全人源胃癌噬菌体单链抗体库的构建与其初步筛选

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1、浙江人学硕一l：学位论文中文摘要全人源胃癌噬菌体单链抗体库的构建及其初步筛选浙江大学医学院：生物化学与分子生物学硕士研究生：杨乃娣导师：詹金彪中文摘要研究目的-构建全人源胃癌噬菌体单链抗体库，并从中筛选出与人胃癌细胞特异结合的单链抗体，并对其进行鉴定分析。研究方法和研究结果：首先分离胃癌病人外周血淋巴细胞，提取总RNA，RT—PCR合成cDNA第一链，采用兼并引物PCR扩增抗体重链(VH)和轻链可变区(VL)基因。经重叠PCR(SOE．PCR)，将VH和VL连接成单链抗体(seFv)基因，并连接到噬菌粒载体p

2、CANTAB．5E中，转化至EcoliTGl，经辅助噬菌体超感染，构建了全人源胃癌噬菌体单链抗体库，库容为2．6x106，质粒片段插入率95％．单链抗体库经与正常人胃黏膜上皮细胞GES．1孵育后，去除与正常细胞结合的噬菌体单链抗体，再以人胃癌细胞MGC．803为靶标对抗体库进行4轮富集筛选，结果显示富集了50倍。选取第四轮筛选后的菌落制备单克隆抗体，并进行ELISA特异性鉴定，获得五株与人胃癌细胞MGC．803有较强特异性结合的噬菌体抗体．送5株抗体进行测序，选取阳性值最高的单克隆对其序列进行分析并预测其空间

3、结构。结果表明：可变区基因与人胚系基因有很高的同源性(大于90％)．结论：成功构建了库容为2．6×106的全人源胃癌噬菌体单链抗体库，抗体库多样性良好。获得的噬菌体单克隆抗体具有较强的特异性，为胃癌的诊断和治疗提供了新载体。关键词：抗体库，胃癌，噬菌体展示，单链抗体浙江人学硕：Ij学位论文英文摘要Constructionand；creeningof．。．，～playedscFvonstructionanscreeHinolaDhage-disscPvDIaVedantibodylibraryagainsthu

4、mangastriccancersZhejiangUniversityMedicalSchoolSpeciality：BiochemistryandMolecularBiologyMSStudent：YangNaidiSupervisor：Prof．ZhanJinbiaoAbstractObjective：Toconstructaphage-displayedlibraryofhumansingle—chainantibodiesandidentifyspecificantibodiesassociatedw

5、ithhumangastricCanCerS．Methodsandresults：ThecDNAlibraryofantibodyvariableregionswasclonedfromperipheralbloodlymphocytesofpatientswithgastriccancersbyRT-PCR．Theheavy-cham(V．)andlight—chainvariableregiongenes(VL)oftheantibodieswereamplifiedbyPCR．TheVgeneswere

6、assembledtoformscFvbyoverlappingPCRandclonedintophagemidpCANTAB一5E．thentransformedintoEcoilTGl．ThescFvgenelibrarywasrescuedbyM13K07helperphage．Toidentifyspecificantibodiesagainstgastriccancels，wedevelopedastrategytoremoveantibodiescross—reactingwithcommonan

7、tigensonnormalcellsbypanningonnormalhumangastricmucosalepithelialcells．Afterfiveroundsofsubtractivepanning，positivecloneswereconfirmedbyELISAtodeterminethespecificityofphageantibodies．Wefinallyidentified5clonesthatboundselectivelytoMGC-803cells．Thecorrespon

8、dingscFvsequenceswerededucedbyDNAsequencingandthetertiarystructureswerepredictedusingUCSFChimera．III浙江火学硕：I：学位论文英文摘要Summery：Wesuccessfullyconstructedahumanphagesingle—chainantibodylibraryandisolatedspe