Pen a 1表位抗原多克隆抗体的制备及鉴定

【目的】建立一种识别、检测致敏蛋白的新方法。【方法】Pen a 1为虾中主要的致敏蛋白，从其5个主要IgE结合区中选择一段具有代表性的（85—105位）含21个氨基酸的多肽序列，进行化学合成，将多肽分别与匙孔血蓝蛋白（KLH）和牛血清白蛋白（BSA）偶联，制得免疫原和包被原，免疫原免疫新西兰纯种白兔得到多克隆抗体。以刀额新对虾蛋白、卵清蛋白、花生蛋白和牛奶蛋白为样品，免疫印迹鉴定多克隆抗体对刀额新对虾中Pen a 1蛋白的特异性。【结果】经Ellman试剂测定多肽与KLH、BSA的偶联比分别为 12﹕1和8﹕1。间接非竞争ELISA测定多克隆抗体的效价达1.024×106，间接竞争ELISA(icELISA)测定该多克隆抗体对多肽的IC50和IC10分别为0.4324 μg•mL-1和0.0004 μg•mL-1，表明多克隆抗体对多肽具有较强的灵敏性。免疫印迹试验结果表明，此多克隆抗体仅可识别刀额新对虾蛋白中的Pen a 1蛋白，对所选其它物种蛋白无响应。【结论】通过人工合成多肽制备的抗体可用于目标致敏蛋白质的检测分析，该方法快捷灵敏，且具有较高的特异性。

Preparation and Identification of Polyclonal Antibody of One Pen a1 Epitope Peptide

【Objective】The study was conducted to develop a new method to identify and detect the shrimp allergic protein.【Method】 Pen a 1 is the main allergic protein of shrimp, and one of its five epitope peptides was chosen and synthesized using Fmoc Method, which site in Pen a 1 was 85-105. The peptide was conjugated to keyhole limpet (KLH) and bovine serum (BSA) by glutaric dialdehyde method to get artificial immune and coating antigen, respectively. The New Zealand white rabbits were immunized with the peptide-KLH to get the antibody, and the binding ratio of artificial antigens was detected by Ellman reagent. The ic-ELISA was conducted to determine the inhibition ratio between the peptide with the antiserum. Western blot was conducted among four different protein samples to identify the specificity of the antibody to Pen a 1.【Result】The binding ratios of the artificial immune and the coating antigen were 12﹕1 and 8﹕1, respectively. The titers of the antibody reached 1.024×106, and the IC50 and the IC10 between the antibody and the peptide were 0.4324 μg•mL-1 and 0.0004 μg•mL-1, respectively. Western blot result demonstrated that the antibody could identify the allergic protein of Metapenaeus ensis (Pen a 1).【Conclusion】Antibody prepared with synthetic peptide is credible and sensitive enough for analysis and detection of targeted allergic protein.