新型α-芋螺毒素Di1.1的克隆、合成及功能研究

Cloning, Synthesis and Functions of a Novel α-Conotoxin Di1.1

目的:从中国南海长距芋螺中克隆出新的芋螺毒素序列并用固相方法合成该毒素,测定其折叠后的二硫键配对方式并初步研究其药理学特性。方法:根据芋螺毒素A超家族保守的信号肽序列设计引物,通过3'-RACE扩增,从芋螺毒腺管中克隆出新的毒素基因;采用Fmoc-固相法合成线性多肽,通过空气氧化折叠获得含二硫键的折叠产物,用两步氧化折叠法测定多肽的二硫键连接方式;用双电极电压钳技术初步研究其药理学特性。结果:发现一种新的α-芋螺毒素Di1.1的cDNA序列,其成熟肽序列为CCVIESCHSNHIDECES;该肽二硫键连接方式以C1-C4、C2-C3为主,以C1-C3、C2-C4连接为辅,对烟碱型乙酰胆碱受体各亚型活性较弱。结论:Di1.1是一种新的α4/7型芋螺毒素,其折叠方式以C1-C4、C2-C3连接为主。

Objective： To from CoMus distans derived of conotoxin A-superfamily, Fmoc solid-phase method, two-step oxidative folding clamp technique was used clone, synthesize, characterize the disulfide bridges and functions of a new conotoxin from the South China Sea. Methods： Based on the conserved signal peptide sequence a new α-conotoxin was cloned by 3＇-RACE. The linear peptide was synthesized using which was oxidized by air to give the product containing disulfide bridges. Then was employed to determine the disulfide bridge connections. Two electrode voltage to determine its binding to neuronal nicotinic acetylcholine receptor subtypes. Results：A novel ct-eonotoxin designated as Dil.1 （CCVIESCHSNHIDECES） was obtained, which its disulfide connectivity is mainly C1-C4,C2-C3, not the general C1-C3, C2-C4, and its binding to neuronal nicotinic acetylcholine receptor subtypes is not strong. Conclusion： Dil.1 is a novel α-conotoxin with a unique disulfide-bridge framework.