攜帶新城疫病毒融合蛋白基因的重組減毒單增李斯特菌構(gòu)建及其免疫原性.pdf

1、浙江大學(xué)碩士學(xué)位論文攜帶新城疫病毒融合蛋白基因的重組減毒單增李斯特菌構(gòu)建及其免疫原性姓名：柯春林申請(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：方維煥20050501AbstractListeriamdnocytogenesisafacultativeintracellulargrampositivepathogenthatCallenterandmultiplywithinbothphagocyticandnon—phagocyticce

2、llsofhumansandanimalsItisamajorcauseoflife—threatening，food—borneillnessinimmunocompromisedandpregnantindividualsListeriamonocytogeneshastobeattenuatedforvaccinationpurposesbecauseofitshighpathogenicityTwodifferentapproa

3、cheshavebeenpursuedforattenuationofLmonocytogenes：inactivationoflisterialvirulencefactorsorconstructionofauxotrophicmutantstrainsNewcastlediseaseisahighlycontagiousandeconomicallyimportantdiseaseofpoultryNewcastledisease

4、virus(NDV)belongstothegenusRuhulaviruswithintheParamyxoviridaefamilyofnegative—StrandedRNAviruses，Thefusionglycoprotein(F)andhemagglitinin(IfN)arethemajorvirulencefactorsofthevirustowhichthehostrespondswellwithprotective

5、antibodyproductionTheycarlelicitagoodprotectiveimmuneresponsesWeattemptedtoconstructtheattenuatedLmonocytogenesmutantcarryingNDVfusionproteingeneandexaminetheimmunogenicityinmouseandchickenmodelsThetargetgenes卯a(chǎn)ndNDVfwer

6、efusedtotheactAgenefragmentdownstreamofitspromoterandsignalsequencebyhomoiogousrecombinationThreerecombinantListeriamonocyogenesmutants(LM—dactA，LM—zlactAgypandLM—AactANDVF)wereconstructedTheLM—AactA—gypandLM—AacM—NDVFmu

7、tantsexpressgreenfluorescentprotein(GFP)andnewcastlediseasevirusfusionprotein(F)asshownbyfluorescentmicroscopy，SDS—PAGEandwesternblowingThemutantsrecoveredfrominvivoexperimentsandinvitrosubculturesweregZPandNDV—fpositive

8、byPCRandrestrictiondigestionanalysisExponential—phasecellsof三monocytogeneswildtypeandmutantstrainsgrownat37。CshowedanapproximatereductionoftheirviablecountsduringexposuretopH55thanpH72atthesalnetemperatureTherecombinantL