多表位惡性瘧人工抗原M.RCAg-1的免疫保護(hù)效果評(píng)價(jià)及其免疫機(jī)制的研究
發(fā)布時(shí)間：2018-07-17 20:12
【摘要】： 瘧疾是目前世界上對(duì)人類危害最嚴(yán)重的蚊媒寄生原蟲病。全世界每年有5億人受到瘧原蟲感染，約三百萬(wàn)人死于惡性瘧疾。其中80％是五歲以下的兒童和孕婦。由于惡性瘧原蟲對(duì)現(xiàn)有藥物的抗藥性不斷出現(xiàn)和蔓延，蚊媒對(duì)殺蟲劑抗藥性的產(chǎn)生等因素影響，瘧疾的防治成為世界性的大難題之一。由于瘧原蟲生活周期復(fù)雜，抗原具有階段特異性、蟲株差異性和高度變異性等特點(diǎn)，長(zhǎng)期以來(lái)形成研制抗瘧原蟲疫苗的技術(shù)瓶頸。因此，針對(duì)惡性瘧原蟲不同生活時(shí)期的免疫反應(yīng)特點(diǎn)，選取誘導(dǎo)產(chǎn)生多種免疫反應(yīng)類型的不同抗原構(gòu)建多表位疫苗已經(jīng)成為研究抗瘧疫苗的熱點(diǎn)。但是，目前對(duì)多抗原表位疫苗的研究仍然是以人工合成多表位肽疫苗或單一固定的串聯(lián)多表位DNA疫苗為主，前者成本昂貴，后者免疫原性低，難以達(dá)到預(yù)期的免疫反應(yīng)多樣性和令人滿意的保護(hù)效果。 本課題組前期工作借鑒了分子育種技術(shù)(即DNA改組)的隨機(jī)重組原理，利用表位改組技術(shù)構(gòu)建了多表位基因庫(kù)，并用庫(kù)免疫血清篩選出高抗原性的陽(yáng)性克隆，發(fā)現(xiàn)其中VR312(改名為M．RCAg-1)基因克隆不僅能在小鼠模型誘導(dǎo)交叉免疫保護(hù)而且能在家兔模型中誘導(dǎo)抗惡性瘧原蟲抑制性抗體。 本研究在前期工作的基礎(chǔ)上，，開展了M．RCAg-1基因和蛋白質(zhì)疫苗在不同種屬的哺乳動(dòng)物模型中的免疫原性和保護(hù)性研究，以及對(duì)M．RCAg-1蛋白質(zhì)進(jìn)行相關(guān)流行病學(xué)分析，并且對(duì)利用核酸分子探針檢測(cè)蟲血率的流式細(xì)胞術(shù)的建立做了相關(guān)的探索，取得以下的結(jié)果： 1．M．RCAg-1蛋白質(zhì)抗原能被不同疫區(qū)的病人血清識(shí)別，提示M．RCAg-1多表位蛋白質(zhì)疫苗可能能克服蟲株之間差異導(dǎo)致的免疫逃逸； 2．非洲喀麥隆疫區(qū)居民血清IgG抗體識(shí)別M．RCAg-1多表位蛋白質(zhì)和年齡呈現(xiàn)顯著的正相關(guān)，提示兒童時(shí)期接種M．RCAg-1多表位蛋白質(zhì)疫苗可能降低惡性瘧導(dǎo)致的高病死率； 3．M．RcAg-1多表位蛋白質(zhì)免疫小鼠、新西蘭白兔和恒河猴均能獲得高滴度特異性抗體水平和誘發(fā)明顯的CD4~+T細(xì)胞反應(yīng)，而且產(chǎn)生的特異性抗體能在體外有效抑制惡性瘧原蟲的生長(zhǎng)。提示產(chǎn)生的特異性抗體中抑制性抗體的比例較大，這可能和M．RCAg-1多表位蛋白質(zhì)的優(yōu)勢(shì)構(gòu)象有關(guān)； 4．M．RcAg-1多表位基因疫苗在小鼠和家兔動(dòng)物模型能產(chǎn)生高滴度抗體水平，但此特異性抗體親合力不高，體外生長(zhǎng)抑制效果不佳。在恒河猴動(dòng)物模型中，單純的基因免疫很難誘導(dǎo)高水平的抗體，而采用基因初免-蛋白質(zhì)加強(qiáng)的方案能有效的誘導(dǎo)出高水平的特異性抗體，然而和小動(dòng)物模型一樣，體外抑制原蟲生長(zhǎng)的效果不明顯。這可能和基因免疫的表達(dá)呈遞模式有關(guān)，從而產(chǎn)生和蛋白質(zhì)免疫不同比例和不同親合力的抗體類型； 5．分析M．RcAg-1蛋白質(zhì)免疫小鼠的細(xì)胞免疫實(shí)驗(yàn)結(jié)果，發(fā)現(xiàn)具有相同特異性的細(xì)胞可以分化為不同亞群的輔助性T細(xì)胞，這主要取決于抗原的構(gòu)象和周圍分泌的細(xì)胞因子類型； 6．利用Hydroethidine流式細(xì)胞術(shù)可以客觀快速相對(duì)定量瘧原蟲蟲血率，而且能較好地評(píng)價(jià)原蟲同步化的效果。
[Abstract]:Malaria is the most serious mosquito parasitic protozoa in the world at present. 500 million people are infected with malaria parasites every year in the world and about three million people die from malarial malaria. 80% of them are children under five years of age and pregnant women. The prevention and control of malaria has become one of the most difficult problems in the world. Due to the complexity of the life cycle of the Plasmodium, the antigen has the characteristics of stage specificity, strain difference and high variability, it has long been a technical bottleneck for the development of Plasmodium antimalarial vaccine. Therefore, the immunological response to Plasmodium falciparum in different life periods is special. The selection of multiple epitopes with different antigens inducing various types of immune responses has become a hot spot in the study of antimalarial vaccines. However, the study of multiple epitope vaccines is still mainly based on synthetic multi epitope vaccine or single fixed series multi epitopes DNA vaccine, the former is expensive and the latter has low immunogenicity. It is difficult to achieve the desired immune response diversity and satisfactory protective effect.
In the previous work, we used the principle of random recombination of molecular breeding technology (DNA regrouping), and constructed a multi epitope gene pool using epitope modification technique, and screened the positive clones with high antigenicity with the immune sera of the library. It was found that VR312 (renamed M.RCAg-1) gene clones could not only induce cross immunization protection in mice model. It can also induce Plasmodium falciparum inhibitory antibodies in rabbit models.
On the basis of the previous work, the immunogenicity and protection of M.RCAg-1 gene and protein vaccine in the mammalian model of different species were carried out, and the related epidemiological analysis of M.RCAg-1 protein was carried out, and the establishment of the flow cytometry with nucleic acid probe for detecting the blood rate of the insect was related. The following results are obtained:
The 1.M.RCAg-1 protein antigen can be identified by the serum of patients in different epidemic areas, suggesting that the M.RCAg-1 polyepitope protein vaccine can overcome the immune escape caused by the difference between the strains of the insect.
2. the M.RCAg-1 polyepitope protein identified by the serum IgG antibody in the Cameroon epidemic area of Africa showed a significant positive correlation, suggesting that the vaccination of M.RCAg-1 polyepitopes in children may reduce the high mortality caused by falciparum malaria.
3.M.RcAg-1 multi epitopes protein immunized mice, New Zealand white rabbits and Ganges RIver monkeys can obtain high titer specific antibody level and induce obvious CD4~+T cell response, and the specific antibodies can effectively inhibit the growth of Plasmodium falciparum in vitro. This suggests that the proportion of the specific antibodies in the specific antibodies is larger. It may be related to the dominant conformation of M.RCAg-1 multi epitope protein.
The 4.M.RcAg-1 multi epitope gene vaccine can produce high titer antibody level in mice and rabbit models, but the specificity of the specific antibody is not high and the inhibition effect is not good in vitro. In the Ganges RIver monkey animal model, the simple gene immunity is difficult to induce the high level of antibody, and the scheme of the genetic primer protein strengthening can be effective. High level specific antibodies were induced, but like small animal models, the effect of inhibition of protozoa growth in vitro was not obvious. This may be related to the expression pattern of gene immunization, thus producing different proportion of protein immunization and the type of antibody with different affinity.
5. the results of cellular immunity test in M.RcAg-1 protein immunized mice showed that the cells with the same specificity could differentiate into the auxiliary T cells of different subgroups, which mainly depends on the conformation of the antigen and the type of cytokines secreted around.
6. Hydroethidine flow cytometry can objectively and rapidly quantify the blood flow rate of malaria parasites, and can better evaluate the synchronization effect of protozoa.
【學(xué)位授予單位】：中國(guó)協(xié)和醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2007
【分類號(hào)】：R392
本文編號(hào)：2130805
[Abstract]:Malaria is the most serious mosquito parasitic protozoa in the world at present. 500 million people are infected with malaria parasites every year in the world and about three million people die from malarial malaria. 80% of them are children under five years of age and pregnant women. The prevention and control of malaria has become one of the most difficult problems in the world. Due to the complexity of the life cycle of the Plasmodium, the antigen has the characteristics of stage specificity, strain difference and high variability, it has long been a technical bottleneck for the development of Plasmodium antimalarial vaccine. Therefore, the immunological response to Plasmodium falciparum in different life periods is special. The selection of multiple epitopes with different antigens inducing various types of immune responses has become a hot spot in the study of antimalarial vaccines. However, the study of multiple epitope vaccines is still mainly based on synthetic multi epitope vaccine or single fixed series multi epitopes DNA vaccine, the former is expensive and the latter has low immunogenicity. It is difficult to achieve the desired immune response diversity and satisfactory protective effect.
In the previous work, we used the principle of random recombination of molecular breeding technology (DNA regrouping), and constructed a multi epitope gene pool using epitope modification technique, and screened the positive clones with high antigenicity with the immune sera of the library. It was found that VR312 (renamed M.RCAg-1) gene clones could not only induce cross immunization protection in mice model. It can also induce Plasmodium falciparum inhibitory antibodies in rabbit models.
On the basis of the previous work, the immunogenicity and protection of M.RCAg-1 gene and protein vaccine in the mammalian model of different species were carried out, and the related epidemiological analysis of M.RCAg-1 protein was carried out, and the establishment of the flow cytometry with nucleic acid probe for detecting the blood rate of the insect was related. The following results are obtained:
The 1.M.RCAg-1 protein antigen can be identified by the serum of patients in different epidemic areas, suggesting that the M.RCAg-1 polyepitope protein vaccine can overcome the immune escape caused by the difference between the strains of the insect.
2. the M.RCAg-1 polyepitope protein identified by the serum IgG antibody in the Cameroon epidemic area of Africa showed a significant positive correlation, suggesting that the vaccination of M.RCAg-1 polyepitopes in children may reduce the high mortality caused by falciparum malaria.
3.M.RcAg-1 multi epitopes protein immunized mice, New Zealand white rabbits and Ganges RIver monkeys can obtain high titer specific antibody level and induce obvious CD4~+T cell response, and the specific antibodies can effectively inhibit the growth of Plasmodium falciparum in vitro. This suggests that the proportion of the specific antibodies in the specific antibodies is larger. It may be related to the dominant conformation of M.RCAg-1 multi epitope protein.
The 4.M.RcAg-1 multi epitope gene vaccine can produce high titer antibody level in mice and rabbit models, but the specificity of the specific antibody is not high and the inhibition effect is not good in vitro. In the Ganges RIver monkey animal model, the simple gene immunity is difficult to induce the high level of antibody, and the scheme of the genetic primer protein strengthening can be effective. High level specific antibodies were induced, but like small animal models, the effect of inhibition of protozoa growth in vitro was not obvious. This may be related to the expression pattern of gene immunization, thus producing different proportion of protein immunization and the type of antibody with different affinity.
5. the results of cellular immunity test in M.RcAg-1 protein immunized mice showed that the cells with the same specificity could differentiate into the auxiliary T cells of different subgroups, which mainly depends on the conformation of the antigen and the type of cytokines secreted around.
6. Hydroethidine flow cytometry can objectively and rapidly quantify the blood flow rate of malaria parasites, and can better evaluate the synchronization effect of protozoa.
【學(xué)位授予單位】：中國(guó)協(xié)和醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2007
【分類號(hào)】：R392
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相關(guān)期刊論文 前2條
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2 林澄濤,姜燕芳,陰彬,董敏,何湘云,王恒;同尾酶技術(shù)在構(gòu)建瘧疾多價(jià)重組DNA疫苗中的應(yīng)用[J];中國(guó)生物化學(xué)與分子生物學(xué)報(bào);1999年06期
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