结核亚单位疫苗Mtb8.4-HspX和HspX-Mtb8.4表达纯化及免疫保护效果研究
发布时间：2018-12-11 04:31
【摘要】：目的：结核病是困扰人类健康的重要疾病,研制新型有效、安全的结核疫苗成为国内外学者共同关注的一个重要课题。结核亚单位疫苗成为最有发展前景的新型疫苗之一,同时也存在着很多影响亚单位疫苗免疫保护效率的因素,如融合蛋白的组成顺序与接种剂量。研究亚单位疫苗的蛋白组成顺序和接种剂量与免疫保护效率的关系,对合理设计疫苗和优化免疫策略有重要的意义。 方法：有两个结核疫苗候选抗原被用于我们的实验研究中,分别为Mtb8.4和HspX。利用生物工程的原理和方法,我们成功制备了两个融合蛋白Mtb8.4-HspX和HspX-Mtb8.4,分别简称为8.4H和H8.4。用8.4H和H8.4体外刺激结核病人及结核分支杆菌潜伏感染人群的外周血淋巴细胞,ELISPOT方法检测各组IFN-Y分泌的水平。将佐剂DDA和poly(I:C)与助溶剂明胶按照一定比例混匀制成复合佐剂,简称DPG。复合佐剂与融合蛋白8.4H和H8.4混合制成均一稳定的亚单位疫苗,分别简称8.4H-DPG和H8.4-DPG,而后免疫C57BL/6小鼠,进行免疫检测和攻毒实验,比较两组疫苗的免疫保护效率和组织病理学差异。鉴于明胶对免疫保护效率的负影响和佐剂DDA与poly(I:C)的混合问题,我们仅用佐剂DDA分别与蛋白8.4H和H8.4制成亚单位疫苗,分别称为8.4H-DDA和H8.4-DDA,而后免疫C57BL/6小鼠,ELISA方法检测各疫苗组Thl型的细胞因子IFN-γ、TNF-a和IL-2和Th2型细胞因子IL-4及IgG的水平,并分别比较8.4H-DDA组和H8.4-DDA组及8.4H-DDA高剂量组和低剂量组小鼠细胞免疫和体液免疫应答水平。 结果：融合蛋白8.4H和H8.4均能够刺激人PBMC分泌高水平的IFN-Y,且蛋白H8.4的免疫原性较8.4H强；对比8.4H-DPG和H8.4-DPG免疫保护效率的差异,发现H8.4-DPG显示出更好的免疫保护效果；比较H8.4-DDA组小鼠和8.4H-DDA组小鼠体液免疫应答水平,发现H8.4-DDA组小鼠产生IgG1的水平要显著高于H8.4-DDA组,而IgG2c/IgG1的值明显低于后者；对比8.4H-DDA组小鼠和H8.4-DDA组小鼠细胞免疫应答水平,发现8.4H-DDA组小鼠产生IFN-γ、TNF-α的水平显著高于H8.4-DDA(?)且,而两组分泌IL-2的水平相近,且均未检测到IL-4的分泌；比较8.4H-DDA高剂量组和低剂量组小鼠的体液免疫应答水平,发现高剂量组小鼠产生IgG2b和IgG2c的水平显著高于低剂量组,且IgG2c/IgG1的值明显高于后者；比较8.4H-DDA高剂量组和低剂量组小鼠的细胞免疫应答水平,发现高剂量组小鼠分泌IFN-γ、TNF-α的水平显著高于低剂量组,而产生IL-2的水平刚好相反。 结论：实验室成功制备了融合蛋白Mtb8.4-HspX (8.4H)和HspX-Mtb8.4(H8.4)且能够刺激结核病患者分泌较高水平的IFN-γ；结核亚单位疫苗Mtb8.4-HspX和HspX-Mtb8.4的蛋白组成顺序和接种剂量与Th1型的免疫应答面有着密不可分的关系,因此也影响着亚单位疫苗的免疫保护效果。
[Abstract]:Objective: tuberculosis is an important disease that puzzles human health. The development of new effective and safe TB vaccine has become an important issue that scholars at home and abroad pay attention to. Tuberculosis subunit vaccine has become one of the most promising new vaccines, and there are many factors that affect the immune protection efficiency of subunit vaccine, such as the sequence of fusion protein composition and the dose of vaccine. It is of great significance to study the relationship between the sequence of protein composition and the dose of subunit vaccine and the efficiency of immune protection for the rational design of vaccine and the optimization of immunization strategy. Methods: two TB vaccine candidate antigens were used in our experimental study, Mtb8.4 and HspX., respectively. Using the principles and methods of bioengineering, we successfully prepared two fusion proteins Mtb8.4-HspX and HspX-Mtb8.4, called 8.4H and H8.4respectively. Peripheral blood lymphocytes were stimulated with 8.4H and H8.4 in vitro in patients with tuberculosis and those with latent infection of Mycobacterium tuberculosis. The levels of IFN-Y secretion in each group were detected by ELISPOT method. Blend adjuvant DDA and poly (I: C) with gelatin in a certain proportion to form a composite adjuvant, DPG. for short The hybrid adjuvant was mixed with fusion protein 8.4H and H8.4 to prepare homogeneous and stable subunit vaccine, which was referred to as 8.4H-DPG and H8.4-DPGrespectively, then C57BL/6 mice were immunized. The immune protection efficiency and histopathological difference of the two groups were compared. In view of the negative effect of gelatin on immune protection efficiency and the mixing of adjuvant DDA and poly (I: C), we only use adjuvant DDA and protein 8.4H and H8.4 to prepare subunit vaccine, which is called 8.4H-DDA and H8.4-DDA. Then C57BL/6 mice were immunized. The levels of Thl type cytokines IFN- 纬, TNF-a, IL-2 and Th2 type IL-4 and IgG were detected by ELISA method in each vaccine group. The cellular and humoral immune responses of 8.4H-DDA group, H8.4-DDA group, high dose 8.4H-DDA group and low dose group were compared respectively. Results: the fusion proteins 8.4H and H8.4 could stimulate human PBMC to secrete high levels of IFN-Y, and the immunogenicity of H8.4 was stronger than that of 8.4H. Compared with the difference of immune protection efficiency between 8.4H-DPG and H8.4-DPG, it was found that H8.4-DPG showed better immune protection effect. By comparing the humoral immune response between H8.4-DDA group and 8.4H-DDA group, it was found that the level of IgG1 production in H8.4-DDA group was significantly higher than that in H8.4-DDA group, and the IgG2c/IgG1 level was significantly lower in H8.4-DDA group than in H8.4-DDA group. Compared with the level of cellular immune response in 8.4H-DDA group and H8.4-DDA group, it was found that the levels of IFN- 纬 and TNF- 伪 in 8.4H-DDA group were significantly higher than those in H8.4-DDA group. Moreover, the level of IL-2 secretion in the two groups was similar, and the secretion of IL-4 was not detected in the two groups. The humoral immune response level of mice in 8.4H-DDA high dose group and low dose group was compared. It was found that the level of IgG2b and IgG2c in high dose group was significantly higher than that in low dose group, and the value of IgG2c/IgG1 in high dose group was significantly higher than that in low dose group. The level of cellular immune response in 8.4H-DDA high dose group and low dose group was compared. It was found that the levels of IFN- 纬 and TNF- 伪 in high dose group were significantly higher than those in low dose group, but the level of IL-2 production was just the opposite. Conclusion: the fusion proteins Mtb8.4-HspX (8.4H) and HspX-Mtb8.4 (H8.4) have been successfully prepared in laboratory and can stimulate TB patients to secrete higher levels of IFN- 纬. The sequence of protein composition and dose of Mtb8.4-HspX and HspX-Mtb8.4 of TB subunit vaccine are closely related to the immune response surface of Th1 type, so it also affects the immune protection effect of subunit vaccine.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392
本文编号：2371874
[Abstract]:Objective: tuberculosis is an important disease that puzzles human health. The development of new effective and safe TB vaccine has become an important issue that scholars at home and abroad pay attention to. Tuberculosis subunit vaccine has become one of the most promising new vaccines, and there are many factors that affect the immune protection efficiency of subunit vaccine, such as the sequence of fusion protein composition and the dose of vaccine. It is of great significance to study the relationship between the sequence of protein composition and the dose of subunit vaccine and the efficiency of immune protection for the rational design of vaccine and the optimization of immunization strategy. Methods: two TB vaccine candidate antigens were used in our experimental study, Mtb8.4 and HspX., respectively. Using the principles and methods of bioengineering, we successfully prepared two fusion proteins Mtb8.4-HspX and HspX-Mtb8.4, called 8.4H and H8.4respectively. Peripheral blood lymphocytes were stimulated with 8.4H and H8.4 in vitro in patients with tuberculosis and those with latent infection of Mycobacterium tuberculosis. The levels of IFN-Y secretion in each group were detected by ELISPOT method. Blend adjuvant DDA and poly (I: C) with gelatin in a certain proportion to form a composite adjuvant, DPG. for short The hybrid adjuvant was mixed with fusion protein 8.4H and H8.4 to prepare homogeneous and stable subunit vaccine, which was referred to as 8.4H-DPG and H8.4-DPGrespectively, then C57BL/6 mice were immunized. The immune protection efficiency and histopathological difference of the two groups were compared. In view of the negative effect of gelatin on immune protection efficiency and the mixing of adjuvant DDA and poly (I: C), we only use adjuvant DDA and protein 8.4H and H8.4 to prepare subunit vaccine, which is called 8.4H-DDA and H8.4-DDA. Then C57BL/6 mice were immunized. The levels of Thl type cytokines IFN- 纬, TNF-a, IL-2 and Th2 type IL-4 and IgG were detected by ELISA method in each vaccine group. The cellular and humoral immune responses of 8.4H-DDA group, H8.4-DDA group, high dose 8.4H-DDA group and low dose group were compared respectively. Results: the fusion proteins 8.4H and H8.4 could stimulate human PBMC to secrete high levels of IFN-Y, and the immunogenicity of H8.4 was stronger than that of 8.4H. Compared with the difference of immune protection efficiency between 8.4H-DPG and H8.4-DPG, it was found that H8.4-DPG showed better immune protection effect. By comparing the humoral immune response between H8.4-DDA group and 8.4H-DDA group, it was found that the level of IgG1 production in H8.4-DDA group was significantly higher than that in H8.4-DDA group, and the IgG2c/IgG1 level was significantly lower in H8.4-DDA group than in H8.4-DDA group. Compared with the level of cellular immune response in 8.4H-DDA group and H8.4-DDA group, it was found that the levels of IFN- 纬 and TNF- 伪 in 8.4H-DDA group were significantly higher than those in H8.4-DDA group. Moreover, the level of IL-2 secretion in the two groups was similar, and the secretion of IL-4 was not detected in the two groups. The humoral immune response level of mice in 8.4H-DDA high dose group and low dose group was compared. It was found that the level of IgG2b and IgG2c in high dose group was significantly higher than that in low dose group, and the value of IgG2c/IgG1 in high dose group was significantly higher than that in low dose group. The level of cellular immune response in 8.4H-DDA high dose group and low dose group was compared. It was found that the levels of IFN- 纬 and TNF- 伪 in high dose group were significantly higher than those in low dose group, but the level of IL-2 production was just the opposite. Conclusion: the fusion proteins Mtb8.4-HspX (8.4H) and HspX-Mtb8.4 (H8.4) have been successfully prepared in laboratory and can stimulate TB patients to secrete higher levels of IFN- 纬. The sequence of protein composition and dose of Mtb8.4-HspX and HspX-Mtb8.4 of TB subunit vaccine are closely related to the immune response surface of Th1 type, so it also affects the immune protection effect of subunit vaccine.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392
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相关期刊论文 前3条
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