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为进一步丰富鱼类MHC class Ⅱ基因的研究,同时也为进一步探讨低磷饲料中添加维生素D3对鱼类免疫功能可能的影响,实验利用RACE(Rapid-amplification of c DNA ends)即c DNA末端快速扩增技术,成功克隆出黄颡鱼(Pelteobagrus fulvidraco)主要组织相容性复合体(Major histocompatibility complex,MHC)class Ⅱ抗原基因,全长1074 bp,其中ORF(Open reading frame)708 bp,编码236个氨基酸,5′UTR(5′端非翻译区)78 bp,3′UTR(3′端非翻译区)259 bp。进行氨基酸序列比对分析得到:黄颡鱼MHC class Ⅱ基因ORF氨基酸序列与长吻逘(Leiocassis longirostris)的氨基酸序列相似度最高为69.5%,与锦鲤(Cyprinus carpio)的氨基酸序列相似度最低为50.4%。利用q PCR对黄颡鱼MHC class Ⅱ基因进行组织表达分析,结果表明MHC class Ⅱ在小肠、肝脏、鳃中表达较高;在肌肉、鳍条中表达较低;而在肾、脾脏、脑、头肾中表达量极低(几乎检测不到)。在低磷饲料中添加维生素D3显著诱导了该基因的上调表达。研究结果展示了黄颡鱼MHC class Ⅱ抗原基因的分子结构、组织表达以及维生素D3的作用,在降低磷排放的同时,为今后黄颡鱼免疫抗病及分子选育等方向的深入研究及免疫型饲料的使用奠定了基础。
In order to further enrich the study of fish MHC class Ⅱ gene and to further explore the possible impact of adding vitamin D3 to fish immune function in low-phosphorus feedstuffs, the rapid amplification of cDNA ends (RACE) (MHC) class Ⅱ antigen gene of Pelteobagrus fulvidraco, which is 1074 bp in length, including an open reading frame (ORF) of 708 bp and a coding sequence of 236 Amino acids, 5 ’UTR (5’ untranslated region) 78 bp, 3 ’UTR (3’ untranslated region) 259 bp. The results of amino acid sequence alignment showed that the amino acid sequence of the ORF of MHC class Ⅱ gene of yellow catfish had the highest similarity of 69.5% with that of Leiocassis longirostris and the lowest similarity with Cyprinus carpio 50.4%. The results of qPCR analysis showed that MHC class Ⅱ was expressed in small intestine, liver and gill, but lower in muscle and ray, while in kidney, spleen, brain, Very low expression in head kidney (almost undetectable). Addition of vitamin D3 to low-phosphorus diet significantly induced upregulation of this gene. The results show the molecular structure, tissue expression and vitamin D3 of the MHC class II antigen in catfish, and in the future, reduce the phosphorus emission and further study on immune, disease resistance and molecular breeding of catfish The use of type feed has laid the foundation.