無序區(qū)段對瘧原蟲裂殖子表面蛋白2（MSP2）聚集和與膜相互作用的影響
發(fā)布時間：2019-05-07 08:52
【摘要】：瘧疾是世界上最嚴重的傳染性疾病之一,即使得瘧疾的風險從20世紀的開始就大幅度的降低,但是每年仍有上百萬人死亡。青蒿素等藥物在治療瘧疾時已經表現(xiàn)出了很大的效果,但是,在根除這種疾病時,瘧原蟲對這些抗瘧疾藥物的抗藥性是必須要克服的難題之一。一個可供選擇的可能更為有效的方法是利用疫苗在早期就阻止其傳染。但是已有的疫苗候選物表現(xiàn)出較低的效力和較短的時效性,且只對部分瘧原蟲株有效。目前仍沒有疫苗能夠有效的填補這些疫苗的缺陷。MSP2是惡性瘧原蟲(Plasmodium falciparum)裂殖子表面第二豐富的蛋白,已有研究顯示MSP2可能參與瘧原蟲粘附和入侵宿主紅細胞,因此是潛在的抗瘧疾疫苗候選物或藥物靶標。已開發(fā)的MSP2疫苗——有些已達到臨床二期試驗階段——顯示其對阻遏部分瘧原蟲入侵紅細胞有顯著效果。像其它的瘧疾疫苗候選物一樣,這些MSP2疫苗候選物只對部分瘧原蟲株有效,而基于蛋白質多態(tài)性的復合MSP2疫苗相對更有效。為提升MSP2作為抗瘧疾疫苗的潛能,有必要對其性質和功能的分子機制進行深入探究。MSP2是一種糖基磷脂酰肌醇(GPI)錨定蛋白,包括保守的N端和C端以及多變的中間可變區(qū)。根據(jù)中間可變區(qū)將MSP2分成兩大家族:FC27-MSP2和3D7-MSP2。MSP2的功能目前還并不清楚。前期的研究表明MSP2大部分區(qū)段是無序的,但N端區(qū)段呈現(xiàn)出更高的螺旋結構的傾向性且可與膜相互作用。當與膜結合時,MSP2的N端區(qū)段被誘導形成更多的螺旋結構。另外,MSP2在體外易于形成淀粉樣纖維,并且在裂殖子表面也呈現(xiàn)聚集狀態(tài)。據(jù)此推斷MSP2可能像其它淀粉樣纖維蛋白Aβ和α-synuclein一樣,以聚集體形式與膜相互作用,在膜上形成孔道或者通過其它機制來破壞膜,從而參與入侵宿主細胞。在MSP2的聚集中,N端保守區(qū)也是聚集區(qū)域,它構成了聚集體的核心�？傊�,相對有序的N端保守區(qū)是MSP2的關鍵區(qū)段。那么,相對無序的其它區(qū)段特別是可變的中間區(qū)在MSP2中扮演什么角色?解答這個問題有助于我們更好地理解MSP2的功能機制并據(jù)此開發(fā)更高效的疫苗。在本研究中,我們利用濁度、透射電鏡、核磁共振、圓二色譜以及熒光泄露等實驗檢測不同長度的3D7-MSP2片段——N段保守的25肽、包含中間可變區(qū)的N端肽段及3D7-MSP2全長蛋白——的聚集和與膜的相互作用,以揭示無序區(qū)段對有序區(qū)段性質的影響。其中濁度和透射電鏡用于監(jiān)測小肽和蛋白的聚集(包括聚集動力學和聚集體形態(tài));圓二色譜和核磁共振用于檢測小肽和蛋白的結構(整體結構和原子分辨結構);而熒光泄露實驗則用于監(jiān)測膜的完整性。結果顯示:1,隨著所含無序區(qū)段長度的增加,3D7-MSP2的聚集逐次受到抑制;2,隨著所含無序區(qū)段長度的增加,3D7-MSP2與膜的相互作用也逐次受到抑制。另外,還有一些現(xiàn)象值得注意:1,3D7-MSP2與膜的相互作用依賴于膜的形態(tài),膠束可誘導MSP2形成螺旋結構并抑制其聚集,而脂質體則不誘導螺旋結構而且對聚集的作用是特異性的;2,3D7-MSP2與膜的相互作用依賴于膜的組成,PG促進聚集而PC抑制聚集;3,MSP2對膜結構有破壞作用,很可能是通過其寡聚體形式。根據(jù)以上結果,我們對MSP2在瘧原蟲表面的狀態(tài)及其參與入侵宿主細胞的作用機制做出了推斷。
[Abstract]:Malaria is one of the world's most serious infectious diseases, and even the risk of malaria has been significantly reduced from the beginning of the 20th century, but a million people die each year. Drugs such as artemisinin have shown a great effect in the treatment of malaria, but in the eradication of such diseases, the resistance of plasmodium to these antimalarial drugs is one of the challenges that must be overcome. A more effective way to choose from is to use the vaccine to prevent its infection in the early days. However, the vaccine candidate has a low efficacy and a short time-effectiveness, and is only effective for a part of the plasmodium. There is still no vaccine to effectively fill the gaps in these vaccines. MSP2 is the second rich protein of the merozoite surface of the Plasmodium falciparum, and it has been shown that MSP2 may be involved in the adhesion and invasion of the host red blood cells of the plasmodium, thus being a potential anti-malarial vaccine candidate or drug target. The developed MSP2 vaccine _ some have reached the second stage of the clinical phase _ show that it has a significant effect on the suppression of the invading red blood cells of the plasmodium. As with other malaria vaccine candidates, these MSP2 vaccine candidates are only valid for some of the plasmodium, whereas the protein-based complex MSP2 vaccine is relatively more effective. In order to increase the potential of MSP2 as an anti-malaria vaccine, it is necessary to explore the molecular mechanism of its character and function. MSP2 is a sugar-based phospholipid polymyo-inositol (GPI) anchor protein, including a conserved N-and C-terminal and a variable intermediate variable region. According to the intermediate variable region, MSP2 is divided into two large families: FC27-MSP2 and 3D7-MSP2. The function of MSP2 is not clear at present. Previous studies have shown that most of the sections of MSP2 are out-of-order, but the N-terminal section exhibits a higher tendency to spiral structure and can interact with the membrane. When combined with the membrane, the N-terminal section of the MSP2 is induced to form more helical structures. In addition, the MSP2 is easy to form a starch-like fiber in vitro and also exhibits an aggregation state on the merozoite surface. It is thus concluded that MSP2 may interact with the membrane in the form of an aggregate, in the form of an aggregate, in the form of an aggregate, or by other mechanisms, as in other amyloid A-and HCO3-synclinin, thereby participating in the invasion of the host cell. In the aggregation of MSP2, the N-terminal conserved region is also an aggregation region, which constitutes the core of the aggregate. In summary, the relatively ordered N-terminal conservative region is the key segment of MSP2. So, what role is the relatively unordered other section, especially the variable middle area, in MSP2? The solution to this problem will help us better understand the functional mechanism of MSP2 and develop a more efficient vaccine accordingly. In this study, we used the experiments of turbidity, transmission electron microscopy, nuclear magnetic resonance, circular dichroism and fluorescence leakage to detect the conserved 25-peptide of the 3D7-MSP2 fragment _ N section of different length, including the N-terminal peptide section of the middle variable region and the aggregation of the full-length protein of the 3D7-MSP2 and the interaction with the membrane, In ord to reveal that effect of the out-of-order segment on the nature of the ordered segment. In which turbidity and transmission electron microscopy are used to monitor the aggregation of small peptides and proteins (including aggregation kinetics and aggregate morphology); circular dichroism and nuclear magnetic resonance are used to detect the structure (overall structure and atomic resolution structure) of the small peptides and proteins; while the fluorescence leakage experiments are used to monitor the integrity of the membrane. The results show that, with the increase of the length of the out-of-order segment, the aggregation of 3D7-MSP2 is suppressed, and the interaction between the 3D7-MSP2 and the membrane is inhibited as the length of the disordered segment is increased. In addition, it is worth noting that the interaction of 1,3-D7-MSP2 with the membrane depends on the morphology of the membrane, the micelle can induce the MSP2 to form the spiral structure and inhibit the aggregation, and the liposome does not induce the spiral structure and is specific to the effect of the aggregation; 2. The interaction of 3D7-MSP2 with the membrane depends on the composition of the membrane, PG promotes aggregation and the PC inhibits aggregation;3, MSP2 has a destructive effect on the membrane structure, possibly through its oligomer form. Based on the above results, we have made an inference on the status of MSP2 on the surface of the plasmodium and its mechanism involved in the invasion of the host cell.
【學位授予單位】：安徽大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R382
本文編號：2470941
[Abstract]:Malaria is one of the world's most serious infectious diseases, and even the risk of malaria has been significantly reduced from the beginning of the 20th century, but a million people die each year. Drugs such as artemisinin have shown a great effect in the treatment of malaria, but in the eradication of such diseases, the resistance of plasmodium to these antimalarial drugs is one of the challenges that must be overcome. A more effective way to choose from is to use the vaccine to prevent its infection in the early days. However, the vaccine candidate has a low efficacy and a short time-effectiveness, and is only effective for a part of the plasmodium. There is still no vaccine to effectively fill the gaps in these vaccines. MSP2 is the second rich protein of the merozoite surface of the Plasmodium falciparum, and it has been shown that MSP2 may be involved in the adhesion and invasion of the host red blood cells of the plasmodium, thus being a potential anti-malarial vaccine candidate or drug target. The developed MSP2 vaccine _ some have reached the second stage of the clinical phase _ show that it has a significant effect on the suppression of the invading red blood cells of the plasmodium. As with other malaria vaccine candidates, these MSP2 vaccine candidates are only valid for some of the plasmodium, whereas the protein-based complex MSP2 vaccine is relatively more effective. In order to increase the potential of MSP2 as an anti-malaria vaccine, it is necessary to explore the molecular mechanism of its character and function. MSP2 is a sugar-based phospholipid polymyo-inositol (GPI) anchor protein, including a conserved N-and C-terminal and a variable intermediate variable region. According to the intermediate variable region, MSP2 is divided into two large families: FC27-MSP2 and 3D7-MSP2. The function of MSP2 is not clear at present. Previous studies have shown that most of the sections of MSP2 are out-of-order, but the N-terminal section exhibits a higher tendency to spiral structure and can interact with the membrane. When combined with the membrane, the N-terminal section of the MSP2 is induced to form more helical structures. In addition, the MSP2 is easy to form a starch-like fiber in vitro and also exhibits an aggregation state on the merozoite surface. It is thus concluded that MSP2 may interact with the membrane in the form of an aggregate, in the form of an aggregate, in the form of an aggregate, or by other mechanisms, as in other amyloid A-and HCO3-synclinin, thereby participating in the invasion of the host cell. In the aggregation of MSP2, the N-terminal conserved region is also an aggregation region, which constitutes the core of the aggregate. In summary, the relatively ordered N-terminal conservative region is the key segment of MSP2. So, what role is the relatively unordered other section, especially the variable middle area, in MSP2? The solution to this problem will help us better understand the functional mechanism of MSP2 and develop a more efficient vaccine accordingly. In this study, we used the experiments of turbidity, transmission electron microscopy, nuclear magnetic resonance, circular dichroism and fluorescence leakage to detect the conserved 25-peptide of the 3D7-MSP2 fragment _ N section of different length, including the N-terminal peptide section of the middle variable region and the aggregation of the full-length protein of the 3D7-MSP2 and the interaction with the membrane, In ord to reveal that effect of the out-of-order segment on the nature of the ordered segment. In which turbidity and transmission electron microscopy are used to monitor the aggregation of small peptides and proteins (including aggregation kinetics and aggregate morphology); circular dichroism and nuclear magnetic resonance are used to detect the structure (overall structure and atomic resolution structure) of the small peptides and proteins; while the fluorescence leakage experiments are used to monitor the integrity of the membrane. The results show that, with the increase of the length of the out-of-order segment, the aggregation of 3D7-MSP2 is suppressed, and the interaction between the 3D7-MSP2 and the membrane is inhibited as the length of the disordered segment is increased. In addition, it is worth noting that the interaction of 1,3-D7-MSP2 with the membrane depends on the morphology of the membrane, the micelle can induce the MSP2 to form the spiral structure and inhibit the aggregation, and the liposome does not induce the spiral structure and is specific to the effect of the aggregation; 2. The interaction of 3D7-MSP2 with the membrane depends on the composition of the membrane, PG promotes aggregation and the PC inhibits aggregation;3, MSP2 has a destructive effect on the membrane structure, possibly through its oligomer form. Based on the above results, we have made an inference on the status of MSP2 on the surface of the plasmodium and its mechanism involved in the invasion of the host cell.
【學位授予單位】：安徽大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R382
【引證文獻】
相關碩士學位論文 前1條
1 盧承會;抗菌肽Uperin 3.5和Uperin 3.6的聚集及其與膜相互作用機制的初步研究[D];安徽大學;2018年
，本文編號：2470941
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