结核杆菌ESAT-6抗原Th1型表位与FL重组DNA疫苗免疫效果及其保护作用的研究

发布时间：2018-04-30 18:03

本文选题：结核杆菌 + ESAT-6　；参考：《南京医科大学》2011年硕士论文

【摘要】：结核病是一种严重危害人类健康的重要传染性疾病,近年来结核病在世界范围内重新流行。根据WHO最新报告,目前全球超过1/3的人口感染结核杆菌(约20亿),在HIV感染患者中发病率则更高。我国是全球22个结核高负担国家之一,也是耐药结核菌疫情最严重的国家之一,疫情形势严峻,防治任务艰巨。卡介苗( Mycobacterium bovis Bacillus Calmette-Guérin, BCG )是当今唯一的预防结核杆菌(Mycobacterium tuberculosis, M. tb )感染的疫苗,它对婴幼儿有较好的保护作用,但对成年人却效果不佳,其保护效率仅在0%-80%之间。因此,发展新型抗结核病疫苗已成为医学界亟待解决的一项重要课题。本实验室前期已成功构建了结核杆菌6kD早期分泌蛋白(early secreted antigenic target, ESAT-6)抗原Th1型优势表位与fms-like tyrosine kinase 3 ligand(FL)重组的DNA疫苗,本课题旨在对该疫苗的免疫效果和保护效率进行鉴定与评判,因此,实验主要围绕以下两方面进行。第一部分结核杆菌ESAT-6抗原Th1型表位与FL重组DNA疫苗prime-boost策略免疫效果的鉴定目的:研究结核杆菌ESAT-6抗原Th1型表位与FL重组质粒(pIRES-TH-FL)对小鼠免疫功能的影响。方法:将质粒pIRES-TH-FL采用prime-boost策略免疫小鼠,检查小鼠体内特异性淋巴细胞增殖;脾细胞培养上清和肺泡灌洗液中Th1与Th2型细胞因子(IFN-γ、IL-12、IL-4、IL-10)的分泌;小鼠血清ESAT-6特异性IgG型抗体的水平;IFN-γ+T细胞的频率,以及体内CTL的杀伤功能。结果:联合ESAT-6蛋白的Th1型表位与FL的DNA疫苗能诱导偏向Th1型的免疫应答,prime-boost免疫方案可诱导出更优于BCG的免疫效果。结论:结核杆菌ESAT-6抗原Th1型表位与FL重组DNA疫苗能够明显上调小鼠体内的细胞免疫应答水平。第二部分结核杆菌ESAT-6抗原Th1型表位与FL重组DNA疫苗prime-boost策略保护作用的评价目的:研究结核杆菌ESAT-6抗原Th1型表位与FL重组质粒(pIRES-TH-FL)对小鼠抵抗结核杆菌的保护作用。方法:将质粒pIRES-TH-FL免疫C57BL/6小鼠后,再用结核杆菌标准株H37Rv对其进行攻击,从菌落计数和病理损害方面评价疫苗的保护作用。结果:结核杆菌ESAT-6抗原Th1型表位与FL重组DNA疫苗免疫小鼠后,小鼠肺和脾中的荷菌量显著减少;HE染色和免疫组化结果显示,其炎症浸润程度明显减轻。Prime-boost组与单独的DNA疫苗免疫组相比,保护效果更为明显。结论:用ESAT-6抗原Th1型表位与FL重组DNA疫苗免疫小鼠能够抵抗结核杆菌的攻击,提供有效的保护作用,而prime-boost策略则能进一步增强这种保护效果。
[Abstract]:Tuberculosis (TB) is an important infectious disease that seriously endangers human health. According to the latest WHO report, more than a third of the world's population is currently infected with Mycobacterium tuberculosis (2 billion kgs), with a higher incidence among patients with HIV infection. China is one of the 22 countries with high TB burden in the world and one of the most serious cases of drug-resistant tuberculosis. The epidemic situation is severe and the task of prevention and control is arduous. BCG vaccine (Mycobacterium bovis Bacillus Calmette-Gu 茅 rin, BCG) is the only vaccine to prevent Mycobacterium tuberculosis, M. tb) infection from Mycobacterium tuberculosis. BCG vaccine has a good protective effect on infants, but not on adults. The protective efficiency of BCG vaccine is only between 0% and 80%. Therefore, the development of new anti-tuberculosis vaccine has become an important subject to be solved. In our laboratory, we successfully constructed the recombinant DNA vaccine of Th1 type epitope and fms-like tyrosine kinase 3 ligand FLL, the early secreted antigenic target, ESAT-6) antigen of Mycobacterium tuberculosis. The purpose of this study was to identify and evaluate the immune effect and protective efficiency of this vaccine. Therefore, the experiment mainly revolves around the following two aspects. Part I Identification of Th1 epitopes of Mycobacterium tuberculosis ESAT-6 antigen and prime-boost strategy of FL recombinant DNA vaccine Aim: to study the effect of ESAT-6 antigen Th1 epitope of Mycobacterium tuberculosis and recombinant plasmid pIRES-TH-FLL on immune function in mice. Methods: plasmid pIRES-TH-FL was used to immunize mice with prime-boost strategy to examine the specific lymphocyte proliferation in vivo, the secretion of Th1 and Th2 type cytokine IL-12, IL-4 and IL-10 in the supernatant of splenocyte culture and alveolar lavage fluid, and the expression of IL-10 in spleen cell culture supernatant and alveolar lavage fluid. The frequency of IFN- 纬 T cells and the killing function of CTL in vivo were determined by the level of serum ESAT-6 specific IgG antibody in mice. Results: the combination of Th1 epitope of ESAT-6 protein and DNA vaccine of FL could induce a prime-boost immune response in favour of Th1 type, which was better than that of BCG. Conclusion: Mycobacterium tuberculosis ESAT-6 antigen Th1 epitope and FL recombinant DNA vaccine can obviously upregulate the level of cellular immune response in mice. The second part: evaluation of Th1 epitope of Mycobacterium tuberculosis ESAT-6 antigen and the protective effect of prime-boost strategy of FL recombinant DNA vaccine Aim: to study the protective effect of ESAT-6 antigen Th1 epitope of Mycobacterium tuberculosis and recombinant plasmid pIRES-TH-FLL on resistance to Mycobacterium tuberculosis in mice. Methods: C57BL/6 mice were immunized with plasmid pIRES-TH-FL and then attacked with the standard H37Rv strain of Mycobacterium tuberculosis to evaluate the protective effect of the vaccine from the aspects of colony count and pathological damage. Results: after immunizing mice with Th1 epitopes of Mycobacterium tuberculosis ESAT-6 antigen and FL recombinant DNA vaccine, the amount of mycobacterium in the lung and spleen of mice decreased significantly, and the results of HE staining and immunohistochemistry showed that: 1. The degree of inflammatory infiltration was significantly reduced in the. Prime-boost group, and the protective effect was more obvious than that in the single DNA vaccine immunization group. Conclusion: mice immunized with ESAT-6 antigen Th1 epitope and FL recombinant DNA vaccine can resist the attack of Mycobacterium tuberculosis and provide effective protection, and prime-boost strategy can further enhance this protective effect.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

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