CDNA cloning, mRNA expression and recombinant expression of a cathepsin L-like cysteine protease from Neobenedenia melleni (Monogenea: Capsalidae)

Abstract

The monogenean Neobenedenia melleni is a worldwide virulent pathogen to many species of sea-caged fishes. Its outbreaks have caused fearful economic losses in fish mariculture. Cysteine proteases play vital biological roles in various important physiological and pathological processes. In the present paper the full-length cDNA of cathepsin L-like cysteine protease was isolated from the N. melleni using rapid amplification of cDNA ends (RACE). It is 1070 bp long and contains an open reading frame (ORF) of 1008 bp. The prepro-cathepsin L consists of a signal peptide of 17 amino acids (aa), a pro-region peptide of 100 aa and a mature peptide of 218 aa. The consensus motifs flanking conserved catalytic active sites (C 25, H 159 and N 175, papain numbering), which are present in all papain-like cysteine proteases, were identified in the sequence. The cathepsin L gene comprises three introns and four exons. mRNA expression of cathepsin L was determined using semi-quantitative RT-PCR analysis. The cathepsin L mRNA was expressed in oncomiracidia, larvae and adult, but not in newly shed eggs and encysted oncomiracidia. The coding region of cathepsin L cDNA for the mature cathepsin L protein was subcloned into an expression plasmid pET-22b(+) and fused with a 6-His-Tag. Moreover, we have successfully developed an expression system in Escherichia coli to overproduce the recombinant cathepsin L of N. melleni. The present study is the first report of the cloning and description of cDNA encoding a cysteine protease from a monogenean, and provides essential information for further studies at the protein level, such as the function, regulation mechanism and possibility for use as a target molecule for an anti-monogenean drug.