抗阴道毛滴虫AP33单克隆抗体的制备及其功能初探

Preparation of Monoclonal Antibodies Against the Adhesion Protein 33 of Trichomonas vaginalis

目的制备阴道毛滴虫(T.υ317株)黏附蛋白抗原(AP33)单克隆抗体并初步分析鉴定其功能。方法将制备和纯化的融合黏附蛋白33(rAP33)为抗原,免疫BALB／c小鼠,共免疫3次(抗原含量分别为100、50和100μg),每次间隔2周,末次免疫后3 d取小鼠脾细胞及SP2／0骨髓瘤细胞在聚乙二醇(PEG1500)作用下进行细胞融合,筛选出高滴度分泌的McAb杂交瘤细胞株,测定其免疫球蛋白亚类及其效价,蛋白质印迹(Western blotting)分析其特异性,间接免疫荧光实验(IFAT)进行定位,并初步探讨其体外对阴道毛滴虫黏附HeLa细胞的抑制作用。结果经筛选获得能稳定分泌抗AP33单克隆抗体的5株(4A2,4F11,4F8,4E7和4H11)杂交瘤细胞株,经免疫球蛋白类型和亚型鉴定为IgG1; ELISA和Western blotting分析显示,5株单抗均能与重组阴道毛滴虫AP33发生特异性结合;IFAT显示其中4株(4F11, 4F8,4E7,4H11)可识别培养的阴道毛滴虫,体外滴虫黏附抑制实验显示终浓度分别为200、200、400和200μg/ml,该4株单抗体外对滴虫黏附HeLa细胞的抑制率分别为50．08％,65．03％、50．70％和49．08％。结论制备的抗重组AP33单克隆抗体,体外对阴道毛滴虫黏附有较好的抑制功能。

Objective To prepare and characterize the monoclonal antibodies （McAbs） against recombinant adhesion protein 33 （AP33） of Trichomonas vaginalis. Methods The purified recombinant fusion protein AP33 was used as antigen to immunize BALB/c mice. Sp2/0 myeloma cells were fused with the splenocytes from immunized BALB/c mice. After ELISA screening and 4 times of limited dilution, 5 positive hybridoma cell lines were obtained, and the biological properties of the McAbs were identified by Western blotting. Indirect immunofluorescent antibody test （IFAT） was performed and the inhibition effect of McAbs on the cytoadherence of T.richomonas vaginalis to HeLa cell was assayed. Results Western blotting demonstrated that 5 McAbs, designated as 4A2, 4F11, 4F8, 4E7 and 4H11, specifically combined with the recombinant AP33 of T.vaginalis. The McAbs were IgG1 isotypes. Four of them （4F11, 4F8, 4E7 and 4H11 ） showed parasite recognition by IFAT. Parasite cytoadherence to a monolayer of HeLa cells was inhibited in vitro with a inhibition rate of 50.08%, 65.03%, 50.70% and 49.08% by the 4 McAhs under a concentration of 200, 200, 400 and 200μg/ml, respectively. Conclusions The prepared McAbs against the recombinant AP33 show a protective inhibition on cytoadherence of Trichomonas vaginalis in vitro.