结核杆菌ESAT-6重组核酸疫苗的制备及其免疫效果的初步研究
发布时间：2018-07-31 08:40
【摘要】： 结核病是一种古老的传染性疾病，20世纪90年代中期以来，随着流动人口的剧增、结核菌／艾滋病毒双重感染和耐药结核菌的出现，结核病在全球范围内又重新流行。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)，其中约10％的人口将发展为活动性结核，在HIV感染患者中发病率则更高。结核病已与艾滋病、血吸虫感染并列为世界三大传染性疾病。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严重的国家之一，，疫情形势严峻，防治任务艰巨。卡介苗作为目前唯一的预防结核杆菌感染的疫苗其保护效率仅在0％～85％之间。因此，发展新型抗结核病疫苗已成为医学界急待解决的一项重要问题。 结核杆菌是一种胞内寄生菌。机体细胞免疫应答能力的提高在抗结核菌感染中发挥着至关重要的作用。因此，如何提高结核杆菌疫苗的免疫效果，尤其是细胞免疫应答的作用是研究抗结核杆菌感染疫苗的关键所在。本研究基于上述考虑，开展了下列研究工作： 目的：构建可同时表达结核杆菌6kD早期分泌蛋白(ESAT-6)和Flt3配体(flt3-ligand，FL)的重组pIRES质粒，并在大鼠肾小球系膜细胞(GMC)中表达，为进一步研究结核杆菌DNA疫苗奠定实验基础。方法：采用PCR的方法，分别扩增出结核杆菌ESAT-6全长和人FL的胞外段基因。分别将这两部分基因定向克隆入真核双表达载体pIRES。用脂质体转染质粒至GMC中，Western blot鉴定其能否在GMC中表达。结果：核酸序列测定证实重组质粒构建正确，该重组质粒在体外GMC中能表达ESAT-6和FL两种蛋白。结论：成功构建了结核杆菌ESAT-6和FL双顺反子真核表达质粒，并在体外实现了两者的共表达。 第二部分结核杆菌ESAT-6和Flt3配体重组DNA疫苗诱导C57BL／6小鼠免疫应答的初步研究 目的：研究结核杆菌ESAT-6及FL共表达质粒对小鼠的免疫功能的影响。方法：将重组质粒免疫小鼠，检测小鼠体内特异性淋巴细胞增殖、Th1与Th2型细胞因子(IFN-γ、IL-2、IL-4、IL-10)分泌以及小鼠血清ESAT-6特异性IgG型抗体的水平。结果：联合ESAT-6与FL的DNA疫苗免疫效果高于单纯ESAT-6的DNA疫苗。以质粒pIRES-ESAT6-FL初次免疫，再用ESAT-6_(1-20)多肽加强的异源性组合免疫方案诱导的免疫效果最好。结论：结核杆菌ESAT-6及FL胞外段共表达质粒能够提高小鼠体内的细胞免疫功能。
[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.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392
本文编号：2155040
[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.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392
【参考文献】
相关期刊论文 前1条
1 杨永林,张学光,黄祖瑚;乙型肝炎病毒核心抗原及Flt3配体双表达核酸疫苗的构建与体外表达[J];南京医科大学学报(自然科学版);2005年05期
本文编号：2155040
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