1、新疆農業(yè)大學碩士學位論文華東地區(qū)豬繁殖與呼吸綜合征病毒GP5基因特征及流行毒株的分離鑒定姓名：扈鴻霞申請學位級別：碩士專業(yè)：預防獸醫(yī)學指導教師：冉多良方維煥20080601AbstractPorcinereproductiveandrespiratorysyndrome(PRRS)ischaracterizedbyseverereproductivefailureinSOWS(1ate—termabortions，dead，weakan

2、dmummifiedpiglets)，respiratorydiseaseandincreasedpreweaningmortalityThePRRSVpossessesfourcharacteristicsthatmaycontributetodifficultiesinpreventionandtreatmentofthedisease，includingproductionofeffectivevaccinesTheseinclu

3、detropismformacrophagesormacrophagelineagecells，remarkableantigenicvariationsamongPRRSVfieldisolates，antibodydependentenhancementofthevirusinfection(ADE)，andabilitytoestablishpersistentinfectionThemainobjectivesofthepres

4、entstudyare(1)toanalyzethevariationsoftheGP5genesofPRRSVisolatesfromsomepigfarmsinsoutheasternChina(2)toexpressandpurifytheGP5proteinforELISAcoatingandpreparationofpolyclonalantibodiestoGP5forindirectimmunofluorescentdet

5、ectionofPRRSVand(3)toidentifyandrecoverafieldPRRSVstrainfrominfectedtissuesReversetranscriptionPCRwasusedtoamplifytheGP5genefromthetissuesamplesofpigssuspectedofhavingPRRSVinsoutheasternChineseprovinces，andgenefragmentsw

6、ereclonedandsentforsequencingPhylogeneticanalysisindicatesthatallisolatesfromtheregionfrom2004to2007belongtosubgroup1ofNorthAmericantypeTheisolatesfromthisstudysharedidentitiesof896100％atthenucleotideleveland907995％atthe

7、aminoacidlevelTheirnucleotideidentitywas881902％andaminoacidsimilarity858—896％totheclassicalNorthAmericanisolateVR2332ThegeneencodingtruncatedGP5wasclonedintotheexpressionvectorpET030a，therecombinantproteinwasefficientlye

8、xpressedinEcoilRosettaintheformofinclusionbodiesafterinductionwithIPTGasrevealedbySDSPAGE，andtheproteinyieldafterpurificationwas875mgperlitercultureoftherecombinantbacteriaWesternblottingandELISAindicatedthatthisrecombin

9、antproteincouldreactwithPRRSVpositiveserumTherecombinantwasusedtogeneratetheantiGP5polyclonalantibodyinrabbitwithtiterashighas1：32768whichwasusedforidentificationofPRRSVbyindirectimmunofluorescentassayThetissuessamplesth

10、atwerePRRSVpositivebyRTPCRwerecollectedandusedtoinoculateontoporcinealveolarmacrophage(PAaVl)monolayersReplicationofPRRSVinthecellsWasconfmnedbyRTPCRfragmenttargetingGP5，indirectimrnunofluorescentassayandtransmissionelec

11、tronmicroscopyThevirushaddeletionof29aminoacidsinNSP2andcouldinducethecytopathiceffectaftersubculturinginMarc145celllinewichTCID50ofabout104卯after10passagesThefindingsinthisstudyprovidesomebasisforflLrtherresearchintothe