1、甘肅農(nóng)業(yè)大學碩士學位論文豬圓環(huán)病毒ⅡRep啟動子功能分析及感染性克隆的構(gòu)建姓名：孫海亮申請學位級別：碩士專業(yè)：預防獸醫(yī)學指導教師：劉磊陳溥言20080601甘肅農(nóng)業(yè)大學2008屆碩士學位論文摘要Summary[Objective】PorcinecircoviruscontainstwoserotypesPCVlandPCV2SomekindsofdiseasesarerelatedwithPCV2inporcinegroupsEspec

2、iallyPMWSisthemostseriousandhasmadegreateconomiclossesinthewordSo，researchingthemechanismofpathogenesisofPCV2isverysignificantforpreventionandtreatmentofdiseaseswhicharerelatedwithPCV2Inthisreseach，PCV2DTwasgoingtobeisol

3、ated，andRepandCappromotorsofPCV2wasgoingtobelocated，andthefunctionofvlSREwhichliesinReppromotorwasgoingtobeanalyzedandinfectiouscloneofPCV2andrecombinantvirusofPCV2withHATagweregoingtobeconstructedThisresearchwouldlayafo

4、undationforstudyingmultiplicationinvitro，pathogenicityofPCV2，pathogenicmechanism，vaccinedevelopment，moleculardiagnosis[Methods】(1)PCV2wasisolatedfromtissueofpigwhichcamefromDTcityanddiedofPMWSbyusingPK15cell，anditsgenomi

5、csequencewasanalyzed。Pigletsof42dayswhichwerefreeofPCVl、PPV、PRV、PRSVwereinoculatedwithPCV2DTvirus(2)PCV2promotorswerepredictedbyusingpromoterpredictionsoftwareandthesequencespredictedwereamplifiedbyPCRandclonedintopGL3Ba

6、sictoconstructpGL3Bpl、pGL3Bp2、pGL3Bp3、pGL3Bp4aswellaspGL3Bp5whichcontainsknownPCVlReppromoterAllrecombinatedplasmidsweretransfectedintoPK15cell，andtheactivityofpromoterswasdetectedbyusingLuciferaseAssaySystem(3)PGL3一Bp2w

7、hichcontainsPCV2ReppromoterandPGL3Bp5whichcontainsPCVlReppromoterandPGL3BpmwhichwasconstructedthroughmutatingPCV2ReppromoterinvlSREwererespectivelytransfectedintoPK15cellAfterbeingstimulatedbydifferentdoseaIFN，thechanges

8、ofactivityofpromotersweredetected(4)TwocopygeneofPCV2wastandemLyclonedintopSK()toconstructpSK2PCV2AndpSK02PCV2wastransfectedintoPK15cell，after4passages，theinfectedcellwasdetectedbyIFAandRTPCR(5)HATaggenewasclonedintoCapg

9、eneendbySOE—PCRThePCV2genewithHATagwascyclizedandampliedThen，circo—DNAHAwastransfectedintoPK15cellAfter4passages，thecellwasdetectedbyRTPCR、IPMA、Amplifyingcorrespondingfragmentandsequencing、HAexperimentThen，thestabilityof

10、HATagoftherecombinatedviruswasdetectedbyrestrictionenzymeNcoIdigestion【Results】(1)PCV2DTissucceedlyseparatedComparingwith35domesticandabroadstrains，thehomologyofnucleotidesequenceareallover95％，andonlysomesitesmutatedThep

11、hylogenetictreedemonstratesthatthereisnoobviousregionalismbetweendifferentstrainsofPCV2AfterbeinginoculatedwithPCV2DTforl0days，thepigletsemergedthesymptomsofPMWS(2)PCV2Reppromotorislocatedin1705nt1968nt；PCV2Cappromotoris

12、locatedin834nt1309ntTheactivitiesofRepandCappromoteraremorestrongerthancontrolSV40promotor(3)Theactivitiesofpromotorsareinhibitedbya—IFNindifferentextentTheactivitychangeofPCV2Reppromoterispositivelycorrelatedwiththedose

13、ofotIFNButdifferentdosesofotIFNhadnoobviousinfluenceontheactivityofPCVlReppromoterAftervlSREbeingmutated，theactivityofPCV2ReppromoterweakensandbecomessensitivetoaIFNWhethermutatedornot，theactivitiesofpromotorswereheavily

14、inhibitedbyhighdoseaIFN(4)PCV2isdetectedinPK15transfectedwithPSK2PCV2for4passagesbyRTPCRandIFA(5)RecombinantviruswithHA—Tag(PCV2HA)isdetectedinPK15transfectedwithCircoDNA—HAfor4passagesbyRTPCRandIPMA，butPCV2一HAhasnoactiv