桶形芋螺毒液蛋白质组分析及二硫键异构酶的鉴定

摘要

蛋白质二硫键异构酶(PDI)是内质网新生肽链折叠中一个重要的折叠酶.在热带药用海洋生物芋螺的毒液中,富含PDI酶,该酶对于毒液中芋螺毒素神经肽的体内氧化折叠至关重要.研究上样量、水化方式与时间、除盐步骤、聚焦电压和时间、平衡时间、凝胶电泳方法和染色方法等方面,优化并建立了较为理想的芋螺毒液蛋白样品双向电泳的实验条件与方法; 通过双向电泳和MALDI-TOF-MS质谱分析技术,从海南产桶形芋螺(Conus betulinus Linnaeus)毒液蛋白中成功分离鉴定出PDI等9种蛋白; 通过双向电泳和PDQuest软件分析了并比较了三种不同大小桶形芋螺毒液总蛋白的差异性,并质谱鉴定出5个明显的差异蛋白点.研究建立了桶形芋螺毒液蛋白双向电泳分析及质谱鉴定的技术方法,为后续大量分离纯化出天然的芋螺PDI酶以及利用蛋白质组学技术方法深入研究芋螺毒液特征提供了重要的基础.%Conopeptides is one kind of small precious nerve toxin peptides, which is excreted from Conus snails. Because the post translational processing of conopeptides is complicated, it is very difficult to synthesize conopeptides peptide with biological activity by the method named chemical synthesis. Many proteins from Conus snails, such as protein disulfide isomerase (PDI), prolylhydroxylase, proline isomerase, and so on, which promote the synthesis and activity formation of conopeptides. The protein disulfide isomerase (PDI) is an important foldase in folding of the nascent polypeptide chain of the endoplasmic reticulum (ER). The venom of tropic officinal marine cone snails is abundant with PDI which is important in assisting the folding and maturation of secretory conopeptides in vivo. As an effective and direct approach of research in all proteins, proteomics represents a novel methodological way to investigate the expression of all proteins of a cell, body fluid or organism, as well as to analyze their structure, function, interaction and modification. Proteomics has been widely applied in studying regulation of metabolism, genetics and development, physiology and biochemistry, pharmacology and toxicology. And many technologies of proteomics were available to research Conus snails venom protein in integer protein level. The report was to analyze the total proteins by the widely use technologies including two-dimensional electrophoresis (2-DE), mass spectrum, chromatogram analysis and biology informations, so as to lay a good foundation for further research on Proteomics of Conus venom. Previous trials on different electrophoresis methods showed that SDS-PAGE electrophoresis is difficult to detect protein with low molecular weight, while Tricine-SDS-PAGE electrophoresis can detect low molecular weight protein clearly. But in the 2-DE experiment, the Tricine-SDS-PAGE electrophoresis makes protein point deform and gets a blurry 2-DE map for taking more time than SDS-PAGE electrophoresis. So in this 2-DE study of Conus snails venom protein, we use the concentration of 15% SDS-PAGE gel to prepare the second PAGE gel. Such as parameters of sample preparation, isoelectric focusing and gel electrophoresis were explored and optimized to establish a better experimental method for two-dimensional electrophoresis (2-DE) analysis on the venom proteome of Conus betulinus Linnaeus native to Hainan. Molecular weights of the total proteins of Conus betulinus Linnaeus venom indicated most proteins located in three areas: 45—66.2 kD, 18.4—25 kD and under 14.4 kD. The PDI and other eight kinds of protein were isolated by 2-DE and confirmed by MALDI-TOF-MS successfully. Differential proteins in three different growing stages of Conus betulinus Linnaeus were studied and compared by 2-DE and PDQuest software, and five of them were confirmed by MALDI-TOF-MS. The technique of isolation and identification of venom protein by 2-DE and MALDI-TOF-MS was established, which would be the basis for isolating and purifying natural PDI from the venom of Conus betulinus and characterizing Conus snails venom proteomics.