摘要
目的制备重组阴道毛滴虫(T.υ)黏附蛋白33(AP33)单克隆抗体(McAb)并进行初步鉴定。方法将pET32(a)-AP33重组质粒转化大肠埃希菌(E.coli)BL21(DE3),用0.5 mmol/L IPTG诱导表达,超声破菌后获得可溶性表达产物,通过Ni-NTA法纯化,以纯化的融合蛋白33为抗原,免疫BALB/c小鼠,采用杂交瘤技术制备McAb,用ELISA和有限稀释法筛选出分泌高滴度McAb杂交瘤细胞株,测定其免疫球蛋白亚类及其效价,免疫印迹分析其特异性。结果共筛选出能稳定分泌抗AP33单克隆抗体的5株(4A2,4F11,4F8,4E7,4H11)杂交瘤细胞株,抗体重链均为IgG1;腹水的上清效价为1∶40 000-1∶80 000。免疫印迹分析显示,5株单抗均能与重组阴道毛滴虫AP33发生特异性结合;与阴道毛滴虫抗原分子量为33kDa抗原分子结合。结论制备的抗重组AP33杂交瘤细胞株能分泌识别重组AP33的高特异性单抗,为进一步研究其在阴道毛滴虫病免疫诊断中的应用奠定了基础。
To prepare the monoclonal antibodies （McAbs） against recombinant adhesion protein 33 （AP33） of Trichomonas vaginalis and to identify its biological properties. The recombinant plasmid pET32a-ap33-BL21（DE3） was induced by 0. 5mmol/L IPTG, and purified with nickel-affinity chromatography column. The recombinant fusion protein AP33 was used as antigen to immunize BALB/c mice and Sp2/0 myeloma cells were fused with the splenic cells from BALB/c mice immunized. After ELISA screening and 4 times of limited dilution, 5 positive strains of hybridom cells were obtained, and the biological properties of the McAbs were identified by Western blotting. Western blotting demonstrated that these 5 McAb, designated as 4A2, 4F11, 4F8, 4E7 and 4H11, could specifically combine with recombinant AP33 and 33kDa protein of Trichomonas vaginalis. As demonstrated by ELISA the titers of the specific antibody in ascites fluid were found to be 1 ： 40 000, 1： 80 000,1 ： 80 000,1 ： 40 000,1 ： 80 000 respectively. The chromosomal numbers of hybridoma cells were 90-108. The McAb were IgG1 isotypes. McAb against recombinant AP33 have been established successfully,thus providing a basis for the immtmodiagnosis of T. vaginalis and the function of the AP33 in the trichomonas vaginalis diagnosis.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2007年第3期282-285,共4页
Chinese Journal of Zoonoses
基金
浙江省科技厅社会发展重点项目(No.2005C23032)
