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目的原核表达猪链球菌7型临床分离株WC-SS7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κ链。结论本研究成功表达了WC-SS7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。
Objective To express the GDH protein of prokaryotic Streptococcus suis serotype 7 clinical isolates WC-SS7 and prepare monoclonal antibody (mAb) against GDH protein. Methods GDH gene of Streptococcus suis serotype 7 strain WC-SS7 was cloned into expression vector pET-30a and transformed into E.coli. The GDH gene was induced by IPTG and immunized with GDH protein purified by Ni-Sepharose (Pierce) BALB / c mice were immunized with SP2 / 0 myeloma cells. The positive clones were screened by indirect ELISA and the McAbs against WC-SS7GDH were identified and their subtypes were identified. Results After SDS-PAGE analysis of the expressed product, a clear induction band was observed at about 52KD, indicating that GDH protein was correctly expressed. After three cell clones were positive for cell fusion, three monoclonal screening , And finally got six positive clones that can react with GDH to secrete monoclonal antibodies. The results of Western blot showed that these McAbs showed good specificity. The identification of six monoclonal antibody subtypes showed that they were subtype All of IgG1 type, and the light chains are both κ chains. Conclusion The GDH protein of WC-SS7 was successfully expressed in this study, and six positive clones that can react with GDH protein and can secrete monoclonal antibodies were obtained successfully. The obtained recombinant GDH protein and specific monoclonal antibody can be used in The study on the structure and function of Streptococcus suis laid a foundation for the establishment of serological diagnostic methods for clinical infection of Streptococcus suis.