新型结核疫苗p846保护作用及其机制探讨-protective effect and mechanism of new tuberculosis vaccine p846.docx

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2018-06-03 发布于上海
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新型结核疫苗p846保护作用及其机制探讨-protective effect and mechanism of new tuberculosis vaccine p846.docx

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新型结核疫苗p846保护作用及其机制探讨-protective effect and mechanism of new tuberculosis vaccine p846

AnovelDNAvaccinep846conferseffectiveprotectionagainstMycobacteriumtuberculosisinfectionAbstractObjectiveWeaimtogenerateanovelMycobacteriumtuberculosis(M.TB)DNAvaccinecoexpressingmycobacterialtripleantigensRv3615c,Mtb10.4andRv2660c,andevaluateitsprotectiveefficacyagainsttuberculosisinfection.Furthermore,wealsowanttoexaminethepotentialmechanismoftheDNAvaccinefortheprotectivefunctioninBALB/cmice.MethodsWeconstructedanovelmycobacterialDNAvaccinep846expressingmycobacterialtriplefusionantigensRv3615c-Mtb10.4-Rv2660c,anditsindividualantigenbased-vaccinepcDNA3.1-Rv3615c(pRv3615c),pcDNA3.1-Mtb10.4(pMtb10.4),pRv2660c(pRv2660c).Theserecombinantplasmidswerethentransfectedinto293Tcellsandtheexpressionoftheseantigensweredetectedbywesternblot.Atthesametime,wealsodevelopedtheprokaryoticexpressionvectorspGEX-5X-1-Rv3615c,pGEX-5X-1-Mtb10.4,pGEX-5X-1-Rv2660candpet28a-846,andpurifiedtheRv3615c,Mtb10.4,Rv2660candTFP846proteinsfromEscherichiacoli.Theimmunizationprotocolwasshownasfollowing:6to8-week-oldfemaleBALB/cmicewererandomlydividedinto6groups(20ineachgroup):p846group,pRv3615cgroup,pMtb10.4group,pRv2660cgroup,vectorpcDNA3.1groupandBCGgroup(positivecontrol),respectively.Allmicewereadministratedintramuscularlywith50μgrecombinantplasmidsinthehindlimb.Theimmunizationwasrepeatedthreetimeseverytwoweeks.AsingledoseofBCG(5×106CFU/mouse)wasinjectedsubcutaneouslyonlyonceat0week.Serumswerecollectedevery2weeksbyretro-orbitalbleedingandsubjectedtoELISAassays.Twoweeksafterthefinalimmunization,fivemiceofeachgroupwereusedforcellularimmunologyanalysis(proliferation,IFN-γELISPOT,CTLactivityandsoonwereevaluated);another5micewerekeptformemoryTcellsdetecting.Theother10wereusedforBCGchallenge英文摘要新型结核疫苗p846的保护作用及其机制探讨experimentineachgroup.Results1.Wesuccessfullyconstructedp846,pRv3615c,pMtb10.4,andpRv2660ceukaryoticexpressingplasmids,andsodidthepGEX-5X-1-Rv3615c,pGEX-5X-1-Mtb10.4,pGEX-5X-1-Rv2660candpet28a-846recombinantprokaryoticexpressionplasmids,andpurifiedthesefourproteinsfrombacteriallysates.2.Theres