弓形虫新基因表位疫苗免疫效果的观察

The protective immunity induced by the epitope vaccines from two new novel genes of Toxoplasma gondii in mice

目的观察弓形虫新基因WX、WX2的表位疫苗对小鼠的保护作用。方法将昆明小鼠分成5组,分别用pcDNA3-W2b、pcDNA3-W4a、pcDNA3-W2b4a质粒及pcDNA3和NS,肌注3次,每次间隔2周。免疫完成后ELISA法检测血清抗体水平,取脾细胞用流式细胞仪检测CD4+与CD8+淋巴细胞比值,PCR检测肌肉组织中重组质粒。免疫后第3周,小鼠经腹腔注射弓形虫速殖子500个,观察发病情况和存活时间。30d后仍存活的小鼠,取组织匀浆后进行小鼠盲传。结果免疫后第3周,pcDNA3-W2b组小鼠血清抗体水平显著高于pcDNA3和NS对照组(P<0.05);用PCR法从pcDNA3-W2b、pcDNA3-W4a和pcDNA3-W2b4a质粒组小鼠肌肉组织中成功检测到各表位疫苗质粒,且各组小鼠脾脏CD4+与CD8+T淋巴细胞比值显著低于pcDNA3组和NS组(P<0.05)。pcDNA3-W2b、pcDNA3-W2b4a组小鼠存活时间与pcDNA3组及NS组比较明显延长(P<0.05)。结论弓形虫新基因WX、WX2表位疫苗能够诱导小鼠产生抗弓形虫感染保护性免疫,提示DNA类表位疫苗的研制可作为弓形虫疫苗研究的策略之一。

Objective In order to observe the protective immunity induced by the epitope vaccines from two new novel gene WX and WX2 in mice. Methods Kunming mice were divided into five groups, each group was inoculated intramuscularly with pcDNA3-W2b, pcDNA3-W4a, pcDNA3-W2b4a, pcDNA3 and NS third （2 weeks apart） respectively. Three weeks after the last immunization, splenocytes, sera and muscle tissues were respectively collected from each group. Leukomonocyte subpopulation was analyzed with flow cytometer, specific antibody levels were measured by ELISA and the intramusclular plasmid DNA was detected by PCR technique. Mice in each group were challenged with Toxoplasma gondii tachyzoites, 500 each, and then the survival time was compared. 30 days post challenge, the tissues from the survive mice were homogenated and transferred blindly to normal mice. Results Three weeks after the last immunization, ELISA showed levels of specific antibody in sera of mice inoculated with pcDNA3-W2b were significantly higher than those in pcDNA3 and NS control group mice. The intramusclular plasmid DNA in mice immunized with pcDNA3-W2b, pcDNA3-W4a and pcDNA3-W2b4a plasmids were detected by PCR successfully and the ratios of CD4^＋ T and CD8^＋ T of immunized mice were significantly lower than those in control group. After challenging with violent virulence strain of tachyzoites, the mean survival time of immunized mice in pcDNA3-W2b and pcDNA3-W2b4a group were significantly longer than those in pcDNA3 and NS group. Conclusion The epitope vaccines from two new novel genes WX and WX2 can induce protective immunity in mice, indicating that the eukaryotic expression vectors with DNA encoding epitopes is a new method for development of T. gondii vaccine.