克氏原螯虾新型过敏原血蓝蛋白的鉴定及性质研究  

Identification and Characterization of Hemocyanin, a Novel Allergen of Procambarus clarkii

作　　者：陈亨莉[1] 曹敏杰[1] 蔡秋凤[1] 高榕 张凌晶[1] 朱妙婷[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 [2]杏林出入境检验检疫局,福建厦门361022

出　　处：《中国食品学报》2014年第2期240-247,共8页Journal of Chinese Institute Of Food Science and Technology

基　　金：国家自然科学基金项目(31171660);福建省自然科学基金项目(2010J06012);福建省高校新世纪优秀人才计划(NCETFJ-2007)

摘　　要：以克氏原螯虾为研究对象,鉴定血蓝蛋白的理化性质及其过敏原性。酶联免疫吸附试验结果表明:6份甲壳类过敏患者血清与克氏原螯虾血淋巴发生特异性IgE反应,利用离心和柱层析方法从血淋巴中分离纯化到目的蛋白。SDS-PAGE分析显示纯化的目的蛋白由6个亚基(依次为68,72,76,82,84和88 ku)组成,采用兔抗凡纳滨对虾血蓝蛋白多克隆抗体的免疫印迹试验证实目的蛋白为血蓝蛋白。免疫印迹试验结果显示,纯化的血蓝蛋白与甲壳类过敏患者血清出现了特异的杂交显色条带,表明血蓝蛋白具有IgE结合活性。与克氏原螯虾主要过敏原(原肌球蛋白)相比,血蓝蛋白的糖含量为1.99%,热稳定性好,pH稳定性及对胃蛋白酶的耐受性不如原肌球蛋白,但比原肌球蛋白更耐胰液消化。综合血清学及理化性质分析结果,提示血蓝蛋白是克氏原螯虾的1种新型过敏原。This study aimed to characterize hemocyanin from crayfish Procambarus clarkii and identify its IgE-binding activity. ELISA analysis of sera from crustacean-allergic patients showed hemolymph from crayfish Procambarus clarkii had the specific IgE-binding activity to six sera. Then hemocyanin was purified from hemolymph via Sephacryl S-200 HR chromatography and revealed six submits with molecular masses of 68, 72, 76, 82, 84, and 88 ku on SDS-PAGE. Western-blotting using anti-shrimp hemocyanin polyclonal antibody confirmed that the purified protein was hemocyanin. Western-blotting analysis of hemocyanin presented the specific IgE-binding activity to the sera from crustacean-allergic patients. Hemocyanin was a glycoprotein with 1.99% carbohydrate which was detected by the method of phenol-sulfuric acid. Analysis of the physicochemical properties of hemocyanin and tropomyosin（the major allergen of Procambarus clarkii） showed that the two proteins were both stable to heating treatment; hemocyanin was less stable to acidic treatment and pepsin digestion, but more resistive to SIF digestion. Both the serum experiment and the analysis of physicochemical property inferred that hemocyanin is a novel allergen of crayfish Procambarus clarkii.

关 键 词：克氏原螯虾 血蓝蛋白 新型过敏原 IgE结合活性 稳定性 

分 类 号：TS254.7[轻工技术与工程—水产品加工及贮藏工程]