结核杆菌蛋白亚单位疫苗的免疫学评价

发布时间：2018-04-30 17:00

本文选题：结核分枝杆菌 + 融合蛋白　；参考：《复旦大学》2011年硕士论文

【摘要】：结核病是一种古老的疾病,早在十七世纪就已经存在。目前,由于全世界感染人口基数大,耐药结核病例大大增加和HIV/TB共感染情况严重,结核病正对人类构成巨大的威胁。为了预防结核病,全球已经半个多世纪前广泛接种了卡介苗BCG,但是BCG的免疫保护效果并不理想。随着生物技术的发展,很多新型疫苗正在陆续开发,例如重组卡介苗,减毒疫苗,蛋白疫苗和DNA疫苗等。他们各自都有不同的优势,但是否可以超越BCG的免疫保护效果还有待临床证明。在人群中给予结核杆菌疫苗免疫是防治结核感染、控制局部爆发流行最有效的措施。结核疫苗主要有两大类：活的结核杆菌疫苗和亚单位疫苗。结核亚单位疫苗虽然免疫原性没有活的结核杆菌疫苗强,但其免疫效力不受预先接触环境分枝杆菌和结核分枝杆菌的影响,而且背景清楚,安全性好,制备简单,可以用于成人强化免疫,是控制结核病爆发的理想疫苗。亚单位疫苗有三种形式：蛋白质疫苗、DNA疫苗和以病毒为载体的疫苗[1,2],其中蛋白质疫苗相对最为安全,易于被人们接受。目前,已经有超过20种的不同抗原用于诱导抗结核的免疫应答[3,4]. 分泌性抗原Ag85a属于Ag85蛋白家族,被认为具有较好的免疫原性。结核杆菌是一种巨噬细胞内寄生菌。结核病的发生与免疫系统密切相关。故人体对结核杆菌感染的免疫,以细胞免疫为主。在抗结核感染中,CD4+和CD8+T细胞起主要作用。结核杆菌活化的T细胞所释放出的淋巴因子,包括白介素-2(IL-2),γ-干扰素(IFN-γ),它们直接或间接杀伤结核杆菌。本研究采用来源于结核杆菌的重要抗原Ag85a和结核免疫相关的细胞因子IFN-y与IL-2,以结核分枝杆菌标准株H37R v基因组DNA为模板,扩增Ag85a基因,分别与鼠源IFN-y和IL-2的基因融合与表达质粒pET28a构建成重组质粒pet28a-Ag85a-IFN-γ和pet28a-Ag85a-IL-2。在大肠杆菌BL21(DE3)中表达去除信号肽的融合蛋白,纯化得到Ag85a—IFN-γ和Ag85a—IL-2的融合蛋白。用该蛋白皮下免疫C57BL/6小鼠,并进行免疫效果评价,以期筛选高效的加强免疫的蛋白亚单位疫苗。通过ELISA方法检测血清中IgG,IgG1和IgG2c水平,我们发现融合蛋白Ag85a-IFN-γ和Ag85a-IL-2实验组的免疫球蛋白水平均高于单独抗原,并且融合蛋白组IgG2c/IgG1的比值大于单独抗原,并且Ag85a—IL-2的免疫保护效果优于Ag85a-IFN-γ;流式细胞术检测胞内特异性CD4+和CD8T+细胞,Ag85a-IFN-γ和Ag85a-IL-2实验组CD8+/CD4+比例高于单独抗原。本实验成功构建并表达了Ag85a-IFN-γ和Ag85a-IL-2的融合蛋白,融合蛋白的免疫效果优于单独Ag85a抗原,并且Ag85a—IL-2的免疫效果优于Ag85a—IFN-y。
[Abstract]:Tuberculosis is an ancient disease that existed as early as the seventeenth century. At present, because of the large number of infected people in the world, the increase of drug-resistant tuberculosis cases and the serious co-infection of HIV/TB, tuberculosis is posing a great threat to human beings. In order to prevent tuberculosis, BCG was widely inoculated around the world more than half a century ago, but the immune protection effect of BCG is not satisfactory. With the development of biotechnology, many new vaccines are being developed, such as recombinant BCG vaccine, attenuated vaccine, protein vaccine and DNA vaccine. They each have different advantages, but whether they can outperform the immune protection of BCG has yet to be clinically proven. Immunization with Mycobacterium tuberculosis vaccine in the population is the most effective measure to prevent and treat tuberculosis infection and to control the local outbreak. There are two main types of TB vaccine: live Mycobacterium tuberculosis vaccine and subunit vaccine. Although the immunogenicity of tuberculosis subunit vaccine is not as strong as that of live mycobacterium tuberculosis vaccine, its immune efficacy is not affected by the preexposure to environmental mycobacterium and mycobacterium tuberculosis, and the background is clear, the safety is good, and the preparation is simple. It is an ideal vaccine to control the outbreak of tuberculosis. There are three forms of subunit vaccine: protein vaccine DNA vaccine and virus-vector vaccine [1], in which protein vaccine is the safest and is easy to be accepted. At present, more than 20 different antigens have been used to induce anti-tuberculosis immune response. Secretory antigen Ag85a belongs to Ag85 protein family and is considered to have good immunogenicity. Mycobacterium tuberculosis is a parasite in macrophages. Tuberculosis is closely related to the immune system. Therefore, the human body to Mycobacterium tuberculosis infection immunity, mainly cellular immunity. CD4 and CD8 T cells play a major role in anti-tuberculosis infection. The lymphoid factors released by activated T cells of Mycobacterium tuberculosis, including interleukin-2, interferon 纬 -IFN- 纬, kill mycobacterium tuberculosis directly or indirectly. In this study, the important antigen Ag85a derived from Mycobacterium tuberculosis and the cytokines IFN-y and IL-2 related to tuberculosis immunity were used to amplify the Ag85a gene using the genomic DNA of Mycobacterium tuberculosis standard strain H37R v as template. The recombinant plasmids pet28a-Ag85a-IFN- 纬 and pet28a-Ag85a-IL-2were constructed by fusion and expression plasmid pET28a with mouse IFN-y and IL-2, respectively. The fusion protein of Ag85a-IFN- 纬 and Ag85a-IL-2 was purified by expressing the fusion protein which removed the signal peptide in E. coli BL21DDE3. The fusion protein of Ag85a-IFN- 纬 and Ag85a-IL-2 was purified. C57BL/6 mice were immunized subcutaneously with this protein, and the immunological effect was evaluated in order to screen highly effective protein subunit vaccine. The levels of IgG1 and IgG2c in serum were detected by ELISA method. We found that the immunoglobulin levels of the fusion protein Ag85a-IFN- 纬 and Ag85a-IL-2 group were higher than those of the single antigen, and the ratio of IgG2c/IgG1 in the fusion protein group was higher than that of the single antigen. The protective effect of Ag85a-IL-2 was better than that of Ag85a-IFN- 纬, and the ratio of CD8 / CD4 in CD4 and CD8T cells detected by flow cytometry was higher than that in Ag85a-IL-2 group. The fusion protein of Ag85a-IFN- 纬 and Ag85a-IL-2 was successfully constructed and expressed in this experiment. The immune effect of the fusion protein was better than that of single Ag85a antigen, and the immune effect of Ag85a-IL-2 was better than that of Ag85a-IFN-y.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

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