Green Fluorescent Protein Recombinant Nisin as a Probe for Detection of Gram-Positive Bacteria

Abstract

A great amount of foodborne pathogens were Gram-positive (G+) bacteria, a threat to public health. In this study, considering the binding ability of nisin towards G+ bacteria and the stable fluorescent ability of EGFP protein, a fluorescent nisin–EGFP protein probe was constructed by a gene engineering method. Nisin and EGFP were used as the receptor and fluorophore, respectively, to detect G+ bacteria. The nisin and egfp gene were amplified separately according to the sequence published in GenBank using unique primers. The two genes were cloned into a pET-28b(+) vector resulting in a pET-28b(+)–nisin–egfp vector. The vector was transferred into Escherichia coli (E. coli) BL21 (DE3) for expression. The expressed protein was extracted, purified by a Ni–NTA column, and then tested by the SDS-PAGE method to confirm its molecular weight. Listeria monocytogenes (L. monocytogenes), Staphylococcus aureus (S. aureus), and Micrococcus luteus (M. luteus) were used as the representations of G+ bacteria. E. coli O157, representing the gram-negative (G−) bacteria, was used as a negative control. The binding specificity of the recombinant protein was performed on two types of bacteria and then detected through fluorescent microscopy. The results indicated that the nisin–EGFP probe could detect G+ bacteria at 10⁸ CFU/mL.

Keywords

Nisin–EGFP / Gram-positive bacteria / Fluorescent detection / Probe

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