抗李斯特菌溶血素单克隆抗体制备及初步鉴定.doc

抗李斯特菌溶血素单克隆抗体制备及初步鉴定 　 作者：董慧, 焦新安, 殷月兰, 潘志明, 黄金林 【摘要】 　　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。 【关键词】 李斯特菌溶血素 产单核细胞李斯特菌 单克隆抗体 　　[Abstract] 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 against LLO were obtained. The immunoglobulin subclasses of the mAbs were IgG1. The ELISA titer of the ascitic fluids of 3B6, 4D1 and 5D10 was 1∶200000, 1∶200000 and 1∶100000, respectively. Western blot analysis confirmed the three mAbs reacted on fusion protein LLOGST but didn’t react on protein GST. DotELISA proved the three mAbs only react on the bacteria expressing LLO. CONCLUSION: The successful preparation of three mAbs specific to protein LLO lays a foundation for further study of the biological characteristics of LLO and the pathogenesis of Listeria monocytogenes. 　　[Keywords]listeriolysin O; Listeria monocytogenes; monoclonal antibody 　　产单核细胞李斯特菌(Listeria monocytogenes, LM)是一种胞内生长、 兼性厌氧的革兰氏阳性杆菌, 是李斯特菌属中致病性最强的一种。LM是一种重要的食源性病原菌, 能引起人兽共患李斯特菌病[1]。在动物中, 对多种畜禽均有较高的致死率; 在人类, 主要表现为脑膜炎、 流产以及免疫缺陷患者和新生儿的