鲈鱼hepcidin原核表达及生物学活性测定

采用RT-PCR方法扩增和克隆了鲈鱼hepcidin(LjFishep)的编码阅读框。该编码阅读框由261个核苷酸组成,编码由86个氨基酸组成的前体蛋白。遗传进化分析表明,LjFishep与条石鲷和河鲈hepcidin的亲缘关系最近。将去除LjFishep信号肽的编码序列克隆到原核表达载体pET-28a(+),实现了LjFishep在大肠杆菌的表达。可溶性分析表明表达蛋白大部分以包涵体形式存在,部分以可溶性形式存在,非变性电泳可见可溶性蛋白存在单体和多聚体组分。镍柱亲和层析法纯化的鲈鱼hepcidin重组表达蛋白(rLjFishep),利用?KTAFPLC(快速蛋白分离纯化系统)进行逐级分离,非变性电泳可见单一rLjFishep可溶性蛋白单体。体外生物学活性分析显示可溶性rLjFishep蛋白单体具有抑制鲈鱼哈维氏弧菌繁殖的能力。这为进一步研究鲈鱼Hepcidin的生物学功能及临床应用奠定了基础。  

PROKARYOTIC EXPRESSION AND FUNCTION ANALYSIS OF LATEOLABRAX JAPONICA HEPCIDIN

The cysteine-rich peptide Hepcidin was known to be an antimicrobial peptide and iron transport regulator that had been found in mammals. Numerous teleost hepcidin sequences had been published and showed that Hepcidin was widespread among fish. The teleost Hepcidins probably had a function in the defense against invading bacteria as sev-eral reports demonstrate an upregulation of the gene expression after treatment with LPS, bacterins or live bacteria. A direct antibacterial effect of some of the synthetic teleost Hepcidins was also demonstrated. Farming of Lateolabrax japonica was a growing industry in china. However, bacterial and viral infections were contributing to reduced produc-tion. The goal of the present study was to molecularly clone the Lateolabraxjaponica hepcidin (LjFishep) open reading frame (ORF), to express the LjFishep ORF in prokaryotic expression, to purify recombinant Lateolabrax japonica hep-cidin (rLjFishep), and to detect the bioactivity of the rLjFishep in vitro, cDNA encoding LjFishep ORF was cloned by RT-PCR. Sequencing results showed that the LjFishep ORF nucleotide sequence was 261 bp in length, encoding a pre-propeptide of 86 amino acids (aa) with a signal peptide of 24 aa. The predicted molecular weight of the peptide is 9.4 kD. A tentative RX(K/R)R motif for propeptide convertases was also identified suggesting a cleavage site located between Arg~(64) and Gla~(65). The deduced mature amino acid sequence of LjFishep was compared with those of the several avian and mammalian species. The results showed that the deduced mature rLjFishep amino acid sequence had 65%, 45.5% and 54.5% identities with Homo sapiens, Xenopus tropicalis and Danio rerio hepcidin in deduced mature amino acid se-quence, and also had 82%-85% identities with other fish Hepcidin in deduced mature amino acid sequence. Phyloge-netie analysis showed that the LjFishep had close relationship with Oplegnathus fasciatus and Percafluviatilis hepcidin. Recombinant expression plasmid of pET-28a(+) was constructed by inserting the LjFishep ORF without the signal pep-tide sequence into the prokaryotic expression vector pET-28a(+). An expected protein band was observed on SDS-PAGE gel, recognized by monoclonal antibody against 6×His in western-blotting assay. In the pET-28a(+) expression system, many of the rLjFishep was found in inclusion bodies with a portion of soluble protein by SDS-PAGE. The monomer and multimers of soluble rLjFishep proteins were observed in native electrophoresis. The rLjFishep soluble protein was iso-lated by a quick protein isolation and purification system of (A)KTA FPLC and identified by native PAGE. Bioactivity analysis showed that the rLjFishep monomer had the antimicrobial activity against Vibrio harveyi from Lateolabrax japonicain in a dose-dependent manner, and the minimal dose for inhibiting Vibrio harveyi was 6.25μg/mL, it could establish a basis for further study the biological function and clinical application of Lateolabrax japonica Hepcidin.