【摘要】 目的:提高hCGβ在真核细胞中的表达效率,观察分子佐剂C3d3增强hCGβ基因疫苗的体液免疫应答。方法:构建带有增强目的基因表达效率的狗胰岛素前原信号肽(DPPISS)的高效真核表达质粒pCMV4hCGβC3d3、pCMV4hCGβ和pCMV4C3d3。分别用pCMV4DPPISShCGβC3d3、pCMV4DPPISShCGβ和pCMV4DPPISShCGβ联合pCMV4DPPISSC3d3免疫BALBc小鼠,各组免疫剂量分别为5、10、50、100、500pmol,间隔3周相同剂量加强免疫1次。间接ELISA法分析免疫小鼠外周血抗hCGβ抗体动态变化以及IgG亚型。结果:含有DPPISS的pCMV4hCGβC3d3较无DPPISS的pCMV4hCGβC3d3在COS7细胞中的表达效率明显提高。50pmol是BALBc小鼠hCGβ基因免疫的最佳免疫剂量。C3d3使得BALBc小鼠hCGβ的免疫原性增强了400倍。hCGβC3d3免疫组IgG1IgG2a比值明显高于hCGβ免疫组和hCGβ与C3d3联合免疫组。结论:分子佐剂C3d3与hCGβ基因融合能够显著增强hCGβ基因避孕疫苗的体液免疫应答及抗体类型转换。更多还原

【Abstract】 Objective:To increase the expression efficiency of hCGβ in eukaryotic cell and to enhance humoral response to the hCGβ DNA vaccine via the molecular adjuvant C3d3.Methods:The eukaryotic expression plasmids of pCMV4-hCGβ-C3d3,pCMV4-hCGβ and pCMV4-C3d3 with dog pre-pro-insulin signal sequence(DPPISS) were successfully constructed by way of molecular cloning.BALB/c mice were inoculated intramuscularly with pCMV4-DPPISS-hCGβ-C3d3,pCMV4-DPPISS-hCGβ alone and pCMV4-DPPISS-hCGβ plus pCMV4-DPPISS-C3d3 respecitively.The immunizing dosage was 5,10,50,100,500 pmol respectively in each group.The immunized mice got a boost immunization in the same dosage and immunizing pathway in 3 week interval.The titers of serum and subclasses of anti-hCGβ IgG antibody were determined by indirect enzyme-linked immunosorbent assay(ELISA).Results:The expression efficiency of pCMV4-hCGβ-C3d3 with DPPISS was higher significantly than that of pCMV4-hCGβ-C3d3 without DPPISS in COS-7 cell.It was found that 50 pmol was the most suitable dosage for hCGβ DNA immunization to BALB/c mice.The anti-hCGβ antibody titers of pCMV4-DPPISS-hCGβ-C3d3 immunization was 400-fold higher than that of pCMV4-DPPISS-hCGβ immunization in BALB/c mice.The IgG1/IgG2a ratio of the hCGβ-C3d3 immunizing group was apparently higher than that of the hCGβ immunizing group and that of the hCGβ plus C3d3 immunizing group.Conclusion:Gene fusion of molecular adjuvant C3d3 to hCGβ can significantly increase the antigen-specific antibody response,and promote antibody IgG subclass turnover in hCGβ DNA vaccine.更多还原