在本計劃中，我們分別將台灣雨傘節β1-Bgt (β1-bungarotoxin) A1 chain及B1 chain cDNA一起裝載至pBridge vector中及分別裝載至pAS2-1 vector中，利用酵母菌雜交系統與Rat brain cDNA library進行β-Bgt binding protein的選殖工作。透過Yeast hybrid system的Protein-Protein interaction作用，我們發現約有5個生長在SD/-Leu/-His/-Trp +3AT培養基上的colony，進一步純化酵母菌中的質體DNA及進行DNA序列分析比對後發現，其中有一段基因序列(編號YB18)經由比對之後與Rat brain的KChIP3 (Potassium channel interacting protein) cDNA序列有極高(90%以上)的相似性，在酵母菌雜交系統中此選殖的YB18基因會與B1 chain有交互作用。我們進一步將此基因裝入pGEX-5X-2表現載體，並Transform至BL21菌種中表現蛋白質，經由SDS-PAGE電泳後染色發現，此基因以Fusion protein方式表現蛋白質，而Fusion protein的表現有利於日後的純化工作。另一方面，我們也參考已知KChIP3 cDNA序列，設計專一性引子，利用RACE-PCR與Nested PCR選殖出完整KChIP3的cDNA序列。由於在先前的研究曾發現單獨的B chain具有阻斷Potassium channel的活性，而在此計畫中發現B chain與Potassium channel interacting protein有分子交互作用之情形，我們在比對DNA序列後更發現KChIP是A type voltage-gate potassium channel的Interacting protein；由於過去的文獻曾指出Dendrotoxin作用的Potassium channel是屬於A type的Potassium channel，因此更引起我們想進一步探討其與β-Bgt及Potassium channel之間的關聯性及其交互作用之機制。目前我們已有KChIP3的cDNA序列，在90年度的工作中我們將以in vivo及in vitro的方法探討KChIP3與B chain交互作用的關聯性及其交互作用後的影響。 Snake presynaptic neurotoxins with phospholipase A/sub 2/ (PLA/sub 2/) activity block nerve terminals in an unknown way. β-bungarotoxin (β-Bgt), the main presynaptic phospholipase A/sub 2/ neurotoxin purified from the venom of Bungarus multicinctus (Taiwan banded krait), consist of two dissimilar polypeptide chains, the A chain and B chains, cross-linked by an interchain disulphide bond. In order to screen the binding protein of β-Bgt, the B1 cDNA of β-Bgt was subcloned into the yeast shuttle vector pAS2-1. A rat brain cDNA library was screened by co-transforming yeast strain YRG-2 with pAS2-1-B1 and rat brain library plasmid. Fortunately, a protein with its high homology nucleotide sequences with KChIP3 was interacted with the β-Bgt B chain by yeast-two hybrid from rat brain. KChIP, the Kv channel-interacting protein was identified and elucidated that which may regulate A-type currents, and hence neuronal excitability, in response to changes in intracellular calcium. In view of the findings of KChIP interact with the B chain of β-Bgt, it is proposed that the activity in blocking voltage-gated potassium channel of β-Bgt which may introduce by these KChIPs. Furthermore, the interaction of B1 chain and KChIP3 and the expression of KChIP3 are now studying.
