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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。
In vitro Simulated Human Intestinal Fluid (SIF) Digestion Analysis of Pen a1 after digestion of immunogenicity changes. After digestion of the shrimp sensitized protein Pen a1 and its epitope polypeptide by SIF, the immunogenicity of Pen a1 and its epitopes was detected by whole antibodies and epitope-specific antibodies, and the SIF digestion stability of the epitope polypeptides was determined Sex. The results showed that the immunogenicity of Pen a1 decreased significantly within 60 min after digestion and decreased slowly after 90 min. The new fragment was still immunogenic, but could not be detected by SDS-PAGE. The immunogenicity of the five epitopes is also similar. Western-blot showed that the binding of the five epitope antibodies to the new protein produced was different. No.3,4,5 antibody bound more than the new protein fragment at 20-30 ku. ELISA tests showed that even after 4 h of digestion, the immunogenicity was reduced by only 80%. The digestive stability of each epitope of Pen a1 was No.2> No.1> No.3> No.4> No.5; the digestion stability of five epitope peptides was No.3> No.1> No.4 > No.2> No.5, negatively correlated with the number of restriction sites on each epitope. It can be concluded that Pen a1 No.2 epitope has the highest digestibility, No.5 epitope table digestion stability of the worst.