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目的探讨重组乙型肝炎病毒核心抗原(HBcAg)的免疫原性及其对重组乙型肝炎病毒表面抗原(HBsAg)的免疫增强作用。方法用透射电镜观察HBcAg、HBsAg及HBcAg+HBsAg混合抗原病毒样颗粒(Virus-like particle,VLP)的形态。将小鼠随机分为6组:阴性对照组(NS)、HBcAg组(C)、HBsAg组(S)、HBsAg+铝佐剂组(S+Al)、HBsAg+HBcAg混合组(S+C)及HBsAg+HBcAg+铝佐剂组(S+C+Al),用相应抗原免疫各组小鼠,分别于第0天、第14天经小鼠双侧大腿肌肉进行初次和加强免疫1次,间接ELISA法检测各组小鼠血清中HBsAg和HBcAg的特异性抗体水平,分析HBsAg特异性抗体亚类,ELISPOT法检测各组小鼠脾细胞中HBsAg特异性IFNγ、HBcAg特异性IFNγ及HBsAg特异性IL-4水平。结果电镜观察可见HBsAg和HBcAg均呈大小均一的VLP结构,二者混合后有聚集现象。初次免疫后,S+C组的HBsAg特异性抗体水平与S组差异无统计学意义(P=0.074),加强免疫后,S+C组HBsAg特异性抗体水平明显高于S组(P=0.002),C组和S+C组均可产生高水平HBcAg特异性抗体。S组HBsAg抗体亚类以IgG1为主,S+C组以IgG2a为主。S+C组HBsAg特异性IFNγ斑点数明显高于S组、S+Al组和S+C+Al组(P<0.05),C组与S+C组HBcAg特异性IFNγ斑点数均较高,与S+C+Al组比较,S+Al组HBsAg特异性IL-4斑点数明显升高(P=0.026)。结论 HBcAg具有良好的免疫原性,可增强HBsAg的免疫反应,使反应向Th1方向极化,为治疗性乙肝疫苗的研发奠定了基础。
Objective To investigate the immunogenicity of recombinant hepatitis B virus core antigen (HBcAg) and its immune enhancement effect on recombinant hepatitis B virus surface antigen (HBsAg). Methods Transmission electron microscopy was used to observe the morphology of HBcAg, HBsAg and HBcAg + HBsAg mixed antigen-like particle (VLP). The mice were randomly divided into 6 groups: NS group, HBcAg group (C), HBsAg group (S), HBsAg + aluminum adjuvant group (S + Al), HBsAg + HBcAg mixed group HBsAg + HBcAg + aluminum adjuvant group (S + C + Al). The mice in each group were immunized with the corresponding antigens. The mice were immunized once and twice with the bilateral thigh muscles on day 0 and day 14. Indirect ELISA The specific antibodies of HBsAg and HBcAg in the serum of each group were detected, and the specific antibody subtypes of HBsAg were analyzed. The levels of HBsAg-specific IFNγ, HBcAg-specific IFNγ and HBsAg-specific IL- 4 levels. Results Electron microscopy showed that both HBsAg and HBcAg were uniform VLP structure, the two aggregation phenomenon. After the first immunization, the level of HBsAg-specific antibody in S + C group was not significantly different from that in S group (P = 0.074). After boosting, the level of HBsAg-specific antibody in S + C group was significantly higher than that in S group ), C group and S + C group can produce high-level HBcAg-specific antibodies. S group HBsAg antibody subclasses to IgG1-based, S + C group with IgG2a-based. The HBsAg-specific IFNγ spots in S + C group were significantly higher than those in S group, S + Al group and S + C + Al group (P <0.05) Compared with S + C + Al group, the HBsAg-specific IL-4 spots in S + Al group were significantly increased (P = 0.026). Conclusion HBcAg has good immunogenicity, which can enhance the immune response of HBsAg and polarize the reaction toward Th1, which lays the foundation for the development of therapeutic hepatitis B vaccine.