表达结核杆菌融合抗原的重组酿酒酵母免疫效果研究

发布时间：2018-04-18 15:53

本文选题：结核病 + 疫苗　；参考：《复旦大学》2010年硕士论文

【摘要】：近年来结核病疫情严重,造成很大危害。疫苗是防治结核病最有效手段。卡介苗(BCG)是唯一得到批准并广泛使用的结核疫苗,但它的保护效果有限而且保护效果随着时间的增加而减弱。因此,开发研制更有效的结核疫苗十分迫切。目前正在研制的结核病疫苗主要有亚单位疫苗和活菌疫苗等,其中多个疫苗已进入临床试验阶段。近年出现了一种新的疫苗形式：重组酿酒酵母疫苗。重组酿酒酵母具有治疗性疫苗潜能,在防治丙型肝炎、结肠癌等传染病和肿瘤方面的应用研究取得了令人嘱目的进展。本研究以结核杆菌重要保护抗原Ag85B, ESAT-6为对象,利用本实验室具有自主知识产权的pHR酿酒酵母表达系统,分别构建了表达融合抗原Interferon-y-ESAT-6-Ag85B (IEA)和ESAT-6-Ag85B(EA)的重组酿酒酵母工程菌Y16/pHR-PAG-IEA (Yeast-IEA)和Y16/pHR-PAG-EA (Yeast-EA)。ELISA证实Yeast-IEA和Yeast-EA有效表达融合抗原IEA和EA。将热失活的重组酵母以皮下注射方式免疫C57/BL6小鼠,研究重组酿酒酵母免疫后对小鼠体液免疫、细胞免疫反应的影响，利用BCG攻击保护模型检测重组酵母疫苗对小鼠的保护作用。主要研究结果如下 (1)表达结核杆菌抗原的重组酵母能够刺激小鼠产生Ag85B特异性抗体IgG,随着免疫次数增加IgG效价明显提高，4周时Yeast-IEA给小鼠IgG效价显著高于Yeast-EA组和BCG组小鼠,对照PBS组和酿酒酵母Y16组小鼠无Ag85B特异性抗体产生。而且随着免疫次数增加,不仅IgG2a、IgG1水平提高,而且IgG2a/IgG1比值也明显增加。免疫4周时Yeast-IEA组IgG2a/IgG1比值显著高于Yeast-EA组和BCG组小鼠。上述结果说明重组酿酒酵母免疫后能够有效刺激小鼠的体液免疫反应,相对于融合抗原EA和BCG,融合抗原IEA更有利于激发小鼠免疫系统Thl型反应。 (2) ELISA检测结果显示重组酵母能够刺激小鼠产生IFN-γ、IL-2,对IL-4没有什么影响,其中Yeast-IEA免疫小鼠IFN-γIL-2显著高于Yeast-EA组和对照BCG组小鼠。与此对应地,流式细胞术检测结果显示Yeast-IEA组小鼠脾细胞中胞内产生IFN-γ或者IL-2的CD4+T淋巴细胞比例明显高于Yeast-EA组和对照BCG组小鼠。说明重组酵母能够刺激小鼠Th1型细胞免疫反应,与Yeast-EA相比,Yeast-IEA的刺激效果更强烈。 (3)重组酵母能够刺激小鼠抗原递呈细胞--树突状细胞(dendritic cell)成熟分化。重组酵母免疫小鼠脾细胞中CD80、CD86、MHC I、MHCII阳性树突状细胞比例明显增加。体外实验也证实酵母与脾脏分离的树突状细胞直接接触作用,导致成熟树突状细胞比例大幅提升。 (4)重组酵母免疫对小鼠感染分支杆菌BCG具有一定保护作用。小鼠经尾静脉大剂量注射BCG,与对照组相比,预先免疫了重组酵母的小鼠肺和脾脏部位感染的细菌数大幅度降低,说明重组酿酒酵母疫苗能够一定程度保护小鼠。上述研究结果表明,表达结核杆菌抗原的重组酿酒酵母皮下注射免疫可以激活小鼠体液免疫和细胞免疫系统,并能够降低分支杆菌感染效率。这些结果为其发展成为新型结核病防治疫苗奠定了基础。
[Abstract]:In recent years, the epidemic situation of tuberculosis is serious, causing great harm. The vaccine is the most effective means to treat TB. BCG (BCG) is the only approved and widely used anti TB vaccine, but its protective effect is limited and the protective effect decreases with the increase of time. Therefore, a more effective vaccine is urgently needed.
TB vaccines currently being developed are the main subunit vaccine and inactivated vaccines, including a number of vaccines have entered clinical trials. In recent years, the emergence of a new vaccine: recombinant yeast vaccine. Recombinant Saccharomyces cerevisiae with therapeutic vaccine potential in the prevention and treatment of hepatitis C, has made a great progress of application study on infectious disease and cancer of colon cancer and so on.
In this study, in order to protect the important antigen Ag85B of Mycobacterium tuberculosis, ESAT-6, pHR in yeast using the laboratory with independent intellectual property rights expression system were constructed. Expression of Interferon-y-ESAT-6-Ag85B fusion antigen (IEA) and ESAT-6-Ag85B (EA) of the recombinant Saccharomyces yeast engineering strain Y16/pHR-PAG-IEA (Yeast-IEA) and Y16/pHR-PAG-EA (Yeast-EA).ELISA confirmed that Yeast-IEA and Yeast-EA the effective expression of IEA fusion antigen and EA.
The heat loss of live recombinant yeast subcutaneously by immune C57/BL6 mice, immune humoral immune of recombinant Saccharomyces cerevisiae, affect the cellular immune response, the protective effect of detection of recombinant yeast vaccine by BCG attack protection model in mice. The main results are as follows
(1) the expression of recombinant Mycobacterium tuberculosis antigen can stimulate mice to produce specific antibodies against Ag85B IgG, with the increase in the number of immune IgG titre was increased, 4 weeks Yeast-IEA mice IgG titer was significantly higher than that of Yeast-EA group and BCG group, control group PBS and Saccharomyces cerevisiae Y16 mice without Ag85B specific antibody production and. With the increase of the immunization times, not only to improve the level of IgG2a, IgG1, and IgG2a/IgG1 ratio also increased significantly. By 4 weeks in group Yeast-IEA, IgG2a/IgG1 ratio was significantly higher than that of Yeast-EA group and BCG group. These results indicated that recombinant Saccharomyces cerevisiae after immunization can effectively stimulate humoral immune response in mice, compared with the EA fusion antigen and BCG fusion antigen, IEA more to stimulate the immune system of mice Thl type reaction.
(2) the results of ELISA showed that the recombinant yeast can stimulate mice to produce IFN- gamma, IL-2, not what impact on the IL-4, the Yeast-IEA IFN- gamma IL-2 immunized mice was significantly higher than that of Yeast-EA group and control group BCG mice. Corresponding with this, the results of flow cytometry showed that the percentage of CD4+T cells producing IFN- or IL-2 of mouse spleen cells in the Yeast-IEA group was significantly higher than that of the intracellular Yeast-EA group and control group. BCG mice showed that the recombinant yeast can stimulate mouse Th1 cell immune response, compared with Yeast-EA, Yeast-IEA stimulation effect is more intense.
(3) recombinant yeast can stimulate mouse antigen-presenting cells, dendritic cells (dendritic cell) differentiation. CD80, recombinant yeast immune spleen cells of mice in CD86, MHC I, the proportion of cells in MHCII positive dendritic increased significantly. In vitro experiments also confirmed the separation of yeast and spleen dendritic cells in direct contact, resulting in the proportion of mature dendritic cells increased dramatically.
(4) the recombinant yeast has certain protective effect on the immunity of mice infected with Mycobacterium BCG. Mice intravenously injected with high dose of BCG, compared with the control group, the number of bacteria in large scale immunization of recombinant yeast mouse spleen and lung infection decreased, indicating recombinant yeast vaccine protects mice to a certain extent.
The results of the study showed that the expression of Mycobacterium tuberculosis antigen immunized by subcutaneous injection of recombinant Saccharomyces cerevisiae can activate both humoral and cellular immune system in mice, and can reduce the efficiency of mycobacterial infection. These results for the development of a new TB vaccine laid the foundation.

【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

【共引文献】