Molecular Cloning and Sequence Analysis at Exon 2 of MHC Class Ⅱ B Gene in Chinemys reevesii

Abstract: The major histocompatibility complex (MHC) is a chromosomal region consisting of closely linked loci which are highly polymorphic, and plays a central role in the immune system. Here we investigated genetic variation at exon2 (including part of the putative antigen-binding site, pABS) of the class Ⅱ MHC B gene in Chinemys reevesii. A pair of degenerate primers was used to amplify 166bp fragments of exon 2 of the class Ⅱ MHC B gene. PCR products were cloned into a pGEM -T Vector after purification. Eight different sequences were obtained from cloning and sequencing. All eight sequences did not show deletions, insertions, or stop codons, and therefore could be assumed functional in vivo. There were 84 variable sites in the nucleotide sequences, the ratio of nonsynonymous substitutions was higher than that of synonymous substitutions (dN/dS=28.533/8.324), which caused 39 variable sites among amino acid sequences. Substitutions of amino acid tended to be clustered around the putative antigen-binding sites, suggesting that the variation at these sites would be responsible for recognition and presentation of different kinds of antigens. Phylogenetic relationships were constructed by neighbor-joining (NJ) method in MEGA 2.1 and maximum parsimony (MP) method in PAUP 4.0. The eight nucleotide sequences can be clustered into two clades in NJ and MP phylogenetic trees. More than twosequences were found in the same individual, suggesting there would be locus duplication in the class Ⅱ major histocompatibility complex B gene of C. reevesii. The high polymorphism of MHC class Ⅱ B gene in C. reevesii was of great benefit to genetic conservation in the wild population of C. reevesii.