特异性水解α_(s1)-酪蛋白过敏原IgE作用表位aa 83~105的蛋白酶筛选  

Screening for Proteases Specifically Hydrolyzing IgE Epitopes aa 83–105 of α_(s1)-Casein Allergen

作　　者：刘迪 吕晓哲 丛艳君[1] 张倩倩 李邻峰[2] 梁萌 高吉安 邱学宇 LIU Di;Lü Xiaozhe;CONG Yanjun;ZHANG Qianqian;LI Linfeng;LIANG Meng;GAO Ji’an;QIU Xueyu(Beijing Advanced Innovation Center for Food Nutrition and Human Health,Beijing Higher Institution Engineering Research Center of Food Additives,School of Food and Health,Beijing Technology and Business University,Beijing 100048,China;Department of Dermatology,Beijing Friendship Hospital,Beijing 100050,China)

机构地区：[1]北京工商大学食品与健康学院,北京食品营养与人类健康高精尖创新中心,北京市食品添加剂工程技术研究中心,北京100048 [2]北京友谊医院皮肤科,北京100050

出　　处：《食品科学》2022年第2期126-133,共8页Food Science

基　　金：“十三五”国家重点研发计划重点专项(2019YFC1605002);国家自然科学基金面上项目(31872886)。

摘　　要：探索特异性水解α_(s1)-酪蛋白作用表位的蛋白酶筛选方法。首先通过固相合成法合成α_(s1)-酪蛋白作用表位aa 83~105,纯化鉴定后,偶联牛血清白蛋白(bovine serum albumin,BSA)制备完全抗原,然后免疫BALB/c小鼠,制备单克隆抗体,进而建立间接竞争酶联免疫吸附测定方法(enzyme linked immunosorbent assay,ELISA),以α_(s1)-酪蛋白制备的单克隆抗体及建立的方法为对照。结果表明:合成作用表位的纯度在90%以上,与BSA偶联比为6.31。单克隆抗体的亚型为IgG_(1),效价可达到1∶320 000,可以与α_(s1)-酪蛋白发生特异性免疫反应,与大豆蛋白无交叉反应。建立的间接竞争ELISA,竞争抑制曲线回归方程为y=-9.22x+100.78(R^(2)=0.989 1),方法重复性、精确性均较好,检出限低于α_(s1)-酪蛋白单克隆抗体建立的方法,初步筛选到木瓜蛋白酶和碱性蛋白酶水解液的抗原残留量较小,需要进一步通过体内实验验证结果。α_(s1)-酪蛋白作用表位aa 83~105 G_(1)型单克隆抗体制备成功,为新型α_(s1)-酪蛋白作用表位aa 83~105低致敏配方粉的开发,提供了ELISA检测高灵敏专用单克隆工具抗体。The aim of this study was to explore a method for screening for proteases specifically hydrolyzing the epitopes ofα_(s1)-casein.First,the epitope aa 83–105 ofα_(s1)-casein was synthesized by solid-phase synthesis method.After purification and identification,the peptide was coupled to bovine serum albumin (BSA) to prepare a complete antigen,and then BALB/c mice were immunized to prepare monoclonal antibodies.In addition,an indirect competitive enzyme linked immunosorbent assay (ELISA) was established.The monoclonal antibody prepared withα_(s1)-casein and the established method were regarded as controls.The results showed that the purity of the synthetic epitope was over 90%,and the coupling rate with BSA was 6.31.The monoclonal antibody belonged to IgG_(1),with a titer of 1:320 000,and it could react specifically withα_(s1)-casein,but did not cross-react with soybean protein.The linear regression equation of the competitive inhibition curve was y=-9.22x+100.78 (R^(2)=0.989 1).The indirect competitive ELISA method showed good repeatability and accuracy,and its detection limit was lower than that of the monoclonal antibody method.The amounts of residual antigen in papain and alcalase hydrolysates were relatively small,which needs to be further verified by in vivo test.The successful preparation of G_(1)monoclonal antibody againstα_(s1)-casein epitope aa 83–105 provides a specialized tool for ELISA detection of antigen residues and for the development of hypoallergenic formula.

关 键 词：α_(s1)-酪蛋白 作用表位 单克隆抗体 抗原残留量 蛋白酶 

分 类 号：TS252.1[轻工技术与工程—农产品加工及贮藏工程]