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目的评价IL-2/IFN-γ融合蛋白基因质粒(pFP)增强HBV DNA疫苗(pS2.S)免疫原性的佐剂作用。方法构建IL-2/IFN-γ融合蛋白和HBV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-γ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ELISA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)mIU/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%]水平和阳性率均显著高于pS2.S+pcDNA3.1组[抗-HBs:(14.8±7.6)mIU/mL,64%;IFN-γT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。
Objective To evaluate the adjuvant effect of IL-2 / IFN-γ fusion protein gene plasmid (pFP) enhancing the immunogenicity of HBV DNA vaccine (pS2.S). Methods The eukaryotic expression plasmid of IL-2 / IFN-γ fusion protein and HBV envelope protein (PreS2 + S) encoding gene was constructed. Based on the sequence of the gene encoding pFP, IL-2 / IFN-γ fusion protein Space structure and detect the expression of the target gene product and its biological activity in transfected cells in vitro. In vivo experiments BALB / c mice immunized with somatic electropulsing (EP) to improve the efficiency of plasmid transfection were immunized with ELISA and enzyme-linked immunosorbent assay The ELISPOT method was used to detect the level of HBsAg-specific IFN-γ-producing lymphocytes in anti-HBs and spleen of mice. Results pFP could express IL-2 and IFN-γ correctly in the form of fusion protein. The activity domain of pFP was relatively independent. The results of molecular simulation confirmed by plasmid in vitro transfection assay. In the EP-mediated HBV DNA immune response, the anti-HBs [(51.2 ± 50.5) mIU / mL, 89%] and the HBsAg-specific IFN- γ-producing lymphocyte response in the pS2.S + ) IFN-rT cells were significantly higher than the pS2.S + pcDNA3.1 group [anti-HBs: (14.8 ± 7.6) mIU / mL, 64% -γ T cells: (84 ± 70) SFCs / 3 × 105 spleen cells, 28%] (P <0.05). Conclusion The experimental results suggest that the fusion protein gene expression plasmids of Th1 cytokines IL-2 and IFN-γ can enhance the immune effect of HBV DNA vaccine and promote the differentiation of primary T cells to Th1 direction.