（毕业设计论文）《BK通道研究》.doc

摘要 大电导,Ca2+-激活的钾通道（BK通道）是钾通道中最复杂的一类，其由电压依赖性钾通道进化而来，可以受到细胞内Ca2+ 和膜电压的双重调控。门控机制的多样性，使得BK通道在细胞的生理活动中占有重要的地位。BK通道是由α亚基和β亚基组成的四聚体，其中4个α亚基是孔道形成单位，而β亚基是调控单位。不同的β亚基与α亚基的结合是构成BK通道电生理多样性的重要基础。β亚基由两个跨膜区段（TM1和TM2）、胞内N-末端和C-末端、以及胞外含有4个保守半胱氨酸残基的loop区组成。 pET-32a质粒载体的His·tag的6个组氨基残基层析挂柱效果不理想，并且thrombin蛋白酶切位点不适合。本课题利用定点突变技术，对pET-32a质粒载体进行了适当的改造。然后将BK通道亚基环状区域hβ3-loop连接到改造后载体上。 本论文对BK通道研究的相关背景进行了综述，并且对实验涉及到主要技术手段进行了探讨，最后获得了比较成功的构建体。 关键词：BK通道 β亚基 pET表达载体 PCR突变技术 Abstract Large conductance，Ca2+-activated K+ channels(BK channels) are the most complicated of the potassium channels, which are evolved from the voltage-dependent potassium channels. BK channels can be gated by voltage signal of the cell membrane and intracellular Ca2+ at the same time, because of this special electrophysiological characteristics (the voltage-dependent gating，the calcium-activated gating),BK channels play a very important role in cells. BK channel is a tetramer formed by four α subunits and several β subunits, the former are basic structural unites of the channel pore, while the latter are the subunits which mainly regulate the functions of BK channels. Different kind and number of the β subunits which combined with α subunits is a very important base of the variety in BK channels. A β subunit has two transmembrane domains(TM1and TM2), intracellular N-terminus and C-terminus, extracellular loop which contains four conservative cys amino acid. His·tag of the pET-32a vector has 6 his amino acid, which cannot provide appropriate affinity Tag for protein purification, furthermore, the site for the thrombin is not fit. With this situation, we used the Site-directed mutagenesis technology to modify the vector system. In this paper, we introduced the background of the BK channels study at first ,then discussed the technology involved in our study . The modified plasmid have laid foundation for our further study of the β subunit. Key word：BK channel β subunit pET vector Site-directed mutagene