β-酪蛋白论文：牛乳中β-酪蛋白构象性表位定位研究.doc

-酪蛋白论文：牛乳中-酪蛋白构象性表位定位研究【中文摘要】牛乳是引起食物过敏的八大类常见食物之一。据报道,牛乳过敏引发的病症在婴幼儿中尤为常见,1岁以下婴幼儿中的发生率约为2-3%。因此,开展牛乳过敏的研究对于提高牛乳过敏人群生活质量具有重要的的现实意义。另外,由于表位是食物过敏反应的物质基础,所以表位定位是牛乳过敏研究的一个核心问题,也是人们认识食物过敏的首要任务。本文以过敏原蛋白质-酪蛋白为研究对象,从鲜牛乳中分离纯化-酪蛋白、制备特异性多克隆抗体、亲和纯化多克隆抗体、建立-酪蛋白构象模型、最终定位出-酪蛋白的构象性表位,主要的研究工作和结论如下。1.采用等电点沉淀法从鲜牛乳中得到酪蛋白,冷冻干燥后的酪蛋白粉末经deae-sepharose fast flow阴离子交换柱分离后,得到了-酪蛋白。经过sds-page电泳和等电聚焦电泳鉴定,结果表明,获得了纯度高于90%的-酪蛋白。2.采用分离纯化的-酪蛋白免疫日本大白耳兔,制备兔抗牛乳-酪蛋白多克隆抗体,免疫过程中利用间接elisa方法监测免疫效价的变化,最终得到效价分别为1：240,000和1：150,000的抗血清各一份。3.利用溴化氰活化的sepharose4b对抗8-酪蛋白兔血清进行亲和纯化,经sds-page电泳检测,结果显示纯化后抗体在泳道中基本上只显示出重链(55kda)和轻链(22kda)2条带,纯化效果较好；同时对纯化后抗体进行间接elisa效价测定,a、b两兔的血清效价分别从亲和纯化上样前的100,000和70,000上升到纯化后的120,000和80,000,表明经过亲和纯化,得到了高纯度和高特异性的多克隆抗体。4.分别利用同源建模、折叠识别法建模、从头预测方法对-酪蛋白结构模型进行构建,并对所建模型进行逐级开放式优化和ramanchandran plot、what_check、errat等方法评估,选择最优模型,结果得到经同源建模并经过优化后的模型为最终的-酪蛋白结构模型；以文献报道的针对-酪蛋白空间构象的实验数据作为参照,将所建模型与kumosinski等人的p-酪蛋白典型模型进行比较,评价本研究工作中建模结果的准确性,结果显示,与kumosinski等人所建模型相比,本实验所建模型更能与-酪蛋白空间构象的实验数据吻合。5.通过噬菌体肽库筛选得到了用于-酪蛋白构象性表位定位的6个模拟肽；同时通过搜索得到晶体结构已解析的抗原抗体的复合物,并提取其中的表位信息,形成数据信息库,然后对该库进行统计分析,得到表位覆盖面积大小、表位氨基酸数目、不同种类表位氨基酸残基的出现频率、表位中起间隔作用的氨基酸残基类型和出现频率以及表位氨基酸残基在不同蛋白质二级结构中出现频率等信息；所得信息结合噬菌体肽库筛选结果,最终定位出-酪蛋白的4个构象性表位。【英文摘要】bovine milk is one of the eight common allergenic foods, it is essential in the different territories and different dietary habits. it is reported that the milk allergy is more prevalent during children, and the incidence in the children aged below 1 year is about 2-3%. undoubtedly, the research on the milk allergy is important to the improvement of the living quality of the people who suffer from milk allergy. since epitope is the binding sites of antibody and antigen, allergen epitopes will play an important role in food allergens, and epitope mapping should be one of the key problems in the milk allergy research.this work focused on beta-casein, one of the magor allergic proteins in bovine milk, and it includes the purification and separation of the beta-casein, the preparing of specific rabit serum against beta-casein, the purification of the polyclonal antibody by affinity chromatography, modelling and epitopes mapping of the beta-casein. the detailed process and conclusions are presented as follow.1. crude caseins were prepared from fresh milk using the pi precipitate method, followed by lyophilization, and the dried caseins powder was then loaded into the columns packed with the deae-sepharose fast flow anion exchange medium. the target eluent then identified by the sds-page and ief page, and it shows the purity of isolated beta-casein was higher than 90%.2. based on the routine way of immunization, specific rbbbit serum against beta-casein was produced. an indirect elisa approach was developed to check the change of immune response titer of two rabbits, and the final antiserums titer are 1:240,000 and 1:150,000, respectively.3. the antiserums loaded to sepharose4b which actived by cnbr, the purified components then identified by sds-page, the result illustrated two bands with the molecular weight were 55 kda and 25 kda respectively; also the indirect elisa be carried through to the purified antibody, then it is come to the conclusion that the polyconal antibody with a higher specificity and higher purity;4. the beta-casein models were obtained using the homology modeling method, fold recognition and ab initio medhod, the models then were optimized and assessed by ramanchandran plot, what_check, errat and the like, the most optimal model compared with the known model created by kumosinski et al, in this process, the experimental data were used as reference and benchmark, from the results, the conclusion can be drawed that homology model is the most optimal model, and this model is more identical with the experimental data than kumosinski et als model;5.6 mimic peptides obtained after the biopanning process, on the other hand, the antibody-antigen complexes which the 3d structures were resolved were filtered, then the epitopes information from the complexes were extracted to constitute the epiotpes information database, the information such as the number of the amino acids included in the epitopes, the amino acids preference in the amino acids, the amino acids which play a interval role in the epitopes, the area the epitopes extend and the like are analysed, finally all these information combined to the pepitides that got from the phage display method for the analysis of epitopes, then four conformational epitopes were obtained in this research.【关键词】-酪蛋白 同源建模 表位 牛乳过敏【英文关键词】-casein homology modeling epitope bovine milk allergy【目录】牛乳中-酪蛋白构象性表位定位研究摘要3-5abstract5-6目录9-14第1章 引言14-261.1 牛乳过敏14-171.1.1 牛乳过敏机理15-161.1.2 牛乳中的主要过敏原161.1.3 牛乳过敏的危害16-171.2 牛乳过敏原的安全管理17-201.2.1 牛乳过敏的检测17-191.2.1.1 血液检测17-181.2.1.2 血液检测181.2.1.3 食物激发试验18-191.2.2 牛乳过敏原的管理19-201.2.2.1 ige介导的牛乳过敏的管理191.2.2.2 非ige介导的牛乳过敏的管理191.2.2.3 针对不同过敏症状的牛乳过敏的管理191.2.2.4 食物避免与营养的管理19-201.2.2.5 食物过敏原标签管理201.3 牛乳中的酪蛋白20-231.3.1 酪蛋白胶束20-211.3.2 牛奶酪蛋白的结构与理化性质21-231.3.2.1 s1-酪蛋白21-221.3.2.2 _(s2)-酪蛋白221.3.2.3 -酪蛋白22-231.3.2.4 -酪蛋白231.4 -酪蛋白的构象性表位23-241.5 研究内容及意义24-26第2章 牛乳中-酪蛋白的分离纯化26-352.1 材料与设备26-272.1.1 试剂与材料262.1.2 设备26-272.1.3 溶液配制272.2 实验方法27-302.2.1 鲜牛奶脱脂272.2.2 等电点沉淀酪蛋白272.2.3 -酪蛋白预处理27-282.2.4 -酪蛋白分离纯化282.2.5 蛋白质及其纯度鉴定28-292.2.5.1 sds-page鉴定28-292.2.5.2 ief-page鉴定292.2.6 蛋白质浓度鉴定29-302.3 实验结果30-332.3.1 -酪蛋白的分离纯化30-312.3.2 蛋白质及其纯度鉴定31-322.3.3 浓度鉴定32-332.4 讨论33-342.4.1 -酪蛋白的离子交换层析条件选择332.4.2 蛋白质鉴定和浓度鉴定33-342.5 小结34-35第3章 抗牛乳-酪蛋白多克隆抗体的制备及亲和纯化35-483.1 材料与设备35-373.1.1 试剂353.1.2 仪器与耗材35-363.1.3 溶液配制36-373.2 实验方法37-403.2.1 多克隆抗体的制备373.2.2 抗体滴度测定的方正滴定373.2.3 间接elisa测定抗体效价37-383.2.4 抗血清的制备383.2.5 亲和层析纯化特异性兔igg38-403.2.5.1 血清的预处理383.2.5.2 亲和层析柱的制备38-393.2.5.3 亲和纯化393.2.5.4 抗体sds-page纯度鉴定393.2.5.5 间接elisa测定抗体效价39-403.3 实验结果40-443.3.1 elisa方法的条件确立及其对效价变化的监测40-413.3.2 亲和层析纯化特异性igg41-443.3.2.1 血清的获取413.3.2.2 偶联效果的检测413.3.2.3 非特异性洗脱过程中蛋白质含量监测41-423.3.2.4 特异性洗脱过程中蛋白质含量监测42-433.3.2.5 浓缩除盐433.3.2.6 纯化前后抗体效价的比较433.3.2.7 纯化后样品的sds-page电泳图43-443.4 讨论44-473.4.1 佐剂的选择44-453.4.2 试验动物的选择453.4.3 多克隆抗体的制备和储存45-463.4.4 多克隆抗体的亲和纯化46-473.4.4.1 配基及载体的选择463.4.4.2 层析条件的选择46-473.5 本章小结47-48第4章 -酪蛋白结构模型的构建48-584.1 实验材料484.2 实验原理和方法48-524.2.1 蛋白质