發(fā)布時(shí)間：2018-03-23 21:50

  本文選題：瘧疾　切入點(diǎn)：候選抗原　出處：《吉林大學(xué)》2016年碩士論文

【摘要】：瘧疾是一種蟲媒性傳染病,主要流行于熱帶及亞熱帶地區(qū),與艾滋病、結(jié)核病一同被認(rèn)為是對(duì)人類危害最為嚴(yán)重的傳染病。據(jù)統(tǒng)計(jì),全世界仍有32億人感染瘧疾,僅2015年就有2.14億新發(fā)病例,約44萬人死亡。目前能感染人的瘧原蟲有五種,其中惡性瘧原蟲感染是造成瘧疾高發(fā)病率和死亡率的主要原因。惡性瘧原蟲是以人和雌性按蚊為宿主,控制瘧原蟲對(duì)人體的致病作用主要是抑制蟲體在人體內(nèi)的發(fā)育繁殖,其中抑制蟲體侵入紅細(xì)胞是防止瘧疾發(fā)生的關(guān)鍵節(jié)點(diǎn)之一。因此紅細(xì)胞內(nèi)期瘧疾疫苗的研究成為了控制和消滅瘧疾的熱點(diǎn)。肝素類分子是瘧原蟲識(shí)別紅細(xì)胞的重要受體,不僅能介導(dǎo)玫瑰花環(huán)形成、染蟲紅細(xì)胞與宿主細(xì)胞黏附,還參與瘧原蟲侵入紅細(xì)胞的過程。研究發(fā)現(xiàn)一部分惡性瘧原蟲的蛋白質(zhì)能與肝素發(fā)生特異性結(jié)合,且與入侵相關(guān)的蟲體蛋白質(zhì)多為肝素結(jié)合蛋白。本實(shí)驗(yàn)室通過親和純化和高通量蛋白質(zhì)質(zhì)譜分析,成功篩選出PF3D7_1361800和PF3D7_0811600這兩個(gè)與肝素結(jié)合肽段數(shù)較多的蛋白質(zhì)。研究發(fā)現(xiàn)這兩個(gè)蛋白質(zhì)定位于裂殖子表面,并且具有良好的免疫原性,其特異性抗體對(duì)裂殖子侵入紅細(xì)胞具有明顯抑制作用,極有可能成為潛在的瘧疾疫苗候選抗原。為進(jìn)一步驗(yàn)證其作為抗瘧疾疫苗候選抗原的可行性,本實(shí)驗(yàn)首先通過生物信息學(xué)分析顯示,PF3D7_1361800和PF3D7_0811600在瘧原蟲中種間保守。進(jìn)一步篩選出與之同源的鼠瘧原蟲P.bergheiANKA株的PBANKA_13780和PBANKA_142590蛋白質(zhì)作為研究對(duì)象,分析其在鼠內(nèi)的抗原特性。首先選取編碼PBANKA_113780和PBANKA_42590的羧基端的基因片段,連接至表達(dá)載體pET-28a, pGEX-4T-1,進(jìn)行原核表達(dá)和純化,通過親和層析方法成功獲得了重組蛋白質(zhì)rPB ANKA_113780-963-His和rPBANKA_142590-660-His、 rPBANKA_113780-963-GST和rPBANKA_142590-660-GST,并通過間接ELISA和Western Blot驗(yàn)證了rPBANKA_113780-963-His和rPB ANKA_142590-660-His具有免疫原性和反應(yīng)原性；以純化獲得的His標(biāo)簽重組蛋白質(zhì)與弗氏佐劑等體積混合乳化,對(duì)BALB/c小鼠進(jìn)行免疫保護(hù)試驗(yàn),評(píng)價(jià)重組蛋白的免疫保護(hù)效果,結(jié)果表明重組蛋白對(duì)感染P.bergheiANKA株的小鼠具有一定的保護(hù)作用,染蟲率和死亡率明顯降低；最后通過血清治療試驗(yàn)檢驗(yàn)免疫保護(hù)性是否來源于抗體,結(jié)果與免疫保護(hù)試驗(yàn)結(jié)果相符,表明對(duì)感染P.bergheiANKA株的小鼠的保護(hù)性來自于免疫重組蛋白后誘導(dǎo)產(chǎn)生的抗體。通過以上結(jié)果表明惡性瘧原蟲PF3D7_1361800和PF3D7_0811600作為瘧疾疫苗候選抗原具有較好的前景。
[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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R392

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