抗弓形虫单克隆抗体的制备鉴定及应用

Preparation and identification of monoclonal antibody against Toxoplasma gondii

目的制备抗弓形虫全抗原、天然P30、重组P30的单克隆抗体(McAb),为抗原提纯、弓形虫病诊断及亚单位疫苗的研制奠定基础。方法用弓形虫全抗原、天然P30、重组P30分别免疫BALB/c小鼠,取脾细胞与骨髓瘤细胞融合,筛选稳定分泌高滴度McAb的杂交瘤细胞株。用ELISA法测定McAb亚类和效价;通过SDS-PAGE和Westernblot进行特异性鉴定;Giemsa染色观察杂交瘤细胞的染色体数目;利用电镜及IFAT观察McAb对弓形虫的杀伤作用及其作用位点。结果筛选出3株(4B10、2B3、1H6)特异性较好的杂交瘤细胞株。Westernblot结果显示,2B3、1H6产生的McAb与弓形虫全抗原的阳性反应带均在30kDa处,4B10反应带主要在22kDa处;2B3,1H6,4B10McAb的效价(ELISA)分别为:1∶102400、1∶51200、1∶12800;2B3、1H6为IgM,4B10为IgG2b;3株细胞的染色体数均在100条以上。电镜观察到McAb作用后的弓形虫虫体聚集、肿胀,表面出现缺口和空洞,虫体变形破碎、膜变厚。抗弓形虫全抗原及天然P30的McAb能准确识别重组体pET-30a-ROP2、pET-30a-P30的表达蛋白。结论抗弓形虫全抗原、P30抗原的McAb均对弓形虫具有明显的杀伤作用,能准确识别P30抗原,可应用于弓形虫病诊断、抗原鉴定及亚单位疫苗的研制。

Objective To prepare and identify strains of monoclonal antibodies （McAb） against Toxoplasma gondii （T. gondii） crude antigen, natural P30 antigen and recombinant P30 antigen to lay foundations for purification of antigens, diagnosis and preparation for subunit vaccine of toxoplasmosis. Methods BALB/c mice were immunized with T. gondii crude antigen, natural P30 antigen and recombinant P30 antigen, respectively. Splenocytes of these mice were fused with myeloma cells. Then many hybridoma cell strains which could stably secrete high titer monocolonal antibodies against T. gondii were screened. Subtypes and titer of these McAb were assayed by ELISA, and their specificities were identified by SDS-PAGE and Western blot. The numbers of hybridoma cell chromosomes were checked through Giemsa dying. The katogenes and effective sites of McAb to T. gondii were observed through electronic microscopy and IFAT. Results Three strains of hybridoma cells including 4B10, 2B3 and 1H6 which had good specificity against T. gondii were screened. Western blot showed that a band of 30 kDa in T. gondii crude antigens reacted with McAb produced by 2B3 and 1H6. The main band produced by 4B10 was 22 kDa. Titers of these McAbs were 1 ： 102 400 （2B3）, 1 ： 51 200 （1 H6） and 1 ： 12 800 （4B10）, respectively. The antibody subtypes of 2B3 and 1H6 were IgM, and that of 4B10 was IgG2b. The number of chromosome of each of the three hybridoma cell strains was more than one hundred. Electron microscope observation showed that the bodies of T. gondii effected with McAb were accumulative, swollen, with nicks and cavitates in the surfaces of bodies. Many of these polypides deformed and crushed and the membrane of which was incrassate. Expressed proteins of recombinants pET-30a-ROP2, pET-30a- P30 were correctly identified by McAb against T. gondii crude antigens and natural P30 antigen. Conclusions All McAbs against T. gondii crude antigens and P30 antigens have obvious katogene to T. gondii and can correctly identify P30 antigen of T. gondii, and these McAbs can be helpful to identification of antigens, diagnosis of toxoplasmosis and preparation for subunit vaccine.