Japanese Schistosome 22.6kda Antigen Gene Sequence Of The Immunosuppressive Fragments Determined

【Objective】To investigate the suppressive oligodeoxynucleotides (ODNs)probably existing in the sequence encoding Schistosoma japonicum 22.6kDa antigen(Sj22.6).【Methods】Several ODNs within the DNA sequence encoding Sj22.6kDa antigen were synthesized.ODN CpG 1826 was used as stimulator and proper controls were set up for subsequent experiments. Splenocytes separated from normal mice were stimulated with optimal immunostimulatory CpG 1826 in the absence or presence of different synthetical ODNs.The suppressive efficacy of each ODN was examined by ~3H-TdR incorporation method.After that,the ODNs with suppressive capabilities were screened and determined.The dose-effect and time-effect relations of inhibitory ODNs were detected,and the inhibitory roles of ODNs on the stimulation caused by ConA were also examined by ~3H-TdR incorporation.The Th1 cytokines IFN-γand IL-12 in the cultural supernanant were measured by ELISA.Finally,the influences of the suppressive ODNs on splenocytes on cell-surface binding and internalization of CpG 1826 were evaluated by flow cytometry and the gene expression levels of TLR9 of splenocytes were detected by RT-PCR.【Results】(1)ODN F546 and F311 which existed in the DNA sequence encoding Sj22.6kDa antigen could inhibit lymphocyte proliferation induced by CpG 1826.When final concentration of each ODN was 1μM, the inhibiting rate of ODN F546 and ODN F311 was 87%and 11% respectively.(2)When the amount of CpG 1826 was constant, suppressive ODNs inhibited lymphocyte proliferation in a dose-dependent manner.When ratio of suppressive ODN:CpG 1826 was 1:1,the maximal inhibition capability was observed as suppressive ODNs were co-administered or pre-administered with CpG 1826.(3) Suppressive ODN F546 could block a majority of the IFN-γand IL-12 production induced by CpG 1826 in vivo and in vitro.(4)FACS analysis demonstrated that both cell surface binding and internalization of FITC-labeled CpG 1826 was significantly reduced by suppressive ODN F546.(5)In vitro,CpG 1826 elevated TLR9 mRNA expression significantly,which could be down-regulated by ODN F546 intervention.【Conclusion】Suppressive ODN F546 and F311 which exist in the DNA sequence encoding Sj22.6kDa antigen could inhibit lymphocyte proliferation induced by CpG 1826.The inhibition was specific to stimulation caused by CpG ODN and showed dose- and time-dependent manner.The ODN F546 could block a majority of the IFN-γand IL-12 production induced by CpG 1826.The decreased cell surface binding and internalization of CpG 1826,as well as the diminished TLR9 mRNA expression,may be associated with the inhibitory roles of ODN F546. The results suggested that the inhibitory ODNs existed in the DNA sequence encoding Sj22.6kDa antigen may be relevant to the ineffectiveness of Sj22.6 DNA vaccines.