目的 :研究弓形虫 SAG1基因在 He L a细胞中的表达 ,为研制弓形虫疫苗奠定基础。方法 :采用磷酸钙 -DNA共沉淀转化法将重组质粒 pc DNA3- SAG1导入 He L a细胞 ,用 G418进行选择培养 ,筛选出表达阳性的细胞克隆 ,再通过培养和加压 ,建立稳定分泌 SAG1抗原的阳性 He L a细胞克隆株 ,分离表达产物 ,进行 SDS- PAGE、Western blot分析。结果 :采用钙沉淀法成功地将重组质粒 SAG1导入 He L a细胞 ,通过 G418选择和加压培养 ,获得稳定分泌 SAG1抗原的阳性 He L a细胞 ,表达的蛋白经 SDS- PAGE、Western blot分析 ,显示其分子量 30 k Da蛋白 ,与理论值相符。结论 :成功构建重组质粒 pc DNA3- SAG1,建立稳定分泌 SAG1抗原的阳性 He L
Objective To observe the immune responses elicited in BALB/c mice by a DNA vaccine. A gene encoding rhoptry protein 1 (ROP1) from Toxoplasma gondii (T. gondii) was cloned into vector pcDNA3. Methods Amplifyied gene fragments coding for ROP1 from the genomic DNA of T.gondii ZS2 were inserted into cloning vector, pUC18, and sub-cloned into pcDNA3. Mice were injected at a dosage of 100?μg recombinant plasmid DNA by intramuscular injection and boosted after 2 weeks. pcDNA3 and normal saline were used as control. 30, 50 and 70 days after the second immunization, NK cell activity, T lymphocyte proliferation and sub-clusters and serum IgG antibody were assayed.Results The specific gene fragment coding for ROP1 was amplified and a pcROP1 recombinant was constructed. At 30 days after immunization, the spleens of the mice were obviously enlarged evidently. NKC activity and the proliferation of spleen T lymphocytes seen on MTT assay were higher in pcROP1 group than in the controls. The number of CD4+ T cells exhibited no obvious increase compared with that of the control, but CD8+ T cells were obviously increased (P<0.05). At 90 days after vaccination, the titer of IgG antibody in the serum of vaccinated mice was positive (1∶100). Conclusion pcROP1 was constructed and it could elicit both cellular and humoral immune responses in immunized mice.
