Molecular Characterization and Antimicrobial Activity Analysis of Liver-Expressed Antimicrobial Peptide 2 (LEAP-2) Gene in Russian Sturgeon (Acipenser gueldenstaedti)

Antimicrobial peptides (AMPs) are an important part of the innate immune defense system; they can effectively kill viruses, fungi, and bacteria, preventing infection (and even sepsis). AMPs also have other functional roles in immune regulation, anti-tumor activity, angiogenesis, and wound healing. Liver-expressed antimicrobial peptide 2 (LEAP-2) is an AMP that has been extensively studied in mammals, birds, and fishes. The earliest fish LEAP-2 study reported the cloning of LEAP-2A and LEAP-2B of rainbow trout. So far, LEAP-2 has been studied in various fishes, including blunt snout bream, grass carp, golden pompano, ayu, and large yellow croaker. These studies found that LEAP-2 generally contains four highly conserved cysteine residues and two disulfide bonds; it can disrupt the structural integrity of bacterial cell membrane, revealing its antibacterial activity with an important role in the innate immune system of fishes. Russian sturgeon (Acipenser gueldenstaedtii) is cultivated on a certain scale in China, and Aeromonas hydrophila is its main pathogen. Hemorrhagic ascites and tissue hemorrhage appear after infection, causing huge economic losses to Russian sturgeon aquaculture. As an important innate immunity component, LEAP-2 research is of great significance. At present, studies on AgLEAP-2 have not been reported. To study the molecular characteristics and the transcriptional expression patterns of AgLEAP-2, as well as its antibacterial activity in vitro, the full-length AgLEAP-2 cDNA sequence was obtained by RACE. AgLEAP-2 was cloned and its sequence characteristics were analyzed by bioinformatics. The qRT-PCR method was used to detect AgLEAP-2 in 13 different tissues (liver, intestine, spleen, head kidney, blood, gill, skin, ovary, brain, heart, stomach, metanephros, and muscle) of healthy Russian sturgeon, and the AgLEAP-2 transcriptional expression pattern in immune tissues after infection with A. hydrophila. The AgLEAP-2 prokaryotic expression vector was constructed and the recombinant AgLEAP-2 protein (designated rAgLEAP-2) was purified. The antibacterial activity of rAgLEAP-2 was preliminarily detected by the agar dilution method. The results showed that the full-length cDNA of the AgLEAP-2 gene was 622 bp in length, of which the 5'-UTR was 184 bp, the 3'-UTR was 192 bp, and the ORF was 246 bp in length, encoding an 81-amino acid peptide. The molecular mass of the rAgLEAP-2 protein was predicted to be 11.2 kDa, and the theoretical isoelectric