隐孢子虫保护性单克隆抗体的制备

Preparation of Protective McAbs against Cryptosporidium parvum

应用从鼠粪便中分离的小球隐孢子虫(Cryptosporidium parvum)卵囊,经阿拉伯胶梯度离心后,超声粉碎制成可溶性抗原.按常规方法免疫BALB/c小鼠,经融合、克隆筛选建立了5个单克隆抗体株(1A_8、2B_2、3C_4、1A_3和2C_3).通过免疫荧光和免疫组化检测,发现1A_8、2B_2和3C_4克隆株针对子孢子,而lA_3和2C_3针对卵囊壁.1A_8、2B_2和3C_4株单抗腹水分别以10%、20%和30%的量与小球隐孢子虫子孢子(约4×10~4个)共同孵育1h后,接种到人胎肺细胞上培养,结果观察到30%1A_8株腹水、10%2B_2株腹水和20%3C_4株腹水对子孢子有明显细胞毒作用.进而观察了2B_2株单抗腹水对培养虫体的反应性,发现其对裂殖体期虫体亦有作用;进行了2B_2株腹水对BALB/c 小鼠的被动保护性试验,发现以1mL/只剂量分别于攻虫前后1d接种,其对5×10~5个卵囊攻击的BALB/c鼠有明显的保护作用.Western印迹结果表明,2B_2株单抗是针对小球隐孢子虫抗原的28000蛋白质分子.

The oocysts of Cryptosporidium parvum were isolated from feces of infected mice and purified by gum acasia discontinuous gradient centrifugation. The soluble oocyst antigen was prepared by repeatedly ultrasonication and ultracentrifugation. Using the prepared soluble oocyst antigen to immunize BALB/c mice and their spleen cells were then fused with Sp2/0 myeloma cells. After several cycles of screening and cloning, 5 hybridoma cell lines (1A8, 2B2, 3C4, 1A3 and 2C3) secreting monoclonal antibodies to this parasite were obtained. The antibody classes were determined to be IgG1 via Ouchterlony analysis. By immunofluorescene and immunohisto-chemistry assays, it was shown that the sporozoites were specifically recognized by McAbs 1A8, 2B2, and 3C4 and the oocyst wall by 1A3 and 2C3. Mixing separately the sperozoites (c. 4 × 104 with 10%, 20% and 30% of 1A8, 2B2 and 3C4 ascites, incubating the mixtures for 1 h, and then inoculating them into HFL cell cultures, the distinct cytotoxicities of 30% 1A8, 10% 2B2 and 20% 3C4 ascites to sporzoites were found. Additionally, 2B2 ascites may react with the cultivated meronts. The passive protection test indicated that the mice could be protected from challenge of 5×105 oocysts when they received 2 doses (each 1 mL/mouse) of 2B2 ascites at 1 day before and after challenge. Western blotting showed that 2B2 McAb reacts with the parasite antigen located in 28000 region.