Cloning Of The Immunoglobulin Heavy Chain Genes For The Grass Carp, Ctenopharyngoden Idellus

The grass carp (Ctenopharyngoden idellus) is an important species in aquaculture industry in China. The understanding of its immune system is essential for its aquaculture. The present study aims to clone immunoglobulin heavy chain genes, such as IgM, IgZ and a new Ig chimera, and to study their expression in different tissues.The IgM cDNA full sequence of grass carp has been cloned using RACE-PCR and PCR. Its open reading frame has 1731 nucleotides which encodes a 576 amino acid peptide. The coding region consists of leader segment, variable region and constant region. The variable region can be divided into four framework regions and three complementarity-determining regions. The deduced amino acid sequence of grass carp IgM was compared with those from other fish species, and the conserved regions, i.e. GKGLEW, YYCAR and FDYWGKGT-VTV-S were found in FR2, FR3 and FR4, respectively. The cysteines and tryptophans in constant regions were identified at conserved locations. The real-time PCR analysis revealed a higher level of IgM gene expression in head kidney, kidney and spleen and lower expression in liver, gill, thymus and intestines and almost none in heart and brain.An Ig cDNA sequence similar to the reported IgZ of zebrafish Danio rerio or IgT of rainbow trout Oncorhynchus mykiss has been cloned in grass carp using degenerate primers and RACE-PCR, and a Ig chimera also has been cloned in this fish. They encode 487 and 402 amino acids peptides, respectively. The IgZ or IgT similar IgH chain has a high similarity with IgZ from zebrafish and common carp Cyprinus carpio. The first two constant domains of the Ig chimera heavy chain are similar to IgM, and the latter two to IgZ, being a structure ofμ1 +μ2 +ζ3 +ζ4. The expression primers were coded for the open reading frame of IgZ and recombinant IgZ was expressed in Escherichia coli M15. The expressed product was 31 kD fusion protein, and was purified in the denatured condition. Rabbit polyclonal antisera were then...