Preliminary Study On A Papain-like Protease From Cryptocaryon Irritans

Cryptocaryon irritans is a parasitic ciliate from marine,which causes ' marine white spot disease' of fish,it's life cycle is complex,and undergoes parasitic stage in teleost.As the increasing density of marine aquaculture,C.irritans spreads in short days around a vast culture area resulting in huge economic lost.While no available effective method to prevent the fish disease,so it's urgent to develop appropriate method to control the disease.A gene coding papain-like protease as predicted was successful amplified by 5' RACE(Rapid Amplification of 5'-End of cDNA).Then the molecular characterization was done to obtain the knowledge of the molecular weight,location and potential functions,which gives us the inspiration of the prevention against C.irritans.The ORF from CiPCP contains 984 bp,which encoded 327 amino acid,and 14 rare codons included in the sequence.The rare codons were modified to adapt to E.coli codon usage by the chemical synthesis.Then a truncated ORF,ranging from the 301th bp downstream of 5' end to the terminal codon was subcloned into pET 28a,an expression vector,and transformed to E.coli BL-21(DE3).The recombinant protein fused with His-tag(rCiPCPt)was expressed,extracted and purified from the recombinant bacteria.And the anti-sera against rCiPCPt were collected from mice immunized with rCiPCPt.Subsequently,western blotting analysis was carried out.The result revealed that good immunogenicity of the rCiPCPt,the antibody to which recognized the native CiPCP from all stages of C.irritans life cycle.Meanwhile,it indicated that the molecular mass of the native mature CiPCP protein was about 26 kDa in all stages of the ciliate and the propeptide contained about 100 amino acids were excised during the protease mature process.The indirect immunofluorescent test implied the native CiPCP is a secreted protein,which may play vital roles in the invasion to,and migration from hosts,digestion of host tissues,and excystment during the life cycle of the parasitic ciliate.Then rCiPCPt was used to immunize Larimichthys crocea by intraperitoneal injection.The immunized fish gained partial immune protection against the challenge by lethal dose of C.irritans,with the rate of 54%.Among the detected immune related genes,the expression of Interleukin-1? was up-regulated.The results suggested preliminarily that CiPCP may have potential for vaccine development,which would shed light on the research for the control of cryptocaryonosis.