旨在探究乌鳢（Channa argus）MHC基因的分子特征、表达方式及多态性。应用抑制差减杂交（SHH）和快速扩增cDNA末端（RACE）技术克隆并鉴定了乌鳢全长MHC I cDNA序列Char-Ia-1、Char-Ia-2和Char-Ib，推测氨基酸序列与已知硬骨鱼MHC I基因同源。Char-Ia-1和Char-Ia-2 cDNA序列包含1167和1083 bp的开放阅读框，分别编码388和360 aa的膜型Ⅰ类分子；而Char-Ib cDNA序列包含978 bp的开放阅读框，编码325 aa，显著截短的羧基末端显示Char-Ib为潜在的分泌型Ⅰ类分子。比较发现Char-Ia与Char-Ib在3'非转录区存在显著差异，在细胞外区、跨膜区和细胞质区的氨基酸序列同源性均较低，推测二者来自不同的MHC I基因座。氨基酸序列比对显示乌鳢MHC I分子与抗原肽结合的关键氨基酸残基较保守，在α1和α3结构域均出现硬骨鱼特征性的氨基酸残基缺失。进化树分析表明Char-Ia-1和Char-Ia-2聚为一簇，与Char-Ib处于不同的进化分支上，进一步证实Char-Ia与Char-Ib分别由不同MHC I基因座编码。RT-PCR分析显示乌鳢MHCI在组织中呈现组成型表达。设计基因专一性引物检测乌鳢Char-Ia与Char-Ib两类MHC I基因在组织中的表达水平，结果显示Char-Ia以较低的浓度表达于所有被检测组织，而Char-Ib主要表达于脾、肠、鳃和外周血，呈现明显的组织表达特异性，提示两类MHC I分子在鱼类免疫反应中发挥不同的生理功能。%By the methods of suppression subtractive hybridization(SSH)and rapid amplification of cDNA ends(RACE), 3 full-length sequences,Char-Ia-1,Char-Ia-2 andChar-Ib of major histocompatibility complex(MHC)I from snakehead(Channa argus)were cloned and characterized. The sequences of these clones were predicted to be in a high degree of homology with known teleost’s MHC I.Char-Ia-1 andChar-Ia-2 contained an open reading frame(ORF)of 1 167 and 1 083 bp, and encoded a putative peptide of 388 and 360 amino acid respectively. However, the cDNA ofChar-Ib contained an ORF of 978 bp encoding putative peptide of 325 amino acid, and a significantly shorter carboxyl terminal indicated that it was a molecule of secreting type I. ComparingChar-IaandChar-Ib demonstrated significant difference in 3' untranslated region, and low homology of amino acid’s sequences in extracellular domains, transmembrane and cytoplasmic regions. These suggested thatChar-IaandChar-Ib were encoded by two different loci. Alignment of amino acid sequence indicated that key antigenic peptide-anchoring residues binding with snakehead’s MHC I were conserved, and there was amino acid deletion inα1 andα3 domains as the same characteristics of teleost. A phylogenetic analysis indicated that Char-Ia-1 and Char-Ia-2 branched together in the tree, and away from Char-Ib, this further demonstrated that they were from two different loci. RT-PCR analysis showed that snakehead MHC I gene was constitutivelyexpressed in all detected tissues. Moreover, we designed specific primers to determine the expression level ofChar-Ia andChar-Ib. The results illustrated that Char-Ia was ubiquitously expressed at low level, whereas theChar-Ib was predominantly expressed in spleen, intestine, gill and peripheral blood, suggesting the significant specificity of expression in tissue, which indicated thatChar-Ia andChar-Ib played the different physiological functions in the immune responses of fish.
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机译：杂交鳢(乌鳢♂ × 斑鳢♀)及其亲本血清胆红素和尿素氮的比较分析 Comparative Analysis on the Serum Bilirubin and BUN of Hybrid Snakehead (Channa Argus♂ × Channa Maculate♀) and Its Parents
