龙虾主要过敏原的筛选及粘蛋白介导的致敏机理研究

High quality food allergen is the important guarantee for diagnosis of allergic disease and food processing labeling. Research had confirmed that the recombinant allergen was better than the crude extract, which could increase the diagnostic specificity and be also good for allergen standardization. Panulirus stimpsoni as China's important food allergen, the current clinical application is still its crude extract or mixed allergens of crab, shrimp and lobster. Our previous study showed that compositions of crude extracts of Panulirus stimpsoni were complex, and allergens of Panulirus stimpsoni had 23000 D, 26000 D, 32000 D, 34000 D, 48000 D and 64000 D positive protein band by immunoblotting. Cloning and expression for Panulirus stimpsoni revealed a pan allergen of 34 000 D. Those strongly suggest lobster may have multiple allergens and their corresponding allergen genes. Therefore, based on our previous work, we will construct the cDNA expression library of Panulirus stimpsoni, immunologically screen the allergen proteins and their genes with application of specific IgE. Then for obtaining major and minor restructuring lobster allergens, cloning, expression and characterization of allergen genes, animal experiment and antigen presenting analysis will be proceed. The mechanism of mucins-mediated lobster food allergy will also be studied . Those provide theoretical and experimental basis for vaccine preparation of lobster recombinant allergen, clinical diagnosis of lobster food allergy, allergen standardization and food processing labeling.

高质量过敏原是食物过敏性疾病诊断及食物加工标签标注的重要保证。研究证实重组过敏原优于粗浸液过敏原，能提高诊断特异性和过敏原标准化，是过敏反应的重要研究方向。龙虾作为我国重要的食物过敏原，目前临床使用的仍为粗浸液或蟹/虾/龙虾的混合过敏原粗浸液。我们前期研究发现龙虾粗浸液成份复杂，免疫印迹出现23、26、32、34、48和64kDa阳性蛋白带；对龙虾Pan s 1基因进行克隆表达，显示泛过敏原为34kDa。提示龙虾具多种过敏原蛋白及相对应的过敏原基因。为此，本项目拟在前期工作基础上，构建龙虾cDNA表达文库，应用特异性IgE进行免疫学筛库，筛选出多个过敏原基因，克隆表达并进行特性分析、动物实验及抗原提呈分析，以获取龙虾的主要及次要重组过敏原，并探讨粘蛋白介导食物致敏机理，为龙虾过敏原诊断试剂开发、过敏原的标准化及食品加工的标签标注提供理论依据。

过敏原研究是食物过敏性疾病诊断及食物加工标签标注的重要保证。研究证实重组过敏原优于粗浸液过敏原，能提高特异性诊断和过敏原标准化，是过敏反应的重要研究方向。龙虾为我国重要的食物过敏原之一，但目前临床实验的仍为虾/蟹混合过敏原粗浸液，或从虾肌肉组织中直接纯化，耗时费力。我们的前期研究提示龙虾具有多种过敏原蛋白及相对应的过敏原基因，提示龙虾的过敏原复杂。本项目利用基因工程技术对斑节对虾中主要过敏原Pen m3和Pen m6，小龙虾中主要过敏原精氨酸激酶（AK），钙结合蛋白（SCP），甘油醛-3-磷酸脱氢酶（GAPDH）的基因序列进行克隆表达, 转化进入大肠杆菌Rosetta(DE3)中进行诱导表达, 经Ni-NTA亲和柱纯化后得到了纯度较高的过敏原蛋白。并分别用临床病人的血清对其免疫学特性进行了鉴定。与此同时，我们还筛选并合成出了小龙虾精氨酸激酶中的T细胞表位肽，通过表面修饰有免疫刺激剂CpG的纳米材料包载，构建出针对小龙虾食物过敏的特异性免疫多肽纳米疫苗，并在小鼠体内进行了药效学考察和机制探讨。该实验, 一方面为进一步研究小龙虾致敏机制和食物过敏源检测试剂开发提供了基础, 另一方面也为制备能供临床上使用的高纯度小龙虾重组过敏原蛋白进行诊断和特异性免疫治疗奠定了基础。