摘要
目的:从牛乳酪蛋白中纯化获得α_(S1)-酪蛋白并对其进行综合鉴定,以及制备特异性识别α_(S1)-酪蛋白的兔多克隆抗体。方法:采用DEAE Sepharose Fast Flow阴离子交换层析色谱对α_(S1)-酪蛋白进行分离纯化,利用酪蛋白的理化性质(等电点和含量)、免疫学技术和质谱技术对纯化的α_(S1)-酪蛋白进行综合鉴定,然后通过透析和冷冻干燥获得高纯度的α_(S1)-酪蛋白,最后免疫新西兰大白兔制备多克隆抗体,并分析其特异性。结果:在4种酪蛋白中,α_(S1)-酪蛋白在阴离子交换层析色谱中的出峰时间最晚、峰面积最大,在电泳图中的位置最高,最终获得纯度高达94.26%的α_(S1)-酪蛋白,得率为27.19%。免疫5次后,两只兔子的抗血清效价分别为128万和32万,抗血清除了与大豆蛋白存在轻微交叉反应(<0.25%)外,与α-乳白蛋白、β-乳球蛋白、蛋清蛋白、花生蛋白均无交叉反应,表明特异性高。结论:本研究制备了高纯度的α_(S1)-酪蛋白及其高特异性的多克隆抗体,为过敏原蛋白的纯化与综合鉴定提供了思路,为α_(S1)-酪蛋白免疫学检测方法的建立提供了物质基础。
Objective:To purify and comprehensive identify α_(S1)-casein from bovine caseins and prepare rabbit polyclonal antibody againstα_(S1)-casein.Method:DEAE Sepharose Fast Flow anion exchange chromatography was used to separate and purifyα_(S1)-casein.The physicochemical property(isoelectric point and protein content),immunological technique and mass spectrometry were used to comprehensively identifyα_(S1)-casein.Then theα_(S1)-casein was obtained by dialysis and lyophilization,polyclonal antibody was prepared by immunizing New Zealand white rabbit with the purifiedα_(S1)-casein,and the specificity of polyclonal antibody was analyzed.Result:Among the four kinds of caseins,α_(S1)-casein had the latest peak time,the largest peak area in anion exchange chromatography and the highest position in the electrophoretic diagram.Finally,the purity ofα_(S1)-casein was 94.26%,and the yield was 27.19%.The titers of sera from two rabbits inoculated with purifiedα_(S1)-casein for five times reached 1280000 and 320000,respectively.In addition to the slight cross reaction with soybean protein(<0.25%),the antiserum showed no cross reaction withα-Lactalbumin,β-Lactoglobulin,egg white protein and peanut protein,indicating high specificity.Conclusion:This study prepared high purityα_(S1)-casein and high specificity polyclonal antibody againstα_(S1)-casein,providing a new train of thought forα_(S1)-casein purification and comprehensive identification,and also providing material basis for the development of immunologic method for detection ofα_(S1)-casein.
作者
何圣发
龙彩云
王娇
熊梦
赵江强
燕燕
董亚萍
李欣
陈红兵
HE Shengfa;LONG Caiyun;WANG Jiao;XIONG Meng;ZHAO Jiangqiang;YAN Yan;DONG Yaping;LI Xin;CHEN Hongbing(School of Public Health and Health Management,Gannan Medical University,Ganzhou 341000,China;State Key Laboratory of Food Science and Technology,Nanchang University,Nanchang 330047,China;Ganzhou Center for Disease Control and Prevention,Ganzhou 341000,China;School of Food Science&Technology,Nanchang University,Nanchang 330047,China;Jiangxi-OAI Joint Research Institute,Nanchang University,Nanchang 330047,China)
出处
《食品工业科技》
CAS
北大核心
2022年第15期106-114,共9页
Science and Technology of Food Industry
基金
赣南医学院博士启动课题(QD201809)
赣南医学院重点科研课题(ZD201816)
南昌大学食品科学与技术国家重点实验室开放课题(SKLFKF-202003)
江西省自然科学基金项目(20202BAB215013)
国家自然科学基金项目(32160606)。
