Cloning and tissue expression of hemocyanin gene in Cherax quadricarinatus during white spot syndrome virus infection

The role of hemocyanin in terms of immune defense was explored in Cherax quadricarinatus infected by WSSV. The cDNA of a hemocyanin gene (CqHC) was cloned from the hepatopancreas of C. quadricarinatus (crayfish) by the Rapid Amplification Complementary DNA Ends (RACE) method. The full-length cDNA of CqHC consisted of 2167bp with a 2043bp open reading frame encoding a 680 amino acid protein with a predicted signal peptide of 19 amino acids. The deduced CqHC protein sequence was found to contain two conserved copper-binding sites and to share 74%, 73%, 72%, 71% and 71% similarity with Penaeus monodon, Marsupenaeus japonicus, Fenneropenaeus chinensis, Metacarcinus magister and Litopenaeus vannamei, respectively. Phylogenetic analysis revealed that CqHC and HC from F. chinensis, M. japonicus and P. monodon clustered in the same phylogenetic branch. Real-time PCR analysis showed that CqHC was widely distributed, with the highest level in hepatopancreas, at lower amounts in hemocytes, gills and antennal gland. HC levels in hepatopancreas, hemocytes and gills from C. quadricarinatus were also compared after white spot syndrome virus (WSSV) infection. After 24h of infection, CqHC levels rose significantly higher than that of the controls (P<0.05) but then sharply declined over the infection period. The HC gene expression in hemocytes and gills (but not hepatopancreas) of crayfish injected with polysaccharides before infection were higher than that of the non-polysaccharide injected infected groups, showing an immunoprotective rate of 51.86% at 7days after exposure, indicating that polysaccharide can act as immunopotentiator and could improve the antiviral ability of C. quadricarinatus against WSSV.