人宫颈癌基因蛋白单克隆抗体的制备、鉴定及初步应用-论文.pdf

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1、·3212·现代生物医学进展www3henuyixue．c0mProgressinModernBiomedicineVoL14NO．17JUN2014doi：10．13241／j．cnki．pmb．2014．17．003人宫颈癌基因蛋白单克隆抗体的制备、鉴定及初步应用陈锋姥勇陈旭义。樊豪军。侯世科△(1天津医科大学研究生院天津300070；2武警后勤学院训练部天津300309；3武警后勤学院附属医院天津300016)摘要目的：制备人宫颈癌基因蛋白单克隆抗体并初步用于肝癌血清检测。方法：重组表达HCCR．1蛋白胞外区(第167th．360th氨基酸)进行动物免疫，用含有HCC

2、R蛋白抗原表位的融合蛋白筛选阳性克隆。通过ELISA、WesternBlot、免疫组化鉴定抗体性质，并用于肝癌血清样本检测。结果：获得3株抗人HCCR蛋白单克隆抗体，其中B3抗体特异性、灵敏度较好。ELISA结果表明B3只识别GST—HCCRep蛋白和His—HCCR蛋白，与其他蛋白没有交叉反应；B3可检测到10mg／L的GST—HCCRep蛋白；B3抗体亲和力常数Kaff=-1．86x10(L／mo1)；WestemBlot结果表明该抗体可识别天然HCCR蛋白；免疫组化检测表明肝癌组织中有HC—CR蛋白表达而正常组织中未见表达；肝癌患者血清HCCR阳性率达到75％。结论：

3、成功制备了HCCR单克隆抗体，为建立新的肝癌早期诊断方法奠定了基础。关键词：人宫颈癌基因；单克隆抗体；肝癌；早期诊断中图分类号：R737．33文献标识码：A文章编号：1673．6273(2014)17—3212．04Preparation，CharacterizationandApplicationofAnti—HCCRMonoclonalAntibody*CHENFengj。MUYong~,CHENXu-y?,FANHaofut?,HOUShi—k《lGraduateSchoolofTianjinMech'calUniversity,Tianjin,300070,Chin

4、a；2LogisticsUniversityofChinesePeople'sArmedPoliceForces,Tianjin,300309,China；3Afiliatedhospital,LogisticsUniversityofChinesePeople'sArmedPoliceForces,Tianjin,30016：,china)ABS，IRACTObjective：Topreparemonoclonalantibody(mAb1againstHCCR．Methods：TheextracellularsegmentofHCCR-1(aa167to360)wase

5、xpressedandusedtoimmunizeBalb／cmice．AnotherproteinGST—HCCRep，containingHCCRepitope(YLGTRR)wasexpressedandpurifiedtodetectthetitersofserumantibodyandscreenthepositivehybridomas．ThemAbwascharacterizedbyELI—SA，Westernblotandimmunohistochemistry．Results：TheELISAanalysisindicatedthattheB3mAbspe

6、cificallyrecognizedHis—HCCRandGST—HCCRepprotein，andshowednocross·reactionwithotherproteins；thedetectionlimitofB3mAbreached10ng／mlofGST—HCCRepprotein；theafinityconstantofB3mAbwas1．86x10L／mol；HCCRwasfoundhigh-expressedinHCCcelllinesHepG2and7721，aswellashumanHCCtissues．Neithernormalhepaticcel

7、llineL02nornormallivertissuesshowedHCCRpositive．TheresultsshowedthatpositiverateofHCCRinsel'umsamplesofHCCpatientsreached58％．Conclusion：AspecificmAbagainstHCCRwassuccessfullygeneratedandprovidedthefoundationforfuturedevelopmentofgoodmethodsforHCCdiagnosis．Keyw