猪链球菌WC—SS7株GDH基因的表达及GDH蛋白单克隆抗体的制备

目的原核表达猪链球菌7型临床分离株WCSS7的GDH蛋白，制备抗GDH蛋白的单克隆抗体（mAb）。方法将临床分离的猪链球菌7型WC—SS7株的GDH基因克隆入表达载体pET-30a，转入大肠杆菌中，用IPTG诱导表达，用Ni化琼脂糖柱（Pierce）纯化的GDH蛋白免疫BALB／c小鼠，取鼠脾细胞与SP2／0骨髓瘤细胞融合，通过间接ELISA方法筛选阳性克隆，对抗WC—SS7GDH蛋白单克隆抗体进行特异性鉴定及亚型鉴定。结果表达产物经SDS-PAGE凝胶电泳分析后，在约为52KD处可见一条明显的诱导表达带，说明GDH蛋白得到了正确的表达；细胞融合后检测为阳性的细胞株经过3次单克隆筛选，最终得到了6株能和GDH蛋白反应的能稳定分泌单抗的阳性细胞克隆，这些单抗通过Western鉴定结果显示其具有良好的特异性，6株单克隆抗体亚型鉴定结果显示其亚型均为IgG1型，且轻链均为k链。结论本研究成功表达了WCSS7的GDH蛋白，并获得了6株能和GDH蛋白反应的、能稳定分泌单抗的阳性细胞克隆，所获得的重组GDH蛋白及特异性单克隆抗体能用于猪链球菌结构和功能的研究，为猪链球菌临床感染血清学诊断方法的建立奠定基础。

Expression of GDH gene of Streptococcus suis and preparation of monoclonal antibody against GDH protein

In order to express GDH protein of Streptococcus suis in prokaryotic cell and produce monoclonal antibody （mAb） against GDH protein. The GDH gene of Streptococcus suis strain of WC SS7 was truncated and cloned into prokaryotic expression vector PET 30（a）, expressed in E. coli after IPTG induction. Expressed fusion protein was purified by Ni-NTA, BALB/c mice were immunized with purified GDH protein. Murine myeloma cells were fused with the splenoeytes of the immunized mice after the third immunization. An indirect ELISA coated with GDH was used to screen Hybridomas for production of specific antibody in hybridoma culture fluid. The specificity of these mAbs were characterized by western-blot. These mAbs were subtyped by ELISA using a commercial kit. The result showed that fusion protein of 52KD was expressed by SDS-PAGE analysis, 6 hybridomas clones producing mAbs steadily were obtained after 3 cycles of cloning. The specificity of These mAbs were conformed by western blot. All the mAbs belong to IgG1 subtype, and the light chains of the antibodies are k chain. The purified GDH protein of streptococcus suis and 6 hybridomas clones producing mAbs steadily were successfully obtained. The anti GDH mAbs obtained has strong specificity and high tilter, which and GDH protein could be used for further analysis of the structure and function of .Streptococcus suis and lay the foundation for diagnosis assays to serology of Streptococcus suis clinical infection.