EB病毒膜蛋白家兔免疫血清的制备及其特性研究.doc

PAGE PAGE 1 EB病毒膜蛋白家兔免疫血清的制备及其特性研究 【摘要】目的：制备高效价EB病毒膜蛋白抗体，并对其效价及其与EBV结合的特异性进行鉴定。方法：将B95-8细胞培养至3×105/mL时，超声破碎，纯化获得EB病毒膜蛋白抗原；EB病毒膜蛋白抗原经弗氏佐剂乳化，在家兔背部皮下多点免疫注射，间隔2周免疫，共3次，并分别于免疫前和每次免疫后1周取兔血清，检测兔免疫血清抗体IgG；进一步采用Westernblot检测制备的血清抗体与EBV结合的特异性，同时采用ELISA法检测兔血清抗体水平及各周抗体滴度的变化趋势。结果：所有家兔在全程免疫后都产生了高效价膜蛋白的特异性抗体，最高滴度达1:6400。结论：用B95-8细胞制备的EB病毒膜蛋白抗原免疫家兔，可成功制备高效价的特异性血清抗体，为EB病毒膜蛋白抗原的免疫学检测奠定了基础。 【关键词】EBV；抗体；免疫性 PreparationandresearchonimmunologicalcharacteristicsofrabbitserumantibodytoEBVmembraneproteinLULi-jun*，MENGRui-feng，GUOQiang，ZHUshan-Li，ZHANGLi-fang.*InstituteofMolecularVirologyandImmunology，DepartmentofMicrobiologyandImmunology，WenzhouMedicalCollege，Wenzhou，325035 Abstract:Objective:Topreparehigh-titerrabbitserumantibodyagainstEBVmembraneprotein.Methods:B95-8(EBV)cellswerecultivatedandproliferatedincellculturesystemwithRPMI-1640culturemedium(with10%FBSandPen/Strep).Thereafter，thepurifiedB95-8(EBV)washarvestedusingsupercentrifugemethod.PrepareEBVmembraneproteincontainingFreundadjuvantbyroutinemethod.Fourrabbitswereimmunizedwiththepreparedantigenbysubcutaneousinjectionatvarioussitesofskinonbackforeverytwoweeks，threetimesinall.TheseraoftheseimmunizedrabbitswerecollectedseparatelyforpreparationoftherabbitserumIgG.SpecificbindingoftherabbitserumantibodyandtheEBVmembraneproteinwereanalyzedbyWesternblotandthetiterwasassayedbyELISA.Results:Alloftheimmunizedrabbitsproducedhigh-titerserumantibodyafterthesecondbooster.ThehighesttiterofantibodyagainstEBVmembraneproteinwas1:6400.TheresultofWesternblotshowedtherabbitserumantibodycouldbindtotheEBVmembraneproteinspecifically.Conclusion:ThespecificrabbitserumantibodyagainstEBVmembraneantigen，withhightiter，issuccessfullyprepared.ItlaidafoundationofdevelopmentofimmunologymethodfordetectionofEBVmembraneantigen. Keywords:EBV；antibody；immunity EB病毒（Epstein-Barrvirus，EBV）是一含有包膜的DNA病毒，在人群中感染非常普遍，与某些肿瘤的发生和发展密切相关[1]，如Burkitt’s淋巴瘤、鼻咽癌、何杰金病和成人T细胞淋巴瘤等，其中鼻咽癌在东南亚及我国南方地区高发；研究发现，EBV感染尚与胃癌、乳腺癌和肺癌等肿瘤的发病有一定的相关性[2-3]。EBV在感染机体时，可在宿主细胞膜上表达包膜糖蛋白及潜伏膜蛋白（latentmembr