军曹鱼MHC-Ⅱα基因全长cDNA的克隆及其组织表达分析

本研究根据其他鱼类MHC-Ⅱα基因的保守序列设计兼并引物，运用同源克隆和末端快速扩增方法扩增了军曹鱼MHC-Ⅱα基因的全长cDNA序列，并对cDNA及其氨基酸序列进行了分析，比较了军曹鱼和其他物种的MHC-Ⅱα氨基酸序列的差异，分析了军曹鱼MHC-Ⅱα基因的组织分布及经LPS刺激后头肾组织中MHC-Ⅱα基因的表达变化。结果表明，军曹鱼MHC-Ⅱα cDNA全长998 bp，包括53 bp的5′末端非编码区（5′UTR）、234 bp的3′末端非编码区（3′UTR）及711 bp的开放阅读框（ORF），编码236个氮基酸，其蛋白质分子质量约为25.94 ku，等电点为4.39；军曹鱼MHC-Ⅱα蛋白质序列具有一些重要的特征，包括前导肽、α1、α2、CP/TM/CYT区和保守的半胱氨酸等；军曹鱼MHC-Ⅱα与鼠、人及其它鱼类的氨基酸同源性在25.0%～69.5%之间。Real-time PCR检测结果显示，MHC-Ⅱα基因在正常军曹鱼组织中均有表达，但其表达量在各种组织中存在差异，其中较强表达于头肾、鳃，中等程度表达于脾脏、肠，在心脏、脑、肌肉中表达较弱；经LPS刺激后，头肾中MHC-Ⅱα基因表达下调。

Full Length cDNA Cloning and Tissue Expression of Major Histocompatibility Complex (MHC)-Ⅱα from Cobia (Rachycentron canadum)

Degenerate primer was designed according to the conserved part of MHC-Ⅱα gene sequences of other fishes, and the techniques of homology cloning and RACE PCR were used to clone the MHC-Ⅱα gene from cobia Rachycentron canadium). The cDNA sequence and amino acid sequence were analyzed, and the differences between MHC-Ⅱα amino acid sequence of cobia and other species were compared. QRT-PCR assay was developed to assess the mRNA expression of MHC-Ⅱα in normal tissues and the tissues after injected with LPS. The results showed that the full length cDNA of MHC-Ⅱα comprises 998 bp with a 5′untranslated region (UTR) of 53 bp, a 3′UTR of 234 bp and an open reading frame (ORF) of 711 bp, which encoding a polypeptide of 236 amino acid residues with a predicted molecular weight of 25.94 ku and theoretical isoelectric point of 4.39. The putative protein showed homology varying from 25.0% to 69.5% compared to some known MHC-Ⅱα amino acids in other fish, mouse （Mus musculus） and human （Homo sapiens）, respectively. The protein sequence showed that all the important features: leader peptide, α1, α2 and CP/TM/CYT regions, and conserved cysteines. Real-time PCR indicated MHC-Ⅱα gene expressed in all detected tissues with different expression level. High expression was detected in head kidney, gill, moderate expression in spleen and intestine, and low expression in heart, brain and muscle. MHC-Ⅱα expression in the head kidney decreased after LPS stimulation.