Prokaryotic Expression Listeriolysin O From Listeria Monocytogenes And Preparation Of Monoclonal Antibody Against Listeriolysin O

Objectives:To prokaryotic express hly gene from Listeria monocytogenes and prepare specific monoclonal antibody against Listeria monocytogenes and make a basis for the establishment of specific detection methods for Listeria monocytogenes.Methods:The hly gene was amplified by PCR according the templete of LM DNA and was constructed into prokaryotic exoression vector PET28a (+).The recombinant was transformed into E.coli BL21 and expressed optimally. The prokaryotic expression product was purified by nickel affinity chromatography and identified by SDS-PAGE and Westernblotting. The hemolytic activity of the product was identified by hemolytic assay. The Balb/c mice were injected with the purified LLO.The McAbs against LLO were produced by hybridoma technology. The subtype of the McAbs were identified by indirected ELISA. The matching rate of the McAbs were analysed by superposition experiment. The hemolytic inhibition activity of McAbs to LLO was identified by hemolytic inhibition experiment. The cross activity between Listeria monocytogenes and Listeria innocna, Listeria welshimeri, Listeria grayi was identified by indirected ELISA.Results:The 58KD protein of LLO was prokaryotic expressed successfully and got 99% homology in sequence with which was published in Gene Bank. The optimal expression condition was induced for 6 h with 0.1 mmol/L IPTG at the temprature of 28℃. The hemolytic assay showed the product had the hemolytic activity.13 strains of McAbs were obtained,4 of which were IgG1 subtype and 9 were IgM subtype. The McAbs could inhibit the lysis of LLO by inhibiton test of hemolytic activity and showed non cross activity with Listeria innocna, Listeria welshimeri, Listeria grayi, except 3B4C6 and 3B4F6.Conclusion:The listeriolysin 0 was successfully expressed in E.coli BL21 and had hemolytic activity. The McAbs against LLO were successfully prepared and were specific to Listeria monocytogenes. These were the basis of the establishment of specific detection methods for Listeria monocytogenes.