Cloning and bioinformatic analysis of elastase gene from skeletal muscle of yellowfin seabream (Acanthopagrus latus)

Researches show that elastase (Ela) is involved in regulation of bacterial ulcer caused by Aerononas hydrophila, and plays an important role in control of immune diseases during fish culture. The full-length cDNA sequence of elastase gene was cloned from Acanthopagrus latus based on the reported sequencing data of other vertebrates by using RT-PCR and RACE, which was further analyzed by bioinformatics analysis. The cloned AlEla gene was 946 bp, containing a 807 bp open reading frame which encoded 269 amino acids. AlEla protein was a hydrophilic stable extracellular protein containing a signal peptide with 16 amino acid residues, which might be located in extracellular matrix. Furthermore, AlEla protein has 3 predicted O-glycosylation sites and 15 predicted phosphorylation sites. The theoretical molecular weight of mature protein was 27 509 u and its isoelectric point was 6.05. The analysis of amino acid sequence homology showed that it has high similarity with Sparus aurata, Spondyliosoma cantharus, Pagrus major, Scophthalmus maximus, Paralichthys olivaceus (84.76%-99.26%). They were also in the same branch in phylogenetic tree, indicating that they are the close relatives. Moreover, AlEla has the conserved structures of Elas, including conserved region Tryp-SPc superfamily, catalytic triad of serine proteinase active site, conserved cysteine residues and the sequence of “GDSGGPL” in C-terminal, which revealed that AlEla might show serine proteinase catalytic activity. In the secondary structure of AlEla protein, the proportion of alpha helix, beta turn, extended strand and random coil was 17.84%, 7.06%, 24.54% and 50.56%, respectively. The result of predicted tertiary structure of AlEla indicated that alpha helix and extended strand might play an important role in its conformation structure. In the present study, AlEla gene was successfully cloned from Acanthopagrus latus and we analyzed its bioinformatics, providing theoretical basis for further elucidation of mechanism of regulation of fish immune diseases by AlEla protein.