Characterization of recombinant Liver-Expressed Antimicrobial Peptide-2 (LEAP-2) and its antimicrobial activity from olive flounder, Paralichthys olivaceus

Abstract

Teleost belonging to a subclass of vertebrate families have an acquired immune system comparable to higher mammals in addition to innate immune system. Liver-expressed antimicrobial peptide-2 (LEAP-2) is a cysteine-rich peptide known to play an important role in the innate immune system of fish. In this study, RT-PCR was carried out to obtain the gene encoding LEAP-2 of olive flounder, Paralichthys olivaceus, and to analyze its tissue-specific expression pattern. The gene region encoding the except for signal peptide of LEAP-2 was amplified from cDNA synthesized using RNA extracted from liver of olive flounder and was cloned into the expression vector pET-44a(+) with his6-tag at the C-terminus. The rOfLEAP-2 was expressed in Escherichia coli BL21 (DE3 codon plus) upon induction with IPTG. The rOfLEAP-2 was purified (from E. coli) by using nickel column and were subject to various antimicrobial activity analysis against Gram-positive (Bacillus subtilis, Lactococcus garvieae, Streptococcus parauberis) and Gram-negative bacteria (E. coli, Vibrio harveyi, Edwardsiella tarda). The sequence of the gene encoding LEAP-2 from olive flounder was identified to has an open reading frame of 309 bp, which encodes a protein consisting of 102 amino acids. In tissue-specific expression analysis, LEAP-2 gene showed predominant expression in liver and lower expression in intestine. The purified rOfLEAP-2 showed distinguishable antimicrobial activity against all bacteria except E. tarda, tested. Upon testing in combination with ampicillin and LEAP-1, the result showed a synergistic effect against B. subtilis and V. harveyi as verified by the fractional inhibitory concentration index (FICI). The result from outer membrane permeability test performed using N-phenylnaphthalen-1-amine (NPN) showed a distinguishable outer membrane permeability by rOfLEAP-2. The result was also confirmed by scanning electron microscope assay (SEM) showing a disrupted surface morphology of E. coli upon incubation with rOfLEAP-2.