疟疾疫苗新型候选抗原的免疫保护效果评价

发布时间：2018-03-23 21:50

本文选题：疟疾　切入点：候选抗原　出处：《吉林大学》2016年硕士论文

【摘要】：疟疾是一种虫媒性传染病,主要流行于热带及亚热带地区,与艾滋病、结核病一同被认为是对人类危害最为严重的传染病。据统计,全世界仍有32亿人感染疟疾,仅2015年就有2.14亿新发病例,约44万人死亡。目前能感染人的疟原虫有五种,其中恶性疟原虫感染是造成疟疾高发病率和死亡率的主要原因。恶性疟原虫是以人和雌性按蚊为宿主,控制疟原虫对人体的致病作用主要是抑制虫体在人体内的发育繁殖,其中抑制虫体侵入红细胞是防止疟疾发生的关键节点之一。因此红细胞内期疟疾疫苗的研究成为了控制和消灭疟疾的热点。肝素类分子是疟原虫识别红细胞的重要受体,不仅能介导玫瑰花环形成、染虫红细胞与宿主细胞黏附,还参与疟原虫侵入红细胞的过程。研究发现一部分恶性疟原虫的蛋白质能与肝素发生特异性结合,且与入侵相关的虫体蛋白质多为肝素结合蛋白。本实验室通过亲和纯化和高通量蛋白质质谱分析,成功筛选出PF3D7_1361800和PF3D7_0811600这两个与肝素结合肽段数较多的蛋白质。研究发现这两个蛋白质定位于裂殖子表面,并且具有良好的免疫原性,其特异性抗体对裂殖子侵入红细胞具有明显抑制作用,极有可能成为潜在的疟疾疫苗候选抗原。为进一步验证其作为抗疟疾疫苗候选抗原的可行性,本实验首先通过生物信息学分析显示,PF3D7_1361800和PF3D7_0811600在疟原虫中种间保守。进一步筛选出与之同源的鼠疟原虫P.bergheiANKA株的PBANKA_13780和PBANKA_142590蛋白质作为研究对象,分析其在鼠内的抗原特性。首先选取编码PBANKA_113780和PBANKA_42590的羧基端的基因片段,连接至表达载体pET-28a, pGEX-4T-1,进行原核表达和纯化,通过亲和层析方法成功获得了重组蛋白质rPB ANKA_113780-963-His和rPBANKA_142590-660-His、 rPBANKA_113780-963-GST和rPBANKA_142590-660-GST,并通过间接ELISA和Western Blot验证了rPBANKA_113780-963-His和rPB ANKA_142590-660-His具有免疫原性和反应原性；以纯化获得的His标签重组蛋白质与弗氏佐剂等体积混合乳化,对BALB/c小鼠进行免疫保护试验,评价重组蛋白的免疫保护效果,结果表明重组蛋白对感染P.bergheiANKA株的小鼠具有一定的保护作用,染虫率和死亡率明显降低；最后通过血清治疗试验检验免疫保护性是否来源于抗体,结果与免疫保护试验结果相符,表明对感染P.bergheiANKA株的小鼠的保护性来自于免疫重组蛋白后诱导产生的抗体。通过以上结果表明恶性疟原虫PF3D7_1361800和PF3D7_0811600作为疟疾疫苗候选抗原具有较好的前景。
[Abstract]:Malaria is a kind of insect-borne infectious disease, mainly prevalent in the tropics and subtropics. Along with AIDS and tuberculosis, malaria is considered to be the most serious infectious disease to human beings. According to statistics, 3.2 billion people are still infected with malaria in the world. There were 214 million new cases and about 440000 deaths in 2015 alone. There are currently five species of Plasmodium that can infect people. Among them, the infection of Plasmodium falciparum is the main cause of the high incidence and mortality of malaria. Plasmodium falciparum is hosted by Anopheles anthropophagus, and the control of the pathogenicity of Plasmodium falciparum is mainly to inhibit the development and reproduction of the parasite in human body. One of the key nodes to prevent malaria is to inhibit the invasion of parasites into red blood cells. Therefore, the study of intraerythrocyte malaria vaccine has become a hot spot for malaria control and elimination. Heparin molecules are important receptors for the recognition of erythrocytes by Plasmodium. It not only mediates the formation of rosettes and adheres to host cells, but also participates in the process of Plasmodium falciparum invading red blood cells. It has been found that some proteins of Plasmodium falciparum bind specifically to heparin. Most of the invasion-related proteins are heparin-binding proteins. PF3D7_1361800 and PF3D7_0811600, two proteins with high number of heparin binding peptides, were successfully screened out. It was found that the two proteins were located on the surface of merozoites and had good immunogenicity. The specific antibody can inhibit the merozoite invading red blood cells, and it is very likely to be a potential candidate antigen for malaria vaccine, in order to further verify its feasibility as a candidate antigen for anti-malaria vaccine. In this study, bioinformatics analysis showed that PF3D71361800 and PF3D7_0811600 were conserved among the species of Plasmodium falciparum. The PBANKA_13780 and PBANKA_142590 proteins of the homologous P.bergheiANKA strain of Plasmodium falciparum were selected as the research objects. Firstly, the carboxyl terminal gene fragments encoding PBANKA_113780 and PBANKA_42590 were selected and ligated to the expression vectors pET-28a, pGEX-4T-1 for prokaryotic expression and purification. The recombinant proteins rPB ANKA_113780-963-His and rPBANKAChe 142590-660-His142590-660-GSTs were successfully obtained by affinity chromatography. The immunogenicity and immunogenicity of rPBANKA_113780-963-His and rPB ANKA_142590-660-His were verified by indirect ELISA and Western Blot. The purified recombinant protein labeled by His was emulsified with Freund's adjuvant in volume to evaluate the immune protective effect of recombinant protein on BALB/c mice. The results showed that the recombinant protein had a protective effect on the mice infected with P.bergheiANKA strain, and the infection rate and mortality rate were significantly decreased. Finally, the serum therapy test was used to test whether the immune protection originated from the antibody, and the results were consistent with the results of the immune protection test. The results showed that the protection of mice infected with P.bergheiANKA strain came from the antibody induced by recombinant protein. The results indicated that PF3D7_1361800 and PF3D7_0811600 of Plasmodium falciparum had a good prospect as candidate antigens for malaria vaccine.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R392

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