新型乙肝dna疫苗pt--lm的构建与表达及诱导小鼠免疫应答的初步实验

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1、温州医学院硕士学位论文新型乙肝DNA疫苗PT--L/M的构建与表达及诱导小鼠免疫应答的初步实验姓名：晏永波申请学位级别：硕士专业：药理学指导教师：林绍强2012-05-22中表达目的蛋白。4．PTL共表达质粒没能引起小鼠特异性抗体的产生，PTM双质粒能诱导小鼠产生初步的免疫应答，刺激小鼠产生抗一HBs抗体。结论成功构建了两种新型乙肝DNA疫苗PTL和PTM，为进一步研究该类新型乙肝DNA疫苗在体内的免疫应答特点和免疫保护效果奠定基础。关键词乙型肝炎；DNA疫苗；人T—bet基因；HBV—L；HBV—M。温州医学院硕士学

2、位论文ConstructionandExpressionofHepatitisBVirusDNAVaccinePT-L／MandDNA—mediatedImmunizationAbstractObjectiveInordertoobtaineffectiveHBVDNAvaccine，recombinantplasmidpIRES--T-bet—-HBV-LcontainingtheHBV-LandhT-betanddoublerecombinantplasmidPTMcontainingtheHBV-MandhT—b

3、etwereconstructedandexpressedinvitro．TheywereusedtostudyhumoralimmuneresponseinBALB／cmice．Methods1．HumanT-betandHBV-LgeneswereobtmnedbyPCRandclonedintoeukaryoticexpressionplasmidplREStoconstructrecombinantplasmidplRES·T-bet—HBV-L．HumanT-betandHBV-Mgeneswereobtmn

4、edbyPCRandclonedintoeukaryoticexpressionplasmidpCDNA3．1respectivelytoconstructrecombinantplasmidpCDNA3．1-T-betandpCDNA3．1一HBV—M．2．Allplasmidsweretransfectedinto293TcellsandtheexpressionoftargetgenesweredetectedbyRT-PCR，immunofluorescenceassayandELISA．3．PTL／Mwasu

5、sedimmunizetheBALB／cmiceandthelevelsofanti．HBswasdeterminedbyELISA．Results1．RestrictionenzymesanalysisandsequencingresultsshowedmattherecombinantplasmidplRES—T-bet-HBV-L．pCDNA3．1-T·betandpCDNA3．1一HBV—Mwasconstructedsuccessfully．2．ThemRNAofT-betandHBV-Lcouldbedet

6、ectedbyRT-PCR．TheT-betandHBVproteincouldbedetectedbyimmunofluorescence．TheHBV-MproteincouldbedetectedbyELISA．3．PTLplasmiddidnotcausethegenerationofspecificantibodiesinmice，thePTMplasmidsCaninducemicetoproduceinitialimmuneresponse，stimulatemicetoproduceanti—HBsan

7、tibodies．Conclusion4塑!!!垦堂堕堡．!堂鱼笙奎．——SuccessfullyconstructedtwonovelhepatitisBvirusDNAvaccinePTLandPTM，tolaythefoundationforfurtherstudythecharacteristicsofthenewclassofhepatitisBDNAvaccineinthebody’simmuneresponseandimmuneprotection·KeywordsHepatitisb；DNAvaccin

8、e；hT-bet；HBV-L；HBV-M·5温州医学院硕士学位论文前言1研究背景乙型肝炎是由乙型肝炎病毒引起的一种传播广泛、流行久远、危害严重的传染性疾病。据世界卫生组织报道，全世界有2．5．3．5亿慢性乙型肝炎病毒感染者【lJ。中国更是慢性HBV感染的高发地区，总的感染人数约有1．3亿【2】，其中慢性肝炎和肝硬化患者约为3000