弓形虫Toxofilin DNA疫苗结合铝盐与脂质体A复合佐剂增强了小鼠的保护免疫性

发布时间：2018-07-06 19:47

本文选题：弓形虫 + 肌动蛋白结合蛋白　；参考：《山东大学》2017年硕士论文

【摘要】：目的:刚地弓形虫疾病在全世界范围内普遍流行,对人类的身体状况造成了严重的伤害。因此,研制弓形虫疫苗治疗弓形虫疾病迫在眉睫。本实验研究目的主要是评估弓形虫肌动蛋白结合蛋白(Toxofilin)基因疫苗对小鼠的免疫效果。此外,我们分别将单佐剂(铝盐佐剂(alum),脂质体A(MPLA)),双佐剂(alum+MPLA)分别与Toxofilin DNA疫苗联合使用注入到宿主体内从而进行筛选宿主抗体表达能力。方法:使用生物信息学的方法,我们分析了 Toxofilin表达蛋白的氨基酸排列序列,并且运用相关生物学软件预测了 Toxofilin蛋白几个潜在的B细胞,T细胞抗原表位。将BALB/c小鼠分为四个实验组(单Toxofilin DNA疫苗,alum佐剂+DNA疫苗,MPLA佐剂+DNA疫苗,双佐剂(alum+MPLA)+ DNA疫苗),并且将上述四组实验试剂连续免疫小鼠三次,通过检测弓形虫总抗体以及细胞因子的含量来评价小鼠产生的免疫反应。待第三次免疫小鼠14天后,将致死性剂量的弓形虫(RH株)速殖子腹腔注射到小鼠体内,通过观察小鼠的存活时间进而来验证DNA疫苗并且结合不同佐剂对小鼠的免疫保护性。结果:所有实验组(Toxofilin,Toxofilin+alum,Toxofilin+MPLA,Toxofilin+alum+MPLA)免疫小鼠后,在其小鼠体内,细胞免疫与体液免疫水平都明显高于对照组(PBS,alum,MPLA)。此外,与单基因组(Toxofilin)疫苗相比,alum+Toxofilin基因疫苗联合使用增强了体液免疫应答与Th2型细胞免疫应答,相反地MPLA+Toxofilin 一起使用增强了体液免疫应答与Thl型细胞免疫反应。更重要地是,与alum+Toxofilin相比,将MPLA与alum+Toxofilin基因疫苗联合使用能够转变alum+Toxofilin基因疫苗中细胞免疫应答的类型,将Th2型细胞免疫转变为Thl型细胞免疫,同时,在alum+MPLA+Toxofilin基因疫苗免疫组中,体液免疫与Thl型细胞免疫应答都显著高于其它实验组与对照组,最后通过弓形虫速殖子攻击实验来评价基因疫苗与不同佐剂对小鼠的免疫保护性,结果表明,与其它实验组与对照组相比,alum+MPLA+Toxofilin基因疫苗明显地延长了小鼠的存活时间。结论:刚地弓形虫Toxofilin基因可以作为一个合适的候选疫苗,将复合佐剂alum+MPLA与Toxofilin基因共同使用,使小鼠体内的免疫应答包括细胞免疫与体液免疫应答都明显地增强与提高。
[Abstract]:Objective: Toxoplasma gondii disease is prevalent all over the world, causing serious damage to human health. Therefore, it is urgent to develop Toxoplasma vaccine to treat Toxoplasma gondii. The aim of this study was to evaluate the immunological effect of Toxofilin gene vaccine on mice. In addition, single adjuvant (aluminum salt adjuvant (alum), liposome A (MPLA), double adjuvant) (alum MPLA) and Toxofilin DNA vaccine were injected into the host to screen the host antibody expression ability. Methods: the amino acid sequence of Toxofilin protein was analyzed by bioinformatics, and several potential T cell epitopes of Toxofilin protein were predicted by relevant biological software. Balb / c mice were divided into four experimental groups (single Toxofilin DNA adjuvant DNA vaccine and double adjuvant (alum MPLA) DNA vaccine). The immune response of mice was evaluated by detecting total antibodies and cytokines of Toxoplasma gondii (Toxoplasma gondii). After the third immunization of mice for 14 days, the lethal dose of Toxoplasma gondii (RH strain) tachyzoites were injected intraperitoneally into mice. The survival time of mice was observed to verify the DNA vaccine and the immune protection of mice combined with different adjuvants. Results: the levels of cellular immunity and humoral immunity in all the experimental groups were significantly higher than those in the control group (PBSalumin MPLA) after the mice were immunized with Toxofilin Toxofilin alum MPLA. In addition, the combination of Toxofilin gene vaccine and Toxofilin gene vaccine enhanced humoral immune response and Th2 type cellular immune response, whereas MPLA Toxofilin combined with MPLA Toxofilin enhanced humoral immune response and Thl type cellular immune response. More importantly, compared with alum Toxofilin, the combination of MPLA and alum Toxofilin gene vaccine can change the type of cellular immune response in alum Toxofilin gene vaccine, transform Th2 type cellular immunity into Thl type cellular immunity, at the same time, In the alum MPLA Toxofilin gene vaccine immunized group, humoral immunity and Thl type cellular immune response were significantly higher than those of other experimental and control groups. Finally, the immune protection of Toxoplasma gondii Tachyzoites was evaluated by Toxoplasma gondii Tachyzoites attack test. The results showed that compared with other experimental groups and control groups, the survival time of mice was significantly prolonged by MPLA Toxofilin gene vaccine. Conclusion: Toxofilin gene of Toxoplasma gondii can be used as a suitable candidate vaccine. The combined adjuvant alum MPLA and Toxofilin gene can significantly enhance and enhance the immune response, including cellular and humoral immune responses in mice.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R392

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