졸복(Takifugu pardalis)과 별불가사리(Patiria petinifera)에서 정제된 생리활성 펩타이드의 분자적 특성 및 재조합 펩타이드 생산 연구

Abstract

Hepcidins are small cysteine-rich antimicrobial peptides that play an important role in fish immunity against pathogens. A hepcidin-like antimicrobial peptide (TpHAMP) identified from the mucus of puffer fish (Takifugu pardalis) had been reported previously. In this study, the full nucleotide sequence of TpHAMP, which was 576 bp, including a 5’-untranslated region (UTR) of 90 bp, a 3’-UTR of 219 bp, and an open reading frame (ORF) of 267 bp was determined. The ORF encoded a polypeptide of 88 amino acids (AAs), which includes a predicted signal peptide of 24 AAs, a prosequences of 41 AAs, and Hepcidin domain of 23 AAs. Recombinant TpHAMP was produced using an expression vector modified to fuse Thioredoxin A (TrxA) to mature TpHAMP (pET-28a (+)-TrxA-TpHAMP) in a heterogeneous expression system. rTpHAMP showed antimicrobial activity against Gram-negative and Gram-positive bacteria including fish pathogens. Real-time quantitative-PCR showed that TpHAMP transcripts were most expressed in liver of healthy T. pardalis. The expression levels post immune challenge were elevated in liver and intestine at 16 hr while no significant changes were detected in skin, spleen, head kidney, body kidney, and gills. Moreover, exposure to 1% NaCl increased expression level in liver. Collectively, these results imply that TpHAMP may be important for innate immune system.

An OrexinA-like peptide (named PpOrxA) was isolated from tubefeet of starfish, Patinia petinifera, and its full sequence was obtained previously. PpOrxA was determined to be composed of 33 amino acids and contain amidation at C-terminus. In this study, recombinant PpOrxA (rPpOrxA) was produced using a modified expression vector (pET-28a (+)-TrxA) in a heterogeneous expression system. To produce PpOrxA with C-terminal amidation, the nucleotide sequence of mature PpOrxA was first modified to obtain Leu residue at C-terminus instead of Met and Gly residues encoded in the original sequence. Then after fusion protein TrxA-PpOrxA was separated by Cyanogen bromide digestion, Leu was replaced by amidated Met residue (Met-NH2) using Carboxypeptidse Y. The amino acid sequence of five residues at the N-terminus and the molecular weight was measured by Edman degradation and LC-MS, respectively, to confirm the identity and C-terminal amidation of rPpOrxA. The bioactivity of rPpOrxA was measured by muscle bioassay. Collectively, rPpOrxA with C-terminal amidation was successfully produced using pET-28a (+)-TrxA-PpOrxA plasmid in a prokaryotic expression system and carboxypeptidase Y. Moreover, the C-terminal amidated recombinant peptide was active in the muscle bioassay while recombinant peptide without C-terminal amidation was inactive suggesting C-terminal amidation is important for PpOrxA bioactivity.