Prokaryotic expression and bioactivity identification of hemolysin from Listeria monocytogenes

Abstract:

Designing the primers of hly gene by biological software, amplifying the hly gene from Listeria monocytogenes by PCR and constructing the gene into prokaryotic expression vector PET28a(+), then transforming the gene into E. coli BL21and expressing optimally; purifying the recombinant product LLO by nickel affinity chromatography, testing the immunogenicity by Western blotting and the hemolytic activity by hemolytic assay. It was demonstrated by agarose gel electrophoresis?that the cloned hly gene was 1 590 bp in length and the protein sequence got about 99% homology with that published in GenBank. SDS-PAGE indicated that the molecular weight was about 58 kD and the optimal expression condition was induced for 6 h with 0.1 mmol/L IPTG at the temprature of 28 °C. The result of Western blotting showed the recombinant protein LLO had immunogenicity. The hemolytic assay showed the product had the hemolytic activity, with the hemolytic titre of 1:1 024. These results would provide basis for the further studies on the development of monoclonal antibody against Listeria monocytogenes and the establishment of the dection methods.