弓形虫 SAG1基因的表达及其诱导的细胞免疫应答

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, Western blot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 Western blot法分析转染细胞,发现存在弓形虫感染血清识别的 26 000u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.

The Expression of SAG1 Gene of Toxoplasma gondii and the Cellular Immune Responses in Mice Vaccinated with This Expression Plasmid

Objective To analyze the expression of Toxoplasma gondii SAG1 gene in vitro, and study on the cellular responses in mice vaccinated with pVAX1-SA G1. Methods The recombinant plasmid pVAX1-SAG1 was transiently transfected into vero cells and the expression of SAG1 was detected by Western blot. Large-scal e recombinant plasmid pVAX1-SAG1 and pVAX1 were prepared and injected into the BALB/c mice via muscles. One-booster injections were given at the 4th week. The proliferation rate of spleen cells was tested by MTT assay, and T cell subgroup CD4+, CD8+were tested by the direct immune fluorescent assay. The positive co ntrol mice were infected with tachyzoites of RH strain of Toxoplasma gondii. Res ults Western blot analysis indicted that the molecular weight of the expressed p rotein of SAG1 in transfected vero cells was about 26000u, similar to the predic ted value. The proliferative responses of immunized and control mice spleen cell s were positive when stimulated by antigen and ConA, and there was a remarkable difference between control group and immunized group(P< 0.05) when stimulated by antigen but not significant(P >05) when stimulated by ConA. The number of CD4 +and CD8+T cells in the immunized group was remarkable different from that of the control one(P1< 0.05,P2< 0.05). The survival time of mice in the immunized group was prolonged and the difference was not obviously between immunized and c ontrol groups (P >0.05). Conclusion The results confirmed that the recombinant p lasmid pVAX1-SAG1 was successfully expressed into vero cells and could elicit B ALB/c mice to generate cellular immune responses.