H3N2亞型豬流感病毒HA1蛋白間接ELISA方法的建立及NP蛋白單克隆抗體的制備.pdf

1、中國農業(yè)科學院碩士學位論文H3N2亞型豬流感病毒HA1蛋白間接ELISA方法的建立及NP蛋白單克隆抗體的制備姓名：劉天強申請學位級別：碩士專業(yè)：預防獸醫(yī)學指導教師：童光志20080601AbstractUsingapairofspecificprimersdesignedaccordingtotherelevantnucleotidesequenceofA／Swine／Heilongiiang／I／05(H3N2)fromGenBank

2、，theHAlgeneofH3N2subtypeswineinfluenzavirus(SIV)wasamplifiedwithRT-PCRmethodThePCRproductWasclonedintopET-32a()andarecombinantplasmidpET-HA1WasgeneratedThetargetgenewasexpressedinthehostcellBL2lsuccessfullywheninducedwit

3、hIPTGTheexpressionWasoptimizedwithproperinducingconditionof08mmol／LIPTGand4hoursSDS—PAGEresultshowedthattheHAlgeneWasexpressedinprokaryoticexpressionsystem，andthetargetproteintaketheproportionof40％(42KD)inthewholeprotein

4、byscanningassayWesternblotanalysisprovedtherecombinantproteinhasgoodreactivityagainstH3N2subtypeSIVpositiveserumTheindirectELISAWasestablishedtodetectantibodyofH3subtypeSIVusingpurifiedproteinTheoptionalworkingcircumstan

5、cesfortheindirectHA-ELISAassay(antigenconcentration：3751ag／ml；serumdilution：l：100)wasconfirmedTheworkingconcentrationofHRP—labeledrabbitanti-swineIgGWasl：l000ThepositivecriterionofthisELISAassayisODthetestedserum0254nete

6、stsshowednocross-reactivitywithantibodiestoH1NlSIV；H5NISIV；H9N2SIVSFV：PRRSVandPRVComparedwithHI，thediagnosticsensitivity,specificity,andaccuracyofHAlELISAweremoreoptimizationafterdetecting309serumsamplescollectedfromdiff

7、erentcoughingherdsinChinaThecoincidencerateWas89％betweenindirectandHItestindicatingtherecombinantHA1proteincouldbeusedforprimarydiagnosesofpiginfectionwithH3subtypeSIVInthisexperiment,severalhybridomacellstrainsweredevel

8、opedbyfusionofSP2／0mousemyelomacellswithspleencellsisolatedfromaBALB／CmousewhichWasimmunizedbythepurifiedSIV(SGD／l6406)FourMcAbcellstrainsweredeterminedbyindirectELISAcoatedwithpurifiedvirus：D4E11，B8G6，BSH5，C8G8Thesubtyp

9、esofNPMcAbsweredeterminedbyWesternblotanalysisusingpurifiedSIV(SGD／16406)asantigenTheresultofWesternblotshowthatthereisonlyonestripin56KDthespecificialMcAbsofNP(ELISAtiters)were：l：320001：320001：80001：16000thenumberofchro

10、mosomewereabout98tol06andisthesameastotalchromosomenumberofSP2／0mousemyelomacellsandspleencellsTheMcAbs，B8G6andB8H5wereprovedtobelongtOimmunoglobulinsubclassIgG2D4EI1andC8G8wereprovedtobelongtoimmunoglobulinsubclass111et