本文選題：結(jié)核病 + 疫苗��； 參考：《復(fù)旦大學》2010年碩士論文

【摘要】：近年來結(jié)核病疫情嚴重,造成很大危害。疫苗是防治結(jié)核病最有效手段。卡介苗(BCG)是唯一得到批準并廣泛使用的結(jié)核疫苗,但它的保護效果有限而且保護效果隨著時間的增加而減弱。因此,開發(fā)研制更有效的結(jié)核疫苗十分迫切。 目前正在研制的結(jié)核病疫苗主要有亞單位疫苗和活菌疫苗等,其中多個疫苗已進入臨床試驗階段。近年出現(xiàn)了一種新的疫苗形式：重組釀酒酵母疫苗。重組釀酒酵母具有治療性疫苗潛能,在防治丙型肝炎、結(jié)腸癌等傳染病和腫瘤方面的應(yīng)用研究取得了令人囑目的進展。 本研究以結(jié)核桿菌重要保護抗原Ag85B, ESAT-6為對象,利用本實驗室具有自主知識產(chǎn)權(quán)的pHR釀酒酵母表達系統(tǒng),分別構(gòu)建了表達融合抗原Interferon-y-ESAT-6-Ag85B (IEA)和ESAT-6-Ag85B(EA)的重組釀酒酵母工程菌Y16/pHR-PAG-IEA (Yeast-IEA)和Y16/pHR-PAG-EA (Yeast-EA)。ELISA證實Yeast-IEA和Yeast-EA有效表達融合抗原IEA和EA。 將熱失活的重組酵母以皮下注射方式免疫C57/BL6小鼠,研究重組釀酒酵母免疫后對小鼠體液免疫、細胞免疫反應(yīng)的影響，利用BCG攻擊保護模型檢測重組酵母疫苗對小鼠的保護作用。主要研究結(jié)果如下 (1)表達結(jié)核桿菌抗原的重組酵母能夠刺激小鼠產(chǎn)生Ag85B特異性抗體IgG,隨著免疫次數(shù)增加IgG效價明顯提高，4周時Yeast-IEA給小鼠IgG效價顯著高于Yeast-EA組和BCG組小鼠,對照PBS組和釀酒酵母Y16組小鼠無Ag85B特異性抗體產(chǎn)生。而且隨著免疫次數(shù)增加,不僅IgG2a、IgG1水平提高,而且IgG2a/IgG1比值也明顯增加。免疫4周時Yeast-IEA組IgG2a/IgG1比值顯著高于Yeast-EA組和BCG組小鼠。上述結(jié)果說明重組釀酒酵母免疫后能夠有效刺激小鼠的體液免疫反應(yīng),相對于融合抗原EA和BCG,融合抗原IEA更有利于激發(fā)小鼠免疫系統(tǒng)Thl型反應(yīng)。 (2) ELISA檢測結(jié)果顯示重組酵母能夠刺激小鼠產(chǎn)生IFN-γ、IL-2,對IL-4沒有什么影響,其中Yeast-IEA免疫小鼠IFN-γIL-2顯著高于Yeast-EA組和對照BCG組小鼠。與此對應(yīng)地,流式細胞術(shù)檢測結(jié)果顯示Yeast-IEA組小鼠脾細胞中胞內(nèi)產(chǎn)生IFN-γ或者IL-2的CD4+T淋巴細胞比例明顯高于Yeast-EA組和對照BCG組小鼠。說明重組酵母能夠刺激小鼠Th1型細胞免疫反應(yīng),與Yeast-EA相比,Yeast-IEA的刺激效果更強烈。 (3)重組酵母能夠刺激小鼠抗原遞呈細胞--樹突狀細胞(dendritic cell)成熟分化。重組酵母免疫小鼠脾細胞中CD80、CD86、MHC I、MHCII陽性樹突狀細胞比例明顯增加。體外實驗也證實酵母與脾臟分離的樹突狀細胞直接接觸作用,導(dǎo)致成熟樹突狀細胞比例大幅提升。 (4)重組酵母免疫對小鼠感染分支桿菌BCG具有一定保護作用。小鼠經(jīng)尾靜脈大劑量注射BCG,與對照組相比,預(yù)先免疫了重組酵母的小鼠肺和脾臟部位感染的細菌數(shù)大幅度降低,說明重組釀酒酵母疫苗能夠一定程度保護小鼠。 上述研究結(jié)果表明,表達結(jié)核桿菌抗原的重組釀酒酵母皮下注射免疫可以激活小鼠體液免疫和細胞免疫系統(tǒng),并能夠降低分支桿菌感染效率。這些結(jié)果為其發(fā)展成為新型結(jié)核病防治疫苗奠定了基礎(chǔ)。
[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.

【學位授予單位】：復(fù)旦大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R392

【共引文獻】

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