无序区段对疟原虫裂殖子表面蛋白2（MSP2）聚集和与膜相互作用的影响
发布时间：2019-05-07 08:52
【摘要】：疟疾是世界上最严重的传染性疾病之一,即使得疟疾的风险从20世纪的开始就大幅度的降低,但是每年仍有上百万人死亡。青蒿素等药物在治疗疟疾时已经表现出了很大的效果,但是,在根除这种疾病时,疟原虫对这些抗疟疾药物的抗药性是必须要克服的难题之一。一个可供选择的可能更为有效的方法是利用疫苗在早期就阻止其传染。但是已有的疫苗候选物表现出较低的效力和较短的时效性,且只对部分疟原虫株有效。目前仍没有疫苗能够有效的填补这些疫苗的缺陷。MSP2是恶性疟原虫(Plasmodium falciparum)裂殖子表面第二丰富的蛋白,已有研究显示MSP2可能参与疟原虫粘附和入侵宿主红细胞,因此是潜在的抗疟疾疫苗候选物或药物靶标。已开发的MSP2疫苗——有些已达到临床二期试验阶段——显示其对阻遏部分疟原虫入侵红细胞有显著效果。像其它的疟疾疫苗候选物一样,这些MSP2疫苗候选物只对部分疟原虫株有效,而基于蛋白质多态性的复合MSP2疫苗相对更有效。为提升MSP2作为抗疟疾疫苗的潜能,有必要对其性质和功能的分子机制进行深入探究。MSP2是一种糖基磷脂酰肌醇(GPI)锚定蛋白,包括保守的N端和C端以及多变的中间可变区。根据中间可变区将MSP2分成两大家族:FC27-MSP2和3D7-MSP2。MSP2的功能目前还并不清楚。前期的研究表明MSP2大部分区段是无序的,但N端区段呈现出更高的螺旋结构的倾向性且可与膜相互作用。当与膜结合时,MSP2的N端区段被诱导形成更多的螺旋结构。另外,MSP2在体外易于形成淀粉样纤维,并且在裂殖子表面也呈现聚集状态。据此推断MSP2可能像其它淀粉样纤维蛋白Aβ和α-synuclein一样,以聚集体形式与膜相互作用,在膜上形成孔道或者通过其它机制来破坏膜,从而参与入侵宿主细胞。在MSP2的聚集中,N端保守区也是聚集区域,它构成了聚集体的核心。总之,相对有序的N端保守区是MSP2的关键区段。那么,相对无序的其它区段特别是可变的中间区在MSP2中扮演什么角色?解答这个问题有助于我们更好地理解MSP2的功能机制并据此开发更高效的疫苗。在本研究中,我们利用浊度、透射电镜、核磁共振、圆二色谱以及荧光泄露等实验检测不同长度的3D7-MSP2片段——N段保守的25肽、包含中间可变区的N端肽段及3D7-MSP2全长蛋白——的聚集和与膜的相互作用,以揭示无序区段对有序区段性质的影响。其中浊度和透射电镜用于监测小肽和蛋白的聚集(包括聚集动力学和聚集体形态);圆二色谱和核磁共振用于检测小肽和蛋白的结构(整体结构和原子分辨结构);而荧光泄露实验则用于监测膜的完整性。结果显示:1,随着所含无序区段长度的增加,3D7-MSP2的聚集逐次受到抑制;2,随着所含无序区段长度的增加,3D7-MSP2与膜的相互作用也逐次受到抑制。另外,还有一些现象值得注意:1,3D7-MSP2与膜的相互作用依赖于膜的形态,胶束可诱导MSP2形成螺旋结构并抑制其聚集,而脂质体则不诱导螺旋结构而且对聚集的作用是特异性的;2,3D7-MSP2与膜的相互作用依赖于膜的组成,PG促进聚集而PC抑制聚集;3,MSP2对膜结构有破坏作用,很可能是通过其寡聚体形式。根据以上结果,我们对MSP2在疟原虫表面的状态及其参与入侵宿主细胞的作用机制做出了推断。
[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.
【学位授予单位】：安徽大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R382
本文编号：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.
【学位授予单位】：安徽大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R382
【引证文献】
相关硕士学位论文 前1条
1 卢承会;抗菌肽Uperin 3.5和Uperin 3.6的聚集及其与膜相互作用机制的初步研究[D];安徽大学;2018年
，本文编号：2470941
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