Phylogenetic characterization and the expression of recombinant C-reactive protein from the Asian seabass (Lates calcarifer)

The Asian seabass (Lates calcarifer) is one of the major aquaculture species in the Asian-Pacific region, but the development of this industry is affected by serious diseases that cause substantial annual losses worldwide. The C-reactive protein (CRP), a prototypic acute phase reactant, is an important element in the primary immune response in most vertebrates, serves as a good indicator for inflammatory reactions and helps to defend against bacterial infections. We have isolated, characterized, and expressed the CRP gene from the L. calcarifer. The full length cDNA of L. calcarifer CRP consists of 750 base pair encodes for ORF of 223 amino acids with a putative signal peptide of 17 amino acids. The multiple sequence alignment showed that the L. calcarifer CRP sequence was highly conserved among other species studied. A phylogenetic tree of the amino acid sequences of pentraxin (C-reactive protein and serum amyloid P) from fish showed that the L. calcarifer CRP clustered closest to the Chinese perch. We also expressed the recombinant L. calcarifer CRP (rCRP) in Escherichia coli, using the pET-28b (+) vector expression system and E. coli Rosetta-gami 2(DE3) pLysS host. Our results show that the recombinant protein was purified and yielded CRP at a higher concentration and purity than obtained by direct purification from blood serum as reported by previous studies. The purified rCRP was tested against E. coli and Aeromonas hydrophila and showed a potent antimicrobial activity. Results from this study support the importance of the CRP gene in innate immunity and may prove to be a beneficial therapeutic agent.