Prokaryotic expression of MurA protein and development of polyclonal antibody for rapid detecting Listeria monocytogenes using immunomagnetic beads

Abstract:

[Objective] To develop polyclonal antibody against MurA protein and combine immunomagnetic with selective plate to rapidly detect Listeria monocytogenes (LM). [Methods] Prokaryotic expression vector of MurA was constructed and transformed into E. coli to optimally express. Product after expression was purified by nickel affinity chromatography, mass spectrometry analysis was used to identify the recombinant protein; after correct identification, the protein was applied to immune mice in order to prepare polyclonal antibodies. The antibodies we acquired were used to develop immunomagnetic beads, which combined with selective medium to detect artificial contaminated milk samples. [Results] A soluble fusion protein with a molecular weight of 72 kD was expressed in E. coli, this protein was identified as MurA protein; antiserum possessed a titer as high as 10 000, with no cross-reaction to Salmonella typhi, Vibrio prholyticus, E. coli and other pathogenic bacteria. Despite a little cross reaction with Listeria innocua, the method combined specific immunomagnetic beads with selective medium managed to detect LM of a concentration of 103 CFU/mL or above. After nine hours enrichment, milk samples within an original concentration of more than 0.4 CFU/mL were successfully detected, which was 39 hours less than the regular enrichment method. [Conclusion] Recombinant MurA protein was highly expressed in E. coli and showed high purity after purification, the polyclonal antibodies showed high affinity and specificity against LM. The immunomagnetic beads-selective medium method for detecting LM was able to detect milk samples within 24 hours, which was 42 hours less than national standard method with the same sensitivity.