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目的评价弓形虫SAG1与SAG3基因真核表达质粒DNA免疫小鼠的免疫应答效果。方法以PCR方法扩增出SAG1与SAG3目的基因片段,并插入载体pEGFP-N3以构建重组质粒pE- SAG1／SAG3,瞬时转染细胞Cos-7。质粒pESAG1／SAG3以10μg剂量,脂质体介导肌肉注射BALB／c小鼠,免疫3次,最后一次免疫后4周,观察体液和细胞免疫。RT-PCR方法检测注射部位目的基因的转录;斑点杂交方法检测目的基因是否与小鼠染色体基因组整合。结果质粒pESAG1／SAG3转染细胞后观察到绿色荧光;Western blot显示pESAG1/SAG3表达识别条带在相对分子质量(Mr)66．2×103附近。小鼠免疫后,血清产生抗弓形虫速殖子抗体,诱导IFN-γ及IL-2水平高于对照组。RT-PCR显示首次免疫15 d后,目的基因在肌肉组织中仍有转录,斑点杂交显示目的基因未与小鼠的基因组发生整合,混合质粒注射的小鼠抗弓形虫感染的存活时间延长。结论脂质体介导弓形虫SAG1与SAG3复合编码基因质粒接种小鼠能明显诱导体液和细胞免疫应答。
Objective To evaluate the immune response of Toxoplasma gondii SAG1 and SAG3 eukaryotic expression plasmid DNA immunized mice. Methods SAG1 and SAG3 gene fragments were amplified by PCR and inserted into vector pEGFP-N3 to construct recombinant plasmid pE-SAG1 / SAG3. Transient transfection of Cos-7 cells was performed. Plasmid pESAG1 / SAG3 was injected intraperitoneally into BALB / c mice at a dose of 10 μg and liposomes, immunized three times, and four weeks after the last immunization, humoral and cellular immunity was observed. RT-PCR was used to detect the transcription of the target gene at the injection site. Dot blot was used to detect whether the target gene was integrated with the mouse genome. Results Green fluorescence was observed after transfection with plasmid pESAG1 / SAG3. Western blot showed that the band of pESAG1 / SAG3 expression was around 66.2 × 103. Anti-Tachyzoites anti-T.gondii antibody was produced in the serum of the mice after immunization, and the levels of IFN-γ and IL-2 in the serum were higher than those in the control group. RT-PCR showed that the target gene was still transcribed in muscle tissue 15 days after the first immunization. The dot blot showed that the target gene was not integrated with the mouse genome. The anti-Toxoplasma infection in mixed plasmid-injected mice prolonged the survival time. Conclusion Liposome-mediated Toxoplasma gondii SAG1 and SAG3 complex coding plasmid inoculated mice can significantly induce humoral and cellular immune responses.