牛乳中β酪蛋白构象性表位定位的的研究

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1、摘要肽；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B．酪蛋白的4个构象性表位。关键词：B．酪蛋白；同源建模；表位；牛乳过敏IIIABSTRACTBovinemilkisoneoftheeightcommonallergenicfoods，itisessentialinthedifferentterritor

2、iesanddifferentdietaryhabits．Itisreportedthatthemilkallergyismoreprevalentduringchildren，andtheincidenceinthechildrenagedbelow1yearisabout2～3％．Undoubtedly,theresearchonthemilkallergyisimportanttotheimprovementofthelivingqualityofthepeoplewhosufferfrommilkallergy．Sinceepitopeisthebindingsitesofa

3、ntibodyandantigen，allergenepitopeswillplayallimportantroleinfoodallergens，andepitopemappingshouldbeoneofthekeyproblemsinthemilkallergyresearch．Thisworkfocusedonbeta-casein,oneofthemagorallergicproteinsinbovinemilk,anditincludesthepurificationandseparationofthebeta-casein，thepreparingofspecificr

4、abitserumagainstbeta-casein,thepurificationofthepolyclonalantibodybyaffinitychromatography,modellingandepitopesmappingofthebeta-casein．Thedetailedprocessandconclusionsarepresentedasfollow．1．Crudecaseinswerepreparedfrom毹ShmilkusingthePIprecipIitatemethod，followedbylyophilization,andthedriedcasei

5、nspowderWasthenloadedintothecolumnspackedwitIltheDEAE·SepharoseFastFlowanionexchangemedium．ThetargeteluentthenidentifiedbytheSDS-PAGEandIEFPAGE，anditshowsthepurityofisolatedbeta-caseinWashigherthan90％．2．Basedontheroutinewayofimmunization，specificrbbbitserumagainstbeta-caseinwasproduced．Anindire

6、ctELISAapproachwasdevelopedtocheckthechangeofimmuneresponsetiteroftworabbits，andthefinalantiserumstiterarel：240，000and1：150，000，respectively．3．TheantiserumsloadedtoSepharose4BwhichactivedbyCNBr,thepurifiedcomponentsthenidentifiedbySDS．PAGE，theresultillustratedtwobandswiththemolecularweightwere5

7、5Kdaand25Kdarespectively；AlsotheindirectELISAbecarriedthroughtothepurifiedantibody,thenitiscometotheconclusionthatthepolyconalantibody诹mahigherspecificityandhigherpurity；4．Thebeta-caseinmodelswereobtainedusingthehomologymodelingme