结核杆菌和布鲁氏杆菌多价DNA疫苗的研究
发布时间：2018-09-14 07:33
【摘要】： 结核病和布鲁氏病是严重危害人类健康和畜牧业发展的人畜共患传染病。疫苗是控制结核病和布鲁氏病的有效方法。目前,BCG是预防结核病的唯一疫苗,但其免疫效力不稳定,且对成年人无保护;S19疫苗是在牛布鲁氏病中使用的疫苗,但血清学反应强,且安全性受到质疑,有感染人的可能。因此,高效的新型疫苗对于控制结核病和布鲁氏病蔓延至关重要。本论文在阐述结核病和布鲁氏病疫苗研究的基础上,着重分析了DNA疫苗的作用机制、发展现状及其改进策略。 用分子生物学的方法分别构建了编码结核杆菌三种抗原(Ag85B,MPT64和MPT83)和布鲁氏杆菌三种抗原(BCSP31,SOD,和L7/L12)的原核表达载体,诱导表达并纯化后得到抗原蛋白。构建此六种抗原的真核表达载体,进行体外瞬时表达实验,用GFP偶联和RT-PCR等方法确定蛋白表达的部位和表达量。大量提取制备真核质粒作为DNA疫苗,免疫小鼠三次后8周,用高剂量的结核杆菌或布鲁氏杆菌感染小鼠或大动物牛。本文从体液免疫水平、细胞因子水平、分泌细胞因子细胞的数量、T细胞激活比例、T细胞亚型的变化、杀伤性T细胞的定量、结合动物感染后脏器载菌量等多个指标,对各个组合的疫苗的优势及所诱导免疫反应的进行综合评价,以期对DNA疫苗效力及其佐剂触发免疫反应的机制进行诠释。实验结果表明,多价DNA疫苗能够诱导更强的TH1型免疫反应,扩大保护范围,保护效力显著高于单价DNA疫苗。 通过对比联合疫苗与不同矿物质佐剂、基因佐剂共同免疫后所产生的保护效果,证实佐剂基因佐剂IL-2、IL-12、IL-15、和无机佐剂DDA在诱导TH1型细胞反应,增强杀伤性T细胞的功能方面发挥重要作用。尤其是用六种抗原联合DNA疫苗与IL-12共同免疫,产生了“一针治两病”的效果,保护效率显著高于目前使用的BCG和S19传统疫苗。IL-15佐剂显著提高了分泌IFN-γ的CD8+T细胞的数量,从体内、体外实验证实了CD8+T细胞在对抗布鲁氏杆菌过程中的重要作用。多肽佐剂KLKL5KLK提高了APC细胞对抗原的摄取、加工和递呈能力,延长了免疫保护期、增强CD4+T细胞作用和细胞杀伤水平,为临床应用提供了重要证据。 用包裹剂PLGA达到DNA缓释和避免核酸酶对DNA降解的效果以减少免疫剂量,结果表明PLGA在提高抗原表达量,延长表达时间上有显著作用,在免疫攻毒试验中,免疫一次PLGA包裹的多价DNA疫苗产生了与免疫三次不加PLGA的多价DNA疫苗类似的保护效力。BCG疫苗加强策略在大动物牛中的实验表明,加强策略显著提高了免疫牛中CD4+T细胞的比例,诱导了高水平的IFN-γ,且一直持续到攻毒后22周。 用结核杆菌强毒株感染小鼠,构建潜伏期感染的动物模型。感染后8周,用异烟肼和吡嗪酰胺治疗3个月。治疗期间,用编码三种结核杆菌抗原的基因联合治疗,使免疫应答朝TH1型方向进行,产生更多的TH1型的细胞因子如IFN-γ,使吞噬有结核杆菌的巨噬细胞从休眠状态转变为活化型,以杀死结核杆菌,提高治疗效率,降低载菌量,使治疗时间减少一半,克服由于长期使用药物产生耐药性等问题。对开发有实际意义的治疗用核酸疫苗具有重要的启示。
[Abstract]:Tuberculosis and Brucellosis are zoonotic infectious diseases that seriously endanger human health and animal husbandry development. Vaccines are effective ways to control tuberculosis and brucellosis. Currently, BCG is the only vaccine to prevent tuberculosis, but its immune efficacy is unstable and unprotected in adults; S19 vaccine is used in bovine brucellosis, but not in adults. Therefore, efficient new vaccines are very important for controlling the spread of tuberculosis and brucellosis. Based on the study of tuberculosis and brucellosis vaccines, this paper focuses on the analysis of the mechanism of action of DNA vaccines, development status and improvement strategies.
Prokaryotic expression vectors encoding three antigens of Mycobacterium tuberculosis (Ag85B, MPT64 and MPT83) and three antigens of Brucella (BCSP31, SOD, and L7/L12) were constructed by molecular biology methods. The antigen proteins were induced to express and purified. The eukaryotic expression vectors of the six antigens were constructed for transient expression in vitro, coupled with GFP. Eukaryotic plasmids were extracted and prepared as DNA vaccines. After eight weeks of immunization, mice or cattle were infected with high doses of Mycobacterium tuberculosis or Brucella. The humoral immunity, cytokine levels, the number of cytokine-secreting cells and the activation ratio of T cells were studied. For example, the changes of T cell subtypes, the quantification of lethal T cells, and the amount of bacteria carried in the organs of infected animals were used to evaluate the advantages of each combination of vaccines and the immune responses induced by them, so as to interpret the efficacy of DNA vaccines and the mechanism of their adjuvants triggering immune responses. It can induce stronger TH1 immune response and enlarge the protective range. The protective efficacy is significantly higher than that of monovalent DNA vaccine.
By comparing the protective effects of the combined vaccine with different mineral adjuvants and gene adjuvants, it was confirmed that the adjuvant gene adjuvants IL-2, IL-12, IL-15, and the inorganic adjuvant DDA played an important role in inducing TH1 cell reaction and enhancing the function of killer T cells, especially the combination of six antigens DNA vaccine and IL-12. IL-15 adjuvant significantly increased the number of CD8+T cells secreting IFN-gamma. In vivo and in vitro experiments confirmed the important role of CD8+T cells in the process of combating Brucella. Polypeptide adjuvant KL5KLK increased APC cells. The ability of antigen uptake, processing and presentation prolongs the immune protection period, enhances the function of CD4 + T cells and the level of cell killing, which provides important evidence for clinical application.
The results showed that PLGA could significantly increase the expression of antigen and prolong the expression time. In the immune challenge test, the polyvalent DNA vaccine encapsulated by PLGA produced the same protection as the polyvalent DNA Vaccine Immunized with PLGA three times without PLGA. Protective effect. BCG vaccine reinforcement strategy in animal cattle showed that the reinforcement strategy significantly increased the proportion of CD4 + T cells in immunized cattle, induced high levels of IFN - gamma, and lasted until 22 weeks after infection.
Mice were infected with a virulent strain of Mycobacterium tuberculosis to establish an animal model of latent infection. Eight weeks after infection, the mice were treated with isoniazid and pyrazinamide for three months. Bacillus macrophages change from dormant state to active type in order to kill Mycobacterium tuberculosis, improve treatment efficiency, reduce bacterial load, reduce treatment time by half, overcome drug resistance and other problems caused by long-term use of drugs. It has important implications for the development of therapeutic nucleic acid vaccines with practical significance.
【学位授予单位】：北京大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R392
本文编号：2242004
[Abstract]:Tuberculosis and Brucellosis are zoonotic infectious diseases that seriously endanger human health and animal husbandry development. Vaccines are effective ways to control tuberculosis and brucellosis. Currently, BCG is the only vaccine to prevent tuberculosis, but its immune efficacy is unstable and unprotected in adults; S19 vaccine is used in bovine brucellosis, but not in adults. Therefore, efficient new vaccines are very important for controlling the spread of tuberculosis and brucellosis. Based on the study of tuberculosis and brucellosis vaccines, this paper focuses on the analysis of the mechanism of action of DNA vaccines, development status and improvement strategies.
Prokaryotic expression vectors encoding three antigens of Mycobacterium tuberculosis (Ag85B, MPT64 and MPT83) and three antigens of Brucella (BCSP31, SOD, and L7/L12) were constructed by molecular biology methods. The antigen proteins were induced to express and purified. The eukaryotic expression vectors of the six antigens were constructed for transient expression in vitro, coupled with GFP. Eukaryotic plasmids were extracted and prepared as DNA vaccines. After eight weeks of immunization, mice or cattle were infected with high doses of Mycobacterium tuberculosis or Brucella. The humoral immunity, cytokine levels, the number of cytokine-secreting cells and the activation ratio of T cells were studied. For example, the changes of T cell subtypes, the quantification of lethal T cells, and the amount of bacteria carried in the organs of infected animals were used to evaluate the advantages of each combination of vaccines and the immune responses induced by them, so as to interpret the efficacy of DNA vaccines and the mechanism of their adjuvants triggering immune responses. It can induce stronger TH1 immune response and enlarge the protective range. The protective efficacy is significantly higher than that of monovalent DNA vaccine.
By comparing the protective effects of the combined vaccine with different mineral adjuvants and gene adjuvants, it was confirmed that the adjuvant gene adjuvants IL-2, IL-12, IL-15, and the inorganic adjuvant DDA played an important role in inducing TH1 cell reaction and enhancing the function of killer T cells, especially the combination of six antigens DNA vaccine and IL-12. IL-15 adjuvant significantly increased the number of CD8+T cells secreting IFN-gamma. In vivo and in vitro experiments confirmed the important role of CD8+T cells in the process of combating Brucella. Polypeptide adjuvant KL5KLK increased APC cells. The ability of antigen uptake, processing and presentation prolongs the immune protection period, enhances the function of CD4 + T cells and the level of cell killing, which provides important evidence for clinical application.
The results showed that PLGA could significantly increase the expression of antigen and prolong the expression time. In the immune challenge test, the polyvalent DNA vaccine encapsulated by PLGA produced the same protection as the polyvalent DNA Vaccine Immunized with PLGA three times without PLGA. Protective effect. BCG vaccine reinforcement strategy in animal cattle showed that the reinforcement strategy significantly increased the proportion of CD4 + T cells in immunized cattle, induced high levels of IFN - gamma, and lasted until 22 weeks after infection.
Mice were infected with a virulent strain of Mycobacterium tuberculosis to establish an animal model of latent infection. Eight weeks after infection, the mice were treated with isoniazid and pyrazinamide for three months. Bacillus macrophages change from dormant state to active type in order to kill Mycobacterium tuberculosis, improve treatment efficiency, reduce bacterial load, reduce treatment time by half, overcome drug resistance and other problems caused by long-term use of drugs. It has important implications for the development of therapeutic nucleic acid vaccines with practical significance.
【学位授予单位】：北京大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R392
【引证文献】
相关博士学位论文 前1条
1 蔺国珍;布鲁氏菌病LAMP检测方法的建立及双基因共表达分子疫苗研究[D];中国农业科学院;2012年
，本文编号：2242004
本文链接：https://www.wllwen.com/yixuelunwen/shiyanyixue/2242004.html
最近更新
教材专著
