PRRSV GP3和GP4的免疫活性及其在感染細(xì)胞內(nèi)的分布研究.pdf

1、河南農(nóng)業(yè)大學(xué)碩士學(xué)位論文PRRSVGP3和GP4的免疫活性及其在感染細(xì)胞內(nèi)的分布研究姓名：王中明申請(qǐng)學(xué)位級(jí)別：碩士專業(yè)：預(yù)防獸醫(yī)學(xué)指導(dǎo)教師：夏平安崔保安20090601河南農(nóng)業(yè)大學(xué)2009屆預(yù)防獸醫(yī)學(xué)碩士畢業(yè)論文TheimmunocompetenceandthedistributionofGP3andGP4intheinfectedcellsbyPRRSVAbstract：Porcinereproductiveandrespirator

2、ysyndromevirus(PRRSV)isoneofthemajorpathogencausedreproductivedisturbanceinthepigfarmsSowscanbeshownfever，anorexia，prematurebirth，miscarriage，fetalmummies，stillbirths，pigletsCanbeshownhighmortalityrates，andallswineinever

3、ystagecallbedyspneicrespiration。PRRShasbeencausingtremendouseconomiclossestotheswineindustrythroughouttheworldPRRSVbelongedtotheArteriviridaefamily，hassixkindsofstructuralprotein，whichareGP3，GP4，GP5，M，andNTheresearchonPR

4、RSVinrecentyearsmainlyconcentratedintheGP5，MandNproteins，andtherewerenotmanystudiesforthesecondarystructureofproteinsGP2，GP3，GP4SoitsnotabsolutelycleartheirroleisinthelifecycleofPRRSVInthisstudy，theGP3，GP4proteingeneofHe

5、nanHn一I／06strainwereexpressedinvitrobytheprokaryoticexpressionsystem，andthemouseantirecombinantTheimmunogenicityofGP3，GP4proteinantibodiesweredetectedbyindirectimmunofluorescenceassay(IFA)andindirectimmunosorbentassay(in

6、directELISA)whichhadbeenestablishedbeforeInthisstudy，thenucleotidefragmentwhichisabout963bp，676bpcontainingORF3，ORF4geneofHnI／06strain，WasamplificatedbyRT—PCRThenthenueleotidefragmentclonedintovectorpTG19TweresequencedIn

7、accordancewithORF3，ORF4geneandtheiraminoacidsequencem，twopairsofprimersweredesignedOnepairwasusedforamplifingthedORF3whichhasBamHI，HindIIIrestrictionsites，andtheotheronewasusedforamplifingthedORF4whichhasBamHI，XhoIrestri

8、ctionsitesThendORF3anddORF4wereclonedintotheprokaryoticexpressionvectorpET32atoconstructtherecombinantexpressionplasmidpETORF3andpET—ORF4ThenthetargetproteinwerepurifiedbythebuffercontainedureaWestern—blotwasperformedtoc

9、onfirmthattheexpressedfusionproteincouldspecificallyreactwithantiserumagainstPRRSVThentheinclusionbodies，denaturedproteinandrefoldedprotein&fusionproteinGP3andGP4wereimmunizedmicetoobtainthemurineantibodiesagainstGP3，GP4

10、fusionproteinThemurineantibodiesweretestedbyindirectELISAandIFA，andthedistributionofGP3，GP4ininfectedMarc一145cellsweredetectedtooTheresultsshowsthatthetiterofmurineantibodiestiterwasl：20andhaverioneutralization；GP3，GP4ca