牛坏死杆菌43K OMP单克隆抗体的制备及鉴定

Preparation and identification of monoclonal antibody against 43K outer membrane protein of Fusobacterium necrophorum

目的制备牛坏死杆菌43K OMP单克隆抗体,并鉴定其生物学特性。方法经IPTG诱导表达重组43K OMP,纯化后免疫BALB/c小鼠,将小鼠脾细胞与SP2/0细胞融合,筛选分泌抗43K OMP特异性抗体的杂交瘤细胞,制备腹水,对抗43K OMP单克隆抗体进行效价、亚类、免疫原性及特异性鉴定。结果获得3株稳定表达抗43K OMP单克隆抗体的杂交瘤细胞株,取其中效价最高的1株命名为O43,其重链为IgG1,轻链为κ链,上清和腹水效价分别为1∶25 600和1∶105;该单克隆抗体可与重组43K OMP发生特异性反应,且与转染后瞬时表达43K OMP的BHK-21细胞特异性结合,发生荧光反应。结论成功表达并纯化了43K OMP,制备了牛坏死杆菌43K OMP单克隆抗体,为研究坏死杆菌的感染机制及发病机理奠定了基础。

Objective To prepare the monoclonal antibody(McAb) against 43K outer membrane protein(OMP) of Fusobacterium necrophorum and identify its biological characteristics.Methods The 43K OMP of F.necrophorum was expressed under induction of IPTG and purified,with which BALB/c mice were immunized.The spleocytes of immunized mice were fused with SP2/0 cells,and the positive hybridoma cells were screened by using hybridoma technique,with which the McAb was prepared and tested for titer,subclass,immunogenicity and specificity.Results Three hybridoma cell strains stably secreting McAb against 43K OMP were screened,of which the one secreting the McAb at the highest titer was named as O43.The heavy and light chains of McAb secreted by O43 were IgG1 and kappa chain,while the titers in supernatant and ascite were 1∶25 600 and 1∶105,respectively.The McAb showed specific reaction with recombinant 43K OMP and specific binding to the BHK-21 cells transiently expressing 43K OMP after transfection.Conclusion The 43 K OMP was successfully expressed and purified,against which the McAb was prepared.It laid a foundation of study on the mechanism in infection and pathogenesis of F.necrophorum.