發(fā)布時(shí)間：2018-04-28 10:52

  本文選題：弓形蟲 + 致密顆粒蛋白(GRA24��； 參考：《安徽農(nóng)業(yè)大學(xué)》2016年碩士論文

【摘要】：剛地弓形蟲(T.gondii)是一種重要的人獸共患寄生性原蟲。作為一種專性細(xì)胞內(nèi)寄生原蟲,弓形蟲可入侵包括人類在內(nèi)的幾乎所有溫血動物的有核細(xì)胞。全球約有三分之一的人群被感染,在中國約有8%的人口感染。弓形蟲感染可引起孕婦流產(chǎn)、胎兒畸形,免疫機(jī)能低下者甚至死亡,對人類的生命和健康造成了嚴(yán)重的影響;同時(shí),其也給畜牧業(yè)生產(chǎn)帶來巨大的經(jīng)濟(jì)損失。目前,因無根治弓形蟲病的特效藥,故疫苗的研制備受關(guān)注。但尚未研制出現(xiàn)安全、高效的疫苗,因此,篩選弓形蟲疫苗的候選抗原分子顯得尤為重要。致密顆粒蛋白(dense granule proteins,GRAs)均是分泌排泄抗原(ESA),ESA均具有高度免疫源性,可通過抗體依賴或細(xì)胞介導(dǎo)的免疫反應(yīng)誘導(dǎo)機(jī)體產(chǎn)生免疫保護(hù)作用。GRA24一旦分泌進(jìn)入宿主細(xì)胞,可持續(xù)性地引發(fā)宿主細(xì)胞的自身磷酸化和宿主細(xì)胞p38αMAP激酶的核易位。GRA24可控制弓形蟲在小鼠腸道中的感染能力。它還具有增加宿主趨化因子的分泌和調(diào)節(jié)弓形蟲速殖子早期復(fù)制的能力。GRA24可刺激活化型T細(xì)胞增殖,促進(jìn)Th0細(xì)胞向Th1細(xì)胞分化,誘導(dǎo)Th1型免疫應(yīng)答反應(yīng)。GRA25是弓形蟲至關(guān)重要的毒力因子之一,影響被弓形蟲感染的巨噬細(xì)胞中的趨化因子(CCL2和CXCL1)的產(chǎn)生。本研究分別對來自于不同宿主和地理來源的弓形蟲分離株的GRA24和GRA25基因組序列進(jìn)行克隆,并對其遺傳變異情況進(jìn)行分析。結(jié)果表明:經(jīng)PCR擴(kuò)增后,可明顯擴(kuò)增出目的片段948bp和6264/6261bp,與預(yù)期結(jié)果一致。GRA24基因的核酸變異率僅有0.05-0.2%,使用ML和MP以及BI法所構(gòu)建的GRA24的系統(tǒng)發(fā)育樹,能夠?qū)⒌湫偷腎和III型分開,但不能將II型蟲株聚集在一起。GRA25基因的核酸變異率為0~4.4%,使用ML和MP法所構(gòu)建的GRA25的系統(tǒng)發(fā)育樹,不能區(qū)分弓形蟲基因I、II和III型蟲株。因此,GRA24和GRA25均不能作為有效的遺傳標(biāo)記用于研究弓形蟲的遺傳變異。本研究構(gòu)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,檢測了體液免疫和細(xì)胞免疫的相關(guān)指標(biāo)。結(jié)果顯示:免疫組小鼠血清中的IgG和IgG1、IgG2a的含量都有明顯的上升;CD3+CD4+CD8-輔助性T細(xì)胞以及CD3+CD8+CD4-T細(xì)胞的含量明顯增高,細(xì)胞因子IFN-γ、IL-2、IL-12和IL-23的含量明顯增加,說明pVAX-GRA24和pVAX-GRA25能誘導(dǎo)小鼠產(chǎn)生高水平的Th1型細(xì)胞免疫和體液免疫應(yīng)答反應(yīng)。用弓形蟲強(qiáng)毒RH株速殖子攻擊感染KM鼠,單基因免疫組和聯(lián)合免疫組小鼠存活時(shí)間均有延長。用弱毒PRU株包囊感染KM鼠,各免疫組小鼠的腦組織中包囊減少率明顯增加。以上研究結(jié)果說明弓形蟲致密顆粒蛋白GRA24和GRA25是良好的預(yù)防弓形蟲感染的疫苗候選抗原。
[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 偽 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- 緯, 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.
【學(xué)位授予單位】：安徽農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S852.4

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