产单核细胞李斯特菌溶血素O单克隆抗体的制备与鉴定  

Preparation and identification of monoclonal antibodies against Listeriolysin O

作　　者：孔德壮[1] 张衍海[2] 龚振华[2] 李葳[2] 郑增忍[2] 李玉清[2] 张彦明[1] 

机构地区：[1]西北农林科技大学动物医学院,陕西杨凌712100 [2]中国动物卫生与流行病学中心,山东青岛266032

出　　处：《西北农林科技大学学报（自然科学版）》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)

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...

关 键 词：产单核细胞李斯特菌 李斯特菌溶血素O 单克隆抗体 

分 类 号：Q789[生物学—分子生物学]