三种猫科动物MHC Class Ⅱ DRB等位基因序列变异性分析
Sequence Variability Analysis of MHC Class Ⅱ DRB Alleles in Three Felines
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摘要: 分析了云豹(Neofelis nebulosa)、豹(Panthera pardus)和东北虎(Panthera tigris altaica)等3种猫科动物的主要组织相容性复合物Class Ⅱ DRB座位的等位基因序列变异性。使用一对简并性引物扩增了DRB座位第二外元目标片段。用单链构像多态性分析方法确定不同的单倍型。每个个体挑出15个单克隆用来分离、纯化和测序。实验中从4个个体中获得了8种不同序列。237 bp核苷酸序列中发现有59个变异位点。根据人类的抗原肽结合区推测79个氨基酸位点中存在21个假定的抗原肽结合位点，而且非同义替换率明显高于同义替换率，这可能说明了第二外元的较高变异性是由平衡选择作用来维持的。重建的NJ树和MP树显示豹和东北虎的亲缘关系较近，而两者与云豹的亲缘关系较远。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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