Sequence Variability Analysis of MHC Class Ⅱ DRB Alleles in Three Felines
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Abstract
The variation of the exon 2 of the major histocompatibility complex (MHC) class Ⅱ gene DRB locus in three feline species were examined on clouded leopard (Neofelis nebulosa), leopard (Panthera pardus) and tiger (P. tigris altaica). A pair of degenerated primers was used to amplify DRB locus covering almost the whole exon 2. Exon 2 encodes the β1 domain which is the most variable fragments of the MHC class Ⅱ molecule. Single-strand conformational polymorphism analysis was applied to detect different MHC class Ⅱ B haplotypes. Fifteen recombinant plasmids for each individual were screened out, isolated, purified and sequenced. Totally eight sequence haplotypes of exon 2 were obtained in 4 individuals. Within 237 bp nucleotide sequences from 4 samples, 30 variable sites were found, and 21 putative peptide-binding sites were disclosed in 79 amino acid residues. The ratio of nonsynonymous substitutions (dN) was much higher than that of synonymous substitutions (dS), which indicated that balancing selection probably maintain the variation of exon 2. MEGA neighbor joining and PAUP maximum parsimony methods were used to reconstruct phylogenetic trees among species, respectively. The results displayed a more close relationship between leopard and tiger; however, clouded leopard has a comparatively distant relationship from the other two.
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