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United States Department of Agriculture

Agricultural Research Service

Research Project: MOLECULAR ANALYSIS OF VIRULENCE DETERMINANTS OF SELECT BACTERIA IN FISH DISEASES

Location: Aquatic Animal Health Research

Title: Molecular cloning and expression of channel catfish, Ictalurus punctatus, complement membrane attack complex inhibitor CD59

Authors
item YEH, HUNG-YUEH
item Klesius, Phillip

Submitted to: Veterinary Immunology and Immunopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 16, 2007
Publication Date: October 27, 2007
Citation: Yeh, H., Klesius, P.H. 2007. Molecular cloning and expression of channel catfish, Ictalurus punctatus, complement membrane attack complex inhibitor CD59. Veterinary Immunology and Immunopathology. 120:246-253.

Interpretive Summary: Channel catfish production is the most important aquacultural industry in the southeastern United States. Its annual output reaches more than 400 million dollars. In the course of studying Edwardsiella ictaluri pathogenesis, we cloned and characterized the complement membrane attack complex inhibitor CD59 gene of channel catfish. The deduced amino acid sequence was compared with CD59 of other species deposited in the GenBank database. The sequence of the channel catfish CD59 consists of 1109 nucleotides. Analysis of the nucleotide sequence reveals one open reading frame and 5’- as well as 3’-untranslated regions. The open reading frame potentially encodes 119 amino acids with a calculated molecular mass of 13.2 kDa. It is interesting to note that the number and position of Cys residues were conserved in the mature protein among all species examined, suggesting that although the primary amino acid sequences are divergent, 3D structure of CD59 may be conserved through the evolutionary process. CD59 expressed in all channel catfish tissues studied, suggesting that like mammals, channel catfish CD59 is constitutively expressed and may play important roles in immunity against microbes.

Technical Abstract: The channel catfish, Ictalurus punctatus, complement membrane attack complex inhibitor CD59 gene was cloned and analyzed. Total RNA from tissues was isolated and cDNA libraries were constructed by the rapid amplification cDNA end (RACE) method. The gene-specific primers in conjunction with the RACE primers were used to PCR amplify 5’- and 3’-ends of the CD59 transcript. The complete channel catfish CD59 cDNA comprised 1109 bp including a 132-bp 5’-untranslated, a 360-bp open reading frame and a 617-bp 3’-untranslated regions. The open reading frame encodes a putative protein of 119 amino acid residues with calculated molecular mass (without potential glycosylation) of 13.2 kDa. Besides, the CD59 protein had one potential N-glycosylation site at the Asn35 residue. The degree of conservation of the channel catfish amino acid sequence to mammalian counterparts is 24-32%, while to those of other fish species is 44-54%. One remarkable feature is that the number and position of Cys residues were conserved in the mature protein among all species examined, suggesting that although the primary amino acid sequences are divergent, three-dimensional structure of CD59 via disulfide linkages may be conserved through the evolutionary process. The putative protein could be further divided into three domains: a 21-amino-acid signal peptide at the N-terminus, a 72-amino-acid mature protein and a 26 amino-acid glycosylphosphatidylinositol (GPI) anchoring signal peptide at the carboxyl terminus. CD59 expressed in all channel catfish tissues studied, suggesting that like mammals, channel catfish CD59 is constitutively expressed and may play important roles in immunity against Edwardsiella ictaluri.

Last Modified: 9/29/2014
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