發(fā)布時(shí)間：2018-03-17 03:24

  本文選題：弓形蟲　切入點(diǎn)：Ca　出處：《華中農(nóng)業(yè)大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：頂復(fù)門寄生蟲是一類嚴(yán)格的胞內(nèi)寄生生物,對人和動(dòng)物會造成嚴(yán)重的疾病。頂復(fù)門寄生蟲的模式生物之一——?jiǎng)偟毓蜗x(Toxoplasma gondii),全球大約有超過三分之一的人口被其所感染,免疫能力低下的個(gè)體和新生兒甚至?xí)艿缴耐{。弓形蟲擁有如此廣泛的宿主范圍與它強(qiáng)大的入侵機(jī)制有密切的關(guān)系。鈣離子作為細(xì)胞內(nèi)重要的第二信使,調(diào)控著弓形蟲多個(gè)生命活動(dòng),尤其是蟲體入侵和逸出過程。而鈣離子信號通路發(fā)揮作用需要依賴于各類鈣離子相關(guān)蛋白,但是目前對于這些鈣相關(guān)蛋白知之甚少,它們之間的關(guān)系也尚不清楚,鑒定它們在鈣離子信號通路中的作用,以及它們的互作關(guān)系可以進(jìn)一步解釋弓形蟲的入侵機(jī)制,為藥物靶標(biāo)的篩選提供依據(jù)。本研究以鈣調(diào)蛋白(Calmodulin,簡稱Ca M)為研究對象,利用CRISPR/Cas9和Cre-Lox P系統(tǒng)對Ca M在RH△HX蟲株中進(jìn)行敲除,比較Ca M基因敲除蟲株和RH△HX蟲株在復(fù)制能力和入侵能力上的差異;同時(shí),利用Bio ID技術(shù),篩選Ca M的互作蛋白,試圖發(fā)現(xiàn)更多的鈣離子信號通路中的功能蛋白。具體實(shí)驗(yàn)內(nèi)容如下:(1)Ca M蛋白原核表達(dá)和多克隆抗體的制備構(gòu)建p GEX-KG-Ca M原核表達(dá)質(zhì)粒,該質(zhì)粒在表達(dá)感受態(tài)BL21(DE3)中能夠順利表達(dá),將表達(dá)的蛋白純化后進(jìn)行動(dòng)物免疫,收集免疫后的動(dòng)物血清,用酶聯(lián)免疫吸附法檢測抗體效價(jià),結(jié)果顯示該多克隆抗體具有較高水平的抗體效價(jià)。(2)RH△HX-Lox P-Ca M蟲株的構(gòu)建利用Cre-Lox P系統(tǒng)對Ca M基因的條件性敲除。首先,構(gòu)建能夠識別Ca M基因座的p SAG1::Cas9-U6::sg Ca M的CRISPR質(zhì)粒和同源替換的模板質(zhì)粒p UC19-Ca M-YFP,然后將擴(kuò)增出來的同源替換片段和p SAG1::Cas9-U6::sg Ca M質(zhì)粒共同電轉(zhuǎn)到RH△HX蟲株的速殖子中,用黃嘌呤和霉酚酸進(jìn)行藥物篩選,再通過限制性稀釋的方法進(jìn)行單克隆的篩選,經(jīng)擴(kuò)大培養(yǎng)后用PCR和IFA進(jìn)行鑒定,結(jié)果顯示成功獲得RH△HX-Lox P-Ca M蟲株。(3)Ca M敲除株表型研究在RH△HX-Lox P-Ca M蟲株的基礎(chǔ)上,轉(zhuǎn)染能夠表達(dá)Cre剪切酶的質(zhì)粒pmin-e GFP-Cre對Ca M基因進(jìn)行剪切,從而實(shí)現(xiàn)Ca M的缺失。將Ca M的缺失株與RH△HX蟲株同時(shí)進(jìn)行胞內(nèi)復(fù)制實(shí)驗(yàn)和入侵實(shí)驗(yàn),發(fā)現(xiàn)缺失株和RH△HX蟲株在胞內(nèi)的復(fù)制能力沒有多大的差異;但是缺失株的入侵能力有明顯缺陷,入侵能力較RH△HX蟲株顯著性下降。(4)Bio ID技術(shù)篩選與Ca M的互作蛋白用生物素標(biāo)記經(jīng)過遺傳改造后的蟲株RH△HX-Bir A*-Ca M和原始蟲株RH△HX,經(jīng)過24h的作用后,通過間接熒光免疫和Western-blot檢測到RH△HX-Bir A*-Ca M蟲株有被生物素標(biāo)記上的特異蛋白,為了進(jìn)一步確定這些蛋白,我們進(jìn)行了質(zhì)譜分析。經(jīng)過質(zhì)譜結(jié)果的分析對比,從本實(shí)驗(yàn)得到25個(gè)特異性的蛋白質(zhì)。對鑒定到的這些蛋白質(zhì)進(jìn)行功能和定位的分析,推測內(nèi)膜復(fù)合物(IMC)和果糖1,6-二磷酸醛縮酶與Ca M存在互作的可能性,但它們之間具體的關(guān)系還需要進(jìn)一步實(shí)驗(yàn)驗(yàn)證。本研究主要利用Cre-Lox P條件性敲除系統(tǒng)和CRISPR/Cas9技術(shù)在RH△HX蟲株中對基因進(jìn)行敲除。敲除株和RH△HX蟲株比較得出:敲除株在復(fù)制能力上有缺陷,但是在胞內(nèi)復(fù)制的能力沒有明顯差異。同時(shí)通過Bio ID技術(shù)和LC-MS/MS鑒定出25個(gè)特異性蛋白,以上結(jié)果希望能夠?yàn)楣蜗x入侵機(jī)制的研究進(jìn)一步提供理論基礎(chǔ)。
[Abstract]:Apicomplexan parasites is a strictly intracellular parasite, can cause serious disease to human and animal. One of the model organisms -- Toxoplasma gondii Toxoplasma apicomplexan parasites (Toxoplasma gondii), about more than 1/3 of the population was infected by it, low immunity and neonatal individuals are even life threatening. Toxoplasma gondii invasion mechanism has such a broad host range and strong it has a close relationship. The calcium ion as an important intracellular second messenger, regulates Toxoplasma multiple life activities, especially the parasite invasion and escape process. Calcium signaling pathway depends on various types of calcium ion the related protein, but very little is currently known for these calcium related proteins, the relationship between them is not clear, identify their role in calcium signaling, and interaction between them The invasion mechanism can explain the relationship between Toxoplasma gondii, provide the basis for the screening of drug targets. In this study, calmodulin (Calmodulin, referred to as Ca M) as the research object, the Ca M RH in HX knockout strains using CRISPR/Cas9 and Cre-Lox P system, Ca M gene knockout strain and RH Delta HX control strain differences in the replication ability and invasion ability; at the same time, the use of Bio ID technology, Ca M interacting protein screening, trying to find the function of protein calcium signaling pathway in more. The experimental results are as follows: (1) the expression of Ca M protein in E.coli and preparation of Polyclonal antibody to construct P GEX-KG-Ca M prokaryotic expression plasmid, the plasmid expression in E.coli BL21 (DE3) in a smooth expression, the expressed protein purified immune animal collected after immunization of animal serum, was used to detect antibody titer by ELISA. The results showed that the polyclonal antibody The antibody titer has higher level. (2) based on Cre-Lox P system of Ca conditional knockout of M RH HX-Lox P-Ca M strains. First, construct to identify Ca M loci: Cas9-U6: P SAG1:: CRISPR Ca M plasmid SG and homologous replacement plasmid P UC19-Ca M-YFP template, and then the amplified sequence substitution fragment and P: Cas9-U6:: SAG1: SG Ca M to RH HX plasmid common electric strain tachyzoites, drug screening using xanthine and mycophenolic acid, and then through the method of limiting dilution of monoclonal screening were identified by using PCR and IFA after culture expansion. The results show that the success obtained RH P-Ca M strains. HX-Lox (3) Ca M deletion mutant phenotype based on RH HX-Lox P-Ca M strains, the expression of Cre transfected with plasmid pmin-e GFP-Cre enzyme shear shear on Ca M gene, so as to realize the deletion of Ca M. Ca M. Loss of RH strain and HX strains and intracellular replication experiments and invasion experiment, found no differences in the replication ability of strain in intracellular mutant RH and delta HX mutant worms; but the invasion ability has obvious defects, the invasion ability than RH HX strains significantly decreased (4). Bio ID and Ca M screening interaction protein with biotin labeled genetically modified strains of RH HX-Bir A*-Ca M and the original strain RH HX, after the action of 24h, by indirect immunofluorescence and Western-blot detected RH HX-Bir A*-Ca Delta M strains have been biotinylated on. In order to further determine the different proteins, these proteins, we performed mass spectrometric analysis by mass spectrometry. The comparative analysis results, 25 specific proteins from this experiment. Analyze the function and orientation of these proteins to speculate on the identification, endometrial complex (IMC) and fructose 1,6- two The possibility of interaction the presence of aldolase and Ca M, but the specific relationship between them still need further experimental verification. This research mainly uses the Cre-Lox P conditional knockout system and CRISPR/Cas9 technology in RH HX strains of gene knockout. Knockout strain and RH strain comparison: HX knockout strains the replication capacity is defective, but the ability to replicate in cells had no obvious difference. At the same time through the Bio ID technology and LC-MS/MS identified 25 specific proteins, the above results to study the mechanisms of Toxoplasma gondii invasion and provide further theoretical basis.

【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S852.7

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本文編號：1622958