抗李斯特菌溶血素单克隆抗体的制备及初步鉴定

Preparation and characterization of the monoclonal antibodies against listeriolysin O

目的:研制抗李斯特菌溶血素(LLO)的单克隆抗体(mAb)。方法:通过诱导重组大肠杆菌BL21(pGEX-6p-1-hly),以SDS-PAGE分离菌体蛋白,切割目的蛋白LLO-GST条带,研磨粉碎后免疫BALB/c小鼠,取免疫鼠脾细胞与Sp2/0细胞融合。利用亲和层析法纯化目的蛋白,作为检测抗原进行杂交瘤细胞的筛选。采用间接ELISA法测定腹水效价,Dot-ELISA、Western blot分析mAb的特异性。结果:获得3株稳定分泌抗LLO mAb的杂交瘤细胞株3B6、4D1、5D10,其Ig亚类均为IgG1。3B6、4D1、5D10腹水的ELISA效价分别为1∶2×105、1∶2×105、1∶1×105。Western blot试验中,3株mAb均能与融合蛋白LLO-GST发生反应出现特异性条带,而与纯化蛋白GST不反应。在Dot-ELISA试验中,3株mAb均能与表达LLO的细菌发生特异性反应。结果表明,mAb 3B6、4D1、5D10是针对LLO的特异性mAb。结论:成功地制备了抗LLO的mAb,为进一步研究LLO的生物学特性、产单核细胞李斯特菌的致病机制,以及建立产单核细胞李斯特菌(LM)快速检测技术奠定了基础。

AIM:To prepare the monoclonal antibodies(mAbs)against listeriolysin O(LLO),which is the major virulence factor of Listeria monocytogenes.METHODS:The BALB/c mice were immunized with the SDS-PAGE product of BL21(pGEX-6p-1-hly).The purified LLO-GST protein was used as antigen for detection.mAbs against LLO were prepared by using the lymphocyte hybridoma technique.The specificity of mAbs was characterized by Dot-ELISA and Western blot.RESULTS:Three hybridoma cell lines named 3B6,4D1 and 5D10 secreting mAbs agai...