肾癌肿瘤相关抗原g250及g250蛋白多糖pg区基因的克隆、表达及鉴定

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1、万方数据·1184·肾癌肿瘤相关抗原G250及G250蛋白多糖(PG)区基因的克隆、表达及鉴定刘俊杰郑骏年吴洋李望郑宏祥温儒民刘晓筠毛立军裴冬生孔德领【摘要】目的探讨肾癌肿瘤相关抗原(；250及G250蛋白多糖(Pc)区基因的克隆，蛋白表达及鉴定。方法从肾癌细胞786-0中提取mRNA，采用逆转录．聚合酶链反应(RT．PCR)扩增出(；250及G250PG区eDNA。将获得的eDNA片段插入pET28a(+)表达载体，构建重组质粒pET-28a(+)．(；250／pET-28a(+)-G250PG，转化DI-15a大肠杆菌。通过PCR鉴定筛选出正确的重组子，并用其转化BL21Codon

2、Plus菌。采用IPTG诱导工程菌进行诱导表达，SDS．PAGE及Westernblot鉴定。结果克隆的基因经测序证实，G250／G250PG区序列正确。构建重组质粒pET-28a(+)．(；250／pET-28a(+)一G250PG，并在原核系统中表达(；250／G250PG重组蛋白，目的蛋白均具有较好的抗原性和特异性。结论成功完成(；250／(；250PG区基因克隆及表达，为在G250／G250PG蛋白纯化基础上进行抗体制备和(；250功能研究提供了实验依据。【关键词】肾细胞癌；抗原；蛋白多糖Cloning，expressionandidentiffcationoftherenal

3、cellcardnoma-associatedantigenG250andG250PGre#onUUJun-jie‘，zHE^GJun-nian，WUY￡‘ng．eta1．‘DepartmentofUrology。4t筇U=edHospitalofXuzhouMedicalCollege，Xuzhou221002．ChinaCorrespondingauthor：zHENGJun—nian【Abstract】0bjectiveToclone，expressandidentifythetumor-associatedantigenG250andG250pmteoglyean(PG)reg

4、ion．MethodsThemRNAWasextractedfromhumanrenalcarcinomacellline786．0．GenefragmentseneodingG250andG250PGwereobtainedbyRT．PCR．andclonedintopro-karyotieexpressionvectorpET28a(+)toconstructtherecombinantplasmidpET-28a(+)-G250／pET-28a(+)．G250PG．TherecombinantvectorsweretransfeetedintoE．coliDH5aandPCRas

5、sayWasper-formedtofindouttherightrecombinantclones．Therightclonesweretransfectedintotheexpressionhost(E．coliBL2lCodonPIUS)，andtheengineeringbacteriawereindueedbyI盯G．TheresultswereacquiredbvSDS．PAGEandWesternblot．ResultsDNAsequenceanalysisshowedthatthesepuenceedcondingG250／G250PGregionwasthesamea

6、sthatinGenBank．GeneofG250andG250PGregionWasex．pressedinE．eoliBL21CodonPIUSsuccessfully．WesternblotanalysisshowedthatthetwokindsofreCom．binantproteincouldbespeciallyrecognizedbytheircorrespondingantibodies．Theybothhadbetteranti．鼯Ill‘cityandspecificity．ConclusionThisstudyprovidesexperimentalbasisf

7、orthepurificationofG250／G250PGproteinandthefurtherstudyonG250functionandpreparationofantibody．【Keywords】Renalcellcarcinoma；Antigen；Protcoglyean肾癌相关抗原G250是分布于肾癌细胞膜和细胞核膜的糖蛋白，属于碳酸酐酶家族的异构酶，具有良好的’肾癌特异性。(；250抗原由459个氨基酸构成，其中345个氨基酸