【摘要】 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。更多还原

【Abstract】 Objective To evaluate, in mice, the immune responses induced by experimental DNA construct encoding Toxoplasma gondii SAG1 and SAG3 as a hybrid gene. Methods Truncated S4G1 and SAG3 DNA fragments were PCR amplified and inserted into pEGFP-N3 vector to construct recombinant plasmid pESAG1/ SAG3. Cos-7 mammalian cells were transiently transfected with the DNA construct. Female BALB/c mice were given three intramuscular injections of 10μg plasmid DNA entrapped in liposome. Four weeks after the final booster injection, blood samples were collected and subjected to ELISA to investigate humoral and cell-mediated immune responses. RT-PCR was used to evaluate the transcription of inoculated DNA-liposome complex in the injected site. To detect whether or not the injected DNA incorporate into the genomic DNA of the immunized mice, Dot-blot hybridization was employed. Results Green fluorescence was observed in pESAGl/SAG3 transfected cells. Western blot analysis showed antibody recognition of the expressed SAG1/SAG3 about Mr 66.2×103. No expression was observed in blank control plasmid-transfected cells. Sera of immunized mice exhibited antibody to T. gondii tachyzoites and primarily interferon-γand interlukin-2. RT-PCR found that the duration of transcribed inoculated liposome entrapped DNA in the injected muscular tissue was at least ten days post first injection. Dot blot hybridization revealed the presence of foreign DNA in the splenocytes and blood leukocytes was transient and no foreign DNA inserted into the genomic DNA of mice immunized with pESAG1/SAG3, challenge of mice vaccinated with combined plasmids with ZS2 tachyzoites resulted in a prolonged survival. Conclusion Immunization with a lipo-some-encapsulated DNA construct encoding the T. gondii SAG1 and SAG3 can induce humoral and cell-mediated immune responses.更多还原