應用特異性噬菌體人抗體克隆和篩選新的大腸癌抗原基因.pdf

1、中南大學碩士學位論文應用特異性噬菌體人抗體克隆和篩選新的大腸癌抗原基因姓名：李艷雯申請學位級別：碩士專業(yè)：病理學與病理生理學指導教師：李官成20040518ABSTRACTObjective：ThecDNAlibraryexpression，whichwasconstructedwithhumancolorectalcancercellHRT18，wasscreenedwiththephageantibodyCH209inorderto

2、findnovelcolorectalcarcinomaantigengenesMethods：ThetotalRNAwasextractedfromhumanCRC(colorectalcancer)cell職T18andthem鼬町Awasisolated療omthetotalRNAbvMagneSpheretechnique。andthenthefirst—standcDNAwassynthesizedwitholigo(dT)p

3、rimercontainingsfiIsitewhilethedouble—strandcDNAwasamplifiedthroughLDPCRflongdistancePCR)bvSMARTtechniqueThedoublestrandcDNAwasdigestedbvsfiIfIA&IB)restrictionenzymebeforecDNAsizefractionation，andthedoublestrandcDNAfract

4、ionatedwasligatedintothe九TripIEX2vectorandthenwaspackagedinto九phageinvitroTitratedtheprimarylibraryanddeterminedthepercentageofrecombinantclonesby‘blueandwhitescreening’ThenPCRiSusedtotestcDNAinsertsforligationefficiency

5、ThelibraywasamplifiedandidentifiedAtlast，itwasscreenedwithphagefusionantibodvCH209andthesequenceofthereactedC】oneswasdeterminedResults：Theprimarylibraryconsistsof65x10。pfuJml，andthepercentageofrecombinantscloneswas97％The

6、titeroftheamplifiedcDNAlibrarywas1110’pfu／mlandtheaverageinsertofrecombinantswas11kbTenpositivecloneswereobtainedafterthreecyclesscreeningandtheseqrlencesweredeterminedandanalyzedwimBLASTsoftwareinGenBankAsaresult，positi

7、vecloneswerederivedfrometendifferentgenesFiveofthesegeneswerehighhomologoustogenesknowninGenBankandtherewerealsotheregeneswithlOWhomologytogeneskowninGenBankFurthermore，twogenesmaybenovelgenesbymatchinginGenBankwithBLAST