弓形虫ROP2核酸疫苗免疫保护作用的研究

Studies on the Immuno-Protection of ROP2 Nuclei Acid Vaccine in Toxoplasma gondii Infection

目的研究ROP2核酸疫苗对BALB/c小鼠的免疫保护作用。方法42只BALB/c小鼠随机分为3组:实验组、空质粒组和PBS对照组,各组小鼠分别肌注pc-DNA3-ROP2重组质粒50μg、pc-DNA3空质粒50μg和PBS50μl,共免疫3次,每次间隔3周,末次接种量均加倍。末次免疫后2周,各组分别取4只小鼠的血清、脾和淋巴组织,检测CD4+、CD8+及各细胞因子。其余各组每鼠腹腔攻击感染RH株弓形虫速殖子500个,观察其存活时间等。结果ROP2核酸疫苗能诱发小鼠产生细胞免疫和体液免疫反应,小鼠血清抗体滴度高并能识别由基因重组体外诱导表达的ROP2蛋白抗原;实验组小鼠的CD4+T细胞增殖明显(69.5±3.4)%、CD4+/CD8+比值显著升高(4.69±1.32)%(P<0.01);其脾、淋巴结细胞培养液及血清中白细胞介素-2(IL-2)、IL-4、IL-6、IL-12、γ干扰素(IFN-γ)和肿瘤坏死因子(TNF)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,实验组小鼠免疫保护率为88.9%,与对照组相比,小鼠存活时间明显延长、初始死亡时间明显推迟(P<0.01)。结论ROP2核酸疫苗具有较强的免疫原性,能产生良好的免疫保护作用。

Objective To study the protective effect of ROP2 nuclei acid vaccine in mice, Methods Fortytwo BALB/c mice were divided into three groups. Each mouse in experiment group was injected with 50 μg recombinant plasmid pc-DNA3-ROP2 through musculus quadriceps fexoris. In control groups, each mouse was injected with 50 μg blank plasmid pc-DNA3 and with 50 μl PBS respectively, All mice were immunized for three times with an interval of three weeks, The volume was doubled for the final injection in the two plasmid groups. Blood, spleens and lymph nodes of 4 mice in each group were taken for the detection of CD4^＋, CD8^＋ T cells and cytokines 2 weeks after the final immunization. The rest mice in 3 groups were challenged with 500 tachyzoites of Toxoplasm gondii RH strain for further observation. Results The vaccine induced strong cellular and humoral immune response, The titer of antibody in serum was high after inoculation and recognized ROP2 protein antigen expressed in vitro, The lymphocyte phenotype was analyzed, CD4^＋ T cells proliferated sharply （69.5±3.4）%, and the ratio of CD4^＋/CD8^＋ increased considerably by （4.69±1.32）%（P〈0.01）, The level of IL-2, IL4, IL-6, IL-12, IFN-γ and TNF in serum and cultured supernatant of spleen cells and lymph cells was higher in the experiment group than that in control groups, especially in serum. 88.9% mice in the experiment group were protected 180 hours after the challenge of T.gondii. The death time of mice in experiment group was delayed and the survival time was prolonged in comparison to that in control groups with a significant difference （P〈0.01）, Conclusion The recombinant ROP2 nuclei acid vaccine shows fair immunogeni-city and obviously produces immuno-protection.