利用SPR原理研究mGAT-1基因5’区域和组织核蛋白的相互作用

STUDY ON THE INTERACTION BETWEEN THE 5' PROXIMAL REGION OF mGAT-1 AND NUCLEAR PROTEINS BY THE METHOD OF SPR

用PCR方法扩增到小鼠GAT-1基因5’近侧序列-1775—-1594片段(F182DNA),通过生物素偶联到感应片(SA5)上,利用生物传感器,研究了小鼠肾、肝组织核蛋白提取液和F182DNA的相互作用,实验表明不同浓度的小鼠肾、肝核蛋白与F182DNA片段结合明显,并且它们的表观解离速率常数都是k_d=1.4E-5/秒。实验还发现,在F182序列中一人鼠保守的序列是一个蛋白主要的结合位点,证明这一保守序列在mGAT-1基因调控中可能起重要的作用。

The DNA fragment (named F182) corresponding the position of -1775-1594 in the mouse GABA transporter 1 (mGAT-1) 5' proximal region was amplified by PCR. Then the DNA was immobilized to the surface of sensor chip SA5 via biotin-streptavidin linkage. The interaction between the F182 on SA5 and nuclear proteins from mouse liver and kidney was studied by the method of SPR with Biosensor of BIAcore-1000 respectively. The Binding between F182 and two nuclear proteins was definitely and specifically and both with the apparent dissociation rate of about 1. 4E-5/S. Competitive experiment revealed that a conserved sequence within F182 had the main contribution to the binding event.