拟穴青蟹新型过敏原肌质钙结合蛋白的纯化、鉴定及分子克隆  

Purification, Identification and Cloning of a Novel Crab Allergen, Sarcoplasmic Calcium Binding Protein, from Scylla paramamosain

作　　者：毛海燕[1] 蔡娜[1] 陈亨莉[1] 曹敏杰[1] 蔡秋凤[1] 刘光明[1] 

机构地区：[1]集美大学生物工程学院福建省高校水产科学技术与食品安全重点实验室,福建厦门361021

出　　处：《食品科学》2014年第3期122-127,共6页Food Science

基　　金：国家自然科学基金项目(31171660);福建省杰出青年科学基金项目(2010J06012)

摘　　要：为进一步认识蟹类的过敏原,采用免疫印迹方法,分析发现甲壳类过敏患者血清能与拟穴青蟹肌浆蛋白中分子质量约为21 kD的蛋白质产生特异的IgE结合反应,结果显示该蛋白可能是蟹类新型过敏原。通过硫酸铵盐析、阴离子交换和凝胶过滤柱层析等方法对21 kD-蛋白进行分离纯化,采用Western blotting和基质辅助激光解析电离飞行时间质谱(matrix assisted laser desorption ionization-time of flight-mass spectrometry,MALDI-TOF-MS)确认纯化的21 kD-蛋白为肌质钙结合蛋白(sarcoplasmic calcium binding protein,SCP)。采用SMART-RACE(Switching Mechanism At RNA Termini-Rapid Amplification of cDNA Ends)的方法获得SCP的cDNA序列,该序列全长986 bp,开放阅读框为579 bp,编码193个氨基酸,其理论分子质量21.94 kD,等电点4.44。拟穴青蟹SCP与甲壳类动物SCP具有较高的同源性,与昆虫SCP的同源性较差;三级结构模拟分析显示,SCP含有5个螺旋-转角-螺旋结构区,即EF-手型结构区,并在其中两个手型结构区形成2个钙离子结合位点;进一步预测得到SCP的4个线性抗原表位和3个构象性抗原表位。For acquiring more information about crab allergens, a 21 kD protein was purified from Scylla paramamosain by ammonium sulfate fractionation, anion exchange chromatography and gel filtration column chromatography. The protein was identified as a novel allergen having IgE binding activity with serum from shellfish-allergic patients. According to the results of Western blotting and matrix assis ted laser desorption ionization-time of flight-mass spectrometry（MALDI-TOF/ TOF-MS） studies, the protein was confirmed as a sarcoplasmic calcium binding protein（SCP）. The method of switching mechanism at RNA termin i-rapid amplification of cDNA ends（SMART-RACE） was applied to clone the cDNA of SCP. A full length cDNA with 986 bp was obtained, which included an open reading frame（ORF） coding for 193 amino acid residues with a predicted molecular weight of 21.94 kD and t heoretical isoelectric point of 4.44. The protein had a high homology with SCP from other shellfishes, but the homology with insects was low. Its tertiary structure was constructed using Phyre 2.0 server and the results showed that the SCP had five helix-loop-helix motifs, namely EF-hand domains, two of which formed two Ca2＋ binding sites. Furthermore, bioinformatics analysis predicted that this SCP contained four linear and three conformational epitopes.

关 键 词：拟穴青蟹 新型过敏原 肌质钙结合蛋白 纯化 质谱分析 分子克隆 生物信息学分析 

分 类 号：TS254.2[轻工技术与工程—水产品加工及贮藏工程] Q785[轻工技术与工程—食品科学与工程]