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

发布时间：2018-05-21 09:36

本文选题：结核杆菌 + ESAT-6　；参考：《南京医科大学》2008年硕士论文

【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。
[Abstract]:Part I Construction and in Vitro expression of ESAT-6 Antigen Multiepitope DNA Vaccine of Mycobacterium tuberculosis Aim: to construct three recombinant plasmids containing T cell epitopes and Flt3 ligand genes of ESAT-6 antigen of Mycobacterium tuberculosis and express them in rat glomerular Mesangial cells. Methods: the T cell epitopes of ESAT-6 antigen of Mycobacterium tuberculosis were predicted by computer software. Three T cell epitopes were selected and Ala-Tyr-AAY sequence was used as the junction. The whole gene sequence was synthesized and cloned into eukaryotic double expression vector pIRES and plasmid pIRES-FL. After restriction endonuclease analysis and sequencing, PEI was transfected into GMCs cells and its expression was identified by Western blot in vitro. Results: nucleic acid sequencing confirmed that the recombinant plasmid was constructed correctly and Western blot confirmed that the recombinant plasmid could express fusion protein in GMCs cells in vitro. Conclusion: the recombinant plasmid of ESAT-6 antigen polyepitope gene of Mycobacterium tuberculosis was successfully constructed. The second part: a preliminary study on the immune response of C57BL/6 mice induced by multi-epitope DNA vaccine against ESAT-6 antigen of Mycobacterium tuberculosis Aim: to study the effect of polyepitope gene of ESAT-6 antigen of Mycobacterium tuberculosis and co expression plasmid of extracellular segment of FL on immune function of mice. Methods: the recombinant plasmid pIRES-THS-TH-FL was used to immunize mice. The secretion of Th1 and Th2 type cytokines (IFN- 纬, IL-2IL-4, IL-4, IL-10) and the level of serum IgG2a- IgG1 antibody in mice were detected by immunizing mice with the recombinant plasmid pIRES-TH-FL. Results: the Th1 type response level of mice immunized with pIRES-TH plasmid up-regulated the level of Th2 type response. The immune effect of pIRES-TH-FL recombinant DNA vaccine combined with FL gene was higher than that of DNA vaccine with ESAT-6 epitope alone. Conclusion: the co-expression plasmid of ESAT-6 epitope and extracellular segment of FL can improve the cellular immune function in mice.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

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