Expression Of MurA And Development Of Polyclonal Antibody Applied To Immunomagnetic Enrichment For Rapidly Detect Listeria Monocytogenes

[Objective] Developing polyclonal antibody against MurA protein for rapidlydetect Listeria monocytogenes (LM) by combining immunomagnetic with selectiveplate.[Methods] Prokaryotic expression vector of MurA was constructed andtransformed into E. coli to optimally express. Product after expression was purified bynickel affinity chromatography, mass spectrometry analysis was used to identify therecombinant protein; after correct identification, the protein was applied to immunemice in order to prepare polyclonal antibodies. The antibodies we acquired were usedto develop immunomagnetic beads, which combined with selective medium to detectartificial contaminated milk samples.[Results] A soluble fusion protein with a molecular weight of72kD wasexpressed in E. coli, this protein was identified as MurA protein; antiserum possesseda titer as high as10000, with no cross-reaction to Salmonella typhi, Vibrioprholyticus, E. coli and other pathogenic bacteria. Despite a little cross reaction withListeria innocua, the method combined specific immunomagnetic beads withselective medium managed to detect LM of a concentration of103CFU/mL or above.After four hours enrichment, milk samples within an original concentration of morethan0.4CFU/mL were successfully detected, which was44hours less than theregular enrichment method.[Conclusion] Recombinant MurA protein was highly expressed in E. coli andshowed high purity after purification, the polyclonal antibodies showed high affinityand specificity against LM. The immunomagnetic beads-selective medium method fordetecting LM was able to detect milk samples within24hours, which was42hoursless than national standard method with the same sensitivity.