弓形虫新基因wx2表位疫苗免疫小鼠的保护研究

The Protective Efficacy Induced by The Epitope Vaccines From New Gene wx2 of Toxoplasma gondii in Mice

采用生物信息学方法对弓形虫新基因wx2进行表位分析预测,PCR扩增基因中编码2个表位的片段W2b和W2a,成功构建新基因的单表位疫苗质粒pcDNA3-W2b、pcDNA3-W2a和双表位疫苗pcDNA3-W2b2a,接种小鼠,观察表位疫苗的免疫保护作用.将表位疫苗分别通过肌肉注射免疫小鼠,对照组注射pcDNA3空质粒.ELISA法检测血清IgG抗体水平,取脾细胞用流式细胞仪检测T淋巴细胞亚群.各组小鼠末次免疫后第4周每只小鼠经腹腔注射弓形虫速殖子500个,观察小鼠的生存时间.结果显示,pcDNA3-W2a2b双表位疫苗组小鼠血清IgG抗体水平显著高于对照组(P<0.05),且pcDNA3-W2a2b双表位疫苗组小鼠脾细胞CD4+T与CD8+T淋巴细胞比值明显低于两单表位疫苗组,pcDNA3-W2a2b双表位疫苗组小鼠存活时间明显长于两单表位疫苗组(P<0.05).实验结果表明,弓形虫新基因wx2表位疫苗能够诱导小鼠产生抗弓形虫感染保护性免疫,并且弓形虫pcDNA3-W2b2a双表位疫苗的免疫保护性优于pcDNA3-W2b、pcDNA3-W2a两单表位疫苗.

Epitope prediction of a new gene wx2 in Toxoplasma gondii was analyzed by bioinformatics. The epitope encoding fragments W2b and W2a were amplified from new gene by PCR, respectively. The single-epitope vaccines pcDNA3-W2b, peDNA3-W2a and double-epitope vaccine pcDNA3-W2b2a were successfully constructed. Mice in group pcDNA3-W2b, pcDNA3-W2a, pcDNA3-W2b2a and two control groups, pcDNA3 and NS, were injected intramuscularly with pcDNA3-W2b, pcDNA3-W2a and pcDNA3-W2b2a epitope vaccines, respectively. As controls, mice were inoculated with NS or empty plasmid pcDNA3. The induced immune responses were tested by ELISA detecting IgG, and flow eytometry sorting the subsets of T lymphocyte. All mice were challenged with highly virulent RH tachyzoites to observe the survival time. Results display that the level of IgG in sera of mice inoculated with pcDNA3-W2b2a was significantly higher than those in pcDNA3 and NS control group, CD4^＋T/CD8^＋ T cell proportion of immunized mice was significantly lower than those in control group. After challenged with highly virulent tachyzoites, the mean survival time of immunized mice in pcDNA3-W2b2a group was significantly longer than groups pcDNA3-W2b, pcDNA3-W2a and control group, indicating that the epitope vaccine from new gene wx2 can induce protective immunity in mice. and the protective efficacy of pcDNA3-W2b2a double epitope vaccine elicits better immunoprotectivity than pcDNA3-W2b, pcDNA3-W2a single-epitope vaccine.