摘要
采用Tricine-十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(Tricine-SDS-PAGE)和免疫印迹(Western-blot)方法,通过模拟肠胃液消化实验分析淡水鱼类肌肉中主要过敏原小清蛋白(parvalbumin,PV)的消化特性。Tricine-SDS-PAGE结果显示,纯化的鲫、鲢PV在模拟胃液消化实验中反应5～30min明显分解,而在模拟肠液消化实验中4h仍未见明显分解。鲫、鲢肌浆蛋白在模拟胃液消化实验中,肌浆蛋白中的PV比纯化PV的分解时间明显延长,消化30～60min仍未被完全分解,而其它肌浆蛋白消化10～30min就完全分解。采用抗PV多克隆抗体的Western-blot显示,该抗体能特异识别PV及其降解产物。研究结果表明,淡水鱼类PV相对于非过敏蛋白具有较高的消化稳定性,而胃蛋白酶能较好地分解该过敏原。
The physicochemical properties of most food allergens confer stability to the proteolytic in the digestive tract,which increases the probability of reaching the intestinal mucosa,where absorption and interaction with the immune system can occur.Stability in simulated gastric fluid is supposed to be an important parameter for the estimation of food allergenicity.The digestive stability of a major fish allergen（parvalbumin,PV） and nonallergenic proteins from the muscle of crucian carp （Carassius auratus cuvieri） and silver carp（Hypophthalmichthys molitrix） in a standard simulated gastric fluid（SGF） and simulated intestinal fluid（SIF） digestion assay system was compared.Proteinases including pepsin,trypsin and chymotrypsin from porcine were used to simulate digestive proteinases from human.The results were evaluated by means of Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis（Tricine-SDS-PAGE） and Western-blot.Fish PV was purified to homogeneity by 70%-100%ammonium sulfate fractionation and DEAE-Sepharose following heat treatment and a protein with molecular weight of approximately 10 ku was finally obtained.In SGF assay of purified crucian carp PV and silver carp PV,similar results were obtained,the original PV band was almost completely degraded within 60 min and some stable peptide fragments were observed.In SIF assay of purified PV,both trypsin and chymotrypsin could not degrade PV effectively in 4 h.In SGF digestion on fish sarcoplasmi proteins,nonallergenic proteins were rapidly degraded within a short period of time,while the digestion of PV was prolonged to some degree.Western-blot analysis indicated that the polyclonal antibody against silver carp PV can specifically detect the PV and it degraded products.In conclusion,our present results indicate that fish PV is more resistant to proteinase digestion than non-allergenic proteins and pepsin treatment is more effective than trypsin and chymotrypsin in reducing the hypersensitivity.It should be remembered that our pres
出处
《水产学报》
CAS
CSCD
北大核心
2010年第7期1136-1142,共7页
Journal of Fisheries of China
基金
国家自然科学基金项目(20872049
30871947)
福建省自然科学基金(2008J0067)
福建省高等学校新世纪优秀人才支持计划(NCETFJ-2007)
集美大学创新团队基金(2008A002)
