【作者】 唐权；

【导师】 窦骏；

【作者基本信息】 东南大学 ， 免疫学， 2006， 硕士

【摘要】 结核病是由结核杆菌引起的慢性传染病,其中肺结核病最为常见。据WHO官方网站数据显示,全世界约有1/3的人口感染过结核杆菌,每年患结核病的人数约新增800万人,约有200万人死于结核病。而我国卫生部公布2005年全国法定报告传染病疫情显示:无论是报告发病数还是死亡数,肺结核均列第一位。HIV加速了结核杆菌的传播,而不规范的用药使大量结核耐药菌株出现,造成许多结核病人用现有抗结核药难以治愈。再加上传统疫苗卡介苗(Bacille calmette guerin,BCG)免疫效果总体下降,对其保护效率(0-80%)也存在争议,因此急需研制新型疫苗以及对已研制的疫苗制订新的免疫策略增强抗结核病效应。国内外学者在结核DNA疫苗方面进行了一系列的研究,其中Ag85A作为靶抗原取得了一定的效果,但单独以其作为DNA疫苗的靶抗原,仍需要一定的改进。我们实验室在前期的研究中成功构建了结核杆菌Ag85A和细胞因子GM-CSF共表达DNA疫苗,通过动物实验证实共表达DNA疫苗免疫组小鼠的CTL活性明显增强、特异性淋巴细胞明显增殖,NK细胞杀伤活性与对照组相比有一定提高。在此实验基础上,利用双启动子真核表达质粒pRSC,分别设计与构建新型结核杆菌DNA疫苗,为研究新型有效的结核杆菌DNA疫苗提供新的策略。本实验分为两个部分:一、基于结核杆菌Ag85A的共表达DNA疫苗的设计、构建与鉴定目的:利用双启动子真核表达质粒pRSC,分别构建结核杆菌Ag85A与细胞因子IL21共表达疫苗pRSC-mIL21-Ag85A以及结核杆菌Ag85A与细胞因子GM-CSF共表达疫苗pRSC-mGMCSF-Ag85A ,为研究新型结核杆菌DNA疫苗提供新的策略。方法:(1)pRSC-mIL21-Ag85A的构建,先用PCR方法从本室保存的重组质粒pcDNA3.1-mIL21中扩增出小鼠IL21基因序列并插入到质粒pRSC上成为pRSC-IL21,再用PCR方法从pIRES-Ag85A重组质粒中扩增出Ag85A基因,构建于pRSC-IL21重组质粒上,成为共表达DNA疫苗pRSC-mIL21-Ag85A;(2)pRSC-mGMCSF-Ag85A的构建,用PCR方法从pIRES-Ag85A重组质粒中扩增出Ag85A基因后构建于本室保存的重组质粒pRSC-mGMCSF上,得到共表达DNA疫苗pRSC-mGMCSF-Ag85A。结果:经酶切、基因测序及RT-PCR证实,重组DNA疫苗构建正确并能成功表达目的基因。结论:基于Ag85A与IL21、Ag85A与GM-CSF的共表达DNA疫苗的构建成功,为比较研究不同载体共表达DNA疫苗(pRSC-mGMCSF -Ag85A、pIRES-Ag85A-mGMCSF)以及基于不同细胞因子的共表达DNA疫苗(pRSC-mIL21-Ag85A、pRSC-mGMCSF-Ag85A)的抗结核免疫效应及筛选高效结核杆菌DNA疫苗奠定了基础。二、基于BCG Prime结核杆菌Ag85A/GM-CSF共表达DNA疫苗Boost免疫策略及其效应更多还原

【Abstract】 Tuberculosis(TB) is a chronic respiratory infectious disease that has afflicted humans for thousands of years. For more than 80 years, the bacillus Galmette-Guérin(BCG) vaccine has been the only licensed TB vaccine given to humans covering 86% of the world population. However, despite the use of BCG, TB remains a global epidemic with one-third of the world population being infected and per year morbidity of 8 million new cases and 2 million deaths were happened. Unluckily, incidence of TB is being accelerated by the spread of HIV/AIDS and the increasing Mycobacterium tuberculosis of multiple resistance to drug. Many studies suggests that BCG vaccination is protective against childhood meningeal tuberculosis and systemic forms of the disease. However, protective efficacy is variable against adult pulmonary disease (ranging from 0-80%) and the immunization effect of BCG wanes with time. Furthermore, BCG vaccine may cause severe complications in immunocompromised hosts. Thus, there is an urgent need for developing more safe, effective TB vaccines, new delivery methods and strategy that are able to confer potent protection. According to this thinking, we make our research on TB DNA vaccine in two parts as below.1. Design, construction and identification of eukaryotic co-expression vectors in view of TB DNA vaccineObjective: To Design, construct and identify two kind of eukaryotic co-expression recombinant DNA vaccines, one of which contains mouse interleukin 21(IL-21) and tuberculosis antigen 85A(Ag85A) and another contains mouse Granulocyte macrophage colony stimulating factor(GM-CSF) and Ag85A.Methods: The gene of IL-21 was amplified from recombinant plasmid pcDNA3.1-mIL21 by PCR and cloned directly into the plasmid pRSC, and form recombinant plasmid pRSC-IL21. The gene of Ag85A was amplified from the recombinant plasmid pIRES-Ag85A by PCR and cloned directly into the recombinant pRSC-IL21 and recombinant pRSC-GMCSF respectively again, and formed co-expression DNA vaccine pRSC-mIL21-Ag85A and pRSC-mGMCSF-Ag85A separately finally.Results: The eukaryotic co-expression DNA vaccine plasmid containing IL-21 and Ag85A or the DNA vaccine containing GMCSF and Ag85A had been constructed correctly by the identification of endonuclease digestion and DNA sequencing. The protein expression of co-expression DNA vaccine had been demonstrated correctly by RT-PCR method.Conclutions: The co-expression DNA vaccines of pRSC-mIL21-Ag85A and pRSC-mGMCSF-Ag85A have been constructed successfully. This study provides the possibility of further research on the development of new type anti-tuberculosis vaccines.2. Study of immune strategy and efficiency based on BCG priming and更多还原