本文選題：瘧疾 + 疫苗��； 參考：《第三軍醫(yī)大學(xué)》2014年博士論文

【摘要】：瘧疾目前仍然是最主要的全球健康威脅之，每年因瘧疾感染而死亡的人數(shù)約62.7萬人，至今臨床上尚無高效可行的瘧疾疫苗問世。紅前期階段為瘧原蟲入侵人體的首個(gè)時(shí)期，阻斷瘧原蟲子孢子在肝臟中發(fā)育和繁殖，可防止肝期裂殖子入血感染紅細(xì)胞，從而出現(xiàn)臨床癥狀，針對(duì)紅前期設(shè)計(jì)的疫苗可從源頭上控制瘧疾感染。目前認(rèn)為，最為有效的紅前期瘧疾疫苗是減毒子孢子疫苗，減毒子孢子誘導(dǎo)的CD8+T細(xì)胞免疫反應(yīng)是其最主要的保護(hù)性免疫機(jī)制，也成為衡量瘧疾疫苗有效性的金標(biāo)準(zhǔn)。然而，減毒子孢子的大量獲得及運(yùn)輸、保存方面存在的困難，使其大規(guī)模的生產(chǎn)及現(xiàn)場(chǎng)應(yīng)用受到限制，亞單位疫苗將會(huì)成為新代疫苗的主要研發(fā)方向，但至今僅鑒定出少數(shù)的紅前期疫苗候選抗原，因此，迫切需要發(fā)展新的高效紅前期保護(hù)性抗原篩選方法，篩選出更多介導(dǎo)CD8+T細(xì)胞免疫反應(yīng)的靶抗原，進(jìn)步認(rèn)識(shí)清除感染細(xì)胞的免疫機(jī)制，為研制出高效的紅前期瘧疾疫苗奠定堅(jiān)實(shí)基礎(chǔ)。 本研究首先通過DNA疫苗評(píng)價(jià)了約氏瘧原蟲（Plasmodium yoelii）候選抗原PyTmp21在小鼠體內(nèi)誘導(dǎo)的紅前期保護(hù)作用。其次，，利用肝臟晚期發(fā)育停滯P.yoeliifabb/f-基因減毒子孢子疫苗小鼠保護(hù)性模型，采用水動(dòng)力法尾靜脈注射（Hydrodynamic tail vein injection, HTVI）表達(dá)特異性抗原的熒光素酶報(bào)告融合質(zhì)粒DNA入小鼠肝細(xì)胞內(nèi)，結(jié)合活體成像系統(tǒng)（In Vivo Imaging System, IVIS）實(shí)時(shí)監(jiān)測(cè)熒光素酶報(bào)告基因在免疫小鼠肝細(xì)胞內(nèi)表達(dá)量，從而鑒定減毒子孢子疫苗誘導(dǎo)特異性CD8+T細(xì)胞免疫應(yīng)答殺傷肝細(xì)胞的靶抗原，為進(jìn)步篩選Pyfabb/f-基因減毒子孢子誘導(dǎo)保護(hù)性免疫相關(guān)的紅前期抗原建立技術(shù)平臺(tái)。最后，利用該技術(shù)平臺(tái)探討Pyfabb/f-基因減毒子孢子能否誘導(dǎo)產(chǎn)生針對(duì)新候選抗原PyTmp21的特異性細(xì)胞免疫反應(yīng)并清除表達(dá)該抗原的肝細(xì)胞，從而評(píng)價(jià)PyTmp21是否為潛在的減毒子孢子疫苗誘導(dǎo)的保護(hù)性相關(guān)抗原。主要研究結(jié)果如下： 一、PyTmp21DNA疫苗免疫后可抑制小鼠紅前期瘧原蟲感染的肝臟蟲荷量及誘導(dǎo)消除性保護(hù)作用 為了鑒定新候選抗原PyTmp21能否在體內(nèi)誘導(dǎo)保護(hù)性免疫，我們利用DNA疫苗免疫小鼠后發(fā)現(xiàn)，PyTmp21免疫可顯著減少子孢子攻擊感染小鼠肝臟內(nèi)的瘧原蟲蟲荷量及誘導(dǎo)60%消除性保護(hù)效率。 二、建立HTVI/IVIS篩選Pyfabb/f-基因減毒子孢子誘導(dǎo)的特異性CD8+T細(xì)胞免疫反應(yīng)相關(guān)保護(hù)性抗原的研究平臺(tái) 1、通過HTVI法將PyCSP-Luc重組質(zhì)粒注射入正常小鼠體內(nèi)，IVIS系統(tǒng)觀察證實(shí)質(zhì)粒DNA在小鼠肝臟內(nèi)持續(xù)、穩(wěn)定高表達(dá)，并可實(shí)時(shí)監(jiān)測(cè)與熒光素酶融合的目的蛋白在肝臟的表達(dá)量。通過流式細(xì)胞術(shù)證實(shí)HTVI注射后質(zhì)粒DNA主要表達(dá)在肝細(xì)胞內(nèi)，為目的蛋白的表達(dá)定位提供依據(jù)。 2、利用Pyfabb/f-基因減毒子孢子免疫小鼠誘導(dǎo)產(chǎn)生完全保護(hù)性免疫反應(yīng)，HTVI將PyCSP-Luc質(zhì)粒DNA攻擊免疫小鼠，IVIS觀察證實(shí)免疫小鼠肝臟內(nèi)熒光素酶表達(dá)量減低。進(jìn)步抗體耗竭實(shí)驗(yàn)證實(shí)CD8+T細(xì)胞介導(dǎo)了清除表達(dá)靶抗原的肝細(xì)胞。再者，對(duì)質(zhì)粒攻擊后的免疫小鼠肝臟淋巴細(xì)胞進(jìn)行流式細(xì)胞內(nèi)染色發(fā)現(xiàn)，CSP特異性CD8+T細(xì)胞數(shù)量明顯增加且IFN-γ分泌水平增加。本實(shí)驗(yàn)表明Py fabb/f-基因減毒子孢子免疫小鼠可誘導(dǎo)產(chǎn)生CSP特異性CD8+T細(xì)胞免疫反應(yīng)，并介導(dǎo)清除表達(dá)該抗原表位的肝細(xì)胞，HTVI與IVIS相結(jié)合技術(shù)可作為有效的工具用于篩選基因減毒子孢子誘導(dǎo)CD8+T細(xì)胞清除肝細(xì)胞的靶抗原。 三、利用HTVI/IVIS研究平臺(tái)鑒定Py fabb/f-基因減毒子孢子誘導(dǎo)保護(hù)性免疫相關(guān)的紅前期抗原 為了鑒定基因減毒子孢子疫苗保護(hù)模型誘導(dǎo)的保護(hù)性免疫相關(guān)紅前期抗原，我們利用HTVI/IVIS研究平臺(tái)檢測(cè)Pyfabb/f-基因減毒子孢子能否誘導(dǎo)產(chǎn)生針對(duì)新候選抗原PyTmp21的特異性T細(xì)胞免疫反應(yīng)并殺傷表達(dá)該抗原的肝細(xì)胞。我們發(fā)現(xiàn)2次Pyfabb/f-子孢子同源免疫后，HTVI法將PyTmp21-Luc質(zhì)粒DNA攻擊免疫小鼠，未能觀察到肝臟內(nèi)熒光素酶表達(dá)量減低現(xiàn)象。然而，利用DNA+子孢子或子孢子+DNA異源性免疫-加強(qiáng)免疫策略發(fā)現(xiàn)，PyTmp21-Luc質(zhì)粒DNA攻擊免疫小鼠后其在肝臟的表達(dá)量明顯減低，提示Pyfabb/f-減毒子孢子可誘導(dǎo)針對(duì)PyTmp21抗原的特異性免疫反應(yīng)并殺傷表達(dá)該抗原的肝細(xì)胞。本實(shí)驗(yàn)表明，同源性減毒子孢子免疫小鼠后可能主要誘導(dǎo)產(chǎn)生針對(duì)優(yōu)勢(shì)抗原CSP的有效免疫反應(yīng)，而不足以誘導(dǎo)產(chǎn)生針對(duì)非CSP抗原的免疫反應(yīng)，因此，我們通過異源性免疫-加強(qiáng)免疫策略證實(shí)Pyfabb/f-基因減毒子孢子可誘導(dǎo)產(chǎn)生有效的PyTmp21抗原特異性T細(xì)胞免疫反應(yīng)，PyTmp21抗原為潛在的減毒子孢子疫苗誘導(dǎo)的保護(hù)性相關(guān)抗原。 我們的研究顯示HTVI/IVIS方法可用于檢測(cè)基因減毒子孢子疫苗誘導(dǎo)的CD8+T細(xì)胞特異性殺傷肝細(xì)胞的靶抗原。采用異源性免疫-加強(qiáng)免疫策略證實(shí)Pyfabb/f-基因減毒子孢子疫苗誘導(dǎo)了針對(duì)PyTmp21抗原的特異性免疫反應(yīng)，并清除了表達(dá)該抗原的肝細(xì)胞，PyTmp21作為新的紅前期抗原參與減毒子孢子疫苗誘導(dǎo)的保護(hù)性免疫反應(yīng)。異源性免疫-加強(qiáng)免疫策略結(jié)合HTVI/IVIS技術(shù)可作為有效的工具用于篩選減毒子孢子全蟲疫苗誘導(dǎo)的新紅前期非CSP保護(hù)性抗原，為進(jìn)步認(rèn)識(shí)減毒全蟲疫苗的保護(hù)性免疫機(jī)制、篩選出更多的亞單位疫苗候選抗原并設(shè)計(jì)出高效的瘧疾疫苗奠定基礎(chǔ)。
[Abstract]:Malaria remains a major threat to global health, the number of infections and deaths due to malaria each year about 627 thousand people, so far there are no clinically effective malaria vaccine. The advent of preerythrocytic stage for the first time the parasite invade the body, blocking the sporozoite development and reproduction in the liver, which can prevent the liver stage merozoites infect red blood cells into the blood, and clinical symptoms, according to the design of the vaccine preerythrocytic malaria infection from the source control. At present, the most effective preerythrocytic malaria vaccine is attenuated sporozoite vaccine, attenuated sub spore CD8+T cells induced immune response is the most important protective immune mechanism also, become the gold standard to measure the effectiveness of malaria vaccine. However, a large number of sporozoites obtained reduced toxicity and transportation, preservation of the difficulties, the large-scale production and application is limited, sub unit A vaccine will become the main development direction of the new generation of vaccines, but has only identified preerythrocytic vaccine candidate antigen, few therefore, there is an urgent need to develop protective antigens of early high Xiaohong new screening method, screened more target antigen to CD8+T cells mediated by the immune response, clear progress in understanding the immune mechanism of infected cells, for developed to lay a solid foundation for effective preerythrocytic malaria vaccine.
Plasmodium yoelii evaluation based on the DNA (Plasmodium yoelii) vaccine candidate antigen PyTmp21 in preerythrocytic protective effect of mice. Secondly, the use of advanced liver stagnation of growth of P.yoeliifabb/f- gene attenuated sporozoite vaccine induced protection of mice model, using the method of hydrodynamic tail vein injection (Hydrodynamic tail vein injection, HTVI) expression. The specific antigen of luciferase reporter fusion plasmid DNA into mouse liver cells, combined with in vivo imaging system (In Vivo Imaging System, IVIS) expression of luciferase reporter gene in real-time monitoring of liver cell immunity in mice, thereby reducing identification of target antigen attenuated sporozoite vaccine induced specific CD8+T cell immune response to kill liver cells, for screening progress Pyfabb/f- gene attenuated sporozoites induce protective immune related antigen preerythrocytic technology platform. Finally, using the technology The platform of Pyfabb/f- gene attenuated sporozoites could induce specific cellular immune responses to the new candidate antigen PyTmp21 and clear expression of the antigen in liver cells, so as to evaluate whether PyTmp21 is a potential attenuated sporozoite vaccine induced protective antigen. The main research results are as follows:
1. Immunization of PyTmp21DNA vaccine inhibits the amount of liver worms and induced protective effects of the mice infected with Plasmodium red Plasmodium
In order to identify new candidate antigen PyTmp21 can induce protective immunity in vivo, we use the DNA vaccine in mice and found that PyTmp21 immunization can significantly reduce the sporozoite infection against malaria burden in mouse liver and induced by 60% to eliminate protection efficiency.
Two, establishment of a HTVI/IVIS screening platform for screening specific CD8+T cell immune response related protective antigens induced by Pyfabb/f- gene attenuated sporozoites
1, PyCSP-Luc recombinant plasmid was injected into normal mice by HTVI method, IVIS system observation confirmed that the plasmid DNA sustained in mouse liver, high expression, and real-time monitoring and fusion protein luciferase expression in liver volume. The main expression in liver cells confirmed by HTVI after injection of plasmid DNA by flow cytometry and to provide the basis for the expression and localization of the protein.
2, attenuated sporozoite immunized mice induced by fully protective immune response by Pyfabb/f- gene, HTVI PyCSP-Luc plasmid DNA mice attacked, IVIS observation confirmed the expression decreased luciferase immune mice liver. The progress of antibody depletion experiments confirmed that CD8+T cells mediated by the expression of clear target antigen of liver cells. Furthermore, after the attack on plasmid immunization of mice liver lymphocytes by flow cytometry were found, the number of CSP specific CD8+T cells were significantly increased and IFN- secretion levels increased. The experimental results show that the Py fabb/f- gene attenuated sporozoite immunized mice can induce CSP specific CD8+T cell immune response, and mediated expression of the epitope of clearing liver cells HTVI, combined with IVIS Technology can be used as an effective tool for screening the target antigen gene attenuated sporozoite induced CD8+T cell depletion of liver cells.
Three, using the HTVI/IVIS research platform to identify the protective immune related prophase antigen induced by the sporozoites of the Py fabb/f- gene attenuated
In order to reduce the immunity related gene identification of preerythrocytic antigen protection induced by attenuated sporozoite vaccine protection model, we use the HTVI/IVIS research platform for detection of Pyfabb/f- gene attenuated sporozoites could induce specific T cell immune response to new candidate antigen PyTmp21 and antigen expression of the destruction of liver cells. We found that 2 Pyfabb/f- homologous sporozoite after immunization, HTVI PyTmp21-Luc plasmid DNA attack mice were not observed the expression of luciferase in the liver to reduce phenomenon. However, the use of DNA+ +DNA sporozoites or sporozoites of heterologous immune booster immunization strategy, PyTmp21-Luc plasmid DNA mice attacked the expression in liver was significantly decreased, suggesting that the specific immune response Pyfabb/f- attenuated sporozoites can induce the PyTmp21 antigen expression of the antigen and kill liver cells. The experimental results show that the homology of attenuated Sporozoite immunized mice may induce effective immune response against dominant antigen CSP, but not enough to induce immune responses to non CSP antigens in us by heterologous immunity boost immunization strategy confirmed that the Pyfabb/f- gene attenuated sporozoites could induce PyTmp21 antigen specific T cell immune response effectively, PyTmp21 as a potential antigen attenuated sporozoite vaccine induced protective antigen.
Our study shows that the HTVI/IVIS method can be used to detect target gene by antigen-specific CD8+T cell attenuated sporozoite vaccine induced killer liver cells. Using heterologous immune booster immunization strategy confirmed that the Pyfabb/f- gene attenuated sporozoite vaccine induced specific immune response to PyTmp21 antigens, and clear expression of the antigen in liver cells PyTmp21, as a new preerythrocytic antigen in reducing spore vaccine induced protective immune reaction. Heterologous immune toxin boost immunization strategy combined with HTVI/IVIS technology can be used as an effective tool for the screening of attenuated elicitor spore parasite vaccine new preerythrocytic non CSP protective antigen, understanding the mechanism of protective immunity of attenuated parasite the vaccine for progress, screened more subunit vaccine candidate antigens and designed to lay the foundation for effective malaria vaccine.

【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R531.3

【共引文獻(xiàn)】

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