Cloning and characterization of a surface antigen CiSA-32.6 from Cryptocaryon irritans

Abstract

Cryptocaryon irritans is a ciliated parasite causing cryptocaryosis in marine fish. To isolate functional genes, a cDNA library of C. irritans trophonts was constructed and a gene designated CiSA-32.6 (GenBank ID: JF812643) was cloned and characterized. The full-length cDNA (1158bp) encoded a deduced polypeptide of 330 amino-acid (aa) with a signal peptide of 22 aa. To express the ciliate gene, a truncated open reading frame (CiSA-32.6t) was synthesized to remove fragments encoding the signal peptide and hydrophobic C-terminal and to modify non-universal genetic codes. CiSA-32.6t was subcloned into Escherichia coli DH5α strain using the pGEX-4T-1 vector and then expressed as a glutathione S transferase fusion protein (rCiSA-32.6t). Western blotting analysis showed that sera from mice immunized with rCiSA-32.6t reacted specifically with a native protein (32.6kDa) in parasite lysates. Moreover, rCiSA-32.6t reacted specifically with sera from mice immunized with a C. irritans trophont lysate. Expression of the CiSA-32.6 gene in C. irritans was detected at all developmental stages by reverse transcriptase PCR and Western blotting analysis. This study provides the basis of further investigations into the pathogenic biology of C. irritans and the control of cryptocaryosis.