The Rapid Detection Of Listeria Monocytogenes In Honey And Royal Jelly And The Identification Of Peptidoglycan Hydrolase Function Of Lmo1521Protein

Listeria monocytogenes (LM) survive in our environment widely asfood-borne pathogens. pregnant women, old people and immuncompromised patientslead to abortion, gastroenteritis, septicemia listeriosis, when they eat the foodcontaminated by Listeria monocytogenes. Honey and royal jelly are consumedlargely and exported to many countries every year. Because of honey and royal jellybelong to ready to food without heating process, they are possibly contaminated byListeria monocytogenes. It is necessary to establish the rapid detection of Listeriamonocytogenes in bee products. Autolysin is an peptidoglycan hydrolase that candegrade bacteria cell wall and can inhibit the growth of bacteria. The identifiedautolysins of Listeria monocytogenes have GW anchoring domain and peptidoglycanhydrolysis domain. lmo1521protein which locate on1521of Listeria monocytogenesEGD-e, have similarity structure to autolysin according to bioinformatics.Peptidoglycan hydrolase function of lmo1521protein is identificated, and itsantibacterial activity can control Listeria monocytogenes.In this study, we establish the double PCR and the real time PCR to detectListeria monocytogenes in the bee and royal jelly. We acquire lmo1521protein inBL21(DE3) and verify its autolysis functions by renature SDS-PAGE. The primaryresults are as follows:1. The survival of Listeria monocytogenes in honey and royal jellyThe survival of Listeria monocytogenes in honey and royal jelly were detectedby cell culture, multiplex PCR and the real-time PCR. At room temperature, honeyand royal jelly inoculated by Listeria monocytogenes in high concentration werestored up to4w. Listeria monocytogenes in the honey survived for3days, but theycould not enrich in the royal jelly.2.The rapid detection technology of Listeria monocytogenes in the honey and royal jellyThe multiplex PCR was accomplished using the primers for the target genes ofhly and16sRNA. The hly probes labeled by FAM and TAMRA were usedsuccessfully to develop a real-time PCR. The DNA was abstracted from Listeriamonocytogenes without enrichment which artificially inoculated in the honey androyal jelly by lysozyme and protease K method. In the results, the detection limits ofhoney and royal jelly are102CFU/mL and103CFU/mL respectively.3. The prokaryotic expression of the putative autolysin lmo1521and autolysisactivity analysislmo1521ORFs was acquired from the LM-CMC54002genome, and insertedinto pET-22b. The recombinant plasmid was transformed into the E. coli BL21(DE3). The expression products with6×His lable was identified by western blot.The quantity of soluble protein increased significantly in the28℃and30℃. At28℃, the concentration of IPTG(0.1mmol/L and0.25mmol/L,0.5mmol/L, and0.75mmol/L) cann’t improve the protein solubility. The solublen protein was purified byNI-NTA Resin and the protein have autolysis activity by renature SDS-PAGE.