Establishment Of PCR Detection Method For Listeria Monocytogenes And Cloning And Expression Of Its Listeriolysin O Gene

Listeria monocytogenes (LM) is a zoonotic and food-borne pathogen which belongs to the Listeria family, and has great impact on food safety and public health. Listeriosis has been regarded as one of the four major food-borne diseases by the World Health Organization (WHO). How to establish a rapid, sensitive and specific detection method has been a pressing issue to be resoveld for the health departments in the world.The pathogenicity of LM is related to several virulence factors. Among them Listeriolysin O (LLO) is the main virulence factor, which exists in all of the pathogenic LM. Coded by hlyA gene, LLO is a sulfhydryl-activated cytolysis that could combine with cholesterol.Acording to the hlyA gene of Listeria monocytogenes a pair of primers was desigened and it was used to amplify PCR producuts. Sensitivity and specification of this method were detected. Artificially contaminated food was detected using PCR method after Half-fraser and Fraser enrichment steps. The special 234bp fragments were amplified in LM while none was amplified in other species. The specification is 104cfu/mL.As few as 8cfu/25g can be detected in artificially contaminated food. The PCR procedure has been proved to be a rapid sensitive and specific method suitable for detect Listeria monocytogenes in food.The hlyA gene of Listeria monocytogenes was amplified by PCR and ligated with pMD18-T vector. The recombinant plasmid named pMD18-T-hlyA was certified by restriction analysis and PCR amplification. A new primer with BamHâ… a nd Xhoâ… restriction enzyme sites was used to amplify hlyA without signal peptide. The products were cloned into a prokaryotic expression vector pET32a. After certification by restriction analysis, PCR amplification and sequence analysis, the recombinant expression plasmid pET32a-hlyA was transformed into E.coli BL21(DE3) and induced by IPTG. After soluble analysis the fusion protein was furified. The culture was collected and tested by SDS-PAGE, Western blot.and indirect ELISA. The result shows that in comparison with Gene Bank data the homologies of the nueleofide sequence is 99%. The 80ku fusion protein was existed in the form of inclusion body and can be recognized by the positive serum of Listeria monocytogenes. The successful expression of LLO in E.coli BL21 constituted a solid foundation for further research such as MAb research and establishment of indirect ELISA method.