文章摘要
黄越倩,张佩,钟名其,姚德福,陈洁辉,章跃陵.基于不同纯化策略的4种对虾血蓝蛋白的凝集活性及分子基础对比分析[J].水产学报,2018,42(11):1747~1753
基于不同纯化策略的4种对虾血蓝蛋白的凝集活性及分子基础对比分析
Analysis and comparison of agglutinative activity and the molecular basis among 4 kinds of Litopenaeus vannamei hemocyanin based on different purification strategies
投稿时间:2017-06-14  修订日期:2017-11-06
DOI:10.11964/jfc.20170610873
中文关键词: 凡纳滨对虾  血蓝蛋白  凝集活性  蛋白质组成  糖基修饰  分子基础
英文关键词: Litopenaeus vannamei  hemocyanin  agglutinative activity  protein composition  glycosylation  molecular basis
基金项目:国家自然科学基金(31372558&31672689);广东省自然科学基金(10251503101000002&2017A030311032);广东省高等学校重大科研项目培育计划(2014GKXM043)
作者单位E-mail
黄越倩 汕头大学理学院生物学系, 广东省海洋生物技术重点实验室, 广东 汕头 515063  
张佩 汕头大学理学院生物学系, 广东省海洋生物技术重点实验室, 广东 汕头 515063  
钟名其 汕头大学理学院生物学系, 广东省海洋生物技术重点实验室, 广东 汕头 515063  
姚德福 汕头大学理学院生物学系, 广东省海洋生物技术重点实验室, 广东 汕头 515063  
陈洁辉 汕头大学理学院生物学系, 广东省海洋生物技术重点实验室, 广东 汕头 515063  
章跃陵 汕头大学理学院生物学系, 广东省海洋生物技术重点实验室, 广东 汕头 515063 zhangyl@stu.edu.cn 
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中文摘要:
      既往研究表明,对虾血蓝蛋白(hemocyanin, HMC)是一种具有抗病毒、抗菌等多种免疫学活性的免疫球蛋白超家族(immunoglobulin superfamily, IgSF)分子,但迄今为止,其功能多样性的分子基础尚不是很清楚。本研究以凡纳滨对虾HMC为研究对象,采用亲和层析、凝集素层析技术获得4种HMC成分:A-HMCs、A-HMCl、AL-HMCs和AL-HMCl,发现其对不同细菌的凝集活性存在较大差异。其中,AL-HMCs和AL-HMCl对大肠杆菌和副溶血弧菌的凝集活性明显强于A-HMCs和A-HMCl,前者约为后者的2~32倍。继而,通过双向凝胶电泳(two-dimensional gel electrophoresis, 2-DE)和凝集素印迹技术对不同HMC的蛋白质组成和糖基化修饰水平进行对比分析。结果显示,4种HMC在2-DE图谱上表现为6~7个差异明显的蛋白质点,且与伴刀豆凝集素(concanavalin A, ConA)、花生凝集素(peanut agglutinin, PNA)、荆豆凝集素(ulex europaeus agglutinin, UEA)和双花扁豆凝集素(dolichos bifows agglutinin, DBA)等4种凝集素的识别存在显著性差异。其中,A-HMCl 6个蛋白点均可识别4种凝集素,而A-HMCs 6个蛋白点中仅有4、3个点分别与UEA、DBA反应呈阳性,AL-HMCs和AL-HMCl可以与UEA、PNA特异性显色的点分别为其总蛋白点的3/7、2/6。由此推测,对虾血蓝蛋白功能多样性的分子基础可能与其蛋白质组成和糖基化修饰水平的多样性密切相关。
英文摘要:
      It has been confirmed that hemocyanin (HMC) is a kind of immunoglobulin superfamily (IgSF) protein with antiviral and antibacterial activity. But until now, the molecular basis of its functional diversity is not well understood. In this study, four kinds of HMC fractions from Litopenaeus vannamei, i.e. A-HMCs, A-HMCl, AL-HMCs and AL-HMCl, were purified by affinity chromatography and lectin chromatography, which possessed distinct agglutinative activities against different pathogens. Among them, the agglutination activities of AL-HMCs and AL-HMCl were significantly (about 2–32 times) stronger than those of A-HMCs and A-HMCl against Escherichia coli K12 and Vibrio parahaemolyticus. Furthermore, the protein component and glycosylation modification of the 4 HMCs were analyzed by 2-DE and 2-D lectin blotting. The results showed that they had 6-7 spots in the 2-DE map with significant difference in isoelectric point and molecular weight. In particular, these protein spots could bind with 4 lectins, including concanavalin A (ConA), peanut agglutinin (PNA), ulex europaeus agglutinin (UEA) and dolichos bifows agglutinin (DBA), to different degrees. Of these, all of the 6 protein spots from A-HMCl could bind to 4 kinds of lectins, while only 4 and 3 protein spots from A-HMCs showed positive to UEA and DBA, respectively. AL-HMCs and AL-HMCl could respectively react with PNA and UEA, to produce 3 spots out of 7 total protein spots and 2 spots out of 6 total protein spots, respectively. Collectively, our data demonstrated that the molecular basis of functional diversity of shrimp hemocyanin may be closely related to the diversity of protein composition and glycosylation.
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