疟原虫TatD-like DNase的功能分析及与虫体致病性的相关性分析

发布时间：2018-03-25 15:36

本文选题：疟原虫　切入点：脱氧核糖核酸酶　出处：《吉林大学》2016年博士论文

【摘要】：疟原虫对宿主的危害主要发生在血液期,即虫体在宿主红细胞内的发育繁殖过程。然而,多年来对疟原虫的生物学及致病机理的解析一直存在着很多空白。这影响着疫苗的研制和抗疟药物的开发。在本实验中,我们通过生物信息学分析首先分析潜在的能降解宿主免疫细胞胞外陷阱ETs(Extracellular Traps)结构的分泌性DNase,即Tat D-like DNase,并分析其基序,结构域和序列特性。结果显示,疟原虫的Tat D-like DNAse是一种金属依赖性的脱氧核糖核酸酶,其蛋白质在不同疟原虫中的序列非常保守,并且在N端存在一个20个氨基酸左右的信号肽,这说明该蛋白质具有分泌的潜力。进一步,对Pf Tat D在疟原虫红内期的转录表达水平进行分析。用q PCR的方法对不同时期编码Pf Tat D基因的转录水平进行分析;同时对Pf Tat D在疟原虫红内期的表达水平进行western blot分析。结果显示,Pf Tat D在疟原虫红内期全程转录表达,并且在32小时达到转录高峰。我们采用间接免疫荧光和免疫电镜的方法定位Pf Tat D在染虫红细胞内的位置。定位结果与生物信息学预测相符,蛋白质存在于纳虫溶泡膜与染虫红细胞膜之间,并且具有向外分泌的趋势。在鼠疟原虫相关实验中,我们发现Tat D的转录与表达和虫体的毒力成正相关,这也说明了该蛋白质在虫体致病中的作用。进一步我们采用double crossover的方法敲除了P.berghei ANKA株编码Pb Tat D的基因,获得了缺陷表达Tat D的虫株。对缺陷虫株的毒力分析结果显示,缺陷表达虫株的感染能力和致死性都明显下降。而将缺陷表达虫株与巨噬细胞J774A.1体外共培养,发现该虫株可以诱导巨噬细胞产生ETs结构,并且产生的数量明显高于野生型虫株。这个结果说明了,疟原虫Tat D参与了虫体逃避宿主ETs清除的过程。而在酶活实验中,原核表达的Tat D蛋白质可以体外降解宿主DNA,这也进一步验证了Tat D参与虫体致病性逃避宿主清除机理的假说,即虫体将Tat D蛋白质释放到胞外,通过其脱氧核糖核酸酶的活性降解宿主产生的ETs结构中的DNA骨架来逃避其杀伤性作用。我们通过对小鼠免疫重组Pb Tat D和Pc Tat D蛋白质评价该蛋白质的免疫保护性。结果显示免疫组的染虫率要低于空白组和阴性组,并且小鼠的存活时间更长。而血清治疗实验的结果也与免疫保护试验的结果相符。以上结果显示,该蛋白质作为一个虫体致病相关蛋白质,参与了虫体逃避宿主天然免疫的过程,并且具有作为红内期疫苗的候选抗原的潜力。本研究首次揭示了Tat D-like DNase蛋白质作为疟原虫致病相关蛋白质,参与虫体逃避NETs清除的过程,这对深入理解疟原虫逃避宿主天然免疫具有重要的意义,也为研制疟疾疫苗提供了新的思路。
[Abstract]:The harm of Plasmodium to the host mainly occurs in the blood phase, that is, the development and reproduction of the parasite in the host erythrocyte. However, Over the years, there have been many gaps in the analysis of the biological and pathogenic mechanisms of Plasmodium. This has affected the development of vaccines and antimalarial drugs. By bioinformatics analysis, we first analyzed the secretory DNase, or Tat D-like DNase, which can degrade the host immune cell extracellular trap (ETs(Extracellular traps) structure, and analyzed its motif, domain and sequence properties. The Tat D-like DNAse of Plasmodium is a metal-dependent deoxyribonuclease, its protein is very conserved in different plasmodium, and there is a signal peptide of about 20 amino acids at the N-terminal. This indicates that the protein has the potential to secrete. Furthermore, the transcriptional expression level of PF Tat D in the erythroid phase of Plasmodium is analyzed. The transcription level of PF Tat D gene at different stages is analyzed by Q PCR method. At the same time, the expression of PF Tat D in the erythroid phase of Plasmodium was analyzed by western blot. The results showed that the expression of PF Tat D was transcriptional in the whole process of the erythrocytic phase of Plasmodium falciparum. We used indirect immunofluorescence and immunoelectron microscopy to locate the position of PF Tat D in the erythrocytes of the parasite. The localization results were consistent with the bioinformatics prediction. The protein exists between the foam membrane and the erythrocyte membrane, and has the tendency of exocrine secretion. We found that the transcription and expression of Tat D was positively correlated with the virulence of the parasite in the related experiments of Plasmodium falciparum. Further, we used double crossover method to knock out the gene encoding Pb Tat D of P.berghei ANKA strain, and obtained the strain with defective expression of Tat D. the virulence analysis to the defective strain showed that, The infection ability and lethal ability of the defective expression insect strain were obviously decreased, but co-cultured with macrophage J774A.1 in vitro, it was found that the strain could induce macrophage to produce ETs structure. The results show that the Tat D of Plasmodium parvum is involved in the process of evading host ETs clearance. The prokaryotic expression of Tat D protein can degrade the host DNA in vitro, which further verifies the hypothesis that Tat D is involved in the mechanism of pathogenicity escape from host, that is, the body releases Tat D protein to extracellular. The DNA skeleton in the ETs structure produced by its deoxyribonuclease activity was used to escape its lethal effects. We evaluated the immune protection of the protein by immunizing the recombinant Pb Tat D and PC Tat D proteins in mice. The results showed that the infection rate of immune group was lower than that of blank group and negative group. And the survival time of the mice was longer. And the results of the serum therapy experiment were consistent with the results of the immune protection test. It is involved in the process of parasite escaping from host innate immunity, and has the potential to be a candidate antigen for intrared vaccine. In this study, we first revealed that Tat D-like DNase protein is associated with Plasmodium plasmodium pathogenicity, and participate in the process of evading NETs clearance. This is of great significance to the understanding of malaria parasite escaping from host innate immunity, and also provides a new idea for the development of malaria vaccine.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R382.31

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