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目的用pVACGRA4真核表达重组质粒免疫小鼠,观察诱导的免疫应答及对弓形虫感染的保护作用。方法大量制备pVACGRA4真核表达重组质粒,经基因枪腹部皮内注射免疫BALB/c小鼠3次,以pVAC空质粒及不经任何处理的空白组为对照。于末次免疫4周后作免疫指标测定(包括MTT法测定小鼠脾脏T淋巴细胞增殖活性、间接免疫荧光法测定T淋巴细胞亚群数目、双夹心ELISA法测定细胞因子IFNγ及IL4含量、间接ELISA法测定IgG抗体滴度),并观察攻毒试验后小鼠存活情况。结果脾淋巴细胞增殖各组间差异无显著性(P>0.05);pVACGRA4组CD4+T细胞百分率无明显变化(P>0.05),CD8+T细胞百分率明显增加(与pVAC对照组比较P<0.05,与空白对照组比较P<0.01),CD4+/CD8+比值也较空白对照组明显降低(P<0.01);pVACGRA4免疫组IFNγA值比对照组略高,但差异无显著性(P>0.05),IL4A值各组间无明显变化(P>0.05);pVACGRA4组可诱导产生特异性IgG抗体,但滴度不高;pVACGRA4免疫组小鼠存活率显著高于两对照组,死亡小鼠的平均存活时间延长(与空白对照组比较P<0.05)。结论用pVACGRA4重组质粒DNA免疫小鼠,可诱导产生以细胞免疫为主的免疫应答及对弓形虫攻击感染的部分保护作用。
Objective To immunize mice with eukaryotic expression vector pVACGRA4 to observe the induced immune response and the protective effect against Toxoplasma gondii infection. Methods The eukaryotic expression plasmid pVACGRA4 was prepared in large quantities. The BALB / c mice were immunized intradermally with gene gun for 3 times. The pVAC empty plasmid and blank group without any treatment were used as controls. Immunoassay was performed 4 weeks after the last immunization (including the measurement of the activity of T lymphocyte proliferation in mouse spleen by MTT method, the number of T lymphocyte subsets by indirect immunofluorescence method, the content of cytokines IFNγ and IL4 by double-sandwich ELISA, indirect ELISA Method to determine IgG antibody titer), and observed the survival of mice after challenge test. Results The percentage of CD4 + T cells in pVACGRA4 group was not significantly different (P> 0.05), while the percentage of CD8 + T cells was significantly increased (P <0.05 compared with pVAC control group) (P <0.01), and the ratio of CD4 + / CD8 + was significantly lower than that of the blank control group (P <0.01). The IFNγA level of pVACGRA4 group was slightly higher than that of the control group, but there was no significant difference (P> 0.05) (P> 0.05); pVACGRA4 group could induce specific IgG antibody but the titer was not high; the survival rate of pVACGRA4 immunized group was significantly higher than that of the two control groups, the average survival of the dead mice The time was prolonged (P <0.05 compared with the blank control group). Conclusion The immunization of mice with pVACGRA4 recombinant plasmid DNA can induce partial immune responses that produce cellular immunity and partial protection against Toxoplasma gondii infection.