副猪嗜血杆菌安徽株分型与tbpa基因的克隆表达和免疫保护作用的的研究

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1、pET-32a(+)-tbpA2和pET-32a(+)-tbpA3，转化BL21后进行诱导表达，经SDS-PAGE电泳可见大小分别为62kD、54kD、44kD的目的蛋白。三段重组蛋白经免疫印记检测后显示其均具有免疫反应性，能与兔抗HPS（LJ3株）高免血清发生特异性反应。将三段重组蛋白及HPS（LJ3株）甲醛灭活菌体分别免疫小鼠后，用同源菌株对菌体免疫组、三个重组蛋白免疫组及对照组小鼠腹腔攻毒，菌体免疫组及TbpA1能分别提供40%及20%的免疫保护率，而其他蛋白免疫组及对照组全部死亡。抗体杀菌试验结果同样表明，TbpA1蛋白的抗体具有杀菌作用。实验结果表明重组的TbpA1蛋白具有抗感染保护

2、作用。关键词：副猪嗜血杆菌，转铁结合蛋白A，分型，克隆表达，免疫保护IIAbstractHaemophilusparasuisistheetiologicalagentofGlasser'sdiseaseinpigs,whosemainsymptomsarefibrinouspolyserositis,polyarthritisandmeningitis;furthermore,somestrainscanalsobefoundasacommensaloftheupperrespiratorytractinhealthypigs.Withtherecentchangesinproduction

3、methods,thediseasehasbecomeoneofthemostimportantbacterialdiseasesinthepig-rearingindustryworldwide.Todate,15serovarshavebeenrecognizedwithmostrecentserotypingstudiesnotingtheexistenceofnon-typableisolates,indicatingthepossibilityoffurtherserovars.Thoughsomestudiesweredesignedtodevelopkilledwholecell

4、sorliveavirulentvaccines,theexistenceofdiversegeneticmake-upsmadetheresultsambiguously.Duetolackofthoroughknowledgeofthevirulencefactorsandprotectiveantigens,novelvaccinedevelopmenthasbeenhinderedtoagainstthediseaseandtheepidemiologicalstudy.ThestudyreportedherefocusedontheserovardistributioninAnhui

5、provinceandcloning,expressionthetbpAgeneofHaemophilusparasuistoresearchtheimmunogenicityandprotectiveinmice.Therearetwomainpurpose:(1)UnderstandHPSepidemicinAnhuiserotypes,ERIC-PCRandPCR-RFLPpolymorphism,soastoexploreareasofAnhuiHPSepidemiologyandmolecularepidemiologyofserumlaythefoundationforeffect

6、iveGlasser'sdiseasepreventionandcontrolprovideascientificbasis;(2)SubcloningandexpressionofHPStbpAdiscussedtheroleofsub-protectiveimmunityproteinsforthepreventionandcontrolandthedevelopmentofnewHPSsubunitvaccinebasis.1SerologicalcharacterizationandERIC-PCRandPCR-RFLPgenotypingofHaemophilusparasuisis

7、olatesfromAnhuiProvinceFromdifferentregionsinAnhui,18strainsofHPSwereisolatedfrom2006-2010,basedonmorphologic,culturalandbolchemicalcharacteristics.AllHPSisolateswereserotypedbyKRGgeldiffusiontestandE