Molecular cloning, characterization and mRNA expression of two antibacterial peptides: crustin and anti-lipopolysaccharide factor in swimming crab Portunus trituberculatus.

In this study, the cDNAs of a crustin and an anti-lipopolysaccharide factor (ALF) were cloned from the hemocytes of swimming crab Portunus trituberculatus using rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of P. trituberculatus crustin (denoted as PtCrustin) was of 584 bp, including an open reading frame (ORF) of 333 bp encoding a 21 amino acids signal peptide and a mature polypeptide of 89 amino acids with the predicted molecular mass of 10.08 kDa. The typical conserved cysteine residues containing WAP domain at the C-terminal displayed high similarity to other crustins. Moreover, the full-length cDNA of P. trituberculatus ALF (denoted as PtALF) was of 1050 bp, including an ORF of 372 bp encoding a 26 amino acids signal peptide and a mature polypeptide of 97 amino acids with the predicted molecular mass of 11.35 kDa. PtALF contained two conserved cysteine residues and showed high similarity to other crab ALFs. Using fluorescent real-time quantitative PCR, PtCrustin mRNA was expressed at the highest level in hemocytes and higher level in stomach while extremely low in hepatopancreas, muscle, gill and heart. In contrast, the PtALF mRNA expression was observed in all tissues. The relative expression levels of PtCrustin and PtALF in hemocytes of P. trituberculatus showed a clear time-dependent response after challenge with Vibrio alginolyticus. These results indicate that PtCrustin and PtALF are two acute-phase proteins involved in the immune responses of P. trituberculatus.