产气荚膜梭菌α毒素单克隆抗体的制备

Preparation of monoclonal antibody against α toxin of Clostridium perfringens

为了制备产气荚膜梭菌致病性毒力因子α毒素的单克隆抗体,试验采用PCR法获得α毒素基因,克隆入原核表达质粒p ET-30b,转化至大肠杆菌BL21感受态细胞,构建表达α毒素的重组菌BL21/p ET-30b-α;IPTG诱导重组菌表达,并进行SDS-PAGE分析,以纯化的重组α毒素蛋白为免疫原免疫Balb/c小鼠,经筛选、亚类鉴定及特异性试验检测单克隆抗体。结果表明:重组菌表达的α毒素蛋白分子质量约为43 ku;杂交瘤细胞株3C5能够稳定分泌抗α毒素单克隆抗体,单克隆抗体亚型为Ig G2a型,能够特异识别产气荚膜梭菌天然α毒素蛋白。说明制备的单克隆抗体具有良好的反应原性。

To prepare monoclonal antibodies against α - toxin as the pathogenic virulence factor of Clostridium perfringens, the α - toxin gene was obtained by PCR assay. It was cloned into prokaryotic expression plasmid pET -30b, and transformed into E. coli BL21 competent cells to construct the recombinant bacterium BI21/pET -30b -α expressing α-toxin, and then the expression of recombinant bacterium induced by IPTG was analyzed by SDS - PAGE. The purified α - toxin protein was used as immunogen to immunize Bala/c mice to prepare monoclonal an- tibodies against α - toxin, and then the monoclonal antibodies were detected by screening, identification of subtype, specificity test. The results showed that molecular weight of the α - toxin protein expressed by recombinant bacterium was about 43 ku, and the hybridoma cell line 3C5 could stably secret anti -α -toxin monoclonal antibodies; the subtype of monoclonal antibodies was IgG2a subtype, which could specifically recognize the naturalα - toxin protein of Clostridium peringens. The results indicate that the prepared moneclonal antibodies have good reactio- nogenicity.