TIP樣蛋白在瘧原蟲免疫抑制中功能的初步研究
發(fā)布時間：2018-08-12 07:38
【摘要】：瘧疾是嚴重威脅人類健康的寄生蟲病，WHO統(tǒng)計資料顯示每年確診的瘧疾患者約1億人。宿主感染瘧原蟲后，可獲得一定的免疫力，但瘧原蟲在有免疫力的宿主體內(nèi)仍能生存與繁殖，即瘧原蟲的免疫逃避。瘧原蟲可以通過抗原變異、免疫抑制等多種途徑逃避宿主的免疫攻擊，，因此宿主產(chǎn)生的免疫保護常常是不穩(wěn)固的，但其具體機制仍不甚清楚，給瘧疾的研究特別是瘧疾疫苗的研究造成了很大的困難。因此，對瘧原蟲免疫逃避及其機制的研究歷來是瘧疾防治研究及疫苗研制的熱點和難點之一。 T細胞免疫調節(jié)蛋白（T cell immunomodulatory protein，TIP）是一種新發(fā)現(xiàn)的存在于人和多種動物體內(nèi)的免疫調節(jié)分子，它是由612個氨基酸組成的跨膜蛋白。在急性移植物抗宿主�。╣raft-versus-host disease, GVHD）模型小鼠體內(nèi)的研究發(fā)現(xiàn)，TIP可以明顯保護因移植物排斥反應而引起的小鼠死亡。本研究通過對瘧原蟲基因和蛋白質生物信息的對比分析發(fā)現(xiàn)，多種瘧原蟲均存在與TIP氨基酸序列、結構域和三級結構相似的蛋白（以下統(tǒng)稱瘧原蟲TIP樣蛋白），推測瘧原蟲TIP樣蛋白在抑制宿主對瘧原蟲的免疫攻擊方面可能也起到重要作用。 1．瘧原蟲TIP樣蛋白表達特點及其抗體抑制瘧原蟲增殖作用的研究 本課題組前期根據(jù)Plasmodb數(shù)據(jù)庫最初公布的部分編碼小鼠伯氏瘧原蟲TIP樣蛋白（以下簡稱PbTIP）的1566個堿基序列，構建了帶GST標簽的PbTIP重組原核表達載體。本研究獲得PbTIP-GST重組蛋白的表達后，以純化的重組蛋白免疫家兔，獲得了較高滴度的抗PbTIP-GST兔血清。間接免疫熒光實驗提示PbTIP主要在瘧原蟲胞質中表達且分布較均勻，而瘧原蟲胞核以及被瘧原蟲寄生的紅細胞膜上均未見明顯的PbTIP表達。 由于目前尚未建立成熟的小鼠伯氏瘧原蟲體外培養(yǎng)方法。因此，瘧原蟲生活史不同發(fā)育階段TIP樣蛋白的表達水平檢測只能在惡性瘧原蟲體外同步化培養(yǎng)模型中進行。對惡性瘧原蟲TIP樣蛋白（PfTIP）生活史各期表達量的檢測發(fā)現(xiàn)，滋養(yǎng)體期PfTIP的表達量最高，較裂殖體期高出約6倍多，比環(huán)狀體期高出5倍多，說明PfTIP發(fā)揮功能的主要時期位于紅細胞內(nèi)的發(fā)育中期，隨著瘧原蟲發(fā)育至裂殖體期PfTIP表達量降至最低水平，提示PfTIP在裂殖子入侵紅細胞過程中可能不會發(fā)揮太重要的作用。該實驗結果與兔抗PbTIP多克隆血清對體外培養(yǎng)惡性瘧原蟲的生長僅具有較弱的抑制作用相符。 重要的是，在PbTIP重組蛋白免疫小鼠的瘧原蟲攻擊實驗中發(fā)現(xiàn)，以抗PbTIP抗體阻斷小鼠伯氏瘧原蟲PbTIP蛋白的功能后，小鼠抗瘧原蟲攻擊的能力明顯增強。推測可能是因為抗PbTIP抗體的存在，抑制了小鼠伯氏瘧原蟲PbTIP蛋白對宿主免疫應答的負調控作用，導致宿主對瘧原蟲的免疫應答水平升高，提高了宿主對瘧原蟲的清除效率。 2．伯氏瘧原蟲PbTIP胞外域重組蛋白對T細胞的作用研究 為了明確PbTIP發(fā)揮作用的功能區(qū)域，本研究根據(jù)瘧原蟲數(shù)據(jù)庫Plasmodb最新公布的小鼠PbTIP全基因序列信息，構建并成功表達了PbTIP完整胞外域的重組蛋白。鑒于第一部分實驗中采用pGEX4T-1原核表達載體出現(xiàn)的GST標簽與純化柱結合不良的問題，本研究改用pET32a原核表達載體表達PbTIP胞外域重組蛋白。 雖然PbTIP完整胞外域重組蛋白主要以包涵體形式表達，但經(jīng)過復性后目的蛋白與Ni-NTA Agarose的結合良好，得到了純化的可溶性PbTIP重組蛋白。但原核表達的PbTIP重組蛋白對CTLL-2淋巴細胞分泌IL-4、TGF-β、IL-10等細胞因子及其增殖水平無明顯影響，與預期不符。分析原因可能有兩個方面，一方面可能是原核表達的PbTIP蛋白復性后并未很好地恢復天然PbTIP分子原有的空間構象，從而影響其功能的發(fā)揮；另一方面也可能是CTLL-2淋巴細胞是IL-2依賴的，在IL-2刺激下，淋巴細胞增殖一直處于較高水平，掩蓋了PbTIP蛋白可能的調節(jié)作用。 3．基因敲除伯氏瘧原蟲PbTIP 為了進一步明確PbTIP是否具有非特異性地抑制宿主免疫應答的功能，本研究嘗試敲除編碼小鼠PbTIP的基因，深入研究PbTIP在瘧原蟲免疫逃避中的作用機制。本部分實驗基于同源重組原理，首先構建含有抗性篩選標記tg-dhfr/ts的同源重組載體，再將線性化的同源重組載體與同期構建好的TALENs質粒共轉染伯氏瘧原蟲，在小鼠體內(nèi)通過抗性篩選獲得重組成功的瘧原蟲。 但由于瘧原蟲區(qū)別于一般哺乳動物細胞的基因表達調控特點，雖經(jīng)過多次艱苦的實驗仍未獲得重組成功的瘧原蟲。截止目前，本研究僅獲得外源線性化基因片段以附加體形式存在的瘧原蟲，隨著瘧原蟲的擴增和傳代外源基因片段隨之喪失。當然，我們也不能排除PbTIP基因無法敲除的可能性，即無法排除由于PbTIP基因的敲除而導致瘧原蟲無法繼續(xù)存活的可能性。 對于TIP樣蛋白在瘧原蟲免疫抑制中的功能研究雖然取得了一些初步的研究結果，但實驗中遇到的很多難點尚未攻克，鑒于研究生學習和工作時間有限，相關的研究工作仍在進行中。
[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.
【學位授予單位】：第四軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R531.3
本文編號：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.
【學位授予單位】：第四軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R531.3
【參考文獻】
相關期刊論文 前1條
1 韓志富,邵丁丁,王恒;惡性瘧原蟲類巨噬細胞遷移抑制因子基因的克隆和表達[J];中國醫(yī)學科學院學報;2004年05期
本文編號：2178370
本文鏈接：http://sikaile.net/yixuelunwen/chuanranbingxuelunwen/2178370.html
最近更新
教材專著
