弓形虫表面抗原SAG3在虫体入侵及增殖中的作用

发布时间：2018-06-06 23:23

本文选题：弓形虫 + SAG3基因　；参考：《广东药学院》2009年硕士论文

【摘要】： 刚地弓形虫(Toxoplasma gondii)是一种呈世界性分布的专性细胞内寄生的机会性致病原虫,可引起人兽共患的弓形虫病。正常人感染弓形虫后多呈无症状带虫免疫状态,但免疫力低下的人群(如肿瘤患者、AIDS病人、器官移植病人等)感染后,可引起严重的后果甚至死亡。孕妇感染弓形虫可通过胎盘垂直传播,引起早产、流产、死胎或婴儿发育畸形等。近年来,随着肿瘤患者、AIDS病人及宠物饲养爱好者的逐渐增多,弓形虫的危害也日益突出,但至今仍无治疗弓形虫病的安全有效药物,因此深入了解其致病机制对于弓形虫的防治具有重要意义。弓形虫致病的首要步骤是对宿主细胞的入侵及在宿主细胞内的快速增殖,但迄今为止,人们对入侵和增殖的机制尚未明了。已有的研究表明弓形虫SRS(SAG1-related sequence)蛋白超家族在其中起了重要作用,而SAG3是SRS蛋白超家族中非常重要的一员,其在速殖子、缓殖子及子孢子等多个时期均有表达,且具有较低的免疫原性。虽然已有的研究显示SAG3参与虫体的入侵及增殖,但是目前关于SAG3的研究极少,其在弓形虫致病中的作用和地位并不知晓,因此深入研究SAG3的功能将为研究阐明弓形虫致病机制提供科学资料。研究目的: 本研究利用RNAi技术,研究弓形虫SAG3对虫体入侵和增殖能力的影响,以期能深入了解表面抗原SAG3的功能,为阐明弓形虫致病机制提供科学资料。研究内容: 1、弓形虫SAG3对虫体入侵的作用:用电穿孔的方法将针对SAG3基因3’-UTR区的dsRNA转入弓形虫速殖子体内,转染后的虫体感染Hela细胞,分别于转染后5min、30min、1h和2h提取细胞虫体的总RNA,并用半定量RT-PCR方法分析与弓形虫入侵密切关的MIC2、ROP2和GRA2三个基因的表达量的变化,同时设立无相关对照组(GFP),电转条件对照组和正常对照组。 2、弓形虫SAG3对虫体增殖的作用:用电穿孔的方法将SAG3基因3’-UTR区和CDS区的dsRNA转入弓形虫速殖子体内,转染后的弓形虫感染Hela细胞,分别于转染后12h、24h和48h提取细胞虫体的总RNA并用半定量RT-PCR方法分析弓形虫嘌呤补救合成途径中AK、HXGPRT两个关键酶基因的表达量的变化,同时设立无相关对照组(GFP),电转条件对照组和正常对照组。研究结果 1)转染SAG3基因dsRNA的弓形虫感染Hela细胞后,MIC2、ROP2和GRA2的表达量均低于对照组。 2)转染SAG3基因dsRNA的弓形虫感染Hela细胞后,AK和HXGPRT的表达量低于对照组。结论:弓形虫SAG3基因的表达被抑制后,弓形虫的入侵和增殖能力明显减弱,说明SAG3可能在弓形虫致病中起着重要的作用,为深入了解表面抗原SAG3基因的功能、阐明弓形虫入侵宿主的致病机制奠定了基础。
[Abstract]:Toxoplasma gondii (Toxoplasma gondii) is a worldwide parasitic opportunistic protozoa, which can cause zooplasmosis. The immune status of Toxoplasma gondii infection in normal people is usually asymptomatic, but the people with low immunity (such as tumor patients with AIDS, organ transplant patients, etc.) can cause serious consequences and even death after infection. Infection of Toxoplasma gondii in pregnant women can be transmitted vertically through the placenta, leading to premature delivery, abortion, stillbirth or malformation of the infant. In recent years, with the increasing number of AIDS patients and pet enthusiasts, the harm of toxoplasmosis is becoming more and more serious, but there is still no safe and effective drug to treat Toxoplasma gondii. Therefore, it is important to understand the pathogenesis of Toxoplasma gondii. The first step of Toxoplasma gondii is invasion of host cells and rapid proliferation in host cells, but the mechanism of invasion and proliferation has not been understood. Previous studies have shown that the SRS(SAG1-related sequence protein superfamily of Toxoplasma gondii plays an important role, while SAG3 is a very important member of the SRS protein superfamily, and it is expressed in several stages, such as tachyzoites, bradyzoites and sporozoites. And has low immunogenicity. Although previous studies have shown that SAG3 is involved in the invasion and proliferation of parasites, there are few studies on SAG3, and its role and position in the pathogenesis of Toxoplasma gondii is unknown. Therefore, further study of the function of SAG3 will provide scientific data for elucidating the pathogenic mechanism of Toxoplasma gondii. Objectives of the study: In this study, RNAi technique was used to study the effect of Toxoplasma gondii SAG3 on the invading and proliferating ability of Toxoplasma gondii, in order to understand the function of surface antigen SAG3 and provide scientific data for elucidating the pathogenic mechanism of Toxoplasma gondii. Research content: 1. The effect of Toxoplasma gondii SAG3 on the invasion of Toxoplasma gondii: by electroporation, the dsRNA targeting the 3'-UTR region of the SAG3 gene was transferred into Toxoplasma gondii Tachyzoites and infected with Hela cells. The total RNAs of cytosomes were extracted at 5 mins 30 min and 2 h after transfection, and the expression levels of three genes, MIC2, ROP2 and GRA2, which were closely related to the invasion of Toxoplasma gondii, were analyzed by semi-quantitative RT-PCR method. At the same time, no correlation control group, electroporation condition control group and normal control group were established. 2. The effect of Toxoplasma SAG3 on the proliferation of Toxoplasma gondii: the dsRNA of 3'-UTR and CDS regions of SAG3 gene were transferred into Toxoplasma gondii Tachyzoites by electroporation, and the transfected Toxoplasma gondii infected Hela cells. Total RNA was extracted from the cytosomes of Toxoplasma gondii 12 h after transfection at 24 h and 48 h after transfection. The expression of two key enzyme genes of AKHXGPRT in the rescue pathway of Toxoplasma gondii purine was analyzed by semi-quantitative RT-PCR method. At the same time, no correlation control group, electroporation condition control group and normal control group were established. Research results 1) the expression of roP2 and GRA2 in Toxoplasma gondii infected with SAG3 gene dsRNA was lower than that in control group after infection with Toxoplasma gondii (Toxoplasma gondii). 2) the expression of AK and HXGPRT in Toxoplasma gondii infected Hela cells was lower than that in the control group. Conclusion: after the expression of SAG3 gene of Toxoplasma gondii is inhibited, the invasion and proliferation ability of Toxoplasma gondii is obviously weakened, indicating that SAG3 may play an important role in the pathogenesis of Toxoplasma gondii. It is necessary to elucidate the pathogenic mechanism of Toxoplasma gondii invading the host.
【学位授予单位】：广东药学院
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R382.5

【引证文献】