TIP样蛋白在疟原虫免疫抑制中功能的初步研究
发布时间：2018-08-12 07:38
【摘要】：疟疾是严重威胁人类健康的寄生虫病，WHO统计资料显示每年确诊的疟疾患者约1亿人。宿主感染疟原虫后，可获得一定的免疫力，但疟原虫在有免疫力的宿主体内仍能生存与繁殖，即疟原虫的免疫逃避。疟原虫可以通过抗原变异、免疫抑制等多种途径逃避宿主的免疫攻击，，因此宿主产生的免疫保护常常是不稳固的，但其具体机制仍不甚清楚，给疟疾的研究特别是疟疾疫苗的研究造成了很大的困难。因此，对疟原虫免疫逃避及其机制的研究历来是疟疾防治研究及疫苗研制的热点和难点之一。 T细胞免疫调节蛋白（T cell immunomodulatory protein，TIP）是一种新发现的存在于人和多种动物体内的免疫调节分子，它是由612个氨基酸组成的跨膜蛋白。在急性移植物抗宿主病（graft-versus-host disease, GVHD）模型小鼠体内的研究发现，TIP可以明显保护因移植物排斥反应而引起的小鼠死亡。本研究通过对疟原虫基因和蛋白质生物信息的对比分析发现，多种疟原虫均存在与TIP氨基酸序列、结构域和三级结构相似的蛋白（以下统称疟原虫TIP样蛋白），推测疟原虫TIP样蛋白在抑制宿主对疟原虫的免疫攻击方面可能也起到重要作用。 1．疟原虫TIP样蛋白表达特点及其抗体抑制疟原虫增殖作用的研究 本课题组前期根据Plasmodb数据库最初公布的部分编码小鼠伯氏疟原虫TIP样蛋白（以下简称PbTIP）的1566个碱基序列，构建了带GST标签的PbTIP重组原核表达载体。本研究获得PbTIP-GST重组蛋白的表达后，以纯化的重组蛋白免疫家兔，获得了较高滴度的抗PbTIP-GST兔血清。间接免疫荧光实验提示PbTIP主要在疟原虫胞质中表达且分布较均匀，而疟原虫胞核以及被疟原虫寄生的红细胞膜上均未见明显的PbTIP表达。 由于目前尚未建立成熟的小鼠伯氏疟原虫体外培养方法。因此，疟原虫生活史不同发育阶段TIP样蛋白的表达水平检测只能在恶性疟原虫体外同步化培养模型中进行。对恶性疟原虫TIP样蛋白（PfTIP）生活史各期表达量的检测发现，滋养体期PfTIP的表达量最高，较裂殖体期高出约6倍多，比环状体期高出5倍多，说明PfTIP发挥功能的主要时期位于红细胞内的发育中期，随着疟原虫发育至裂殖体期PfTIP表达量降至最低水平，提示PfTIP在裂殖子入侵红细胞过程中可能不会发挥太重要的作用。该实验结果与兔抗PbTIP多克隆血清对体外培养恶性疟原虫的生长仅具有较弱的抑制作用相符。 重要的是，在PbTIP重组蛋白免疫小鼠的疟原虫攻击实验中发现，以抗PbTIP抗体阻断小鼠伯氏疟原虫PbTIP蛋白的功能后，小鼠抗疟原虫攻击的能力明显增强。推测可能是因为抗PbTIP抗体的存在，抑制了小鼠伯氏疟原虫PbTIP蛋白对宿主免疫应答的负调控作用，导致宿主对疟原虫的免疫应答水平升高，提高了宿主对疟原虫的清除效率。 2．伯氏疟原虫PbTIP胞外域重组蛋白对T细胞的作用研究 为了明确PbTIP发挥作用的功能区域，本研究根据疟原虫数据库Plasmodb最新公布的小鼠PbTIP全基因序列信息，构建并成功表达了PbTIP完整胞外域的重组蛋白。鉴于第一部分实验中采用pGEX4T-1原核表达载体出现的GST标签与纯化柱结合不良的问题，本研究改用pET32a原核表达载体表达PbTIP胞外域重组蛋白。 虽然PbTIP完整胞外域重组蛋白主要以包涵体形式表达，但经过复性后目的蛋白与Ni-NTA Agarose的结合良好，得到了纯化的可溶性PbTIP重组蛋白。但原核表达的PbTIP重组蛋白对CTLL-2淋巴细胞分泌IL-4、TGF-β、IL-10等细胞因子及其增殖水平无明显影响，与预期不符。分析原因可能有两个方面，一方面可能是原核表达的PbTIP蛋白复性后并未很好地恢复天然PbTIP分子原有的空间构象，从而影响其功能的发挥；另一方面也可能是CTLL-2淋巴细胞是IL-2依赖的，在IL-2刺激下，淋巴细胞增殖一直处于较高水平，掩盖了PbTIP蛋白可能的调节作用。 3．基因敲除伯氏疟原虫PbTIP 为了进一步明确PbTIP是否具有非特异性地抑制宿主免疫应答的功能，本研究尝试敲除编码小鼠PbTIP的基因，深入研究PbTIP在疟原虫免疫逃避中的作用机制。本部分实验基于同源重组原理，首先构建含有抗性筛选标记tg-dhfr/ts的同源重组载体，再将线性化的同源重组载体与同期构建好的TALENs质粒共转染伯氏疟原虫，在小鼠体内通过抗性筛选获得重组成功的疟原虫。 但由于疟原虫区别于一般哺乳动物细胞的基因表达调控特点，虽经过多次艰苦的实验仍未获得重组成功的疟原虫。截止目前，本研究仅获得外源线性化基因片段以附加体形式存在的疟原虫，随着疟原虫的扩增和传代外源基因片段随之丧失。当然，我们也不能排除PbTIP基因无法敲除的可能性，即无法排除由于PbTIP基因的敲除而导致疟原虫无法继续存活的可能性。 对于TIP样蛋白在疟原虫免疫抑制中的功能研究虽然取得了一些初步的研究结果，但实验中遇到的很多难点尚未攻克，鉴于研究生学习和工作时间有限，相关的研究工作仍在进行中。
[Abstract]:Malaria is a parasitic disease that seriously threatens human health. WHO statistics show that about 100 million people are diagnosed with malaria every year. After the host is infected with the parasite, certain immunity can be obtained, but the parasite can still survive and reproduce in the immune host, that is, the immune escape of the parasite. The immune protection produced by host is often unstable, but the specific mechanism is still unclear, which makes it difficult to study malaria, especially the malaria vaccine. Therefore, the study of immune escape and its mechanism has always been the research of malaria control and vaccine development. One of the hotspots and difficulties.
T cell immunomodulatory protein (TIP) is a newly discovered immunomodulatory molecule in humans and a variety of animals. It is a transmembrane protein composed of 612 amino acids. In this study, we compared the biological information of genes and proteins of Plasmodium falciparum and found that there were proteins similar to TIP amino acid sequence, domain and tertiary structure (hereinafter referred to as TIP-like protein of Plasmodium) in many kinds of Plasmodium falciparum. The immune attack of malaria parasites may also play an important role.
1. the expression characteristics of TIP like protein in Plasmodium and the inhibition of Plasmodium on the proliferation of Plasmodium falciparum
Our group constructed a Recombinant Prokaryotic expression vector of Plasmodium berghei TIP-like protein with GST tag according to the 1 566 base sequences of the TIP-like protein (hereinafter referred to as PbTIP) which was originally published in Plasmodb database. Indirect immunofluorescence assay showed that PbTIP was mainly expressed and distributed uniformly in the cytoplasm of Plasmodium, but not in the nucleus of Plasmodium and erythrocyte membrane parasitized by Plasmodium.
Because there is no mature method for in vitro culture of Plasmodium berghei in mice, the detection of TIP-like protein expression in different stages of life cycle of Plasmodium berghei can only be carried out in a synchronized culture model of Plasmodium falciparum in vitro. The expression of PfTIP was the highest, about 6 times higher than that in the merozoite phase, and 5 times higher than that in the annular phase. It indicated that the main stage of PfTIP function was in the middle stage of erythrocyte development, and the expression of PfTIP decreased to the lowest level with the development of Plasmodium to the merozoite phase, suggesting that PfTIP might not play too heavy role in the process of merozoite invasion of erythrocyte. The results were consistent with that of rabbit anti-PbTIP polyclonal serum, which had only a weak inhibitory effect on the growth of Plasmodium falciparum in vitro.
Importantly, the ability of mice to resist Plasmodium berghei infection was significantly enhanced by blocking the function of the mouse Plasmodium berghei PbTIP protein with anti-PbTIP antibody. It was speculated that the presence of anti-PbTIP antibody inhibited the host immune response of the mouse Plasmodium berghei PbTIP protein. Negative regulation of the host immune response to Plasmodium increased the host clearance efficiency.
Effect of recombinant protein from 2. extracellular domain of Plasmodium bergi PbTIP on T cells
In order to clarify the functional region of PbTIP, we constructed and successfully expressed the recombinant protein of the complete extracellular domain of PbTIP according to the latest published sequence information of mouse PbTIP gene from Plasmodb, a Plasmodium database. In the first part of the experiment, the GST tag of pGEX4T-1 prokaryotic expression vector was poorly bound to the purified column. In this study, we used pET32a prokaryotic expression vector to express PbTIP extracellular domain recombinant protein.
Although the complete extracellular domain recombinant protein of PbTIP was mainly expressed in the form of inclusion bodies, after renaturation, the recombinant protein of PbTIP was well bound to Ni-NTA Agarose and purified soluble PbTIP protein was obtained. On the one hand, the renaturation of the prokaryotic expression of PbTIP protein may not restore the original spatial conformation of natural PbTIP molecule, thus affecting its function; on the other hand, it may be that CTLL-2 lymphocytes are IL-2 dependent, and the proliferation of lymphocytes stimulated by IL-2. It has been at a high level, covering up the possible regulatory role of PbTIP protein.
3. gene knockout Plasmodium bergi PbTIP
In order to further clarify whether PbTIP can inhibit the host immune response nonspecifically, this study attempted to knock out the gene encoding mouse PbTIP and further study the mechanism of its role in immune escape of Plasmodium. Based on the principle of homologous recombination, a homologous recombinant vector containing resistance screening marker tg-dhfr/ts was constructed. Then the linearized homologous recombinant vector was co-transfected with the TALENs plasmid constructed at the same time, and the recombinant plasmid was obtained by resistance screening in mice.
However, due to the characteristics of gene expression regulation different from that of mammalian cells, the recombinant Plasmodium has not been successfully reconstructed after many painstaking experiments. Up to now, only the extrinsic linearized gene fragments in the form of additives have been obtained. With the expansion and passage of the parasite, the extrinsic gene fragments have been followed. Loss. Of course, we can't rule out the possibility that the PbTIP gene can't be knocked out, that is, we can't rule out the possibility that the Plasmodium can't survive because of the knockout of the PbTIP gene.
Although some preliminary results have been obtained on the function of TIP-like protein in the immunosuppression of Plasmodium, many difficulties encountered in the experiment have not been overcome. In view of the limited study and working time of postgraduates, the related research work is still in progress.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R531.3
本文编号：2178370
[Abstract]:Malaria is a parasitic disease that seriously threatens human health. WHO statistics show that about 100 million people are diagnosed with malaria every year. After the host is infected with the parasite, certain immunity can be obtained, but the parasite can still survive and reproduce in the immune host, that is, the immune escape of the parasite. The immune protection produced by host is often unstable, but the specific mechanism is still unclear, which makes it difficult to study malaria, especially the malaria vaccine. Therefore, the study of immune escape and its mechanism has always been the research of malaria control and vaccine development. One of the hotspots and difficulties.
T cell immunomodulatory protein (TIP) is a newly discovered immunomodulatory molecule in humans and a variety of animals. It is a transmembrane protein composed of 612 amino acids. In this study, we compared the biological information of genes and proteins of Plasmodium falciparum and found that there were proteins similar to TIP amino acid sequence, domain and tertiary structure (hereinafter referred to as TIP-like protein of Plasmodium) in many kinds of Plasmodium falciparum. The immune attack of malaria parasites may also play an important role.
1. the expression characteristics of TIP like protein in Plasmodium and the inhibition of Plasmodium on the proliferation of Plasmodium falciparum
Our group constructed a Recombinant Prokaryotic expression vector of Plasmodium berghei TIP-like protein with GST tag according to the 1 566 base sequences of the TIP-like protein (hereinafter referred to as PbTIP) which was originally published in Plasmodb database. Indirect immunofluorescence assay showed that PbTIP was mainly expressed and distributed uniformly in the cytoplasm of Plasmodium, but not in the nucleus of Plasmodium and erythrocyte membrane parasitized by Plasmodium.
Because there is no mature method for in vitro culture of Plasmodium berghei in mice, the detection of TIP-like protein expression in different stages of life cycle of Plasmodium berghei can only be carried out in a synchronized culture model of Plasmodium falciparum in vitro. The expression of PfTIP was the highest, about 6 times higher than that in the merozoite phase, and 5 times higher than that in the annular phase. It indicated that the main stage of PfTIP function was in the middle stage of erythrocyte development, and the expression of PfTIP decreased to the lowest level with the development of Plasmodium to the merozoite phase, suggesting that PfTIP might not play too heavy role in the process of merozoite invasion of erythrocyte. The results were consistent with that of rabbit anti-PbTIP polyclonal serum, which had only a weak inhibitory effect on the growth of Plasmodium falciparum in vitro.
Importantly, the ability of mice to resist Plasmodium berghei infection was significantly enhanced by blocking the function of the mouse Plasmodium berghei PbTIP protein with anti-PbTIP antibody. It was speculated that the presence of anti-PbTIP antibody inhibited the host immune response of the mouse Plasmodium berghei PbTIP protein. Negative regulation of the host immune response to Plasmodium increased the host clearance efficiency.
Effect of recombinant protein from 2. extracellular domain of Plasmodium bergi PbTIP on T cells
In order to clarify the functional region of PbTIP, we constructed and successfully expressed the recombinant protein of the complete extracellular domain of PbTIP according to the latest published sequence information of mouse PbTIP gene from Plasmodb, a Plasmodium database. In the first part of the experiment, the GST tag of pGEX4T-1 prokaryotic expression vector was poorly bound to the purified column. In this study, we used pET32a prokaryotic expression vector to express PbTIP extracellular domain recombinant protein.
Although the complete extracellular domain recombinant protein of PbTIP was mainly expressed in the form of inclusion bodies, after renaturation, the recombinant protein of PbTIP was well bound to Ni-NTA Agarose and purified soluble PbTIP protein was obtained. On the one hand, the renaturation of the prokaryotic expression of PbTIP protein may not restore the original spatial conformation of natural PbTIP molecule, thus affecting its function; on the other hand, it may be that CTLL-2 lymphocytes are IL-2 dependent, and the proliferation of lymphocytes stimulated by IL-2. It has been at a high level, covering up the possible regulatory role of PbTIP protein.
3. gene knockout Plasmodium bergi PbTIP
In order to further clarify whether PbTIP can inhibit the host immune response nonspecifically, this study attempted to knock out the gene encoding mouse PbTIP and further study the mechanism of its role in immune escape of Plasmodium. Based on the principle of homologous recombination, a homologous recombinant vector containing resistance screening marker tg-dhfr/ts was constructed. Then the linearized homologous recombinant vector was co-transfected with the TALENs plasmid constructed at the same time, and the recombinant plasmid was obtained by resistance screening in mice.
However, due to the characteristics of gene expression regulation different from that of mammalian cells, the recombinant Plasmodium has not been successfully reconstructed after many painstaking experiments. Up to now, only the extrinsic linearized gene fragments in the form of additives have been obtained. With the expansion and passage of the parasite, the extrinsic gene fragments have been followed. Loss. Of course, we can't rule out the possibility that the PbTIP gene can't be knocked out, that is, we can't rule out the possibility that the Plasmodium can't survive because of the knockout of the PbTIP gene.
Although some preliminary results have been obtained on the function of TIP-like protein in the immunosuppression of Plasmodium, many difficulties encountered in the experiment have not been overcome. In view of the limited study and working time of postgraduates, the related research work is still in progress.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R531.3
【参考文献】
相关期刊论文 前1条
1 韩志富,邵丁丁,王恒;恶性疟原虫类巨噬细胞迁移抑制因子基因的克隆和表达[J];中国医学科学院学报;2004年05期
本文编号：2178370
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