esat6ag85a融合基因dna疫苗增强卡介苗初免免疫原性和保护性word格式论文.docx

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2018-05-18 发布于上海
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esat6ag85a融合基因dna疫苗增强卡介苗初免免疫原性和保护性word格式论文.docx

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esat6ag85a融合基因dna疫苗增强卡介苗初免免疫原性和保护性word格式论文

pcDNA3.1(+), resulting in recombinant plasmids named pPro685Aand pcD685A, respectively.TransformtheprokaryoticexpressionrecombinantplasmidpPro685AintothecompetenceEscherichiacoliBL21cells.Theproteinr685Awasinducedandpurified.ProteinpurificationwasperformedusingaNi-NTApurificationsystem.TheseprocesseswereexaminedbySDSandverifiedbyWesternblotting.Thepurifiedproductofr685AproteinwasfurtherquantitatedbyBCAmethod.TransformtherecombinantplasmidpcD685AintothecompetenceEscherichiacoli DH5αcells.PlasmidDNAwasextractedandpurifiedinscale,andtheconcentration wasdeterminedbyspectrophotometricmethod.C57BL/6micewereimmunizedwithaBCGprimeandpcD685Abooster.IgG antibodiesagainstr685AproteinweredeterminedbyELISA,andtheexpressionof IFN-γandIL-10inlungtissuewasdeterminedbyqRT-PCR.Afterimmunization,themicewerechallengedwithM.tbH37Rvstrain,theprotectiveimmunitywasdeterminedbytheorgansbacterialloadsandorganshistopathologicalexaminationofinfectedmice.ResultsTherecombinantprokaryoticplasmidpPro685AandeukaryoticplasmidpcD685A wereconstructedsucessfully.Theexpressionofr685AwasexaminedbySDSandverifiedbyWesternblotting.Thepurifiedproteinofr685AwascongfirmedbySDS.Asexpected,the mostsignificantIgG-specificresponsetor685AproteinwasinducedinBCGprimeandpcD685ADNAboostergroup,whenr685A proteinwasusedfor detection.IFN-γresponseincreasedinallgroupsexceptthevectorcontrolgroup. MicevaccinatedwithBCGpluspcD685Ainducedthehighest levels of IFN-γresponsesinthelungs.CombinationwithBCGincreasedsignificantlytheexpressionofIFN-γwhencomparedwithBCGorpcD685Aalone.Themostsignificantreductioninbacterialloadofbothspleenandlungwasobtainedin micevaccinatedwithBCGprimeandpcD685ADNAboosterwhencomparedwith BCGorpcD685Aalone.AfterchallengedwithvirulentM.tbH37Rv,asshoweninhistopathologicalexamination,themostslightvariationwasinBCGprimeandpcD685ADNAboostergroup.ConclusionThus,ourstudyindicatesthatpcD685AmaybeanefficientboostervaccineagainstTBwithpriorBCGimmunity.Keywords:Mycobacteriumbovis，BCG,Ag85A,ESAT-6,prime-boostregimen.