การทำให้บริสุทธิ์และการศึกษาคุณสมบัติ ของฮีโมไซยานินและการโคลนยีนฮีโมไซยานิน ของกุ้งแชบ๊วย

Abstract

Hemocyanin is a copper binding protein present mainly in hemolymph of crustaceans. It is found to be a precursor of anti-fungal peptides or converted to contain phenoloxidase activity. Physiological role of phenoloxidase is involved in the innate immune system of crustaceans. These suggest that hemocyanin may also play a role in immune response. In this study, hemocyanin (HC) was purified from hemolymph of banana shrimp Penaeus merguiensis by ultracentrifugation and preparative polyacrylamide gel electrophoresis (PAGE). Purified HC showed a single band in Native PAGE and arranged in a doublet of 79.4 and 75 kDa in SDS-PAGE. It was estimated to have M, of 457 kDa by gel filtration chromatography. Polyclonal antibody raised against purified HC was highly specific to HC in hemolymph and used to develop enzyme linked immunosorbent assay (ELISA). By ELISA analysis, HC content was 85% of total hemolymph protein of normal shrimp. HC gene was cloned from hepatopancreas of P. merguiensis by means of reverse- transcription polymerase chain reaction (RT-PCR) and 5' and 3' rapid amplification of cDNA ends (RACE). The full-length cDNA of HC gene consists of 2,128 bp with one 1,983 bp open reading frame, encoding 661 amino acids. Its deduced amino acid sequence contains a putative signal peptide of 20 amino acids and 6 histidine residues that stabilize 2 Cu² binding sites. By BLAST analysis, P. merguiensis HC cDNA showed closely identity to that of Penaeus vannamei (90%), RT-PCR analysis revealed that HC transcript was expressed only in hepatopancreas but not in other tissues. To study the responses of HC gene in Vibro harveyi challenge, the hepatopancreas fragments were incubated with the bacterium. A semi-Quantitative RI-PCR demonstrated that the expression of HC increased and reached the maximum at 2.5 h post-incubation. After challenging whole shrimp by injection with V. harveyi, the expression of HC gene was up-regulated. Moreover, HC protein content in the hemolymph also increased to the highest at 12 h post-injection. These results indicate that HC is inducible and may be involved in a shrimp immune response to pathogenic bacteria.