SITE‐DIRECTED MUTAGENESIS, ISOLATION AND PURIFICATION OF LISTERIA MONOCYTOGENES LISTERIOLYSIN O AND ITS IMMUNOGENICITY

Listeria monocytogenes is a food-borne pathogenic bacterial species that can cause diseases in humans and animals. A rapid and reliable method for detection of L. monocytogenes is crucial in disease control. Listeriolysin O (LLO), encoded by the hly gene, is the most important virulence factor of L. monocytogenes and a useful identifying factor. We studied the protein structure of LLO with the aim of developing an antibody to be used in an enzyme-linked immunosorbent assay (ELISA) for detection of L. monocytogenes. By in silico analysis, a Leu52Phe modification of LLO sequence was predicted to increase its immunogenicity. The corresponding modification in the hly gene sequence was achieved by site-directed mutagenesis. The wild-type and mutated hly genes were cloned and expressed in Escherichia coli; the expression levels were 10 and 12%, respectively. Highly purified protein preparations of LLO and its Leu52Phe variant (∼37 kDa) were obtained and their purity values were both greater than 90%. Both proteins were used independently to immunize rabbits. The average optical density (OD) value obtained with the anti-Leu52Phe variant antibody (P2) was higher by 0.463 OD units compared with the corresponding value for the anti-LLO antibody (P1) by ELISA, which indicated that the mutation had indeed resulted in enhanced immunogenicity of LLO. PRACTICAL APPLICATIONS In this paper, the enzyme-linked immunosorbent assay method developed by using listeriolysin O variant provided a powerful tool for detection of Listeria monocytogenes in raw milk with specificity, sensitivity and rapidity.