| Listeria monocytogenes (LMO), which belongs to a member of Listeria family, is a zoonotic and food-borne pathogen and has great significance in public health. In this study, a pathogen separated from sheep brain issues possibly infected by LMO was primarily identified by general methods such as biochemical tests and virulence test.In this study, with a pair of primers designed to amplify hlyA of Listeria monocytogenes (LMO), a fragment of 722bp in lengh of the Listeria monocytogenes target gene was amplified by PCR and a standard PCR assay was developed for quick detection of Listeria monocytogenes infection .Specificity and sensitivity assays revealed that the assay did not cross with Escherichia coli, Salmonella,Staphylococcus aureus, Streptococcus,and the assay could detect as low as 104cfu/mL . The study supports an important method for specifically diagnosing and detecting LMO.The hlyA of Listeria monocytogenes was amplified with PCR, and ligated with pMD18-T vector. The recombinant plasmid certified by PCR restriction analysis and sequencing. A prokaryotic expression plasmid, named pGEX-4T-hlyA, was constructed by the hlyA and cloned into pGEX-4T-1. The expression was induced by IPTG, then collected the culture and tested them by SDS-PAGE and Western blot. The result shows that the full length of hlyA is 1 594bp,this fragment contained the complete open reading frame(ORF) which is 1 512bp and encodes 504 amino acids. In comparison with GenBank data F5782,the homologies of the nueleofide sequence is 100%. The 86KD fusion protein can be recognized by the positive serum of Listeria monocytogenes .Hemolytic experiment demonstrated the expressed fusion protein can lyse swine red cell.Conclusively,the Listeria monocytogenes hlyA was successfully cloned and expressed in E.coli.The successful expression of LLO in E.coli BL21 constituted a solid foundation for further researches such as pathogenesis and immune mechanism,MAb and vaccine development and the development of indirect ELISA.
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