Cloning And Expression Of Hemolysin Gene Of Listeria Monocytogenes

listeriosis is caused by Listeria monocytogenes(LMO).It is a acute,highly infectious disease.Listeriolysin O(LLO) is one of the important virulence factors.In this assay,hlyA gene was amplified by PCR.prokaryotic expression vector pET-30a-hlyA was constructed.LLO was expressed in E.coli BL21(DE3).The optimal conditions for LLO expression were,IPTG concentration at 1.0mmol/L, temperature at28℃,induction time as 5h.The expressed LLO protein mainly existed in soluble form.Expressed LLO was purified by affinity chromatography,and 500mM imidazole eluate got the best result.Western-blot analysis demonstrated that the expressed LLO protein could be detected by positive serum from mice.Using the purified LLO,mice were immunized.45 days after immunization,mice were chanlleged with 1 lethal dose of LMO.The protective percentage was 100%.Finally ELISA method was established for detection of LMO.