单增李斯特菌溶血素基因的原核表达及其单克隆抗体的制备
发布时间：2018-09-18 14:40
【摘要】：目的：hly是编码单增李斯特菌特有的溶血素蛋白LLO的基因,克隆hly表达LLO蛋白并制备单增李斯特菌特异性的单克隆抗体,为单增李斯特菌特异性检测方法的建立奠定基础。 方法：以单增李斯特菌的DNA为模板,通过PCR扩增出hly基因,与PET28a(+)原核表达载体连接,经测序鉴定后,转化大肠杆菌BL21,并诱导表达。经表达条件优化,大量诱导表达,用镍柱纯化。通过SDS-PAGE和westernblotting鉴定表达的蛋白,通过溶血试验鉴定其溶血活性。用纯化的表达产物免疫Balb/c小鼠,利用杂交瘤技术制备抗LLO的单克隆抗体,通过间接ELISA鉴定其亚型。单克隆抗体叠加实验分析不同单克隆抗体组合的配对效果。溶血抑制实验鉴定单抗克隆抗体能否抑制LLO的溶血活性。间接ELISA检测单克隆抗体与威尔斯李斯特菌、英诺克李斯特菌、格氏李斯特菌的交叉性。 结果：原核表达出58KD的溶血素蛋白,其序列与Gene Bank公布的序列有99%的同源性。其最优化的表达条件是28℃下用0.1 mmol/L IPTG诱导6 h。。溶血实验表明重组表达的LLO具有较强的溶血活性。总共获得13株单克隆抗体,4株为IgGl亚型,9株IgM亚型。溶血抑制试验表明13株单抗均能不同程度地抑制LLO的溶血反应。只有3B4C6、3B4F6与威尔斯李斯特菌、英诺克李斯特菌、格氏李斯特菌有交叉反应,其余11株均无交叉反应。 结论：成功原核表达单增李斯特菌的溶血素蛋白(LLO),获得具有溶血性的LLO。成功制备了抗LLO的单抗克隆抗体,获得具有单增李斯特菌特异性的单抗。为建立单增李斯特菌特异性的免疫学检测方法奠定了基础。
[Abstract]:Objective to clone hly and express LLO protein and prepare monoclonal antibody against Listeria monocytogenes specific to Listeria monocytogenes (Listeria monocytogenes), which lays a foundation for the establishment of a specific detection method for Listeria monocytogenes (Listeria monocytogenes). Methods: using the DNA of Listeria monocytogenes as template, the hly gene was amplified by PCR and ligated with PET28a () prokaryotic expression vector. After sequencing, it was transformed into E. coli BL21, and induced to express. After optimized expression conditions, a large number of induced expression, purified by nickel column. The expressed protein was identified by SDS-PAGE and westernblotting, and its hemolytic activity was determined by hemolysis test. Balb/c mice were immunized with purified expression products. Monoclonal antibodies against LLO were prepared by hybridoma technique and their subtypes were identified by indirect ELISA. The pairing effect of different monoclonal antibody combinations was analyzed by monoclonal antibody superposition experiment. Hemolytic inhibition assay was used to determine whether monoclonal antibody could inhibit the hemolytic activity of LLO. Indirect ELISA was used to detect the cross-crossing of monoclonal antibodies with Listeria Welsh, Listeria Innoxa and Listeria gravis. Results: 58KD hemolysin protein was expressed in prokaryotic cells, and its sequence was 99% homology with that published by Gene Bank. The optimal expression conditions were induced with 0.1 mmol/L IPTG at 28 鈩，
本文编号：2248233
[Abstract]:Objective to clone hly and express LLO protein and prepare monoclonal antibody against Listeria monocytogenes specific to Listeria monocytogenes (Listeria monocytogenes), which lays a foundation for the establishment of a specific detection method for Listeria monocytogenes (Listeria monocytogenes). Methods: using the DNA of Listeria monocytogenes as template, the hly gene was amplified by PCR and ligated with PET28a () prokaryotic expression vector. After sequencing, it was transformed into E. coli BL21, and induced to express. After optimized expression conditions, a large number of induced expression, purified by nickel column. The expressed protein was identified by SDS-PAGE and westernblotting, and its hemolytic activity was determined by hemolysis test. Balb/c mice were immunized with purified expression products. Monoclonal antibodies against LLO were prepared by hybridoma technique and their subtypes were identified by indirect ELISA. The pairing effect of different monoclonal antibody combinations was analyzed by monoclonal antibody superposition experiment. Hemolytic inhibition assay was used to determine whether monoclonal antibody could inhibit the hemolytic activity of LLO. Indirect ELISA was used to detect the cross-crossing of monoclonal antibodies with Listeria Welsh, Listeria Innoxa and Listeria gravis. Results: 58KD hemolysin protein was expressed in prokaryotic cells, and its sequence was 99% homology with that published by Gene Bank. The optimal expression conditions were induced with 0.1 mmol/L IPTG at 28 鈩，
本文编号：2248233
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