CaM在弓形虫入侵过程中的机制研究

发布时间：2018-03-17 03:24

本文选题：弓形虫　切入点：Ca　出处：《华中农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：顶复门寄生虫是一类严格的胞内寄生生物,对人和动物会造成严重的疾病。顶复门寄生虫的模式生物之一——刚地弓形虫(Toxoplasma gondii),全球大约有超过三分之一的人口被其所感染,免疫能力低下的个体和新生儿甚至会受到生命的威胁。弓形虫拥有如此广泛的宿主范围与它强大的入侵机制有密切的关系。钙离子作为细胞内重要的第二信使,调控着弓形虫多个生命活动,尤其是虫体入侵和逸出过程。而钙离子信号通路发挥作用需要依赖于各类钙离子相关蛋白,但是目前对于这些钙相关蛋白知之甚少,它们之间的关系也尚不清楚,鉴定它们在钙离子信号通路中的作用,以及它们的互作关系可以进一步解释弓形虫的入侵机制,为药物靶标的筛选提供依据。本研究以钙调蛋白(Calmodulin,简称Ca M)为研究对象,利用CRISPR/Cas9和Cre-Lox P系统对Ca M在RH△HX虫株中进行敲除,比较Ca M基因敲除虫株和RH△HX虫株在复制能力和入侵能力上的差异;同时,利用Bio ID技术,筛选Ca M的互作蛋白,试图发现更多的钙离子信号通路中的功能蛋白。具体实验内容如下:(1)Ca M蛋白原核表达和多克隆抗体的制备构建p GEX-KG-Ca M原核表达质粒,该质粒在表达感受态BL21(DE3)中能够顺利表达,将表达的蛋白纯化后进行动物免疫,收集免疫后的动物血清,用酶联免疫吸附法检测抗体效价,结果显示该多克隆抗体具有较高水平的抗体效价。(2)RH△HX-Lox P-Ca M虫株的构建利用Cre-Lox P系统对Ca M基因的条件性敲除。首先,构建能够识别Ca M基因座的p SAG1::Cas9-U6::sg Ca M的CRISPR质粒和同源替换的模板质粒p UC19-Ca M-YFP,然后将扩增出来的同源替换片段和p SAG1::Cas9-U6::sg Ca M质粒共同电转到RH△HX虫株的速殖子中,用黄嘌呤和霉酚酸进行药物筛选,再通过限制性稀释的方法进行单克隆的筛选,经扩大培养后用PCR和IFA进行鉴定,结果显示成功获得RH△HX-Lox P-Ca M虫株。(3)Ca M敲除株表型研究在RH△HX-Lox P-Ca M虫株的基础上,转染能够表达Cre剪切酶的质粒pmin-e GFP-Cre对Ca M基因进行剪切,从而实现Ca M的缺失。将Ca M的缺失株与RH△HX虫株同时进行胞内复制实验和入侵实验,发现缺失株和RH△HX虫株在胞内的复制能力没有多大的差异;但是缺失株的入侵能力有明显缺陷,入侵能力较RH△HX虫株显著性下降。(4)Bio ID技术筛选与Ca M的互作蛋白用生物素标记经过遗传改造后的虫株RH△HX-Bir A*-Ca M和原始虫株RH△HX,经过24h的作用后,通过间接荧光免疫和Western-blot检测到RH△HX-Bir A*-Ca M虫株有被生物素标记上的特异蛋白,为了进一步确定这些蛋白,我们进行了质谱分析。经过质谱结果的分析对比,从本实验得到25个特异性的蛋白质。对鉴定到的这些蛋白质进行功能和定位的分析,推测内膜复合物(IMC)和果糖1,6-二磷酸醛缩酶与Ca M存在互作的可能性,但它们之间具体的关系还需要进一步实验验证。本研究主要利用Cre-Lox P条件性敲除系统和CRISPR/Cas9技术在RH△HX虫株中对基因进行敲除。敲除株和RH△HX虫株比较得出:敲除株在复制能力上有缺陷,但是在胞内复制的能力没有明显差异。同时通过Bio ID技术和LC-MS/MS鉴定出25个特异性蛋白,以上结果希望能够为弓形虫入侵机制的研究进一步提供理论基础。
[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.

【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.7

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