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

Abstract:

[Objective] To identify and characterize a bacterial strain S1-2 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 amplication of the iap gene was used to detect strain S1-2. The flaA gene from S1-2 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 β-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.