Molecular Characterization of Actin Depolymerizating Factor 1 from Cryptocaryon irritans and its Application

• 1、Fujian Key Laboratory of Developmental Biology and Neuroscience, College of Life Science, Fujian Normal University, Fuzhou 350117
• 2、Fujian Key Laboratory of Developmental Biology and Neuroscience, College of Life Science, Fujian Normal University, Fuzhou 350117

Abstract：Cryptocaryon irritans is a parasitic ciliate and a pathogen of marine \'white spot disease\' on teleost fishes, which have been causing severe economic loss in mariculture. Actin depolymerizing factors (ADFs) are a family of small actin-binding proteins, regulating actin dynamics, involved in cell motility, development and diseases. In this study, a gene homologous to ADF was cloned from cDNA library of C. irritans trophonts (CiADF1, Genbank accssion number JQ906265), its molecular characterization was analyzed. The result showed that its open reading frame (ORF) was 324 bp, encoding a polypeptide of 107 aa with a calculated molecular weight of 12.6 kDa. There were 4 non-universal codons (3 TAA and 1 TAG) in the ORF which encoded Gln in ciliates. The gene expression of CiADF1 was detected in all developmental stage of C. irritans as detected with reverse transription PCR. After the codon modification, the ORF was inserted into pGEX-4T-1, and a recombinant plasmid pGEX-4T-1/CiADF1\' was constructed and transformed into E. coli. The recombinant protein rCiADF1 expressed by the bacteria was extracted and purified. Mice antisera against rCiADF1 were prepared by immunization of SPF mice with rCiADF1. Western blot analysis using the antisera against rCiADF1 showed that native CiADF1 protein expressed in all stages of C. irritans . The molecular mass of native CiADF1 was about 12.6 kDa, corresponding with the calculated one. The rCiADF1 emulsified with complete Freund\'s adjuvant was used to injected Pseudosciaena crocea ( 20 g/fish) intraperitoneally at a dose of 100 μg/fish . GST was used as a control antigen. The second injection was carried out using the same antigen emulsified with incomplete Freund\'s adjuvant 2 w post the first immunization. Two weeks post 2nd immunization, the fish were challenged with sublethal dose of the infective theronts of C. irritans . Five days post the challenge, the survival rates of rCiADF1-injected group, GST-injected group and untreated group were 78.6% (11/14), 56.5% (13/23) and 39.4% (13/33) respectively. The relative percent survivals were 64.7% between rCiADF1-injected group and the untreated group, and 50.8% between rCiADF1-injected group and GST-injected group. The preliminary result showed that rCiADF1 had certain protective effect for fish against C. irritans challenge. This study will provide the material for further study in pothogen biology of C. irritans and provide references for the control of cryptocaryonosis.

Keywords： molecular characterization Cryptocaryon irritans actin polymerizing factor immunization