Characterization of serum immunoglobulin M of grouper and cDNA cloning of its heavy chain

Abstract

Immunoglobulin M (IgM) from the whole serum of grouper fish, Epinephelus coioides was purified by affinity chromatography using protein A-Sepharose column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions revealed that the relative molecular masses ( M r) of the equimolar heavy and light chains of IgM were 78,000 and 27,000, respectively. The cDNAs encoding IgM heavy chain comprising its variable (V H) and constant (C H) regions have been cloned and sequenced from a grouper kidney cDNA library by antibody screening method. Five V H (130–142 amino acids) and four C H (450–454 amino acids) families were identified. The variable and constant regions were conserved with their putative domains. All the four constant region domains (C H1–C H2–C H3–C H4) contained each three conserved cysteine residues, which are considered to form the inter- and intra-chain disulfide bridges. There were three carbohydrate acceptor sites in the constant region. In general, the pattern of IgM gene organization seems to resemble that of other teleosts. Moreover, the C H genes in grouper IgM occur as multifamily as reported in Atlantic salmon and common carp.