运输应激条件下弓形虫缓殖子向速殖子转化差异蛋白的筛选及鉴定

发布时间：2018-02-28 08:17

本文关键词： 弓形虫速殖子缓殖子应激转化差异蛋白细胞因子　出处：《华中农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：刚地弓形虫(Toxoplasma gondii)是一种专性细胞内寄生的原虫,可以引起严重的人畜共患寄生虫病。弓形虫宿主范围极其广泛,可以感染家禽、家畜、伴侣动物以及人类在内的所有温血动物。对于免疫功能健全的个体,弓形虫的感染不表现出明显的临床症状,虫体多以半休眠的组织包囊形式在宿主脑部、肌肉及眼部定居,与机体长期共存。但是包囊并不是无威胁的存在,当宿主的免疫力低下时,包囊会再活化,进而由缓殖子转变为速殖子,引起急性感染。因此,弓形虫速殖子和缓殖子的转化在弓形虫致病过程中扮演着关键的角色。研究表明,运输应激可以降低宿主的免疫力,从而导致多种疾病的复发,据此推测,弓形虫作为一种机会性致病病原体,运输应激可能诱发弓形虫慢性感染小鼠脑内包囊的再活化。弓形虫速殖子和缓殖子转化过程中,多种期特异性蛋白、热休克蛋白以及代谢酶类的表达发生明显变化,它们对弓形虫的生长以及代谢有着重要的调控作用。因此,本研究针对弓形虫速殖子和缓殖子转化过程中可能的差异蛋白进行筛选及鉴定。本研究首先建立运输应激诱导弓形虫缓殖子向速殖子转化的动物模型,验证了运输应激诱使包囊再活化,即由缓殖子转化成速殖子;随后模拟运输应激,利用细胞因子抗体芯片检测小鼠血清中细胞因子的变化,结果显示小鼠外周血血清中γ干扰素(IFN-γ),白细胞介素(IL-1α),白细胞介素(IL-4),白细胞介素(IL-10),白细胞介素(IL-12)以及巨噬细胞集落刺激因子(M-CSF)的表达水平在应激后显著下降;最后利用双向电泳以及质谱鉴定技术筛选并鉴定出了缓殖子向速殖子转化过程中的47个鼠源蛋白和15个弓形虫蛋白。具体工作包括以下几个方面:(1)弓形虫缓殖子向速殖子转化动物模型的建立利用运输应激成功诱导弓形虫脑包囊的再活化,即由缓殖子转变成速殖子。将弓形虫PRU株慢性感染小鼠模拟运输应激3天后,利用Real-time PCR分别检测弓形虫速殖子期特异性基因SAG1和缓殖子期特异性基因BAG1的m RNA相对表达水平。结果显示在运输应激3天后,BAG1的相对表达水平下降,SAG1的相对表达水平显著上升,表明运输应激诱导包囊的再活化,即从缓殖子向速殖子转化。(2)运输应激影响弓形虫慢性感染小鼠的免疫水平利用弓形虫缓殖子向速殖子转化的动物模型,分别收集运输应激前,应激1天,应激2天,应激3天的小鼠外周血血清,利用细胞因子抗体芯片检测小鼠细胞因子的变化。结果显示,IFN-γ,IL-1α,IL-4,IL-10,IL-12以及M-CSF 6种细胞因子水平随着应激时间的延长逐渐下降,在应激3天后显著下降。表明运输应激降低慢性感染小鼠的免疫水平。(3)弓形虫缓殖子向速殖子转化差异蛋白的筛选及鉴定利用弓形虫缓殖子向速殖子转化的动物模型,分别收集并纯化运输应激前,应激1天,应激2天,应激3天的小鼠脑组织包囊,提取包囊总蛋白,利用双向电泳技术筛选缓殖子向速殖子转化过程中的差异蛋白并进行质谱鉴定。结果显示,47个鼠源蛋白和15个弓形虫相关蛋白的表达量在缓殖子向速殖子转化过程中显著变化;随后利用Real-time PCR验证了弓形虫肌动蛋白(Actin),微线体蛋白13(MIC13),致密颗粒蛋白9(GRA9),致密颗粒蛋白1(GRA1)和烯醇酶1(ENO1)的m RNA水平在运输应激后显著上升,与双向电泳结果一致,表明缓殖子向速殖子转化过程中多种宿主和虫体的蛋白表达发生变化。本研究利用小鼠运输应激模型证实了应激导致机体免疫力下降;成功筛选并鉴定出了弓形虫缓殖子向速殖子转化过程中的47个鼠源蛋白和15个弓形虫相关蛋白,为阐明弓形虫速殖子和缓殖子转化机制提供了理论依据。
[Abstract]:Toxoplasma gondii (Toxoplasma gondii) is an obligate intracellular parasitic protozoa, can cause serious zoonotic parasitic diseases. Toxoplasma gondii can infect a broad host range, poultry, livestock, all warm blooded animal companion animal and human beings. The immune function of healthy individuals, Toxoplasma infection do not show obvious clinical symptoms, the body in the form of semi dormant tissue cysts in the host brain, eye and muscle settled. But long-term coexistence with the body, cyst is not threatened, when the host's immune system is low, will be activated and the cyst, bradyzoite into tachyzoites that caused by acute infection. Therefore, the transformation of Toxoplasma gondii tachyzoite and bradyzoite of plays a key role in the pathogenesis of toxoplasmosis. The results show that transport stress can reduce host immunity, leading to a variety of diseases We speculated that the recurrence of Toxoplasma gondii as an opportunistic pathogen, transport stress may re activation induced by chronic Toxoplasma infected mouse brain cyst. The transformation process of Toxoplasma tachyzoites and in a variety of stage specific protein, heat shock protein and metabolic enzymes expression changed significantly, with regulation an important role of Toxoplasma growth and metabolism. Therefore, screening and identification of possible differences in protein transformation process of this research and the tachyzoites of Toxoplasma gondii tachyzoites in animal models. In this study we developed transport stress induced bow gondii bradyzoite into tachyzoites, verify the transport stress inducing cyst re activation from tachyzoites into tachyzoites; then simulated transport stress, detecting the changes of cytokines in the serum of mice using cytokine antibody array, results showed that the mice of Zhou Xuexue In the interferon gamma (IFN- gamma), interleukin (IL-1 alpha), interleukin (IL-4), interleukin (IL-10), interleukin (IL-12) and macrophage colony-stimulating factor (M-CSF) expression level decreased significantly after stress; finally by two-dimensional electrophoresis and the screening and identification of mass spectrometry and identified 47 mouse protein tachyzoites into tachyzoites of Toxoplasma gondii and 15 protein. The specific work includes the following aspects: (1) Toxoplasma gondii tachyzoites to establish animal model of tachyzoites reactivation induced cerebral Toxoplasma cysts the use of transport stress, from tachyzoites into tachyzoites. The chronic infection of Toxoplasma gondii PRU strain mice simulated transport stress after 3 days, we detected m RNA of Toxoplasma gondii tachyzoite stage specific gene SAG1 and tachyzoites stage specific gene BAG1 the expression level by using Real-time PCR. The results showed In the transport stress after 3 days, the relative expression level of BAG1 decreased significantly increased the relative expression level of SAG1, showed that the reactivation of transport stress induced cysts, which transformed from tachyzoites to tachyzoites. (2) the transport of animal model of chronic infection of Toxoplasma gondii in mice with immune level arch insect bradyzoite into tachyzoites stress effects were collected before 1 days of transport stress, stress, stress for 2 days, the serum of mice 3 days of stress, the changes of cytokines were detected by cytokine antibody array. The results showed that IFN- gamma, IL-1 alpha, IL-4, IL-10, IL-12 and M-CSF 6 kinds of cytokines decreased gradually with the prolonging of stress time, stress in 3 days decreased significantly. That transport stress reduce chronic infection of mice immune level. (3) insect bow shape using screening and identification of Toxoplasma gondii tachyzoites into proteins to tachyzoite to bradyzoite tachyzoites Animal model of transformation, were collected and purified before 1 days of transport stress, stress, stress and stress for 2 days, 3 days in mice brain cyst, cyst extraction of total protein were identified by 2-DE screening bradyzoite into tachyzoites in the process of protein and differences. The results showed that significant changes in the expression of 47 mouse proteins and 15 proteins related to Toxoplasma tachyzoite bradyzoite in transformation process; then using Real-time PCR to verify the Toxoplasma gondii actin (Actin), microneme protein 13 (MIC13), dense granule protein 9 (GRA9), dense granule protein 1 (GRA1) and enolase 1 (ENO1) m RNA levels were significantly increased in the transport stress, whichisinaccordancewith2 dgel results, showed that the expression of bradyzoite into tachyzoites and host insect bodies during protein change. This study using a mouse model confirmed the transport stress stress machine In the process of transformation of Toxoplasma gondii to tachygonite, 47 mouse proteins and 15 Toxoplasma gondii related proteins were successfully screened out and identified, providing a theoretical basis for clarifying the transformation mechanism of Toxoplasma gondii tachygoni and slowly growing organisms.

【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.7

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