Molecular biological characterization and hypoallergenicity of seafood allergens

1.从9种鱼类和食用蛙中分离纯化了小清蛋白（Parvalbumin，PA），PA是9种鱼类和食用蛙的主要过敏原。利用cDNA克隆技术对8种鱼类的PA进行了一级结构分析。重组鲭鱼PA在E.大肠杆菌的IgE结合能力与天然PA相当。2.多种鱼类胶原蛋白具有致敏性。在虹鳟鱼和鲭鱼的情况下，分离的链被证明是所有过敏和交叉反应。在大肠杆菌中表达了5个覆盖虹鳟鱼α2链全序列的重叠蛋白。原肌球蛋白（Tropomyosin，Tm）是甲壳类动物和头足类动物常见的主要过敏原。利用cDNA克隆技术对5种甲壳类和5种头足类Tm的一级结构进行了分析。4.制备了甲壳类和软体类Tm的单克隆抗体和多克隆抗体，并应用夹心ELISA法对甲壳类Tm进行了特异性分析。5.用鲭鱼PA和美洲龙虾Tm免疫BALB/c小鼠，获得过敏原应答性Th 1细胞。Th 1细胞对合成的重叠肽的反应分析显示，鲭鱼PA的T细胞表位包含在肽11- 25、26 -40和56-75中，而美洲龙虾Tm的T细胞表位包含在肽146- 160、196 -210和226-250中。

1.Parvalbumin(PA) was purified from nine species of fish and the edible frog and demonstrated to be a major allergen in all species. Primary structures of PA from eight species of fish were elucidated by a cDNA cloning technique. Recombinant mackerel PA expressed in E. coli was shown to be comparable in IgE binding ability to native PA.2.Collagens from various species of fish were confirmed to be allergenic. In the case of rainbow trout and mackerel, a chains separated were shown to be all allergenic and cross-reactive. Five overlapping proteins that cover the entire sequence of rainbow trout α2 chain were individually expressed in E. coli and the recombinant protein corresponding to the C-terminal region of the α2 chain was found to contain important IgE binding epitopes.3.Tropomyosin(Tm) was established to be a major and cross-reactive allergen in common with various species of crustaceans and cephalopods. Primary structures of Tm from five species of crustaceans and five species of cephalopods were determined by cDNA cloning.4.Monoclonal and polyclonal antibodies against crustacean and molluscan Tm were produced and shown to be applicable to specific analysis of crustacean Tm by sandwich ELISA. The surface plasmon resonance method using monoclonal antibodies raised against fish PA was useful to determine PA in seafood with high sensitivity.5.Allergen-responsive Th1 cells were obtained from BALB/c mice immunized with either mackerel PA or American lobster Tm. Analysis of the Th1 cell response to synthetic overlapping peptides revealed that T cell epitopes of mackerel PA are contained in peptides 11-25,26-40 and 56-75 and those of American lobster Tm in peptides 146-160,196-210 and 226-250.