Purification and molecular cloning of hemocyanin from Fenneropenaeus merguiensis(de Man, 1888): response to Vibrio harveyi exposure

Abstract

Hemocyanin (HC) was purified from hemolymph of Fenneropenaeus merguiensis(de Man, 1888). Purified HC showed one band in non-denaturing PAGE with a molecular mass of 457 kDa, determined by gel filtration. Using an ELISA, HC content was 85.6% of total hemolymph protein. HC’s hexamer form contained no phenoloxidase activity under trypsin or SDS activation. FmHC, cloned from F. merguiensis, had a full-length cDNA comprised of 2128 bp with an open reading frame encoding a peptide of 661 amino acids with two Cu-binding sites. Expression of FmHCwas found only in the hepatopancreas where it was up-regulated after exposure to Vibrio harveyiinjection into shrimp and with tissue incubations. HC protein levels in the hemolymph of bacterial injected shrimp also increased. FmHCexpression was also up-regulated after shrimp were challenged with a lipopolysaccharide, which is a surface component of gram-negative bacteria. These results indicate that HC is inducible and up-regulated in response to pathogenic challenge, confirming that HC participates in the innate immune response in F. merguiensis.