| CpG-ODN, finding in bacteria, the oligodeoxynecleotides containing non-methylated CpG dinucleotides, has immune activation. Accumulating evidence has revealed CpG-ODN activate directly proliferation of B cells, facilitate maturation of dendritic cells and induce secretion of cytokines from APCs. Some researches of application foundation demonstrated the very optimistic application foreground of CpG-DNA as a new kind of adjuvant for vaccination strategies for cancer, allergy and asthma. Since the extensive immunomodulatory effects and application potential of CpG-ODN, the study of intracellular molecular mechanism of CpG-ODN became a hotspot at present, and that focus problem is to search the specific receptor of CpG-ODN on immune cells and elucidate the mechanism of signal transduction transmembrane pathway.Recent researches showed that specific identified receptor of CpG-ODN was very possibly a new member of Toll-Like-Receptors (TLRs) family—TLR9. The evidence comes mainly from gene knocked-out mice, which was knocked out the gene of MyD88, the adaptor molecule involved in the signalling through TLR9 and TLR families. MyD88 mice did not respond to CpG-ODN if MyD88 was knocked out. Furthermore, TLR9, a transmembrane protein, which extracellular part of it contains a homologous DNA binding domain with MBD-1-4, a methylated CpG-DNA binding protein. It suggested that after binding with TLR9, CpG-ODN transfer activating signal through MyD88. However, it is wondering that the specific interaction of CpG-ODN with TLR9 has not been observed directly and there isn' t specific binding site of CpG-ODN on the immune cells membrane. Although ODN could bind to cell membrane, there was no specific binding for CpG-motif. Moreover, some data showed that specific recognition for CpG-ODN and signal triggering didn' t occur on the extracellular membrane in immune cells, but occurred sequentially after cell uptake and endosome acidified.Hereby, we presumed that the specific binding of CpG-ODN with TLR9 and the trigger of activating signal be likely to occur in acidic endosomes after uptaken by cells, not on cells ectoblast. It suggested that the specific binding of CpG-ODN with TLR9 very possibly needs a specifical exterior condition, an acidic pH.In order to approve this assumption, some studies of immunomodulation activity of CpG-ODN and binding activity of CpG-ODN with cell membrane in different pH were compared. It was detected that there was actually no sequence selectivity that binding of ODNs with immune cells membrane at neutral pH, whereas the specific binding was observed at acidic pH. Moreover, the stimulation of CpG-ODN was more sensitivity than ever at slight pH in immune cells. In addition, RT-PCR was performed to amplified mouse TLR9 gene from mouse splenic cells and extra-cellular region of TLR9 was subcloned into pcDNA3.1, and then the recombinant plasimds was transferred instantly into COS7, the expressed interesting protein was identified specifically by anti-mTLR9 antibody. EMSA tested the interaction of ODNs with the expression production at different pH, and found that the binding of ODNs with the expression production become higher affinity at acidic pH (pH6.4) than at neutral pH. Moreover, It suggested that the recombinant expressed extra-cellular region of TLR9 bind with CpG-ODN specifically in pH-dependent way. The results of our studies supported the assumption, so we put forward to a novel signal transmembrane transduction pathway that stimulation from extra-cellular, the hypothesis that the binding CpG-ODN to its specific membrane receptor, TLR9, and subsequently triggering of CpG-related signaling occurred within acidified endosomes. It is obviously different with the convenient pathway that the binding of the extra-cellular stimulation molecular with membrane receptor and the trigger of activated signals occurred on endoblast and then the binding complexes weredismissed and stopped signal transduction after entering the acidic endosomes.
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