Identification of two major histocompatibility (MH) class II A genes and their association to Vibrio anguillarum infection in half-smooth tongue sole (Cynoglossus semilaevis)

Abstract

Major histocompatibility complex class II antigens are important in vertebrate immune system. In the present study, the full cDNA sequence of class II A gene was synthesized by RACE-PCR from half-smooth tongue sole (Cynoglossus semilaevis), and its open reading frame (ORF) polymorphism was studied. The whole cDNA sequence was 992 bp in length, including the ORF with 717 bp. Twenty-five alleles were identified and clustered into two distinct groups according to the specific nucleotides/ amino acids in specific positions. Eleven alleles belonged to Cyse-DAA while fourteen alleles belonged to Cyse-DBA. Four Cyse-DAA alleles were observed in one individual, and three to five Cyse-DBA alleles were observed in each of the three detected individuals, which indicated that at least two loci existed in each gene. Moreover, in order to study the function of the alleles in resistance to infection, 200 individuals were intraperitoneally injected with Vibrio anguillarum and the first 20 dead individuals and 20 surviving ones were selected for genotype analysis. Fifty-six alleles were identified among the 40 individuals. Twenty-nine alleles belonged to Cyse-DAA and the other 27 alleles belonged to Cyse-DBA. Eighteen alleles were selected for studying their function in resistance to infection. Alleles Cyse-DAA*0201,

Duplicated major histocompatibility complex class II genes in the tongue sole (Cynoglossus semilaevis)

“…91, 92, 94, 95, 101 and 103) with gene‐unique amino acid in exon 2 (Figure a). Cyse‐DAA 's unique amino acids in those six sites are Asn 91 , Ser 92 , Ser 94 , Gly 95 , Lys 101 and Lys 103 , while Cyse‐DBA 's unique residues are Thr 91 , Val/Gly 92 , Gln/Glu 94 , Ala 95 , Leu 101 and Asn 103 (Figure a, Cyse‐DBA could be Thr/Ala/Lys/Ile 91 according to alleles isolated before (Li et al., )). Based on the PBRs of HLA‐DRA (Brown et al., ), the putative PBRs in tongue sole class II A are predicted and shown in Figure a, with most of the putative PBRs polymorphic.…”

“…Our previous study suggested that in tongue sole, MHC class II A may have been duplicated into two genes based on the ORF sequences, with most of the polymorphism existing in exons 2 and 3 (Li et al., ). In order to verify this, primers MIIA‐AbFw and MIIA‐AbRv, which could amplify a fragment of around 800 bp long, spanning complete exon 2, intron 2 and exon 3, were used to isolate alleles of class II A from the three families used in this study.…”

“…Tongue sole class II A and B paralogs each have unique amino acid substitutions in exon 2. Regardless of whether exon 2 or ORF sequences are used to construct the phylogenetic tree, two divergent clades are formed, with bootstrap value of 100 when using the ORF (Li et al., , ). Other studies demonstrate that different groups of class II genes exist based on exon 2/ORF sequences, including stone flounder (Jiang, Li, Zhang, & Wang, ) and two percichthyids (Bracamonte et al., ).…”

“…According to our previous study (Li et al., ), primers MIIA‐DNA‐Fw/MIIA‐ORF‐Fw2 and MIIA‐DNA‐Rv (Table ) were designed to amplify an approximate 1.6‐kb genomic sequence of class II A from tongue sole. High fidelity enzyme PfuUltra (Stratagene, San Diego, CA, USA) was used for cloning from three adult individuals.…”

“…During our previous study of tongue sole MHC class II genes and their roles in disease resistance after Vibrio anguillarum infection, we noticed that the open reading frame (ORF) sequences of class II A and B genes both fall into two groups, with unique amino acid residues in certain positions (Li et al., , ). Here, to further analyse the possible gene duplication of MHC in tongue sole, we isolated the genomic sequence of class II genes and characterized their genomic structures.…”