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目的研制抗弓形虫DNA疫苗,评价其免疫原性及免疫保护作用,进而推广应用。方法首先用RH株弓形虫速殖子提取基因组DNA,应用PCR、酶切、连接、测序等技术,扩增棒状体蛋白2(ROP2)基因片段,将其克隆于真核表达载体pc-DNA3质粒中,制备抗弓形虫棒状体蛋白2DNA疫苗。再应用该疫苗免疫小鼠,取其血清、脾和淋巴结培养液检测免疫学指标评价其免疫原性,最后应用弓形虫攻击感染实验评价其免疫保护作用。结果PCR扩增出1.7kbROP2基因片段,克隆、构建出pc-DNA3-ROP2重组DNA疫苗能诱发小鼠产生强烈的细胞免疫和体液免疫反应,小鼠血清抗体滴度高且能正确识别由基因重组体外诱导表达的ROP2蛋白抗原;实验组小鼠CD4+T细胞增殖明显,体液中各细胞因子含量均有不同程度的升高,尤其是血清中升高最为显著。弓形虫攻击感染180h后,疫苗免疫保护率为88.9%,小鼠存活时间明显延长。结论pc-DNA3-ROP2重组DNA疫苗具有较强的免疫原性,安全可靠,能产生良好的免疫保护作用。
Objective To develop anti-Toxoplasma gondii DNA vaccine, evaluate its immunogenicity and immune protection, and then promote its application. Methods Genomic DNA was extracted from tachyzoites of Toxoplasma gondii RH strain. The ROP2 gene fragment was amplified by PCR, restriction enzyme digestion, ligation and sequencing. The recombinant plasmid was cloned into the eukaryotic expression vector pc-DNA3 , An anti-toxoplasma rod-like protein 2 DNA vaccine was prepared. The vaccine was then used to immunize mice and the immunological indexes of serum, spleen and lymph node culture were detected to evaluate the immunogenicity. Finally, the immunological protection effect was evaluated by Toxoplasma gondii infection experiment. Results The 1.7kbROP2 gene fragment was amplified by PCR and cloned. The recombinant plasmid pc-DNA3-ROP2 was used to induce strong cellular and humoral immune responses. The titer of mouse serum antibody was high and it could be correctly identified by gene recombination The expression of ROP2 protein antigen was induced in vitro. The proliferation of CD4 + T cells in experimental group was obvious, and the content of cytokines in body fluid increased to some extent, especially in serum. Toxoplasma gondii attack infection 180h, the vaccine immunization rate was 88.9%, significantly longer survival time in mice. Conclusion pc-DNA3-ROP2 recombinant DNA vaccine has strong immunogenicity, safe and reliable, can produce good immune protection.