本文選題：結(jié)核桿菌 + ESAT-6��； 參考：《南京醫(yī)科大學》2011年碩士論文

【摘要】：結(jié)核病是一種嚴重危害人類健康的重要傳染性疾病,近年來結(jié)核病在世界范圍內(nèi)重新流行。根據(jù)WHO最新報告,目前全球超過1/3的人口感染結(jié)核桿菌(約20億),在HIV感染患者中發(fā)病率則更高。我國是全球22個結(jié)核高負擔國家之一,也是耐藥結(jié)核菌疫情最嚴重的國家之一,疫情形勢嚴峻,防治任務(wù)艱巨�？ń槊�( Mycobacterium bovis Bacillus Calmette-Guérin, BCG )是當今唯一的預(yù)防結(jié)核桿菌(Mycobacterium tuberculosis, M. tb )感染的疫苗,它對嬰幼兒有較好的保護作用,但對成年人卻效果不佳,其保護效率僅在0%-80%之間。因此,發(fā)展新型抗結(jié)核病疫苗已成為醫(yī)學界亟待解決的一項重要課題。 本實驗室前期已成功構(gòu)建了結(jié)核桿菌6kD早期分泌蛋白(early secreted antigenic target, ESAT-6)抗原Th1型優(yōu)勢表位與fms-like tyrosine kinase 3 ligand(FL)重組的DNA疫苗,本課題旨在對該疫苗的免疫效果和保護效率進行鑒定與評判,因此,實驗主要圍繞以下兩方面進行。 第一部分結(jié)核桿菌ESAT-6抗原Th1型表位與FL重組DNA疫苗prime-boost策略免疫效果的鑒定 目的:研究結(jié)核桿菌ESAT-6抗原Th1型表位與FL重組質(zhì)粒(pIRES-TH-FL)對小鼠免疫功能的影響。方法:將質(zhì)粒pIRES-TH-FL采用prime-boost策略免疫小鼠,檢查小鼠體內(nèi)特異性淋巴細胞增殖;脾細胞培養(yǎng)上清和肺泡灌洗液中Th1與Th2型細胞因子(IFN-γ、IL-12、IL-4、IL-10)的分泌;小鼠血清ESAT-6特異性IgG型抗體的水平;IFN-γ+T細胞的頻率,以及體內(nèi)CTL的殺傷功能。結(jié)果:聯(lián)合ESAT-6蛋白的Th1型表位與FL的DNA疫苗能誘導(dǎo)偏向Th1型的免疫應(yīng)答,prime-boost免疫方案可誘導(dǎo)出更優(yōu)于BCG的免疫效果。結(jié)論:結(jié)核桿菌ESAT-6抗原Th1型表位與FL重組DNA疫苗能夠明顯上調(diào)小鼠體內(nèi)的細胞免疫應(yīng)答水平。 第二部分結(jié)核桿菌ESAT-6抗原Th1型表位與FL重組DNA疫苗prime-boost策略保護作用的評價 目的:研究結(jié)核桿菌ESAT-6抗原Th1型表位與FL重組質(zhì)粒(pIRES-TH-FL)對小鼠抵抗結(jié)核桿菌的保護作用。方法:將質(zhì)粒pIRES-TH-FL免疫C57BL/6小鼠后,再用結(jié)核桿菌標準株H37Rv對其進行攻擊,從菌落計數(shù)和病理損害方面評價疫苗的保護作用。結(jié)果:結(jié)核桿菌ESAT-6抗原Th1型表位與FL重組DNA疫苗免疫小鼠后,小鼠肺和脾中的荷菌量顯著減少;HE染色和免疫組化結(jié)果顯示,其炎癥浸潤程度明顯減輕。Prime-boost組與單獨的DNA疫苗免疫組相比,保護效果更為明顯。結(jié)論:用ESAT-6抗原Th1型表位與FL重組DNA疫苗免疫小鼠能夠抵抗結(jié)核桿菌的攻擊,提供有效的保護作用,而prime-boost策略則能進一步增強這種保護效果。
[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- 緯 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.
【學位授予單位】：南京醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R392

【參考文獻】

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1 陳霞;徐聞歡;徐娟;趙聃;王迎偉;;結(jié)核桿菌ESAT-6抗原多表位DNA疫苗的構(gòu)建與表達[J];南京醫(yī)科大學學報(自然科學版);2008年04期

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本文編號：1825599