应用免疫磁珠分离法纯化弓形虫速殖子的研究

Immunomagnetic separation for purification of Toxoplasma gondii tachyzoites

目的用免疫磁珠分离法分选弓形虫速殖子,以去除宿主细胞成分,并尽可能对虫体的生物学特性无不良影响,为弓形虫的基础与临床研究提供技术基础。方法采用弓形虫Wh3株(China 1基因型)速殖子感染小鼠,提取腹腔液,常规方法制备速殖子可溶性抗原,免疫家兔,获得兔抗弓形虫多克隆IgG抗体。用抗体包被的免疫磁珠对小鼠腹腔液内弓形虫速殖子进行纯化,比较其纯度、回收率、虫体活力、毒力与感染性。结果用免疫磁珠分离技术纯化弓形虫速殖子后,其纯度提高到98.2%,细胞清除率为96%,虫体回收率为73.5%;用内盐法(MTS)细胞增殖与毒性检测试剂盒检测分离后的速殖子活性为95.6%。定量分组感染小鼠后死亡时间无显著性差异。结论免疫磁珠分离的速殖子纯度、细胞清除率和虫体回收率较高,能有效去除宿主细胞,且对速殖子的活性和毒力无影响。该法操作简便快速,无需昂贵的仪器设备,具有较大的实用价值。

Objective To obtain the highly purified tachizoites without contamination of host cells and loss of parasite viability for the diverse purposes of Toxoplasma and toxoplasmosis approach. Methods The peritoneal fluids containing both parasites and redundant host cells were harvested from the mouse, that had been peritoneally inocu- lated with Toxoplasma gondii（ T. gondii） Wh3 strain （ genotype China 1 ）. T. gondii soluble antigen was prepared and used to immunize rabbits to obtain polyclonal rabbit anti-Ibxoplasma antibodies. The mouse peritoneal exudate containing parasites was subjected to the antibody-coated immunomagnetic beads. The purity, recovery, viability and virulence to mice were tested. Results Immunomagnetic separation technique gave rise to as high as 98.2% of the tachyzoites purity; the removal rate of host cells reached to 96% and the recovery rate of tachyzoites was 73.5%. The viability of tachyzoites was 95.6% when detected by the MTS kit of cell proliferation and toxicity detection, and no attenuation of viability and virulence to mice was noted. Conclusion A high purity of T. gondii techyzoites are achieved by immunomagnetic technique and no negative impacts of the novel method on the parastes viability and virulence are found, suggesting that the immunomagnetic separation, which is fast and simple to carried out, might be a useful in the immune and molecular approach of Toxoplasma and toxoplasmosis.