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1、浙江人學(xué)碩一l：學(xué)位論文中文摘要全人源胃癌噬菌體單鏈抗體庫的構(gòu)建及其初步篩選浙江大學(xué)醫(yī)學(xué)院：生物化學(xué)與分子生物學(xué)碩士研究生：楊乃娣導(dǎo)師：詹金彪中文摘要研究目的-構(gòu)建全人源胃癌噬菌體單鏈抗體庫，并從中篩選出與人胃癌細(xì)胞特異結(jié)合的單鏈抗體，并對其進(jìn)行鑒定分析。研究方法和研究結(jié)果：首先分離胃癌病人外周血淋巴細(xì)胞，提取總RNA，RT—PCR合成cDNA第一鏈，采用兼并引物PCR擴增抗體重鏈(VH)和輕鏈可變區(qū)(VL)基因。經(jīng)重疊PCR(SOE．PCR)，將VH和VL連接成單鏈抗體(seFv)基因，并連接到噬菌粒載體p
2、CANTAB．5E中，轉(zhuǎn)化至EcoliTGl，經(jīng)輔助噬菌體超感染，構(gòu)建了全人源胃癌噬菌體單鏈抗體庫，庫容為2．6x106，質(zhì)粒片段插入率95％．單鏈抗體庫經(jīng)與正常人胃黏膜上皮細(xì)胞GES．1孵育后，去除與正常細(xì)胞結(jié)合的噬菌體單鏈抗體，再以人胃癌細(xì)胞MGC．803為靶標(biāo)對抗體庫進(jìn)行4輪富集篩選，結(jié)果顯示富集了50倍。選取第四輪篩選后的菌落制備單克隆抗體，并進(jìn)行ELISA特異性鑒定，獲得五株與人胃癌細(xì)胞MGC．803有較強特異性結(jié)合的噬菌體抗體．送5株抗體進(jìn)行測序，選取陽性值最高的單克隆對其序列進(jìn)行分析并預(yù)測其空間
3、結(jié)構(gòu)。結(jié)果表明：可變區(qū)基因與人胚系基因有很高的同源性(大于90％)．結(jié)論：成功構(gòu)建了庫容為2．6×106的全人源胃癌噬菌體單鏈抗體庫，抗體庫多樣性良好。獲得的噬菌體單克隆抗體具有較強的特異性，為胃癌的診斷和治療提供了新載體。關(guān)鍵詞：抗體庫，胃癌，噬菌體展示，單鏈抗體浙江人學(xué)碩：Ij學(xué)位論文英文摘要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浙江火學(xué)碩：I：學(xué)位論文英文摘要Summery：Wesuccessfullyconstructedahumanphagesingle—chainantibodylibraryandisolatedspe
