锦鲤Hepcidin基因的克隆及其组织特异性表达分析

【目的】克隆锦鲤hepcidin全长cDNA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cDNA，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(GenBank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。

Cloning and tissue-specific expression analysis of hepcidin gene in koi (Cyprinus carpio)

Objective] Our aim is to clone cDNA sequence of hepcidin gene (k-hepc) and find its expression pattern in koi (Cyprinus carpio). Methods] In this study, the full-length cDNA sequence of hepcidin gene in koi was cloned by RT-PCR and RACE PCR and sequenced. The liver, spleen, kidney, intestine, brain, heart, muscle and gill were obtained 0, 4, 8, 12, 24 and 48 h after the koi were challenged with Aeromonas veroii and evaluated by real time PCR to find gene expression of k-hepc. Results] The sequence of k-hepc cDNA (GenBank No. KC795559) is 755 bp in length, and ORF is 276 bp long. The deduced amino acid sequence of k-hepc gene consists of 91 amino acids including signal peptide, prodomain and mature peptide. The mature peptide contains 8 cysteines, and is able to form 4 disulfide bonds. The deduced amino acid sequence of k-hepc has 93% similarity to hepcidins of known common carp and 29%?93% similarity to hepcidins of other fish species. Expression of k-hepc is found in all the tissues tested by our lab. The relative expression levels in normal fish showed high basal values in liver and low values in gill. The expression of k-hepc mRNA was significantly increased in the liver and heart but not significantly induced in other tissues after bacterial-challenge. Conclusion] The protein encoded by k-hepc is a member of hepcidin family. Expression of k-hepc is mainly affected by intrinsic regulation factors.