摘要
【目的】Pen a 1是虾中的主要过敏原,已知其5个抗原表位在过敏反应中起着决定性的作用。用全蛋白抗体和表位特异性抗体,检测Pen a 1经体外模拟胃液(simulated gastric fluid,SGF)消化后其抗原表位免疫原性的变化情况,为研究人体消化对Pen a 1的影响机理及脱敏食品的制备提供理论依据。【方法】以刀额新对虾(metapenaeus ensis)为原材料,提取纯化虾过敏原Pen a 1,采用Fmoc化学法合成Pen a 1抗原表位的5个多肽片段(No.1—No.5),分别与载体蛋白KLH和牛血清蛋白BSA偶联制备人工免疫原和包被原。用Pen a 1和制备的多肽免疫原免疫新西兰纯种白兔获得全白蛋抗体和特异性表位抗体。参照美国药典模拟人体胃液的条件消化处理Pen a 1,利用SDS-PAGE和Tricine-SDS-PAGE观察消化后Pen a 1分子量变化;通过免疫印迹(Western-blot)定性观察Pen a 1消化后生成的新蛋白片段与各抗体的结合情况;再经竞争抑制酶联免疫吸附试验(ci-ELISA)定量检测消化后的Pen a 1和表位多肽与各抗体结合能力,从而得出Pen a 1经SGF消化后各抗原表位免疫原性的变化程度。【结果】SDS-PAGE结果显示Pen a 1随SGF消化时间的延长逐渐被降解,在22 kD处生成一些较稳定的新蛋白片段,Tricine-SDS-PAGE结果表明在1.7—18 k D之间无新蛋白片段生成。Western-blot表明消化后分解生成的小分子蛋白与六种抗体发生不同程度结合,其中全蛋白抗体和No.4抗体几乎与生成的所有小分子蛋白结合,在22 k D左右处生成的稳定新蛋白片段均与No.3、4、5抗体有较强的结合,而与No.1、2抗体结合较弱甚至无结合,表明该蛋白携带No.3、4、5表位,而基本无No.1、2表位。ci-ELISA结果显示,Pen a 1经SGF消化后对全蛋白抗体和5个表位抗体的抑制率均显著下降,说明经过消化后Pena1及其抗原表位的免疫原性均明显降低,各抗原表位消化稳定性排序为No.4>No.2>No.1>No.3>No.5,其中No.2、1、3之
【Objective】 Pen a 1 is a major allergen in shrimp,and its epitopes play a decisive role in allergic reactions.In order to evaluate the digestion stability of Pen a 1,the immunogenicity of Pen a 1 and its epitopes digested by simulated gastric fluid（SGF） was detected by antibodies of Pen a 1 and epitopes.It provided a theoretical basis for the study of the human digestive effects on Pen a 1 and the mechanism of desensitization food preparation.【Method】Pen a 1 was purified from Metapenaeus ensis.Five epitope peptides of Pen a 1 were synthesized using Fmoc Method.The peptides were conjugated to keyhole limpet（KLH） and bovine serum（BSA） to get artificial immunity and coating antigen,respectively.The tested serum was prepared by immuning New Zealand rabbits with Pen a 1 and the artificial immune.SGF was prepared according to the method of United States Pharmacopoeia.The changes of molecular weight of Pen a 1 was observed by SDS-PAGE and Tricine-SDS-PAGE.The capacity of Ig E-binding of Pen a 1 and its epitope peptides by SGF digestion was analyzed by means of Western-blot qualitatively and ci-ELISA quantitatively.【Result】SDS-PAGE showed that Pen a 1 was degraded with increasing digestion time and generated a new 22 KD sensitization allergic protein.Tricine-SDS-PAGE showed that there were no protein fragments generated between 1.7-18 KD.Western-blot indicated that after the digestion of Pen a 1,the proteolytic fragments inordinately bound to six antibodies.Antibodies of Pen a 1 and No.4 bound to almost all proteolytic fragments.The fragment of 22 KD was resistant to digestion.This fragment bound to antibody No.3,4,and 5 but almost no reaction to antibodies No.1,and 2,which indicated that the fragment carried No.3,4,and 5 epitopes.The inhibition rate between Pen a 1 after SGF digestion and the antibodies tested by ci-ELISA was significantly decreased,indicating that the immunogenicity of Pen a 1 and its epitopes was decreased significantly.Moreover,the epitope peptides digested by SGF got the same
出处
《中国农业科学》
CAS
CSCD
北大核心
2015年第4期769-777,共9页
Scientia Agricultura Sinica
基金
国家公益性行业(农业)科研专项(201103007)
