IL-2/IFN-γ融合蛋白质粒增强HBV DNA疫苗免疫原性的实验研究

摘要

[Objective] To investigate the adjuvant efficacy of IL-2 and IFN-γ fusion protein gene plasmid (pFP)on HBV DNA vaccination.[Methods] The eukaryotic expression plasmids encoding the IL-2/IFN-γ fusion protein (pFP) and HBV envelope middle protein (pS2.S) were constructed and analyzed by molecular modeling and in vitro ELISA or bioassay for the corresponding expressed proteins after their transient transfection into COS-7 cell line.The in vivo study was conducted in healthy BALB/c mice with or without in vivo electroporation (EP).The immune response of serum anti-HBs and spleen HBsAg-speeifie IFN-γ,T cells were determined by ELISA and ELISPOT,respectively.[Results] The molecular modeling based upon the gene sequence of the IL-2/IFN-γ fusion protein demonstrated the correct construct of pFP releasing either of the cytokines or their bioactive domain separately,which had been confirmed by ELISA and bioassay for detection of the presence and biological activity of either IL-2or IFN-γ in pFP transfected cell culture supematant.In EP-mediated HBV DNA vaccination,the highest response parameter and positive rate of anti-HBs [(51.2±50.5) mIU/mL,89%] or IFN-γ T cells [(207±103) SFCs/3×105splenocytes,78%] were achieved in the group of pS2.S+pFP and were significantly higher than those in the group of pS2.S+pcDNA3.1 [anti-HBs:(14.8-.7.6) mIU/mL,64%;IFN-γ,T cells:(84±70) SFCs/3x105 splenocytes,28%] (P ＜0.05).[Conclusion] The results suggest that IL-2 and IFN-γ,as the Th1-type eytokines in the form of a fusion protein expression plasmid,can enhance the potency of a HBV DNA vaccine,more favorably biased naive T cells towards Th1 differentiation.%目的 评价IL-2/IFN-y融合蛋白基因质粒(pFP)增强HBVDNA疫苗(pS2.S)免疫原性的佐剂作用.方法 构建IL-2/IFN-y融合蛋白和H.BV包膜中蛋白(PreS2+S)编码基因的重组真核表达质粒,根据pFP编码的目的基因序列分子模拟IL-2/IFN-y融合蛋白的空间结构,检测质粒体外转染细胞表达目的基因产物及其生物活性;体内实验采用提高质粒转染效率的在体电脉冲(EP)技术免疫健康BALB/c小鼠,分别以ELISAT及免疫酶联斑点(ELISPOT)方法检测小鼠血清抗HBs及脾脏HBsAg特异性分泌IFN-γ的淋巴细胞应答水平.结果 pFP能够以融合蛋白的形式正确表达IL-2和IFN-γ,其活性域保持相对独立,其分子模拟的结果得到了质粒体外细胞转染检定实验的证实.EP介导的HBV DNA免疫反应中,pS2.S+pFP组血清抗-HBs[(51.2±50.5)mlU/mL,89%]及HBsAg特异性分泌IFN-γ的淋巴细胞应答[(207±103)IFN-rT细胞SFCs/3×105脾细胞,78吲水平和阳性率均显著高于pS2.S+pcDNA3.1组[抗-HBs:(14.8±7.6)mlU/mL,64%;IFN-γ T细胞:(84±70)SFCs/3×105脾细胞,28%(P＜0.05).结论 实验结果提示Th1型细胞因子IL-2和IFN-γ的融合蛋白基因表达质粒可提高HBV DNA疫苗的免疫效果,并促进初始T细胞向Th1方向分化.