摘要
目的 :研究人肾上腺素α2A受体基因 - 1 2 96位G/C多态性对DNA与蛋白质结合的影响。方法 :以重组质粒为模板 ,扩增 - 1 2 96位为G或C的 390bp片段 (- 1 4 1 4 - - 1 0 2 5)bp,分别与细胞核蛋白质结合 ,经非变性聚丙烯酰胺凝胶电泳 ,与地高辛 (Dig)标记的同源DNA片段杂交。另将Dig-dUTP标记的同一片段与经SDS -PAGE转印至膜上的细胞核蛋白质进行DNA -蛋白质结合反应 ,以抗Dig抗体检测结合反应信号。结果 :EMSA结果显示 ,- 1 2 96位为C的片段可与细胞核蛋白质结合 ,X光片上显示一条特异性结合带 ;为G时则无 ;人肾上腺素α2A受体基因 (- 1 4 1 4 - - 1 0 2 5)bp 390bpDNA片段与细胞核提取物杂交结果显示 ,在约 1 5kU位置可见一蛋白质条带。结论 :肾上腺素α2A受体基因 - 1 4 1 4 - - 1 0 2 5bp之间可能存在一转录因子结合位点 ,- 1 2 96位为C时 ,能与一特异性核蛋白结合 ,此蛋白质的相对分子量约为 1 5kU。
AIM:To study the influence of G/C polymorphism of adrenergic receptor alpha-2A gene on DNA binding with protein.METHODS:The 390 bp fragments (-1414--1025 bp) with G or C at site -1296 were amplified using the recombinants as templates and combined with nuclear extract, respectively. The complexes were subjected to electrophoresis and hybridized with the homogenous DNA labeled with Dig-dUTP. The same labeled DNA was hybridized with nuclear extracts, which were electrophoresed and blotted onto membrane. RESULTS:The DNA-protein binding signals were captured by immunological detection. In EMSA, it was found that a DNA-binding protein could bind to the fragment (-1414--1025 bp) with C at site -1296; and the molecular weight of the protein was determined as 15kU by South-Western. CONCLUSION: There was a specific nuclear protein with molecular weight of 15 kU, which could bind with the binding site for transcriptor in the 390 bp fragment (-1414--1025 bp) of adrenergic receptor alpha-2A gene with C at the site -1296 bp.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2002年第11期1400-1403,共4页
Chinese Journal of Pathophysiology
基金
全军"十五"医药卫生基金重点课题 (No.0 1 2 0 1 6)
空军后勤部司令部科研项目(KH0 0 1 90 93)资助
关键词
晕动病
易感性
SNP
肾上腺素α2A受体
基因表达调控
Receptors, adrenergic, alpha
Expression regulation
DNA-binding proteins
Motion sickness
