| 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0.26 μg/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。 |
| 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 |
| DOI： |
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| 基金项目:公益性行业（农业）科研专项经费项目（200903055） |
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| Preparation and identification of monoclonal antibodies against Listeriolysin O |
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| Abstract: |
| 【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 mouse Mab isotyping test kit from HBT;The chromosome was processed for counting the number of chromosome and observing marker chromosome;The specificity of McAbs was characterized by Western blot assay.【Result】2 McAbs against LLO were obtained and named 3F5-G3 and 2B4-C8 respectively;The titre of ascites was up to 1∶（1×105）(3F5-G3) and 1∶(1×107)（2B4-C8）;The number of chromosome was 97±5(3F5-G3) and 93±8(2B4-C8);The affinity constant of McAbs was 0.49 μg/mL(3F5-G3) and 0.26 μg/mL(2B4-C8);The specificity identification showed that the two kinds of monoclonal antibodies could react specifically with LLO protein,but no reaction with vector protein.【Conclusion】The specific McAbs against LLO were prepared in the study. |
| Key words: Listeria monocytogenes Listeriolysin O monoclonal antibody |
