抗弓形虫P30单克隆抗体的制备及其抗虫作用观察

制备抗弓形虫天然P30、重组P30的单克隆抗体(mAb),并研究它们对虫体的影响,为弓形虫病诊断及保护性抗原鉴定奠定基础。采用弓形虫天然P30、重组P30及全虫抗原(对照)分别免疫BALB/c小鼠,取免疫小鼠脾细胞与SP2/0骨髓瘤细胞融合,筛选稳定分泌高滴度mAb的杂交瘤细胞株。用ELISA法测定mAb亚类和效价;通过SDS-PAGE和West-ern blot进行特异性鉴定;Giemsa染色观察杂交瘤细胞的染色体数目;利用扫描电镜及间接荧光抗体试验(IFAT)观察mAb对弓形虫的杀伤作用及其靶抗原定位。结果表明,筛选出2株(2B3、1H6)特异性较好、能稳定分泌mAb的杂交瘤细胞株。Western blot结果显示,2B3、1H6产生的mAb与弓形虫全抗原的阳性反应带均在30 000 Mr处;mAb亚类均属IgM,其效价(ELISA)分别为:2B3 1∶102 400、1H6 1∶51 200;2株细胞的染色体数均在100条以上。电镜观察与mAb作用后的弓形虫虫体聚集、肿胀,膜变厚,表面出现缺口和空洞,甚至虫体变形、破碎。抗弓形虫天然P30的mAb能识别重组体pET-30a-P30的表达蛋白。成功制备了抗弓形虫天然P30、重组P30的mAb,可进一步用于弓形虫病诊断及保护性抗原鉴定研究。

Preparation of the monoclonal antibody against P30 antigen of Toxoplasma gondii and observation on its anti-parasite effect

The monoclonal antibodies against P30 antigen of Toxoplasma gondii were prepared by immunization of BALB/c mice with natural and recombinant P30 antigens of T.gondii.The splenocytes from the immunized mice were collected and fused with SP2/0 myeloma cells of mice.Then many hybridoma cell lines stably secreting high level of T.gondii monoclonal antibodies were screened,and subtypes and titres of these mAb were assayed by ELISA.Meanwhile,their specificity was identified by SDS-PAGE and Western blotting,and the numbers of chromasomes in the hybridoma cells were examined with Giemsa staining.By using scanning electron microscopy and indirect fluorrscent antibody testing(IFAT),the killing effect of mAb upon parasites and the localization of target antigens were observed.Two strains of hybridoma cell lines,2B3 and 1H6,which could steadily secrete mAb against T.gondii with good specifity were obtained in this way.As demonstrated by Western blotting,the positive reaction band produced by mAb 2B3 or 1H6 with crude antigen of T.gondii showed a location at 30 000 Mr.These mAb belonged to IgM with an ELISA titres of 1∶102 400 in 2B3 and 1∶ 51 200 in 1H6.The numbers of chromosomes in these hybridoma cells were more than 100. As observed under electron microscopy,after reacting with mAb,the toxoplasma aggregated together,swollen,thickened in the surface of parasites with formation of cavities,or even the parasites deformed and crushed.It was also found that the mAb against natural antigen of T.gondii could recognize proteins expressed by pET-30a-p30 recombinant.It is concluded that the monoclonal antibodies against the natural and recombinant P30 antigen of T.gondii are successfully prepared in the present study,which can be used for the diagnosis of toxoplasmosis and for the identication of the protective antigens in T.gondii.