摘要
【目的】制备抗产单核细胞李斯特菌溶血素O(LLO)的特异性单克隆抗体。【方法】用LLO重折叠的原核表达产物LLO-his免疫Balb/c小鼠,取其脾细胞,与骨髓瘤细胞在PEG4000作用下融合,经间接ELISA法筛选、多克隆及单克隆分离出阳性细胞株后,再用体内诱生腹水法大量制备单克隆抗体。用辛酸-硫酸铵沉淀法纯化单克隆抗体,间接ELISA法测定其效价及相对亲和常数;用HBT单克隆抗体亚类鉴定试剂盒鉴定抗体亚型;常规方法制备染色体,观察细胞株染色体数目及标志染色体;Western blot检测抗体的特异性。【结果】获得了2株稳定分泌抗LLO单克隆抗体的杂交瘤细胞系,分别命名为3F5-G3和2B4-C8,其腹水效价分别为1∶(1×105)和1∶(1×107);染色体分别为97±5和93±8条,且观察到标志染色体;相对亲和常数分别为0.49和0.26μg/mL;抗体亚类均为IgG1,轻链均为κ链。特异性鉴定结果表明,2种单克隆抗体均可与LLO蛋白发生特异性反应,而与载体蛋白不发生反应。【结论】获得了抗LLO的特异性单克隆抗体。
【Objective】The study was to obtain specific monoclonal antibodies(McAbs)against Listeriolysin O(LLO).【Method】Balb/c mice were immunized with refolding LLO-his expressed in E.coli BL21,and splenocytes of immunized mice were fused with SP2/0 cell;Anti-LLO hybridoma cells were screened by indirect ELISA;McAbs were largely prepared and purified from ascites deposited by octylic acid and ammonia sulfate;The titre and relative affinity of McAbs were identified by indirect ELISA,identifing isotype of McAbs by mous...
出处
《西北农林科技大学学报（自然科学版）》
CSCD
北大核心
2010年第7期39-43,共5页
Journal of Northwest A&F University(Natural Science Edition)
基金
公益性行业(农业)科研专项经费项目(200903055)
