本文選題：弓形蟲 + SAG3基因��； 參考：《廣東藥學(xué)院》2009年碩士論文

【摘要】： 剛地弓形蟲(Toxoplasma gondii)是一種呈世界性分布的專性細(xì)胞內(nèi)寄生的機(jī)會(huì)性致病原蟲,可引起人獸共患的弓形蟲病。正常人感染弓形蟲后多呈無癥狀帶蟲免疫狀態(tài),但免疫力低下的人群(如腫瘤患者、AIDS病人、器官移植病人等)感染后,可引起嚴(yán)重的后果甚至死亡。孕婦感染弓形蟲可通過胎盤垂直傳播,引起早產(chǎn)、流產(chǎn)、死胎或嬰兒發(fā)育畸形等。近年來,隨著腫瘤患者、AIDS病人及寵物飼養(yǎng)愛好者的逐漸增多,弓形蟲的危害也日益突出,但至今仍無治療弓形蟲病的安全有效藥物,因此深入了解其致病機(jī)制對于弓形蟲的防治具有重要意義。 弓形蟲致病的首要步驟是對宿主細(xì)胞的入侵及在宿主細(xì)胞內(nèi)的快速增殖,但迄今為止,人們對入侵和增殖的機(jī)制尚未明了。已有的研究表明弓形蟲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員,其在速殖子、緩殖子及子孢子等多個(gè)時(shí)期均有表達(dá),且具有較低的免疫原性。雖然已有的研究顯示SAG3參與蟲體的入侵及增殖,但是目前關(guān)于SAG3的研究極少,其在弓形蟲致病中的作用和地位并不知曉,因此深入研究SAG3的功能將為研究闡明弓形蟲致病機(jī)制提供科學(xué)資料。 研究目的: 本研究利用RNAi技術(shù),研究弓形蟲SAG3對蟲體入侵和增殖能力的影響,以期能深入了解表面抗原SAG3的功能,為闡明弓形蟲致病機(jī)制提供科學(xué)資料。 研究內(nèi)容: 1、弓形蟲SAG3對蟲體入侵的作用:用電穿孔的方法將針對SAG3基因3’-UTR區(qū)的dsRNA轉(zhuǎn)入弓形蟲速殖子體內(nèi),轉(zhuǎn)染后的蟲體感染Hela細(xì)胞,分別于轉(zhuǎn)染后5min、30min、1h和2h提取細(xì)胞蟲體的總RNA,并用半定量RT-PCR方法分析與弓形蟲入侵密切關(guān)的MIC2、ROP2和GRA2三個(gè)基因的表達(dá)量的變化,同時(shí)設(shè)立無相關(guān)對照組(GFP),電轉(zhuǎn)條件對照組和正常對照組。 2、弓形蟲SAG3對蟲體增殖的作用:用電穿孔的方法將SAG3基因3’-UTR區(qū)和CDS區(qū)的dsRNA轉(zhuǎn)入弓形蟲速殖子體內(nèi),轉(zhuǎn)染后的弓形蟲感染Hela細(xì)胞,分別于轉(zhuǎn)染后12h、24h和48h提取細(xì)胞蟲體的總RNA并用半定量RT-PCR方法分析弓形蟲嘌呤補(bǔ)救合成途徑中AK、HXGPRT兩個(gè)關(guān)鍵酶基因的表達(dá)量的變化,同時(shí)設(shè)立無相關(guān)對照組(GFP),電轉(zhuǎn)條件對照組和正常對照組。 研究結(jié)果 1)轉(zhuǎn)染SAG3基因dsRNA的弓形蟲感染Hela細(xì)胞后,MIC2、ROP2和GRA2的表達(dá)量均低于對照組。 2)轉(zhuǎn)染SAG3基因dsRNA的弓形蟲感染Hela細(xì)胞后,AK和HXGPRT的表達(dá)量低于對照組。 結(jié)論:弓形蟲SAG3基因的表達(dá)被抑制后,弓形蟲的入侵和增殖能力明顯減弱,說明SAG3可能在弓形蟲致病中起著重要的作用,為深入了解表面抗原SAG3基因的功能、闡明弓形蟲入侵宿主的致病機(jī)制奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：廣東藥學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R382.5

【引證文獻(xiàn)】

相關(guān)期刊論文 前1條

1 郭凱;陳興智;;弓形蟲主要抗原的研究進(jìn)展[J];蚌埠醫(yī)學(xué)院學(xué)報(bào);2012年04期

相關(guān)碩士學(xué)位論文 前1條

1 郭凱;弓形蟲緩殖子期特異抗原BSR4的克隆、表達(dá)、純化與鑒定[D];蚌埠醫(yī)學(xué)院;2011年

，

本文編號(hào)：1988551