【作者】 张伟；

【导师】 邓子峰； 戚正武；

【作者基本信息】 同济大学 ， 分析化学， 2006， 硕士

【摘要】 芋螺毒素(conotoxins，CTx)是近年来国际上的一个研究热点。它是肉食性软体动物芋螺(conus)分泌出来的能特异性作用于体内各种离子通道及神经受体的生物活性多肽化合物，通常是由10-46个氨基酸残基组成，具有分子量小、结构多样、富含半胱氨酸、作用靶点广泛、功能专一、组织特异性强等特点。目前发现的芋螺毒素超家族主要有A-，T-，O-，M-，P-，I-和S-超家族等，它们既可以作为体内一些具有重要生理功能靶点的探针和“解密器”，也可被直接开发成为药物或作为新药的先导化合物，因此对芋螺毒素的研究有重要的理论和实际意义。 本文利用RACE方法从大理石芋螺(C．marmoreus)和花玛瑙芋螺(C．achatinus)克隆得到A-超家族芋螺毒素κA-Ac4.1a／b，O-超家族芋螺毒素Mr6.1和T-超家族芋螺Mr5.2，并对之进行序列分析。从大理石芋螺中分离纯化得到3个新的芋螺毒素mr5.2，mr3.5和mr3.6，并对它们进行质谱鉴定和氨基酸序列测定。其中mr5.2属于T-超家族芋螺毒素，有翻译后加工的形式存在，两个Glu被加工成γ-羧基谷氨酸(Gla)，推测Gla可能对芋螺肽的折叠起着重要的作用。分离得到的mr3.5和mr3.6具有CC—C—C—CC的半胱氨酸骨架排列方式，但与先前发现的M-超家族芋螺毒素(μ-、ψ-和κM-芋螺毒素)存在显著差异，分子明显偏短，在第三个loop只有1个氨基酸，同时也没有类似于mr3a中的GCxPC motif结构存在，推测mr3.5和mr3.6可能是不同于M-超家族中前两类家族的一个新的家族。 同时本文通过基因工程方法在大肠杆菌BL21(DE3)中表达出带有PDI的Mr6.1融合蛋白，发现芋螺PDI在芋螺毒素表达过程中起到重要的作用，并找到一条简单高效表达并纯化融合蛋白的途径。更多还原

【Abstract】 Conotoxins are biologically active peptides separated from venomous marine cone snails. They are typically small disulfide-rich peptides that potently and specifically interfere with neurotransmission by targeting a variety of proteins expressed on the cell surface,such as the ion channels of Ca²⁺, Na⁺ and K⁺, and the receptors of acetylcholine,NMDA,and neurotensin. So far they have been categorized into seven superfamilies named A, I, M, O, P, S, and T, which are characterized by the number of their cysteine residues, the arrangement of the disulfide bonds and the distinctive and conserved signal region of the toxin precursor. Conotoxins are utilized as reasearch tools in many fields of neurobiology with high-affinity antagonists for receptor and ion channel subtypes, and being small peptides, they are synthesizable and modifiable, thus increasing their availability and potential utility. As mentioned above they can be used both as pharmacological tools and as templates for drug design.In this study, four novel conotoxins κA-Ac4.1a/b, Mr6.1 and Mr5.2, belonging to A-, O-, T-superfamily respectively were cloned from C.marmoreus and C.achatinus with RACE. In addition, three novel conotoxins mr5.2, mr3.5 and mr3.6 were separated and purified from the venom of the mollusc-hunting cone snail C.marmoreus, and their primary structures were identified with mass-spectrometric and protein sequencing technologies. The mr5.2, which is belong to T-superfamily, exists with high degree of posttranslational modifications, contains two γ-carboxylation of glutamyl residuses. It is presumed that the Gla in conotoxins from invertebrate cone snail plays an important role for conotoxins folding. The other two, mr3.5 and mr3.6 have cysteine residue framework of CC-C-C-CC which is belong to O-spuperfamily, however, they were obviously different from the discoveried M-superfamily, the length of molecule is shorter, and there is only one amino acid in the third loop. They don’t have GCxPC motif structure which exist in mr3a. Thus, the mr3.5 and mr3.6 were presumed to be a new family of M-superfamily.The fusion protein of pET-24（+）-PDI-Mr6.1 in E.coli BL21 was expressed with更多还原