乌鳢(Channa argus)MHC Class I 全长cDNA序列的克隆及表达特征

doi:10.13560/j.cnki.biotech.bull.1985.2015.12.024

生物技术通报 ›› 2015, Vol. 31 ›› Issue (12): 167-173.doi: 10.13560/j.cnki.biotech.bull.1985.2015.12.024

乌鳢(Channa argus)MHC Class I 全长cDNA序列的克隆及表达特征

汪强强¹,贾伟章²,黄泽波^1,2

1. 武汉大学药学院，武汉 430071；
2. 广东药学院生物制药研究所，广州 510006

收稿日期:2015-03-23 出版日期:2015-12-19 发布日期:2015-12-19
作者简介:汪强强，男，硕士，研究方向:鱼类分子免疫学；E-mail:wqq@whu.edu.cn
基金资助:
国家自然科学基金项目(30371091)

The Cloning and Expression Characteristics of A Full-length cDNA of MHC I from Snakehead Channa argus

Wang Qiangqiang¹, Jia Weizhang², Huang Zebo^1,2

1. School of Pharmaceutical Sciences，Wuhan University，Wuhan 430071；
2. Institute of Biopharmaceutics，Guangdong Pharmaceutical University，Guangzhou 510006

Received:2015-03-23 Published:2015-12-19 Online:2015-12-19

摘要/Abstract

摘要：

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1 167和1 083 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MHC I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。

关键词: 主要组织相容性复合物, 基因座, 表达特征, 乌鳢

Abstract:

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 and Char-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 and Char-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 of Char-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. Comparing Char-Ia and Char-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 that Char-Ia and Char-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 in α1 and α3 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 constitutively expressed in all detected tissues. Moreover, we designed specific primers to determine the expression level of Char-Ia and Char-Ib. The results illustrated that Char-Ia was ubiquitously expressed at low level, whereas the Char-Ib was predominantly expressed in spleen, intestine, gill and peripheral blood, suggesting the significant specificity of expression in tissue, which indicated that Char-Ia and Char-Ib played the different physiological functions in the immune responses of fish.

Key words: major histocompatibility complex, loci, expression characteristics, snakehead Channa argus

汪强强,贾伟章,黄泽波. 乌鳢(Channa argus)MHC Class I 全长cDNA序列的克隆及表达特征[J]. 生物技术通报, 2015, 31(12): 167-173.

Wang Qiangqiang, Jia Weizhang, Huang Zebo. The Cloning and Expression Characteristics of A Full-length cDNA of MHC I from Snakehead Channa argus[J]. Biotechnology Bulletin, 2015, 31(12): 167-173.

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乌鳢(Channa argus)MHC Class I 全长cDNA序列的克隆及表达特征

The Cloning and Expression Characteristics of A Full-length cDNA of MHC I from Snakehead Channa argus

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