SAG1-MIC8复合DNA基因疫苗免疫小鼠诱导抗弓形虫感染的保护性研究(英文)

Protective Immunity Induced in Mice by Multiantigenic DNA Vaccine with Genes Encoding SAG1 and MIC8 of Toxoplasma gondii

目的观察弓形虫SAG1-MIC8复合DNA基因疫苗对C57BL/6J小鼠的免疫保护作用。方法构建重组质粒pcDNA3.1-SAG1、pcDNA3.1-MIC8和pcDNA3.1-SAG1-MIC8,并分别转染Hela细胞体外表达蛋白,蛋白质印迹(Westernblotting)分析鉴定。将70只小鼠随机均分为5组,分别为PBS组、空质粒组、pcDNA3.1-SAG1组、pcDNA3.1-MIC8组和pcDNA3.1-SAG1-MIC8组,每2周肌注免疫(100μg/只)1次,共3次,于免疫前和初次免疫后13、27、41和55d采血分离血清。初次免疫后56d,每组小鼠中7只剖杀后分离脾细胞,另7只经腹膜感染弓形虫RH株速殖子(1×104/只),观察各组生存时间。ELISA法分别检测各组小鼠血清IgG、IgG1、IgG2b和IgG2c水平,以及干扰素γ(IFN-γ)和白介素4(IL-4)水平。放射法检测T淋巴细胞增殖情况。结果Westernblotting分析结果显示,重组质粒pcDNA3.1-SAG1、pcDNA3.1-MIC8和pcDNA3.1-SAG1-MIC8均能在Hela细胞表达,蛋白相对分子质量(Mr)分别为34000、74000和109000。pcDNA3.1-SAG1-MIC8组初次免疫后41d和55d血清中IgG,末次血清中IgG2b、IgG2c和IFN-γ,以及T淋巴细胞增殖能力均显著高于其他各组(均P<0.05)。各组的末次血清中IgG1和IL-4水平差异均无统计学意义(均P>0.05)。5组小鼠感染弓形虫速殖子后生存时间中位数分别为3、4、7、7和10d,各组间差异有统计学意义(P<0.01)。结论弓形虫SAG1-MIC8复合DNA抗原较SAG1和MIC8单基因抗原有更好的免疫保护性。

Objective To observe the immunoprotection induced by multiantigenic SAG1-MIC8 DNA vaccine of Toxoplasma gondii in C57BL/6J mice. Methods The sequences of genes encoding SAG1 and MIC8 protein were inserted into the eukaryotic expression vector pcDNA3.1 and the multiantigenic recombinant plasmid pcDNA3.1-SAG1- MIC8 was constructed. Then the recombinant plasmid was transfected into Hela cells to test its expression and the recombinant protein characterized by Western blotting. 70 mice were divided into 5 groups randomly： PBS, pcDNA3.1, pcDNA3.1-SAG1, pcDNA3.1-MIC8 and pcDNA3.1-SAG1-MIC8. Each mouse was injected intra-muscularly by 100 μg recombinant plasmid for 3 times every two weeks. Mice were bled on day 0, 13, 27, 41, and 55. Four weeks after the final inoculation （on day 56）, spleens from seven immunized mice per group were collected. Another seven immunized mice per group were intraperitoneally challenged with 1×104 tachyzoites of RH T. gondii and the survival time was observed. Serum IgG antibody and cytokines IFN-γ and IL-4 were demonstrated by ELISA and the T lymphocyte proliferation assay were carried out with 3H-TdR incorporation. Results Western blotting showed that the mature protein extracts in Hela cells upon transfection with pcDNA3.1-SAG1 （Mr 34 000）, pcDNA3.1-MIC8 （Mr 74 000） and pcDNA3.1-SAG1-MIC8 （Mr 109 000） were effectively expressed in cells. The results of IgG antibodies （on day 41 and 55）, IgG2b, IgG2c, IFN-γ （on day 55） and T lymphocyte proliferation assay （on day 56） were more obvious in mice immunized with pcDNA3.1-SAG1-MIC8 multiantigenic DNA vaccine than those in mice with single-gene plasmids （P0.05）. There was no significant difference in IgG1 and IL-4 levels between vaccinated and control mice after the final inoculation （on day 55） （P0.05）. The median survival time was 3, 4, 7, 7, and 10 d, respectively, with considerable difference among the groups （P0.01）. Conclusion The multiantigenic DNA vaccine elicits a stronger immuno-protection in mice than the monovalent DNA vaccine.