esat 6 ag85a融合基因dna疫苗增强卡介苗初免的免疫原性和保护性word格式论文.docx

4．利用BCG（中国株）初免pcD685A增强的方式免疫C57BL/6小鼠。通过ELISA法检测外周血抗r685A蛋白IgG抗体，qRT-PCR检测肺脏表达的IFN-γ和IL-10的手段，来评价在小鼠模型中的免疫原性。5．对免疫后小鼠用M.tbH37Rv毒株攻击实验进行攻击，测定组织荷菌量、组织病理学改变等指标评价疫苗的保护性。三结果：1. 成功的构建了esxA-fbpA融合基因的真核表达载体和原核表达载体。2. SDS和Westernblotting证实r685A原核表达和纯化成功。纯化的r685A经SDS证实。3.pcD685A诱导表达了特异性IgG的抗体反应，BCG初免pcD685A增强免疫组明显高于其它组。qRT-PCR检测肺脏细胞因子，除了空载体组，其他各组的IFN-γ反应水平均增加。pcD685A增强BCG初免组诱导小鼠肺脏产生了最高剂量的IFN-γ，并比单独BCG组或者pcD685A组IFN-γ有显著提高。4. 更重要的是pcD685A增强免疫BCG初免与单独BCG或pcD685A组相比，能明显的减少肺脏和脾脏的荷菌量。5. 小鼠用M.tb H37Rv毒株攻击后，肺脏组织病理学检查结果显示BCG初免pcD685A增强组的肺脏病理变化最轻微。四结论：因此，我们的研究显示pcD685A可能将是一种有效地增强BCG免疫的抗TB疫苗。【关键词】结核分支杆菌,卡介苗,Ag85A,ESAT-6,初免增强方案。Immunogenicity and protectiveefficacyagainstmurine tuberculosis ofa prime-boostregimen with BCGanda DNA vaccine expressingESAT-6 andAg85Afusion protein Candidate:ZhiguangZhouSupervisor:ChangxueCai,Af.XionglinFan,Prof.AbstractBackgroundandObjectivesHeterologousprime-boostregimensutilizingBCGasaprimevaccineprobablyrepresentthebesthopefortheimmunitymeasureofnovelanti-tuberculosis.ESAT-6andAg85AhavebeenthoughtastwomajorimmundominantantigenforTBvaccine.Inthisstudy,wefirstlytheclonedfusiongeneofESAT-6andAg85Aencodingexpressingthefusionproteinof Ag85AandESAT-6(r685A).Then,weconstructedeukaryoticexpressionplasmidpcD685A andprocaryotic expression plasmidpPro685A.TheImmunogenicity andprotective efficacy againstmurinetuberculosisofaprime-boostregimenwithBCGandaDNAvaccineexpressingESAT-6andAg85Afusionproteinwasinvertigated.MethodsThegenefbpAandesxAencodingM.tuberculosisAg85AandESAT-6proteinwereamplifiedbyPCRfromM.tuberculosisH37RVstrainrespectively.ThefusiongeneesxA-fbpAwasgeneratedbygenesplicingwiththeoverlapextension(GeneSOEing)method.Therecombinantr685Agenewasclonedintothecorrespondingsitesof prokaryoticexpression vector pProEXHTb and eukaryotic expression vectorpcDNA3.1(+), resulting in recombinant plasmids named pPro685Aand pcD685A, respectively.TransformtheprokaryoticexpressionrecombinantplasmidpPro685AintothecompetenceEscherichiacoliBL21cells.Theproteinr68