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采用同源克隆和末端快速扩增(RACE)的方法,得到1161bp的军曹鱼(Rachycentron canadum)MHC-Ⅱβ全长cDNA片段。该序列包括20bp的5′末端非编码区(UTR),394bp的3′UTR及747bp的开放阅读框(ORF),编码248个氨基酸,预测其蛋白质分子量约为27.99ku,等电点为6.21。通过构建MHC-Ⅱβ氨基酸序列的系统进化树,并进行氨基酸相似性比对,结果表明,军曹鱼和已知鱼类、鼠(Musmusculus)、鸡(Gallus gallus)及人类(Homo sapiens)MHC-Ⅱβ氨基酸的同源性在28.5%~77.5%之间。所推测的蛋白序列具有一些重要的特征,包括:前导肽、β1、β2和CP/TM/CYT区,保守的半胱氨酸等。Real-time PCR检测结果显示,MHC-Ⅱβ基因在正常军曹鱼组织中均表达,但表达量在各个组织中不同,其中,头肾表达较强,鳃、脾、肠表达程度中等,在心、脑、肌肉中表达较弱。
Using homologous cloning and rapid amplification of end (RACE) method, a full-length cDNA fragment of 1161 bp of MHC-Ⅱβ from Rachycentron canadum was obtained. The sequence contains 20bp 5 ’untranslated region (UTR), 394bp 3’UTR and 747bp open reading frame (ORF), which encodes 248 amino acids. The predicted protein molecular weight is about 27.99ku and the isoelectric point is 6.21. The phylogenetic tree of amino acid sequence of MHC-Ⅱβ was constructed, and amino acid similarity comparison was performed. The results showed that there were significant differences between the cobia and the known fish, Musmusculus, Gallus gallus and MHC-Ⅱβ amino acids of human (Homo sapiens) The homology between 28.5% ~ 77.5%. The putative protein sequence has some important features including: leader peptide, β1, β2 and CP / TM / CYT regions, conserved cysteines and the like. Real-time PCR results showed that MHC-Ⅱβgene was expressed in normal cobia tissues, but its expression was different in different tissues. Among them, the expression of MHC-Ⅱβ was strong in head and kidney, moderate in gill, spleen and intestine, Weak expression in muscle.