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 IgD, and express IgZ heavy chain in E.coli.
The IgM cDNA full sequence of grass carp has been cloned using RACE-PCR and PCR. Its open reading frame has 1731 nucleotides which encode 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 hypervariable 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 Zebra fish (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 acid peptides, respectively. The IgZ or IgT similar IgH chain has a high similarity with IgZ from zebrafish and common carp ( Mus musculus). The first two constant domains of the chimeric Ig heavy chain are similar to IgM, and the latter two to IgZ, being a structure of μ1 + 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 developed against the purified protein. A higher level of IgZ expression was detected by using Western blotting analysis in head kidney, thymus, gills and heart, and lower level in spleen and kidney and almost none in liver and brain.