关键词: 斑点叉尾鮰, LEAP2成熟肽, RT-PCR, 融合表达

Abstract: Antimicrobial peptides generally are some small molecule peptides with strong ability to fight against microbial organisms.They play an important role in the host’s immune system and are considered to be good substitutes for traditional antibiotics. A cDNA fragment（mLEAP2）encoding the LEAP2（liver-expressed antimicrobial peptide 2）mature peptide consisted of 41-amino-acid was cloned from the liver of channel catfish（Ictalurus punctatus）by RT-PCR. When the amino acid sequence of channel catfish mLEAP2 was compared with those of poikilothermal fish and other species, fourteen residues were observed at conserved positions, especially four highly conserved cysteine residues occurred at C-terminal regions of these mLEAP2s, suggesting that two disulfide bridges resulted from these four cysteines possibly related to antimicrobial activity of LEAP2. Subsequently, the mLEAP2 gene was fused with a trxA partner gene with a 6×His-tag and an enterokinase site to construct the recombinant expression plasmid pET32a-mLEAP2. The fusion protein “trxA-mLEAP2” was successfully expressed in E .coli BL21（DE3）at 25℃ after a 16 h induction with 0.7 mmol/L IPTG. Tricine-SDS-PAGE showed that the fusion protein existed in both the supernatant and inclusion body after sonication and centrifugation. The purified fusion protein was successfully obtained by immobilized metal affinity chromatography（IMAC）.