白-1 酶联免疫吸附实验 receptor adrenergic α1A bone morphogenetic proteins enzyme-linked immunosorbent assays
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