| Recently, two natural receptors(CMG2 and TEM8) of anthrax toxin have been confirmed.But nature function and distribution in vivo of them hasn't been known.Some research had showed the affinity between CMG2 and PA was extremely higher than that of TEM8 and PA. Studies related to recombinant proteins of their extracellular region had demonstrated that cytoprotection activity of CMG2 is higher by hundreds folds than that of TEM8.At present,it has not been known that which of both receptors is more importance in the anthrax toxin pathopoiesis.Therefore,it became very significant to make clear the distribution in vivo and the diference of molecular structures of the proteins. More research is needed to do for us comprehending anthrax toxin pathopoiesis and designing new inhibitors to anthrax toxin.In order to develop rapid assays for detecting anthrax toxin receptors(ATRs) and provide tools for investigating the pathogenesis of anthrax toxin, monoclonal antibodies against two types of ATRs were preparated and characterized. Five hybridoma cell lines secreting McAbs against CMG2 and TEM8 were obtained, named as 4B5, 4G3, 2G4, 2D6 and 4B9 respectively. 4B5 and 4G3 is specifically against CMG2, 2D6 and 4B9 is specifically against TEM8, 2G4 can bind both CMG2 and TEM8. Only 4B5 exhibited the cell protective activity against anthrax lethal toxin. The McAbs obtained were characterized and 4B5 and 2D6 were used to detect ATRs. We had applyed western blotting and immunohistochemistry and detected nine tissues/ organs of BALB/c mice.The results showed CMG2 and TEM8 were both widespread in many tissues/ organs of mouse,but there is some difference in important tissues.Thus, it is diffcult to decide which of both receptors is the main receptor.Those difference of distribution suggested that different ATR may play a role in different route of anthrax infection.CMG2 and TEM8 may be important receptors in inhalational anthrax ,and TEM8 may be more important in cutaneous anthrax and intestine anthrax..Genes encoding six truncated domains of CMG2 were cloned into pQE30 and pET21a(+) respectively and induced to express by IPTG in order to analyze the structure and function of CMG2. The recombinant proteins were purified by chromatography and then identified by cytoprotection assay and western blotting. All recombinant proteins obtained can bind to McAb 4B5 ,but four proteins CMG2 (40-133) ,CMG2 (40-159) ,CMG2 (40-186) and CMG2 (40-202) lost the activity of cytoprotection ,while two proteins CMG2 (40-212) and CMG2 (40-207) still kept the activity. From the result, it was supposed that the binding site of McAb 4B5 is among 40-133 amino acids of CMG2 and FQALK sequence is important structure in the interaction of ATR with PA.Peptide 12-mer Phage Display Library was screened by 4B5 as target molecule.Those peptides obtained were analyzed and showed that they could bind 4B5 specially as well as they could be inhibited by CMG2.By aligning and comparing,a concensus sequence was definited among eight peptides obtained, which is YI---LK at 119-125 of CMG2. This sequence close to T118 which is one of the key amino acids in CMG2 MIDAS domain but the sequence is not exist in TEM8.These distinction may explain the great difference in the activity of cytoprotection between CMG2 and TEM8.These datas would be basis to design new therapy strategy in destroy the interaction of ATR with PA.
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