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【目的】克隆锦鲤Hepcidin全长c DNA序列(k-hepc),并获得此基因在鱼体内的表达模式。【方法】利用RT-PCR和RACE PCR的方法,从锦鲤肝脏中克隆锦鲤Hepcidin的全长c DNA,进行序列测定和分析;锦鲤经肌肉注射维氏气单胞菌0、4、8、12、24和48 h后,分别取其肝、脾、肾、肠、脑、心、肌肉和鳃组织,采用实时荧光定量PCR的方法,以β-actin为内参基因,检测k-hepc基因的表达量。【结果】锦鲤抗菌肽(Gen Bank登录号KC795559)全长755 bp,编码序列276 bp,编码91个氨基酸,包括信号肽、原肽和成熟肽,成熟肽C端含有8个半胱氨酸,可形成4个分子内二硫键。与已报道的普通鲤鱼Hepcidin氨基酸序列的一致性为93%,与其他鱼类Hepcidin氨基酸序列的一致性为29%-93%。在本研究所检测的正常锦鲤的组织中,k-hepc均有表达,其中在肝组织中表达量最高,鳃组织中表达量最低。经维氏气单胞菌感染后,k-hepc在肝和心组织中的表达量明显增加,在其余组织中变化不显著。【结论】k-hepc编码的蛋白是Hepcidin家族的成员之一。锦鲤Hepcidin的表达主要受内在调节因素影响。
【Objective】 The full length c DNA sequence of koi Hepcidin was cloned and the expression pattern of this gene in fish was obtained. 【Method】 The full-length c DNA of Koi carp Hepcidin was cloned from the liver of Koi by RT-PCR and RACE PCR, and sequenced and analyzed. Koi muscle was injected with Aeromonas sobria 0,4,8 After 12, 24 and 48 h, the liver, spleen, kidney, intestine, brain, heart, muscle and gill tissues were harvested. The real-time quantitative PCR was used to detect the k-hepc gene The amount of expression. 【Result】 The results showed that the antimicrobial peptide (GenBank accession number KC795559) was 755 bp in length with a coding sequence of 276 bp and encoded 91 amino acids including signal peptide, pro-peptide and mature peptide. C-terminal mature peptide contained 8 cysteines , Can form four intramolecular disulfide bonds. It was 93% identical to the reported Hepcidin amino acid sequence of common carp and 29% -93% identical to the Hepcidin amino acid sequence of other fish. In our study, k-hepc was expressed in the tissues of normal koi carps, with the highest expression in liver tissue and the lowest in gill tissue. The expression of k-hepc in liver and heart tissue increased significantly after infected with A.ivoltamicum, but not in other tissues. 【Conclusion】 The k-hepc-encoded protein is a member of the Hepcidin family. The expression of Koipi Hepcidin is mainly affected by the internal regulation factors.