炭疽毒素受體的組織分布和活性位點的結構與功能研究
發(fā)布時間：2019-06-04 08:56
【摘要】： 目前已確認炭疽毒素在哺乳動物細胞上有兩種天然受體CMG2與TEM8，但是這兩種受體在體內的天然功能，以及在不同組織的分布還不十分清楚。已有的研究表明，CMG2與炭疽保護性抗原PA的親合力比TEM8大的多，重組表達CMG2胞外區(qū)比重組表達TEM8胞外區(qū)的細胞保護活性高一百多倍，究竟CMG2是起主要作用的受體，還是TEM8是主要受體，或兩者同樣重要，也是未知。弄清兩者的組織分布和分子結構上的差異將為正確理解毒素致病機理與設計新型毒素抑制劑提供依據。 為了解炭疽毒素受體的組織分布，本研究制備了兩種受體的單克隆抗體，獲得了針對CMG2的單克隆抗體4B5和4G3，針對TEM8的單克隆抗體2D6和4B9，以及可同時結合兩者的單克隆抗體2G4，其中4B5還具有一定的細胞保護活性。經鑒定4B5和2D6可用于ATR體內外分布的檢測，在對小鼠的9個組織器官進行了Western blotting和免疫組化檢測后發(fā)現，CMG2和TEM8廣泛分布于各組織器官，在一些關鍵的組織中有明顯的差別，不能確定兩種受體在機體內的分布誰強誰弱，，但是在分布上的差異可能暗示了在不同的炭疽感染途徑中是不同的受體在起作用，肺炭疽時可能CMG2和TEM8均起作用，皮膚炭疽和腸炭疽時則可能是TEM8為主。 出于探索炭疽毒素受體的結構與功能，本研究還用大腸桿菌表達系統(tǒng)克隆表達了六個CMG2胞外區(qū)截短片段，經蛋白純化后，進行了Western blotting和細胞保護性實驗分析。結果顯示六個截短的CMG2蛋白均可與4B5單抗結合，其中CMG2(40-133)、CMG2(40-159)、CMG2(40-186)和CMG2(40-202)失去了細胞保護活性，CMG2(40-212)、CMG2(40-207)保持了很強的細胞保護活性。經綜合分析，可以初步確定，4B5的結合位點在CMG2上40-133氨基酸之間，CMG2的203-207段FQALK氨基酸殘基可能是PA與CMG2相互作用的關鍵序列。 此外，以單抗4B5為靶分子對噬菌體隨機12肽庫進行了篩選，希望得到一批特異性結合4B5的噬菌體肽，用來分析CMG2活性位點的組成和結構。結果顯示，隨機挑選的8個陽性克隆能夠特異性結合4B5，并被CMG2所抑制，表現了它們之間的結構相關性。經序列對齊比較，8個陽性克隆與CMG2(40-217)可確定一個共有序列：YI—LK，位于CMG2上的119-125處，與MIDAS基序中的關鍵氨基酸T118相臨。此共有序列在TEM8中沒有被發(fā)現，而這種不同也許就是它們在細胞保護性方面差異比較大的原因，為我們設計新的治療策略破壞PA與ATR相互作用提供了依據。
[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
本文編號：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
本文編號：2492610
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