α-芋螺毒素Lt1.1的克隆、合成及靶点鉴定  

Cloning,synthesis and target identification of a novel α-conotoxin Lt1.1

作　　者：宁胡莹 李靓[1] 张龙霄 刘珠果[1] 戴秋云[1] 

机构地区：[1]军事医学科学院生物工程研究所,北京100071

出　　处：《军事医学》2017年第5期334-337,共4页Military Medical Sciences

基　　金：国家自然科学基金资助项目(81473192);国家973计划资助项目(2010CB529802)

摘　　要：目的发现作用于神经元烟碱型乙酰胆碱受体(nAChR)的拮抗剂,为研制新型镇痛药和神经生物学工具奠定基础。方法根据芋螺毒素A超家族DNA中保守的内含子及3'端非转录区(3'UTR)设计引物,从中国南海信号芋螺(Conus litteratus)中克隆获得新芋螺毒素Lt1.1序列。固相法合成Rink树脂肽,经裂解、空气氧化折叠、纯化后获得目的肽,利用两步氧化折叠法鉴定其二硫键连接方式。将神经元nAChR各亚基的cRNA在爪蟾卵母细胞中表达,利用双电极电压钳检测通道电流。结果获得一种新α-芋螺毒素Lt1.1序列(GCCSHPACNVNNPDICNH2),其二硫键连接方式为"C1-C3、C2-C4",作用靶点为nAChR α3β2和α3β4亚型,IC50分别为166.76和190.00nmol/L。结论 Lt1.1是一种典型的4/7型α-芋螺毒素,其选择性抑制了nAChR α3β2和α3β4亚型。Objective To discover novel conopeptides which are the antagonists of neuronal nicotinic acetylcholine receptors （nAChRs） in order to contribute to the development of novel analgesic drugs and neuropharmacological probes. Methods Based on the conserved untranslated region and intron of A-superfamily conotoxins, a novel α-conotoxin Lt1. 1 was cloned from Conus litteratus. The peptide-resin was synthesized using the solid-phased method and was cleaved. The resulting linear peptide was oxidized by air to give the product containing disulfide bridges. The folding product was finally purified by HPLC. The disulfide bond connectivity was determined using the two-step oxidative folding methods. The cRNA of rat nAChRs was expressed on the membrane of Xenopus oocyte. Membrane currents were recorded using the two electrode voltage-clamp technique. Results A novel α-conotoxin designated as Lt1. 1 （ GCCSHPACNVNNPDIC-NH2 ） was cloned and its disulfide connectivity was ＂C1-C3, C2-C4＂. Lt1. 1 selectively inhibited the α3β2 and α3β4 nAChRs with an IC50 of 166.76 and 190.00 nmol/L, respectively. Conclusion Lt1. 1 is a novel 4/7 α-conotoxin that selectively targetsα3β2 and α3β2 nAChRs.

关 键 词：α-芋螺毒素 Lt1.1 克隆 合成 烟碱型乙酰胆碱亚型 

分 类 号：R91[医药卫生—药学]