Screening CpG ODN And Studying On The Promoting Immunostimulatory Effects Of CpG Complex

Objective:Screened oligodeoxynucleotides containing unmethylated CpG dinucleotides (CpG ODN) which was optimal stimulation on neonate, in order to improve the immunostimulatory effects of CpG ODN, CpG complex{CD40L (CD40 ligand) -EDC{ (l-Ethyl-3[(3-dimethylamino)propyl ] carbo-diimide methiodide }-pLL (poly-L-lysine) -CpG ODN}was synthesized to research immune activity, especially it's target to B lymphocyte.Methods:1. Screening CpG ODNAccording to the relationship between CpG ODN structure and immune activity , comparing with CpG2006 and T8, 15 pieces of CpG ODN were designed to stimulate neonate umbilical mononucleocytes, used MTT method to assess their proliferation of mononucleocytes2. Applied receptor-conjugated technology to synthesize CpG2006 complexEDC catalysed CD40L and pLL to be CD40L-pLL, which carried positive charge, then CD40L-pLL combined with CpG2006 which carried negative charge to be CpG2006 complex (CD40L-pLL-CpG2006). Used MTT method to assess the proliferation of neonate mononucleocytes stimulated by CpG2006 complex , used2004ELISA method to assess the level of supernate IgG antibody. Applied FACS to assess the uptake rate of CpG2006 and to analysis the expression level of CD 19, CD20, CD22 and CDS. Then compared the immunostimulatory effects of CpG2006 complex(CD40L-pLL-CpG2006), CpGl complex(CD40L-pLL-CpGl) and CpG6 complex(CD40L-pLL-CpG6).Results:1. The proliferation of CpGl and CpG6 on neonate mononucleocytes were stronger than that of T8 , and no less than that of CpG20062. The synthesis of CpG2006 complexThe result of SDS-PAGE showed that CD40L-pLL was blocked at the beginning of the sample point, without electrophoretic band on the CD40L level . 1% agarose electrophoresis detected that the CpG2006 complex which was composed of CD40L-pLL diluted 16 times and 10uL(165ng/mL) CpG2006 is the best match group.The uptake rate on CpG2006 of mononucleocytes simulated by CpG2006 complex group was markedly higher than that of CpG2006 group (p<0.05). The proliferation of mononucleocytes on CpG2006 complex group was notably stronger than that of CpG2006 group (p<0.05). The level of antibody of CpG2006 complex is also higher than that of CpG2006 group. The proportion of CD19+, CD20+ and CD22+ accounted for mononucleocytes in CpG2006 complex were all higher than that of CpG 2006 group, however , there are no different on CD3+cells among them .3. Comparing the immunostimulatory effects of CpGl complex, CpG6 complex and CpG2006complexThe uptake rate of mononucleocytes on the three kinds of CpG complex were all above 80 per cent, which were all higher than that of pure CpG ODN group , among the CpG ODN group , the uptake rate of CpG6 was higher than that of CpG2006 and CpGl groups.The proportion of CD19+ accounted for mononucleocytes in CpG complex were2004both higher than that of CpG ODN groups (P<0.05) . The level of IgG antibody in CpGl complex group and CpG6 complex group were both notably higher than that of CpGl group and CpG6 group (P<0.05). The level of IL-12 in the three CpG complex groups are higher than that of negative comparing group (P<0.05 ) .Conclusion:1. CpG6 and CpGl have optimal stimulation on neonate mononucleocytes, CpG6 has stronger immunostimulatory activity on neonate B lymphocytes .2. We applied receptor-conjugated technology to successfully synthesize CpG complex, which could improve the uptake rate of CpG ODN, target to B lymphocytes, trigger B lymphocytes proliferating, differentiating and maturing. The receptor-conjugated technology is an effective method to improve the cellular uptake rate of CpG ODN and to improve the stimulatory effects of CpG ODN.3. The immunostimulatory of CpG6 complex is stronger than that of CpG2006 complex.