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目的分析弓形虫SAG1基因真核表达质粒的体外表达及其诱导小鼠的细胞免疫应答。方法重组表达质粒pVAX1-SAG1瞬时转染vero细胞,Westernblot法检测在细胞中的表达。将重组真核表达质粒pVAX1-SAG1及空质粒pVAX1于0、4周分别经肌注免疫BALB/c小鼠;第8周杀鼠,取脾,分离淋巴细胞,分别经ConA和抗原刺激,用MTT法测定免疫鼠脾脏T淋巴细胞转化率;使用直接免疫荧光法,用流式细胞仪对CD4+、CD8+T细胞亚群进行测定;两组小鼠经弓形虫速殖子攻击感染,计算存活期。结果Westernblot法分析转染细胞,发现存在弓形虫感染血清识别的26000u的特异带,与理论值相符;抗原刺激小鼠脾T淋巴细胞发生增殖反应,免疫组与空质粒对照组间差异有显著性(P<0.05),但ConA刺激小鼠后,两组间差异无显著性(P>0.05);T细胞亚群CD4+、CD8+动态分析,CD4+、CD8+T细胞数量均升高,免疫组与空质粒对照组差异均有显著性(P1<0.05,P2<0.05)。弓形虫攻击感染,免疫组鼠的平均存活时间较空质粒对照组延长。结论真核表达质粒pVAX1-SAG1在vero细胞中获得表达,且能诱导BALB/c小鼠产生细胞免疫应答。
Objective To analyze the expression of SAG1 gene eukaryotic expression plasmid in vitro and the cellular immune response induced by it in mice. Methods The recombinant plasmid pVAX1-SAG1 was transiently transfected into vero cells, and the expression of pVAX1-SAG1 was detected by Western blot. The recombinant eukaryotic expression plasmids pVAX1-SAG1 and empty plasmid pVAX1 were respectively immunized BALB / c mice by intramuscular injection at 0 and 4 weeks. The mice were killed at the 8th week, and the spleens were removed and lymphocytes were stimulated with ConA and antigens. The rate of T lymphocyte transformation was measured by MTT assay. The direct immunofluorescence assay was used to detect the CD4 + and CD8 + T cell subsets by flow cytometry. Toxoplasma gondii infection was used to calculate the survival rate period. Results Western blot analysis of transfected cells and found that the presence of Toxoplasma gondii recognized serum 26000u specific band, in line with the theoretical value; antigen-stimulated splenic T lymphocyte proliferation reaction in mice immunized group and empty plasmid control group differences were significant (P <0.05). However, there was no significant difference between the two groups after ConA stimulation (P> 0.05). The dynamic changes of CD4 + and CD8 + in T lymphocyte subsets and CD4 + and CD8 + There was significant difference between the control group and empty plasmid (P <0.05, P <0.05). Toxoplasma gondii attack infection, the average survival time of immunized mice than the empty plasmid control group extended. Conclusion The eukaryotic expression plasmid pVAX1-SAG1 is expressed in vero cells and can induce cellular immune responses in BALB / c mice.