体外模拟人体肠液（SIF）消化分析Pen a1消化后免疫原性的变化规律。虾致敏蛋白Pen a1及其表位多肽经SIF消化后，用全抗体和表位特异性抗体检测Pen a1及其各抗原表位的免疫原性的变化，并测定表位多肽的SIF消化稳定性。结果表明，Pen a1的免疫原性在消化60 min内下降显著，在90 min后下降缓慢，生成的新片段仍具有免疫原性，但会逐步完全分解至SDS-PAGE无法检测出；Pen a1中5个抗原表位的免疫原性变化也类似。Western-blot表明五个表位抗体与生成的新蛋白结合程度不同，No.3、4、5抗体与20~30 ku处的新蛋白片段结合多于No.1、2。ELISA检测表明即使经过4 h的消化，免疫原性也只是降低了80%。Pen a1各表位消化稳定性为No.2>No.1>No.3>No.4>No.5；5个表位多肽的消化稳定性为No.3>No.1>No.4>No.2>No.5，与各表位上的酶切位点的个数呈负相关。可以得出Pen a1中No.2表位具有最高的消化稳定性，No.5表位表消化稳定性最差。

Simulated intestinal fluid (SIF) was used to digest Pen a1, and changes in the immunogenicity of Pen a1 after digestion were analyzed. After digestion of shrimp antigen Pen a1 and the epitope peptides of Pen a1, a Pen al antibody and epitope-specific antibody were used to measure changes in the immunogenicities of Pen a1 and its epitopes, and the digestive stabilities of the epitope peptides of Pen a1 were determined. The results showed that the immunogenicity of Pen a1 decreased significantly within 60 min of digestion and continued to decrease slowly after 90 min. The generated protein fragments remained immunogenic, but were gradually degraded until they could no longer be detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Similar changes were observed for the immunogenicities of five epitopes of Pen a1. Western blot results indicated that the binding degrees of the five epitope antibodies with the proteolytic fragments of Pen a1 after the SIF digestion were different. The numbers of 20~30 ku fragments bound to antibody No. 3, 4, and 5 were greater than those bound to antibody No. 1 and 2. Enzyme-linked immunosorbent assay results indicated that after 4-h digestion, the immunogenicity had only decreased by 80%. The digestive stabilities of the five epitopes of Pen a1 were as follows: No. 2 > No. 1 > No. 3 > No. 4 > No. 5. In contrast, the digestive stabilities of the five epitope peptides of Pen a1 were in the following order: No. 3 > No. 1 > No. 4 > No. 2 > No. 5, which was negatively correlated with the number of cleavage sites on the epitope peptide. In conclusion, epitope No. 2 showed the highest digestive stability, while epitope No. 5 showed the lowest digestive stability.

农业部公益性行业科研专项（201103007）