特异性靶向犬细小病毒融合DNA疫苗的构建及免疫效果的研究

摘要

To design chimeric DNA vaccine targeted to antigen-presenting cells with enhanced effi-cacy to induce immunization.The plasmid containing the gene encoding the extracelluar domain of canine cytotoxic T-lymphocyte antigen 4(CTLA-4 1 2 5 ),was directly fused to the gene fragment for major antigenic epitopes of VP2(VP2 228 )by molecular engineering technology.The plasmid con-taining VP2 228 alone was also constructed to serve as control and transfection of COS-7 cells with the two resultant plasimds was performed respectively,followed by assay of Western blotting. The mice were immunized with the constructed two plasimds,respectively.After immunization, the antibodies anginst CPV in the immunized mice at different times were measured by HI.The spleen lymphocyte proliferation response was determined by lymphocyte proliferation assay,and the interferon-γ(IFN-γ)expression level of the mouse lymphocytes were measured by ELISA. The eukaryotic expression plasimds of CTLA-4 1 2 5-VP2 228 fusion protein or VP2 228 alone were cloned.Western blotting showed that the two recombinant proteins could be expressed.Immuni-zation results showed that the antibody levels in serum of CTLA-4 1 2 5-VP2 228-immunized mice were significantly higher than that of VP2 228-immunized mice (P <0.05).The lymphocyte stimulation indexes and secreted IFN-γ levels of the CTLA-4 1 2 5-VP2 228-immunized mice were significantly higher than that of VP2 228-immunized mice (P < 0.05 and P < 0.01 ),respectively.CTLA-4 1 2 5-VP2 228 chimeric DNA vaccine stimulates strong immune response in mice,making it possible for further exploration into chimeric DNA that target the antigen to APCs.%研究旨在构建特异性靶向犬细小病毒(canine parvovirus,CPV)融合 DNA 疫苗,探讨其诱导机体产生免疫应答的效果。试验用重组技术构建了含细胞毒性 T 淋巴细胞抗原-4胞外区(CTLA-4125)与犬细小病毒 VP2的主要抗原表位区域(VP2228)融合表达质粒 pVAX1-CTLA-4125-VP2228,同时构建了不含 CTLA-4125的 pVAX1-VP2228,体外转染 COS-7细胞,Western blotting 检测其表达产物；将 pVAX1-CTLA-4125-VP2228、pVAX1-VP2228分别免疫小鼠,免疫后进行抗体水平测定和抗体亚型分析；通过淋巴细胞增殖试验和 ELISA 分别检测淋巴细胞刺激指数和γ-干扰素表达水平。结果显示成功构建了 pVAX1-CTLA-4125-VP2228和 pVAX1-VP2228,并能在 COS-7细胞中正确表达；抗体检测结果显示 pVAX1-CTLA-4125-VP2228免疫组抗体水平显著高于 pVAX1-VP2228免疫组(P ＜0.05)；淋巴细胞增殖试验显示 pVAX1-CTLA-4125-VP2228免疫组的刺激指数显著高于 pVAX1-VP2228免疫组(P ＜0.05)；γ-干扰素表达水平测定显示 pVAX1-CTLA-4125-VP2228免疫组极显著高于 pVAX1-VP2228免疫组(P ＜0.01)。结果表明,CTLA-4125-VP2228融合 DNA 能有效增强动物对 VP2228抗原的免疫应答,为进一步研究融合 DNA 疫苗特异性的靶向递呈机制奠定基础。