【摘要】： 結(jié)核病是一種古老的傳染性疾病，20世紀(jì)90年代中期以來(lái)，隨著流動(dòng)人口的劇增、結(jié)核菌／艾滋病毒雙重感染和耐藥結(jié)核菌的出現(xiàn)，結(jié)核病在全球范圍內(nèi)又重新流行。根據(jù)WHO最新報(bào)告，目前全球超過(guò)1／3的人口感染結(jié)核桿菌(約20億)，其中約10％的人口將發(fā)展為活動(dòng)性結(jié)核，在HIV感染患者中發(fā)病率則更高。結(jié)核病已與艾滋病、血吸蟲(chóng)感染并列為世界三大傳染性疾病。我國(guó)是全球22個(gè)結(jié)核高負(fù)擔(dān)國(guó)家之一，也是耐藥結(jié)核菌疫情嚴(yán)重的國(guó)家之一，，疫情形勢(shì)嚴(yán)峻，防治任務(wù)艱巨�？ń槊缱鳛槟壳拔ㄒ坏念A(yù)防結(jié)核桿菌感染的疫苗其保護(hù)效率僅在0％～85％之間。因此，發(fā)展新型抗結(jié)核病疫苗已成為醫(yī)學(xué)界急待解決的一項(xiàng)重要問(wèn)題。 結(jié)核桿菌是一種胞內(nèi)寄生菌。機(jī)體細(xì)胞免疫應(yīng)答能力的提高在抗結(jié)核菌感染中發(fā)揮著至關(guān)重要的作用。因此，如何提高結(jié)核桿菌疫苗的免疫效果，尤其是細(xì)胞免疫應(yīng)答的作用是研究抗結(jié)核桿菌感染疫苗的關(guān)鍵所在。本研究基于上述考慮，開(kāi)展了下列研究工作： 目的：構(gòu)建可同時(shí)表達(dá)結(jié)核桿菌6kD早期分泌蛋白(ESAT-6)和Flt3配體(flt3-ligand，F(xiàn)L)的重組pIRES質(zhì)粒，并在大鼠腎小球系膜細(xì)胞(GMC)中表達(dá)，為進(jìn)一步研究結(jié)核桿菌DNA疫苗奠定實(shí)驗(yàn)基礎(chǔ)。方法：采用PCR的方法，分別擴(kuò)增出結(jié)核桿菌ESAT-6全長(zhǎng)和人FL的胞外段基因。分別將這兩部分基因定向克隆入真核雙表達(dá)載體pIRES。用脂質(zhì)體轉(zhuǎn)染質(zhì)粒至GMC中，Western blot鑒定其能否在GMC中表達(dá)。結(jié)果：核酸序列測(cè)定證實(shí)重組質(zhì)粒構(gòu)建正確，該重組質(zhì)粒在體外GMC中能表達(dá)ESAT-6和FL兩種蛋白。結(jié)論：成功構(gòu)建了結(jié)核桿菌ESAT-6和FL雙順?lè)醋诱婧吮磉_(dá)質(zhì)粒，并在體外實(shí)現(xiàn)了兩者的共表達(dá)。 第二部分結(jié)核桿菌ESAT-6和Flt3配體重組DNA疫苗誘導(dǎo)C57BL／6小鼠免疫應(yīng)答的初步研究 目的：研究結(jié)核桿菌ESAT-6及FL共表達(dá)質(zhì)粒對(duì)小鼠的免疫功能的影響。方法：將重組質(zhì)粒免疫小鼠，檢測(cè)小鼠體內(nèi)特異性淋巴細(xì)胞增殖、Th1與Th2型細(xì)胞因子(IFN-γ、IL-2、IL-4、IL-10)分泌以及小鼠血清ESAT-6特異性IgG型抗體的水平。結(jié)果：聯(lián)合ESAT-6與FL的DNA疫苗免疫效果高于單純ESAT-6的DNA疫苗。以質(zhì)粒pIRES-ESAT6-FL初次免疫，再用ESAT-6_(1-20)多肽加強(qiáng)的異源性組合免疫方案誘導(dǎo)的免疫效果最好。結(jié)論：結(jié)核桿菌ESAT-6及FL胞外段共表達(dá)質(zhì)粒能夠提高小鼠體內(nèi)的細(xì)胞免疫功能。
[Abstract]:Tuberculosis is an ancient infectious disease. Since the mid 1990s, with the surge of floating population, tuberculosis / HIV double infection and drug-resistant tuberculosis, tuberculosis is re prevalent worldwide. According to the latest WHO report, over 1 / 3 of the world's population is infected with Mycobacterium tuberculosis (about 2 billion), of which about 2 billion 10% of the population will develop into active tuberculosis, which has a higher incidence in HIV infected patients. Tuberculosis has been listed as the three largest infectious disease in the world with AIDS and Schistosoma infection. China is one of the 22 countries with high tuberculosis burden in the world, and one of the countries with a serious epidemic of drug-resistant tuberculosis. The epidemic situation is severe and the task of prevention and control is arduous. As the only vaccine to prevent Mycobacterium tuberculosis, the protection efficiency of the vaccine is only between 0% and 85%. Therefore, the development of a new type of anti tuberculosis vaccine has become an important problem to be solved urgently in the medical field.
Mycobacterium tuberculosis is a kind of intracellular parasitic bacteria. The enhancement of cellular immune response ability plays a vital role in anti tuberculosis infection. Therefore, how to improve the immune effect of TB vaccine, especially the role of cellular immune response, is the key to the study of the vaccine against Mycobacterium tuberculosis. This study is based on the above considerations. The following research work has been carried out:
Objective: to construct a recombinant pIRES plasmid that can simultaneously express the early secretory protein (ESAT-6) and Flt3 ligand (Flt3-ligand, FL) of Mycobacterium tuberculosis 6kD and to express it in rat glomerular mesangial cells (GMC), and lay an experimental basis for further study of the Mycobacterium tuberculosis DNA vaccine. Methods: the ESAT-6 full length of Mycobacterium tuberculosis was amplified by PCR. The extracellular segment genes of human FL were cloned into eukaryotic double expression vector, pIRES., transfected into plasmids with liposomes to GMC, and Western blot could be expressed in GMC. Results: nucleic acid sequencing confirmed that the recombinant plasmid was constructed correctly, and the recombinant plasmid could express ESAT-6 and FL two proteins in extracellular GMC. The eukaryotic expression plasmid of ESAT-6 and FL BIS was successfully constructed and co expression was achieved in vitro.
The second part is a preliminary study on the immune response induced by recombinant DNA vaccine of Mycobacterium tuberculosis ESAT-6 and Flt3 ligand in C57BL / 6 mice.
Objective: To study the effect of Mycobacterium tuberculosis ESAT-6 and FL co expression plasmid on the immune function of mice. Methods: the recombinant plasmid was immunized in mice to detect the specific lymphocyte proliferation in mice, the secretion of Th1 and Th2 type cytokines (IFN-, IL-2, IL-4, IL-10) and the level of ESAT-6 specific IgG type antibody in mice blood. The immunization effect of 6 and FL DNA vaccine was higher than that of the DNA vaccine with simple ESAT-6. The immunization with the initial immunization of plasmid pIRES-ESAT6-FL and the heterologous Combination Immunization with ESAT-6_ (1-20) polypeptide was the best. Conclusion: the co expression plasmids of Mycobacterium tuberculosis ESAT-6 and FL extracellular segment can improve the cellular immune function of mice.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類(lèi)號(hào)】：R392

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 楊永林,張學(xué)光,黃祖瑚;乙型肝炎病毒核心抗原及Flt3配體雙表達(dá)核酸疫苗的構(gòu)建與體外表達(dá)[J];南京醫(yī)科大學(xué)學(xué)報(bào)(自然科學(xué)版);2005年05期

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