进口竹荚鱼中单核细胞增生李斯特菌的鉴定及其鞭毛蛋白的原核表达

Identification of Listeria monocytogenes from imported Trachurus japonicas and prokaryotic expression of flagellin

【目的】对进口竹荚鱼中分离的一株病原菌S1-2进行鉴定,并在大肠杆菌中表达其鞭毛蛋白。【方法】采用全自动微生物鉴定仪和革兰氏阳性菌鉴定卡进行生理生化反应测试,利用iap基因实时荧光PCR特异性扩增检测病原菌。通过PCR技术扩增病原菌S1-2的鞭毛蛋白flaA基因,克隆筛选和测序鉴定后,构建该基因的原核表达质粒pET22b-flaA,镍柱法纯化表达产物,通过免疫印迹鉴定其免疫原性。【结果】分离病原菌为革兰氏阳性菌,生理生化特征与单核细胞增生李斯特菌(Listeria monocytogenes)的相似性为99%,协同溶血实验在靠近金黄色葡萄球菌的接种端溶血增强。SDS-PAGE结果表明融合表达产物分子量约为32 kD,Western blot结果表明重组表达的鞭毛蛋白具有免疫原性。【结论】flaA基因的原核表达为制备单核细胞增生李斯特菌的单克隆抗体及其检测方法的建立奠定了基础。

[Objective] To identify and characterize a bacterial strain S 1-2 isolated from the imported Trachurus japonicas and express the flagellin in Escherichia coli BL21. [Methods] The physiological and biochemical characteristics were elucidated by employing VITEK 2 compact automated microbiology system and Gram-positive identification card. Real-time PCR method aiming for the amplieation of the lap gene was used to detect strain S 1-2. The flaA gene from S 1-2 was amplified by PCR and cloned into prokaryotic expression vector pET-22b. The recombinant product was purified by nickel affinity chromatography. Western blot was employed to test the immunogenicity. [Results] Strain S1-2 was identified as Gram-positive and exhibited the highest levels of 99% probability to be Listeria monocytogenes based on the conventional physiological test. An enhanced zone of [3-haemolysis at the intersection of Staphyloccocus aureus was found.SDS-PAGE indicated that the molecular weight was about 32 kD. Western blot showed that the recombinant protein FlaA had immunogenicity. [Conclusion] These results would provide basis for the further studies on the development of monoclonal antibody against Listeria monocytogenes and the establishment of the detection methods.