應(yīng)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(xué)碩士學(xué)位論文應(yīng)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(qǐng)學(xué)位級(jí)別：碩士專業(yè)：病理學(xué)與病理生理學(xué)指導(dǎo)教師：李官成20040518ABSTRACTObjective：ThecDNAlibraryexpression，whichwasconstructedwithhumancolorectalcancercellHRT18，wasscreenedwiththephageantibodyCH209inorderto

2、findnovelcolorectalcarcinomaantigengenesMethods：ThetotalRNAwasextractedfromhumanCRC(colorectalcancer)cell職T18andthem鼬町Awasisolated療o(wú)mthetotalRNAbvMagneSpheretechnique。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