结核杆菌ESAT-6抗原多表位DNA疫苗的制备及其免疫效果的初步研究

【作者】 陈霞；

【导师】 王迎伟；

【作者基本信息】 南京医科大学 ， 免疫学， 2008， 硕士

【摘要】 第一部分结核杆菌ESAT-6抗原多表位DNA疫苗的构建与体外表达的鉴定目的:构建含3个结核杆菌ESAT-6抗原T细胞表位及Flt3配体基因的重组质粒,并使其在大鼠肾小球系膜细胞(GMCs)中表达。方法:用计算机软件预测结核杆菌ESAT-6抗原的T细胞表位谱,选取3个T细胞表位,并以Ala-Ala-Tyr(AAY)序列作为接头,合成全基因序列,定向克隆入真核双表达载体pIRES及质粒pIRES-FL。在酶切分析与序列测定后,用PEI转染至GMCs细胞,并行Western blot鉴定其体外表达。结果:核酸序列测定证实重组质粒构建正确,Westernblot证实该重组质粒能在体外GMCs细胞中表达融合蛋白。结论:成功构建了结核杆菌ESAT-6抗原多表位基因重组质粒。第二部分结核杆菌ESAT-6抗原多表位DNA疫苗诱导C57BL/6小鼠免疫应答的初步研究目的:研究结核杆菌ESAT-6抗原多表位基因及FL胞外段共表达质粒对小鼠的免疫功能的影响。方法:将pIRES-TH、pIRES-TH-FL重组质粒免疫小鼠,检测小鼠体内特异性淋巴细胞增殖、Th1与Th2型细胞因子(IFN-γ、IL-2、IL-4、IL-10)分泌以及小鼠血清ESAT-6特异性IgG2a、IgG1型抗体的水平。结果:经pIRES-TH质粒免疫的小鼠Th1型应答水平上调,Th2型应答水平下降。联合FL基因的pIRES-TH-FL重组DNA疫苗免疫效果高于单纯ESAT-6表位的DNA疫苗。结论:结核杆菌ESAT-6表位及FL胞外段共表达质粒能够提高小鼠体内的细胞免疫功能。更多还原

【Abstract】 PARTⅠConstruction and expression of multiepitope DNA vaccine on ESAT-6 antigen of Mycobacterium tuberculosis in vitroObjective: To construct recombinant plasmids containing 3 T cell epitopes on ESAT-6 antigen of Mycobacterium tuberculosis and extra-cellular fragment of Flt3 ligand (FL) genes, and to express them in rat glomerular mesangial cells (GMCs). Methods: The amino acid sequence of ESAT-6 antigen was analysed using predictive algorithms and 3 T cell epitopes were predicted. The oligonucleotide encoding and the linker Ala-Ala-Tyr (AAY) were synthesized and inserted into the bicistronic vector pIRES and pIRES-FL. The recombinant plasmids were transfected into GMCs, the recombinant proteins expressing in GMCs was examined by Western blot. Results: The recombinant plasmids were verified by sequencing and the recombinant fusion proteins were identified by Western blot. Conclusion: The multiepitope DNA vaccine from ESAT-6 antigen of Mycobacterium tuberculosis was constructed and expressed successfully. PARTⅡImmunol effects of gene vaccine constituted with epitopes of tuberculosis ESAT-6 antigen and Flt-3 ligand in miceObjective: To study the immunol effects of gene vaccine constituted with epitopes of tuberculosis ESAT-6 antigen and Flt-3 ligand in mice. Methods: C57BL/6 mice were inoculated with 100μg of pIRES-TH and pIRES-TH-FL each time and a total of 3 times as a whole. The spleen cell proliferation responses to antigen, the Th1/Th2 responses and the titer of ESAT-6 specific IgG2a/IgGl antibody were examined. Results: The spleen cell proliferation responses, the Th1 responses and the antibody level of ESAT-6 specific IgG2a/IgG1 of the pIRES-TH-FL group are higher than those of control groups. Conclusion: pIRES-TH-FL can effectively elevate the cellular immunity of C57BL/6 mice.更多还原