【作者】 李梦思； 夏菲； 刘光明；

【Author】 Li Mengsi;Xia Fei;Liu Guangming;College of Food and Biological Engineering,Jimei University;Fujian Provincial Engineering Technology Research Center of Marine Functional Food;Fujian Collaborative Innovation Center for Exploitation and Utilization of Marine Biological Resources;

【机构】 集美大学食品与生物工程学院； 福建省海洋功能食品工程技术研究中心；

【摘要】 蟹类作为一种营养价值高、肉质鲜美的水产品而广受消费者喜爱,但随之引起的食物过敏发病率持续上升,主要是由于过敏原序列上存在的抗原表位而导致的。目前,拟穴青蟹肌肉中热稳定的肌原纤维过敏原（原肌球蛋白TM,肌球蛋白轻链MLC）的抗原表位尚无系统性分析。本研究以蟹TM与MLC为研究对象,利用噬菌体展示技术与一珠一肽化合物技术对其抗原表位进行淘选;获得的肽段序列经生物信息学软件分析划分为模拟线性表位或模拟构象表位;合成肽结合血清学实验验证模拟线性表位,定点突变技术结合血清学试验验证模拟构象表位。利用两种淘选技术共获得TM的10条模拟线性表位与3条模拟构象表位,MLC则包含7条模拟线性表位与7条模拟构象表位;随后,抑制性Dot blot试验表明:TM与MLC的模拟线性表位肽段均能不同程度地抑制蛋白与血清中IgE的结合,其中TM线性表位区域R₁₀₀-T₁₁₀与L₁₁₃-R₁₂₅,MLC线性表位区域L₄₅-K₅₈、L₉₂-L₁₀₇与D₁₃₇-K₁₄₈更重要;对TM与MLC构象表位中的关键氨基酸进行突变而获得构象表位突变体。与野生型过敏原相比,圆二色谱与表面疏水性分析发现各个突变体的二级结构与三级结构都呈现不同程度地改变,ELISA试验发现各个突变体的IgE结合能力都有显著下降,进一步说明TM与MLC序列上构象表位的存在。拟穴青蟹TM与MLC抗原表位的定位分析有望为不同物种间存在的临床交叉反应研究奠定基础,同时有望为低致敏性免疫制剂的开发提供理论依据。更多还原

【Abstract】 As a kind of aquatic product with high nutritional value and delicious meat,crab is widely loved by consumers.However,the incidence of food allergy continues to rise,which is mainly caused by the antigen epitope of allergens.At present,there is no systematic analysis of the epitopes in the heat-stabilized myofibril allergens（tropomyosin TM,myosin light chain MLC） from crab.In this study,the epitopes of crab TM and MLC were screening by phage display technology and one bead one peptide compound technology.The obtained peptide sequences were divided into linear mimotopes or conformation mimotopes by bioinformatics software analysis.Linear mimotopes were verified by synthetic peptide combined with serological experiments,and the conformational mimotopes were verified by site-directed mutagenesis combined with serological experiments.Combined with the results of two panning techniques,a total of 10 linear mimotopes and 3 conformation mimotopes were obtained for TM,while 7 linear mimotopes and 7 conformation mimotopes were obtained for MLC.Subsequently,the results of inhibition Dot blot showed that the linear mimotope peptides of TM and MLC can inhibit the binding of protein to serum IgE to varying degrees,among which the linear epitopes R₁₀₀-T₁₁₀,L₁₁₃-R₁₂₅ of TM and L₄₅-K₅₈,L₉₂-L₁₀₇ and D₁₃₇-K₁₄₈of MLC are more important.Conformational epitope mutants were obtained by mutating key amino acids of TM and MLC.Compared with wild-type allergens,the secondary and tertiary structures of each mutant were changed.ELISA experiment indicated that the IgE binding capacity of each mutant was significantly decreased,which further suggested the existence of conformational epitopes on TM and MLC.The mapping analysis of TM and MLC epitopes is expected to lay a foundation for the clinical cross-reaction between different species,and provide theoretical basis for the development of hypoallergenic immunological agents.更多还原