人CpG-寡脱氧核苷酸对质粒DNA免疫刺激活性的影响

目的：研究人CpG寡脱氧核苷酸（CpG-ODN)对DNA疫苗质粒骨架免疫刺激活性的影响。方法：将多拷贝对人和小鼠均有免疫刺激活性的CpG2006序列导入质粒骨架,并在小鼠模型体内外评价重组质粒DNA的免疫刺激活性。结果：首先构建了质粒pMini,仅含有卡那抗性基因和pUC18复制起点,然后向pMini中导入了多拷贝对小鼠具有交叉免疫刺激活性的人CpG2006拷贝数的增加,质粒DNA在体外活化小鼠脾细胞分泌IgM的能力逐步增强,但诱生IFN－γ的能力却依次降低,用重组质粒DNA作为HBsAg疫苗佐剂,免疫BALB／c小鼠,pMini和pCpG11对特异性总抗体以及IgG1亚型抗体水平无明显影响（P＞0．05）,但pCpG26能够使两类抗体水平分别提高3倍（P＜0．001）和2倍（P＜0．02）,所有质粒DNA均能够使IgG2a亚型抗体水平提高5－10倍,但三者之间差异无显著意义（P＞0．05）,结论：向质粒骨架中导入一定数量的人CpG-ODN能够增强质粒DNA的免疫刺激活性。

Augmentation of the immunostimulatory effects of plasmid DNA by incorporation of human CpG-oligodeoxynucletide

OBJECTIVE: To study whether the immunostimulatory activities of DNA vaccine could be enhanced by incorporation of human CpG-oligodeoxynucleotide (ODN) into its plasmid backbone. METHODS: Various copies of human CpG2006 sequence with immonostimulating activity for both human beings and mice were transfected into plasmid. Mouse spleen cells were cultured together with different kinds of plasmid DNA and then the amounts of IgM and interferon gamma (IFN-gamma) in the supernatant were examined by antibody sandwich ELISA. HBsAg plus plasmid DNA, as adjuvant, was injected into the shank muscles of 6 BALB/c mice in experimental group and recombinant HBsAg plus Al (OH)(2) was injected into 6 mice as controls. Four weeks later, the anti-HBsAg, IgG1 and IgG2a in their blood were examined. RESULTS: A minimum plasmid named pMini was constructed, containing only kanamycin-resistant gene and pUC18 replication origin. By incorporating different amounts of CpG2006 into pMini plasmid, two recombinant plasmids, pCpG11 and pCpG26, with 9 and 16 copies of CpG2006 respectively, were derived. With the increase of number of CpG2006 copies, the ability of recombinant plasmid DNA to induce the secretion of IgM by cultured murine splenocytes increased accordingly in sequence of pCpG26 > pCpG11 > pMini, and its ability to induce the secretion of IFN-gamma by cultured murine splenocytes decreased instead in sequence of pCpG26 < pCpG11 > pMini. After the mice were immunized with recombinant HBsAg plus pMini and pCpG1, the anti-HBs total antibodies and IgG1 isotype in their blood remained unchanged (P > 0.05), but pCpG26 increased the levels of anti-HBs total antibodies and IgG1 isotype by 3 times (P < 0.001) and 2 times (P < 0.02) respectively. The three kinds of plasmid DNA increased the level of specific IgG2a isotype by 5 approximately 10 times at similar degrees (P > 0.05). CONCLUSION: The immunostimulatory activities of plasmid DNA can be enhanced by incorporation of human CpG-ODN.