摘要
根据已知鱼类抗菌肽hepcidin的同源性设计简并引物,利用同源克隆结合RACE法从黄鳝(Monopterus albus)肝脏中扩增得到了黄鳝hepcidin基因全长cDNA序列(GenBank accession number FJ436808).结果表明,黄鳝hepcidin基因cDNA全长712bp,5′端非翻译区有133bp,3′端非翻译区有306bp,包含1个273bp的开放阅读框,编码1个长90氨基酸的前体肽.同源性分析表明,黄鳝hepcidin推测的氨基酸序列与其他鱼类报道的hepcidin具有较高的同源性,并具有8个半胱氨酸这一典型特征,是hepcidin家族的1个新成员.采用半定量PCR法对该基因在健康成体不同组织的转录本和脂多糖(LPS)对该基因表达的影响进行了分析.结果发现,该基因在肝脏中表达量最高,其次是肾脏,在心脏、皮肤、脑、血液、小肠、脾脏和胃中的表达量较低,而在肌肉中没有检测到该基因的转录本;LPS注射24h后,各组织的hepcidin基因表达量都有明显升高.
Based on the homology of known hepcidin, a pair of degenerate primers were designed, and a molecular cloning of a hepcidin gene from the liver of a rice field eel was conducted. The full-length cDNA of the gene （GenBank accession number： FJ436808） was identified by RT-PCR and RACE. It is 712 bp long flanked by a 133 bp of 5＇-untranslated region and a 306 bp of 3＇-untranslated region, and containing an ORF of 273 bases encoding a prepropeptide of 90 amino acid residues. Homologous analysis indicated that deduced amino acid sequence from rice field eel was highly homologous to hepcidin of other fishes reported, featuring 8 cysteine, being a membership of hepcidin family. Semi-quantitative RT-PCR was performed and the results demonstrated that hepcidin transcripts are highly abundant in liver, abundant in kidney, less abundant in heart, skin, brain, blood cells, intestine, spleen and stomach, and undetectable in muscle. When challenged with LPS injection 24 h, up-inducible hepcidin mRNA levels were detected in all tested organisms. The cloning of rice filed eel hepcidin provide new candidate gene for fish breeding and basis data for searching alternative therapeutants.
出处
《湖南农业大学学报（自然科学版）》
CAS
CSCD
北大核心
2009年第3期304-308,共5页
Journal of Hunan Agricultural University(Natural Sciences)
基金
湖北省教育厅青年基金项目(Q200712005)
长江大学博士启动基金(2006A0443)
关键词
黄鳝
抗菌肽
HEPCIDIN基因
克隆
表达分析
Monopterus albus
antimicrobial peptides
hepcidin gene
cloning
gene expression
