Fry Michael M, Liggett Jason L, Baek Seung Joon
Department of Pathobiology, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996, USA.
Vet Clin Pathol. 2004;33(4):223-7. doi: 10.1111/j.1939-165x.2004.tb00377.x.
Hepcidin is a recently identified acute phase protein with antimicrobial and iron regulatory functions. It has been suggested that hepcidin may be the key mediator of anemia of chronic disease. Our research group is interested in developing a diagnostic assay to measure hepcidin in dogs.
The objectives of this study were to clone and sequence the canine hepcidin gene and to gather preliminary data about tissue expression of hepcidin in dogs.
RNA was extracted from fresh canine liver tissue and cDNA was generated and amplified. Standard reverse transcription polymerase chain reaction techniques were used with degenerate primers based on sequence homology between several other species. The amino acid (AA) sequence was compared with known sequences in other species. Tissue expression of canine hepcidin was determined by Western blot.
The canine hepcidin cDNA sequence encoded a highly conserved protein of 85 AAs with 8 cysteine residues at the C-terminal end. This protein was likely the precursor form (pro-hepcidin) of a smaller secreted peptide. Phylogenetic analysis showed that human hepcidin was more homologous with canine than with rodent hepcidin. In dogs, as in people, hepcidin was expressed most strongly in the liver. Western blotting showed a clear band of approximately 9 kDa, consistent with pro-hepcidin. Weak expression was also detected in canine kidney and lung tissues.
The results of this study establish the basis for future investigation involving canine hepcidin and suggest that the dog may be a suitable model for studying the role of hepcidin in human health and disease.
铁调素是最近发现的一种具有抗菌和铁调节功能的急性期蛋白。有人提出，铁调素可能是慢性病贫血的关键介质。我们的研究小组有兴趣开发一种诊断检测方法来测量犬类中的铁调素。
本研究的目的是克隆和测序犬铁调素基因，并收集有关犬铁调素组织表达的初步数据。
从新鲜犬肝组织中提取RNA，生成并扩增cDNA。使用基于其他几个物种之间序列同源性的简并引物，采用标准逆转录聚合酶链反应技术。将氨基酸（AA）序列与其他物种的已知序列进行比较。通过蛋白质印迹法测定犬铁调素的组织表达。
犬铁调素cDNA序列编码一种由85个氨基酸组成的高度保守蛋白质，在C末端有8个半胱氨酸残基。这种蛋白质可能是一种较小分泌肽的前体形式（前铁调素）。系统发育分析表明，人铁调素与犬铁调素的同源性高于与啮齿动物铁调素的同源性。在犬类中，与人一样，铁调素在肝脏中表达最强。蛋白质印迹显示一条约9 kDa的清晰条带，与前铁调素一致。在犬肾和肺组织中也检测到弱表达。
本研究结果为未来涉及犬铁调素的研究奠定了基础，并表明犬可能是研究铁调素在人类健康和疾病中作用的合适模型。
