猪霍乱沙门氏菌C78_1株_crp缺失株的构建及其生物学特性初步研究

2010,41(5):587593ActaVeterinariaetZootechnicaSinicaC781crp郁川,程相朝*,赵战勤,张春杰,李银聚,吴庭才(,471003):C781crp,C781PCR,crp(10481743bp),pRE112,320bpcrppREcrp,C781crp,C781,crp,C781,C781750,C781crp,,,:C781;crp;;;crp:S852.612:A:03666964(2010)05058707:20090910::(1985),,,,,Email:[e-mail]*:,Email:[e-mail]terizationofaSalmonellaCholeraesuisC781crpDeletionMutantYUChuan,CHENGXiangchao*,ZHAOZhanqin,ZHANGChunjie,LIYinju,WUTingcai(CollegeofAnimalScienceandTechnology,HenanUniversityofScienceandTechnology,Luoyang471003,China)Abstract:Inthisstudy,acrpmutantofSalmonellaCholeraesuisC781wasconstructedbytheallelicexchangeintroducedbythetransductionofthepRE112suicideplasmidandcharacteristicsofthemutantwerealsodetermined.Firstly,theupstreamanddownstreamfragementsofcrpgenewereamplifiedfromSalmonellaCholeraesuisC781genomeandthensuccessivelysubclonedintosuicideplasmidpRE112toconstructtherecombinantsuicidevectorpREcrpwith320bpdeletedcrpfragment.Then,theC781crpmutantwasconstructedthroughthetwostepmethodintroducedbythetransductionofrecombinantsuicideplasmid.TheOandHantigensofthemutantwas6,7:C:1,5,identicaltotheparentC781strain.Themutantwasstabilewiththerecombinantcrpgeneinvitro.However,fermentationpatternsandgrowthrateofthemutantweredifferedfromthatoftheparentC781strain,obviously.Themouselethaltestshowedthatthevirulenceofthemutantwas750timeslowerthanC781.TheseresultsshowedthattheC781crpmutantwasconstructedsuccessfully.ItislikelythatthiscrpmutantcouldbeadaptedtodevelopattenuatedSalmonellavaccine.Keywords:SalmonellaCholeraesuisC781strain;crpgene;recombinantsuicidevector;conjugationtransfer;acrpmutant41(SalmonellaCholeraesuis),,,[1],,[2][3],,,,,[4],C781,C500[57],,,[8]DNA,,[9],,crp(cAMPreceptorprotein,crp),C781crp,,11.1C781pBluescriptSK(+)JM109;pRE112(oriToriVasdCmrSacB)7213(Thi-1thr-1leuB6fhuA21lacY1glnV44asdA4recA1RP42TcMu[pir]Kmr)Dr.RoyCurtiss!,LB37∀()(),100!g#mL-1(Amplicillin,Amp),25!g#mL-1(Chloramphenicol,Cm),7213(DL∀,#Diaminopimelicacid,DAP),50!g#mL-11.2SS;LBBD;DAPSigmaAldrich;DNAMarkerPyrobestDNAPolymeraseT4DNALigaseTaKaRa;DNA6SPFKM(20g∃2g)1.31GenBankLT2(GenBankNo.AE008859)crp[8]crpinvAPi1Pi2[10],SS1.4pREcrpC781,P1/P21048bpcrpcrp1,pMD18T,BamH%Xba%,crp1pBluescriptSK(+),pSKcrp1P3/P41743bpcrpcrp2,pSKcrp1,pSKcrp,Kpn%Xba%,crp1+crp2crppRE112,7213,320bpcrppREcrp1.5crp[11][12],,7213(pREcrp),C781,100!L,CmLB,Cm(Cmr)5885:C781crp(SUCs),T1/T2NaClLB(NB),10,5%NB,Cm(Cms)(SUCr),T1/T2,T2/T31PCRTable1TheprimersequencesforPCRamplificationGeneamplifiedPrimer(5&∋3&)Sequence(5&∋3&)RestrictionsiteUpstreamofcrpP1P2GCTCTAGAGCTGGATGAGAGTTTTGTGGTCGGATCCCCATTCAAGAGTCGGGTCTXba%BamH%DownstreamofcrpP3P4TTCTCGAGGCTCGTCGCTTACAAGTCACCTGGTACCCAGTAACTGGATGGTGTATAXho%Kpn%crp/crpT1T2TACGCGCATACAACAAAAGTCGCGCCATTCTGACGGAATTAACGGGcrp/crpT3T2TCGCGTACCCATATCAACTTGCCATTCTGACGGAATTAACGGGinvAPi1Pi2CAGGATACCTATAGTGCTGCCGCACCGTCAAAGGAACCGT1.6crpC7811.6.1crpC781crpC781C7811%,,OH1.6.2crpC781crpC781LB20,5101520,T2/T3PCR,crpcrp1.6.3crpC781crpC781C781LB37∀,10,,100!LLB,3,37∀,,,CFU106CFU#mL-1,37∀100r#min-1,1h,1.6.4crpC781crpC781C781LB37∀,1.6.3,5,,5KM,,500!L,30dSS,Pi1/Pi2,,BlissLD5022.1pREcrppREcrp1,Kpn%Xba%2,5170bppRE1122791bpcrpcrp2BamH%,BamH%Xba%3,crp2(1),,pREcrpM.DNAmarker(DL10000);1.pREcrp/BamHI+XbaI;2.pREcrp/KpnI+XbaI1pREcrpFig1EnzymeidentificationofplasmidpREcrp589412.2crpC7817213(pREcrp)C781,,pREcrpC781,CmrSUCspREcrp,crp2,,,T1T22,918bp,599bp(2A)NaClLB(NB),2,2,,599bp;,918bp(2B)T1T2,,T2T3,1412bp,1731bp,pREcrp(2C)PCR,CmsSUCrcrpA.T1T2:1.;2.pREcrp;3.C781;4.crp;M.DL2000DNA;B.T1T2:M.DL2000DNA;1.;2.;3.crp;C.T2T3:M.DL2000DNA;1.pREcrp;2.;3.C781;4.crpA.PCRidentificationofcrpmutantconjugantswithT1andT2.1.ddH2Ocontrol;2.pREcrpcontrol;3.C781control;4.crpmutantconjugants;M.DL2000DNALadder;B.PCRidentificationofcrpmutantswithT1andT2;M.DL2000DNALadder;1.ddH2Ocontrol;2.Widetype;3.crpmutant;C.PCRidentificationofcrpmutantswithT2andT3.M.DL2000DNALadder;1.pREcrpcontrol;2.ddH2Ocontrol;3.C781control;4.crpmutant2PCRcrpFig2PCRidentificationofcrpmutant2.3crpC7812.3.1crpC781C781,crpC781crp,,(3),C781,crpC781crpC781,H2S,(2)crpC7816,7:C:1,5,C7812crpC781C781Table2ComparisonofthebiologicalcharacterizationbetweenthecrpmutantandC781StrainsBiologicalcharacterizationGluMalSucLacFruXylRaManRhaH2SMRVPC781++--+++++++-crpC781+---------+-Glu.Glucose;Mal.Maltose;Suc.Sucrose;Lac.Lactose;Fru.Fructose;Xyl.Xylose;Ra.Raffinose;Man.Mannitol;Rha.Rhamnose;H2Sintriplesugarironagar2.3.2crpC781crpC781LB20,5101520,T2/T3PCR,1412bpcrp,C7811731bpcrp(4),5905:C781crp1.C7811%;2.C781;3.crpC7811%;4.crpC7811.C781onMacConkeyagarplus1%maltose;2.C781onMacConkeyagar;3.crpC781mutantonMacConkeyagarplus1%maltose;4.crpC781mutantonMacConkeyagar3crpC781C781(1%)Fig3ThegrowthofcrpC781mutantandparentC781strainonMacConkeyagarplus1%maltosecrpC781320bpcrpM.DL2000DNA;1.;2.C781;3~6.5101520crpC781M.DL2000DNALadder;1.ddH2Ocontrol;2.C781control;36.The5th,10th,15thand20thgenerationofcrpC781mutant4T2T3PCRcrpC781Fig4PCRidentificationofstabilityofcrpC781mutantwithT2andT32.3.3crpC781crpC781C781,,37∀12h,crpC781C7810.40.8mmcrpC781C781106CFU#m