鼠伤寒沙门菌spvb.spvc基因共同缺陷变异株构建及spvb、spvc基因对中毒症状、免疫功能影响-construction of common defective mutant strain of salmonella typhimurium sp vb.spvc gene and effect of spvb and spvc gene on poisoning symptoms and immune function.docxVIP

ABSTRACTObjective：SincewehadconstructedspvB/spvCgene-deficentstrains,nowweconstructedthespvB.spvCgene-deficentstrainstomaketheexperimentmorecredible.ThewildstrainsandspvB/spvC/spvB.spvCdefectstrainsinfectBALB/cmicerespectively.AnalyzetheeffectofspvB,spvCgenesbyobservethesymptomsandimmuneresponsebetweentheinfectedmice.ThereforeprovidetheoreticalandexperimentalevidencetostudythepathogenicmechanismofthespvB/spvC,andprovidemeansforcellinfectionmodelorvaccinedevelopmentwiththefollowresearch.Methods：Acordingtothesuicideplasmid-mediatedhomologousrecombinationconstructespvB.spvCGene-DeletedMutantsinSalmonellaTyphimurium.CheckouttheSalmonellaspvgenesequence(includespvABCD)intheGeneBankdatabase,designedtwopairsofspecificprimersforspvB/spvC,andplusrestrictionsitesonthe5endoftheprimer.AmplifynucleicacidfragmentofspvBupstreamandspvCdownstreambyPCR,connectethemandclonethemintotothesuicideplasmidpCVD442(pCVD442-ΔspvB.spvC),whichwastransducedintothetargetcellofthewidetypestrainofSalmonellatyphimuriumforhomologousrecombination．ObservethereorganizationbyPCR,ThemutantspvB.spvCwasselectedbyspecificPCR,choosethestablerecombinantstrainconsideredcompletespvB.spvCmutantgenedefects,thesenttoDNAsequencing.StudytheeffectofspvB,spvCgenesbyobservethesymptomsandimmuneresponsebetweentheinfectedmice.AllBALB/cmiceweregivenfreeaccesstofoodandwater,roomtemperature(20-22℃),humidity(50%-55%).TheywererandomizedtoPBS、STM.211、STM.211-ΔspvB、STM.211-ΔspvC、STM.211-ΔspvB.spvCgroup,andwereinfectedwith0.2mlPBS、STM.211、STM.211-ΔspvB、STM.211-ΔspvC、STM.211-ΔspvB.spvCrespectivelybyintraperitoneal.Observedtheinfectionsymptoms(thechangeofthementalstatus、movement、diarrhea、weight、hair).Uponinfection1d、3d、5d、7d、9d,5micesineachgroupwererandomlyselectedtoremoveeyestogetblood.Then,textIL-10,IL-12,IFN-γoftheSerumbyELISA.Atthesametime,isolateliverandspleentoobservethepathologicalchangesoftargetorganwiththegeneralvieworunderthemicroscope.Results：AstablespvB.spvCgenedeletedmutantsinSalmonellatyphimurium(STM.211-ΔspvB.spvC)wasc