炭疽毒素受体的组织分布和活性位点的结构与功能研究
发布时间：2019-06-04 08:56
【摘要】： 目前已确认炭疽毒素在哺乳动物细胞上有两种天然受体CMG2与TEM8，但是这两种受体在体内的天然功能，以及在不同组织的分布还不十分清楚。已有的研究表明，CMG2与炭疽保护性抗原PA的亲合力比TEM8大的多，重组表达CMG2胞外区比重组表达TEM8胞外区的细胞保护活性高一百多倍，究竟CMG2是起主要作用的受体，还是TEM8是主要受体，或两者同样重要，也是未知。弄清两者的组织分布和分子结构上的差异将为正确理解毒素致病机理与设计新型毒素抑制剂提供依据。 为了解炭疽毒素受体的组织分布，本研究制备了两种受体的单克隆抗体，获得了针对CMG2的单克隆抗体4B5和4G3，针对TEM8的单克隆抗体2D6和4B9，以及可同时结合两者的单克隆抗体2G4，其中4B5还具有一定的细胞保护活性。经鉴定4B5和2D6可用于ATR体内外分布的检测，在对小鼠的9个组织器官进行了Western blotting和免疫组化检测后发现，CMG2和TEM8广泛分布于各组织器官，在一些关键的组织中有明显的差别，不能确定两种受体在机体内的分布谁强谁弱，，但是在分布上的差异可能暗示了在不同的炭疽感染途径中是不同的受体在起作用，肺炭疽时可能CMG2和TEM8均起作用，皮肤炭疽和肠炭疽时则可能是TEM8为主。 出于探索炭疽毒素受体的结构与功能，本研究还用大肠杆菌表达系统克隆表达了六个CMG2胞外区截短片段，经蛋白纯化后，进行了Western blotting和细胞保护性实验分析。结果显示六个截短的CMG2蛋白均可与4B5单抗结合，其中CMG2(40-133)、CMG2(40-159)、CMG2(40-186)和CMG2(40-202)失去了细胞保护活性，CMG2(40-212)、CMG2(40-207)保持了很强的细胞保护活性。经综合分析，可以初步确定，4B5的结合位点在CMG2上40-133氨基酸之间，CMG2的203-207段FQALK氨基酸残基可能是PA与CMG2相互作用的关键序列。 此外，以单抗4B5为靶分子对噬菌体随机12肽库进行了筛选，希望得到一批特异性结合4B5的噬菌体肽，用来分析CMG2活性位点的组成和结构。结果显示，随机挑选的8个阳性克隆能够特异性结合4B5，并被CMG2所抑制，表现了它们之间的结构相关性。经序列对齐比较，8个阳性克隆与CMG2(40-217)可确定一个共有序列：YI—LK，位于CMG2上的119-125处，与MIDAS基序中的关键氨基酸T118相临。此共有序列在TEM8中没有被发现，而这种不同也许就是它们在细胞保护性方面差异比较大的原因，为我们设计新的治疗策略破坏PA与ATR相互作用提供了依据。
[Abstract]:It has been confirmed that anthrax toxin has two natural receptors CMG2 and TEM8, in mammal cells, but the natural function of anthrax toxin in vivo and its distribution in different tissues are not very clear. Previous studies have shown that the affinity between CMG2 and anthrax protective antigen PA is much higher than that of TEM8, and the extracellular domain of recombinant CMG2 is more than 100 times more active than that of recombinant expression of TEM8. After all, CMG2 is the main receptor. It is also unknown whether TEM8 is the main receptor, or both. Understanding the differences in tissue distribution and molecular structure between the two will provide a basis for the correct understanding of the pathogenic mechanism of toxins and the design of new toxin inhibitors. In order to understand the tissue distribution of anthrax toxin receptors, monoclonal antibodies 4B5 and 4G3 against CMG2 and 2D6 and 4B9 for TEM8 were prepared. And the monoclonal antibody 2G4, which can bind the two at the same time, in which 4B5 also has certain cell protective activity. It was identified that 4B5 and 2D6 could be used to detect the distribution of ATR in vitro and in vivo. It was found that CMG2 and TEM8 were widely distributed in all tissues and organs in 9 tissues and organs of mice, and there were significant differences in some key tissues. It is not possible to determine who is stronger and who is weak in the distribution of the two receptors in the body, but the difference in distribution may suggest that different receptors play a role in different pathways of anthrax infection, and that both CMG2 and TEM8 may play a role in lung anthrax. Skin anthrax and intestinal anthrax may be dominated by TEM8. In order to explore the structure and function of anthrax toxin receptor, six truncated fragments of CMG2 were cloned and expressed by E. coli expression system. After purification of the protein, Western blotting and cellular protective experiments were carried out. The results showed that all six truncated CMG2 proteins could bind to 4B5 monoclonal antibodies, including CMG2 (40 鈮
本文编号：2492610
[Abstract]:It has been confirmed that anthrax toxin has two natural receptors CMG2 and TEM8, in mammal cells, but the natural function of anthrax toxin in vivo and its distribution in different tissues are not very clear. Previous studies have shown that the affinity between CMG2 and anthrax protective antigen PA is much higher than that of TEM8, and the extracellular domain of recombinant CMG2 is more than 100 times more active than that of recombinant expression of TEM8. After all, CMG2 is the main receptor. It is also unknown whether TEM8 is the main receptor, or both. Understanding the differences in tissue distribution and molecular structure between the two will provide a basis for the correct understanding of the pathogenic mechanism of toxins and the design of new toxin inhibitors. In order to understand the tissue distribution of anthrax toxin receptors, monoclonal antibodies 4B5 and 4G3 against CMG2 and 2D6 and 4B9 for TEM8 were prepared. And the monoclonal antibody 2G4, which can bind the two at the same time, in which 4B5 also has certain cell protective activity. It was identified that 4B5 and 2D6 could be used to detect the distribution of ATR in vitro and in vivo. It was found that CMG2 and TEM8 were widely distributed in all tissues and organs in 9 tissues and organs of mice, and there were significant differences in some key tissues. It is not possible to determine who is stronger and who is weak in the distribution of the two receptors in the body, but the difference in distribution may suggest that different receptors play a role in different pathways of anthrax infection, and that both CMG2 and TEM8 may play a role in lung anthrax. Skin anthrax and intestinal anthrax may be dominated by TEM8. In order to explore the structure and function of anthrax toxin receptor, six truncated fragments of CMG2 were cloned and expressed by E. coli expression system. After purification of the protein, Western blotting and cellular protective experiments were carried out. The results showed that all six truncated CMG2 proteins could bind to 4B5 monoclonal antibodies, including CMG2 (40 鈮
本文编号：2492610
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