TgGRA24和25的基因序列分析及免疫保护性研究

发布时间：2018-04-28 10:52

本文选题：弓形虫 + 致密颗粒蛋白(GRA24　；参考：《安徽农业大学》2016年硕士论文

【摘要】：刚地弓形虫(T.gondii)是一种重要的人兽共患寄生性原虫。作为一种专性细胞内寄生原虫,弓形虫可入侵包括人类在内的几乎所有温血动物的有核细胞。全球约有三分之一的人群被感染,在中国约有8%的人口感染。弓形虫感染可引起孕妇流产、胎儿畸形,免疫机能低下者甚至死亡,对人类的生命和健康造成了严重的影响;同时,其也给畜牧业生产带来巨大的经济损失。目前,因无根治弓形虫病的特效药,故疫苗的研制备受关注。但尚未研制出现安全、高效的疫苗,因此,筛选弓形虫疫苗的候选抗原分子显得尤为重要。致密颗粒蛋白(dense granule proteins,GRAs)均是分泌排泄抗原(ESA),ESA均具有高度免疫源性,可通过抗体依赖或细胞介导的免疫反应诱导机体产生免疫保护作用。GRA24一旦分泌进入宿主细胞,可持续性地引发宿主细胞的自身磷酸化和宿主细胞p38αMAP激酶的核易位。GRA24可控制弓形虫在小鼠肠道中的感染能力。它还具有增加宿主趋化因子的分泌和调节弓形虫速殖子早期复制的能力。GRA24可刺激活化型T细胞增殖,促进Th0细胞向Th1细胞分化,诱导Th1型免疫应答反应。GRA25是弓形虫至关重要的毒力因子之一,影响被弓形虫感染的巨噬细胞中的趋化因子(CCL2和CXCL1)的产生。本研究分别对来自于不同宿主和地理来源的弓形虫分离株的GRA24和GRA25基因组序列进行克隆,并对其遗传变异情况进行分析。结果表明:经PCR扩增后,可明显扩增出目的片段948bp和6264/6261bp,与预期结果一致。GRA24基因的核酸变异率仅有0.05-0.2%,使用ML和MP以及BI法所构建的GRA24的系统发育树,能够将典型的I和III型分开,但不能将II型虫株聚集在一起。GRA25基因的核酸变异率为0~4.4%,使用ML和MP法所构建的GRA25的系统发育树,不能区分弓形虫基因I、II和III型虫株。因此,GRA24和GRA25均不能作为有效的遗传标记用于研究弓形虫的遗传变异。本研究构建了基于GRA24和GRA25基因的核酸疫苗pVAX-GRA24和pVAX-GRA25,并用其免疫昆明(KM)鼠,检测了体液免疫和细胞免疫的相关指标。结果显示:免疫组小鼠血清中的IgG和IgG1、IgG2a的含量都有明显的上升;CD3+CD4+CD8-辅助性T细胞以及CD3+CD8+CD4-T细胞的含量明显增高,细胞因子IFN-γ、IL-2、IL-12和IL-23的含量明显增加,说明pVAX-GRA24和pVAX-GRA25能诱导小鼠产生高水平的Th1型细胞免疫和体液免疫应答反应。用弓形虫强毒RH株速殖子攻击感染KM鼠,单基因免疫组和联合免疫组小鼠存活时间均有延长。用弱毒PRU株包囊感染KM鼠,各免疫组小鼠的脑组织中包囊减少率明显增加。以上研究结果说明弓形虫致密颗粒蛋白GRA24和GRA25是良好的预防弓形虫感染的疫苗候选抗原。
[Abstract]:T. gondii (Toxoplasma gondii) is an important zoonotic parasite. As a specific protozoa, Toxoplasma gondii invades nucleated cells of almost all warm-blooded animals, including humans. About 1/3 people worldwide are infected, and about 8% of the population in China. Toxoplasma gondii infection can cause miscarriage of pregnant women, fetal malformation, low immune function and even death, which has a serious impact on human life and health. At the same time, it also brings huge economic losses to animal husbandry production. At present, because there is no cure for toxoplasmosis, the development of vaccine has attracted much attention. However, safe and efficient vaccines have not been developed, so it is very important to screen candidate antigen molecules of Toxoplasma gondii vaccine. Dense granule proteinsm gras are highly immunogenic, which can induce immune protection by antibody dependent or cell-mediated immune response. GRA24 can be secreted into the secreted host cells once it enters the secreted host cells. The self-phosphorylation of host cells and the nuclear translocation of p38 伪 MAP kinase in host cells. GRA24 can control the infection ability of Toxoplasma gondii in the intestinal tract of mice. It can also increase the secretion of host chemokines and regulate the early replication of Toxoplasma gondii tachyzoites. GRA24 can stimulate the proliferation of activated T cells and promote the differentiation of Th0 cells into Th1 cells. GRA25 is one of the most important virulence factors of Toxoplasma gondii, which affects the production of chemokines CCL2 and CXCL1 in macrophages infected by Toxoplasma gondii. In this study, GRA24 and GRA25 genomes from Toxoplasma gondii isolates from different hosts and geographical sources were cloned and their genetic variations were analyzed. The results showed that the target fragment 948bp and 6264 / 6261bp could be amplified by PCR, and the nucleic acid variation rate of .GRA24 gene was only 0.05-0.2. The phylogenetic tree of GRA24 constructed by ML, MP and BI method could separate the typical I from III type. However, the nucleic acid variation rate of GRA25 gene could not be clustered together. The phylogenetic tree of GRA25 constructed by ML and MP method could not distinguish Toxoplasma gondii Igai II from III strain. Therefore, neither GRA24 nor GRA25 can be used as an effective genetic marker to study the genetic variation of Toxoplasma gondii. In this study, the nucleic acid vaccine pVAX-GRA24 and pVAX-GRA25 based on GRA24 and GRA25 genes were constructed and immunized with them. The humoral and cellular immune indexes were detected. The results showed that the contents of IgG and IgG1hIgG2a in serum of immunized mice were significantly increased, and the contents of CD3 CD4 CD8- helper T cells and CD3 CD8 CD4-T cells were significantly increased, and the contents of cytokines IFN- 纬, IL-2-, IL-12 and IL-23 were significantly increased in the immunized mice. These results suggest that pVAX-GRA24 and pVAX-GRA25 can induce high level of Th1 type cellular and humoral immune responses in mice. Km mice were infected with Toxoplasma gondii RH strain tachyzoites. The survival time of mice in single gene immunization group and combined immunization group was prolonged. When km mice were infected with attenuated PRU strain, the decrease rate of cyst in brain tissue of mice in each immunized group was significantly increased. These results suggest that Toxoplasma gondii dense granule protein GRA24 and GRA25 are good vaccine candidate antigens for the prevention of Toxoplasma gondii infection.
【学位授予单位】：安徽农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S852.4

【参考文献】