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目的:探索复苏因子E(RpfE)-DNA疫苗的小鼠免疫效果。方法:利用真核表达载体PCDNA3.1构建PCDNA3.1-RpfE重组质粒。将SPF级BALB/C小鼠随机分为5组,即空质粒组、RpfE质粒组、BCG组、生理盐水组、空白对照组,于0、3、6周先后3次免疫小鼠,在第3次免疫后3周,心脏取血ELISA方法检测血清RpfE IgG抗体效价、干扰素γ(IFNγ)水平;并行脾淋巴细胞培养,以RpfE蛋白刺激淋巴细胞,CCK-8法检测淋巴细胞增殖指数、ELISA方法检测上清诱导IFNγ和白介素12(IL-12)产生水平,乳酸脱氢酶试验检测小鼠脾细胞毒性T淋巴(CTL)杀伤效应。结果:(1)RpfE质粒组小鼠血清中RpfE抗体水平明显高于其他各组(P<0.001);RpfE质粒组IFNγ水平明显高于空质粒组、生理盐水组、空白对照组和BCG组(P<0.01或P<0.05)。(2)RpfE质粒组具有较强的刺激淋巴细胞增殖作用,CCK-8实验表明其增殖指数高于其他各组(P<0.05);上清ELISA检测表明IFNγ和IL-12产生水平也高于其他各组(P<0.05)。(3)RpfE质粒组脾淋巴细胞特异性CTL杀伤性与其他各组比较有统计学意义(P<0.05)。结论:RpfE-DNA疫苗可诱导小鼠的免疫应答,具有良好的体液和细胞免疫原性。
Objective: To explore the immune effect of RpfE-DNA vaccine in mice. METHODS: PCDNA3.1-RpfE recombinant plasmid was constructed by eukaryotic expression vector PCDNA3.1. The SPF BALB / C mice were randomly divided into 5 groups: empty plasmid group, RpfE plasmid group, BCG group, normal saline group and blank control group. The mice were immunized three times at 0, Three weeks after the three immunizations, the serum of rabbits was collected to measure the antibody titer of RpfE IgG and the level of interferon gamma (IFNγ) by ELISA. The lymphocytes were stimulated with RpfE protein and the lymphocyte proliferation index (CCK-8) The levels of IL-12 and IL-12 induced by supernatants were detected by ELISA. The cytotoxic T lymphocyte (CTL) killing effect was detected by lactate dehydrogenase assay. Results: (1) The RpfE antibody level in RpfE plasmid group was significantly higher than that in other groups (P <0.001). The IFNγ level in RpfE plasmid group was significantly higher than that in empty plasmid group, normal saline group, blank control group and BCG group P <0.01 or P <0.05). (2) The RpfE plasmid group had strong stimulation of lymphocyte proliferation. The CCK-8 assay showed that the proliferation index of RpfE plasmid group was higher than that of other groups (P <0.05). The supernatant ELISA showed that the production levels of IFNγ and IL-12 were also higher Other groups (P <0.05). (3) The CTL cytotoxicity of spleen lymphocytes in RpfE plasmid group was statistically significant compared with other groups (P <0.05). Conclusion: RpfE-DNA vaccine can induce immune response in mice with good humoral and cellular immunogenicity.