Amplification of immobilization antigen from Ichthyophthirius multifiliis by degenerate PCR

The immobilization antigen (i-ag) is a predominant component of the Ichthyophthirius multifiliis cilia and is the most important antigen recognized by host immune system. Based on the hydrophobic conserved peptide at the N C terminal of i-ag protein sequences of three Ichthyophthirius multifiliis isolates, a pair of degenerate PCR primers P6/P7 were designed and used to amplify the i-ag gene from the parasite isolate IchFJ9 genomic DNA. The amplified gene is 1 398 bp long with an open-reading-frame (ORF) without TGA stop codon. Sequence analysis showed the amplified gene iagFJ9 had typical i-ag structure with 18 TAA encoding Glutamine (Q), instead of stop codon. The deduced amino acids had 466 aa with six tandem repeats all initiated with CPXGT amino acid residues. Homology analysis indicated that the amplified gene had 88% homology with IAG52A gene. All these prove that the gene is the new discovered i-ag gene and the degenerate PCR is one of the useful tool for searching and discovering the other members of i-ag gene family.