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《β-乳球蛋白抗原表位譜的解析與關鍵抗原表位串聯(lián)體的重組表達與鑒定》由會員上傳分享,免費在線閱讀,更多相關內(nèi)容在學術(shù)論文-天天文庫。
1、天津醫(yī)科大學碩士學位論文最主要表位的氨基酸序列為AA76-95、AA1-20、AA106-125,主要表位的氨基酸序列為AA91-110、AA61-81、AA121-140,次要表位的氨基酸序列為AA46-65、AA31-50、AA151-162。(2)β-乳球蛋白關鍵抗原表位串聯(lián)體重組蛋白的表達及鑒定:通過DNAStar軟件對理論上6種組合方式進行了分析,得到最佳組合方式,成功構(gòu)建了大腸桿菌原核表達載體,IPTG誘導表達后,獲得大小為36kDa左右的融合蛋白,并純化得到單一組分的36kDa大小的重組蛋白,并經(jīng)免疫印
2、跡法、斑點印跡法鑒定其免疫活性,并利用重組蛋白作為檢測抗原建立了一種活性氧傳遞均相發(fā)光免疫測定技術(shù)來檢測血清sIgE。結(jié)論:本研究對牛奶主要過敏原β-乳球蛋白的抗原表位進行了解析,不同牛奶過敏患者血清所識別的抗原表位具有異質(zhì)性。成功表達了β-乳球蛋白關鍵抗原表位串聯(lián)體重組蛋白,并鑒定了關鍵抗原串聯(lián)體重組蛋白具有良好的免疫學活性,在檢測牛奶過敏疾病中有可能代替天然抗原,為新一代診斷試劑的制備奠定基礎。關鍵詞:牛奶過敏β-乳球蛋白特異性IgE抗原表位重組表達II萬方數(shù)據(jù)天津醫(yī)科大學碩士學位論文AbstractObject
3、ive:SerumspecificIgEisacommonmeansofdiagnosisoffoodallergy,StandardizationofknownantigenistheimportantpreconditionfordevelopmentofspecificIgEinvitrodiagnosticreagentsandthedevelopmentoffoodcomponentsofantigenepitoperecombinantisanimportantmeasuretosolvethestand
4、ardizationofantigen.Inthisstudy,themostimportantmilkallergen"β-lactoglobulin"asanexample,startingfromtheanalysisofitsepitopes,andanalyzetheimportantantigenepitopeandthepatientsserumsIgEresponsefrequency,thustoselectkeyepitopesandbuildkeyepitopesrecombinantandtr
5、ythepossibilityofusingepitoperecombinantproteininsteadofnaturalprotein.Methods:(1)β-lactoglobulinkeyepitopesscreeningandserumheterogeneityanalysis:First,intheNCBIdatabasesearchedmilkallergensβ-lactoglobulinaminoacidsequence(162aminoacids),thenentrustedGL(Shangh
6、ai)BiochemLtdsynthesisantigenepitopesbytheendoftheoverlap.20aminoacidsofapolypeptide,twoadjacentoverlappingpeptidesof5aminoacids;ThepolypeptidepointedonnitrocellulosemembraneandthespecificIgEinmilkallergypatientsserumasa"probe".Toobservepolypeptideresponsefrequ
7、encydifferencebetweenpatientswithmilkallergyandpeptideidentificationbyanydifferencesbetweendifferentindividuals.(2)β-lactoglobulinkeyantigenepitoperecombinanttandemofbuildinganditspreliminaryapplicationresearch:Thehighestresponsefrequencyofthreepolypeptideexpre
8、ssedinseries,betweenadjacenttwopeptidesdojointwithaglycine.FirstusingDNAstarbioinformaticssoftwaretoforecastthreetablesof6kindsofcombinationandselectingthebestcombination;Th