鲈鱼hepcidin原核表达及生物学活性测定
PROKARYOTIC EXPRESSION AND FUNCTION ANALYSIS OF LATEOLABRAX JAPONICA HEPCIDIN
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摘要: 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。Abstract: 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 several 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 production. The goal of the present study was to molecularly clone the Lateolabrax japonica hepcidin (LjFishep) open reading frame (ORF), to express the LjFishep ORF in prokaryotic expression, to purify recombinant Lateolabrax japonica hepcidin (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 prepropeptide 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 Arg64 and Gla65. 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 sequence, and also had 82%?85% identities with other fish Hepcidin in deduced mature amino acid sequence. Phylogenetic analysis showed that the LjFishep had close relationship with Oplegnathus fasciatus and Perca fluviatilis hepcidin. Recombinant expression plasmid of pET-28a(+) was constructed by inserting the LjFishep ORF without the signal peptide 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 isolated by a quick protein isolation and purification system of ?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 6.25 μg/mL, it could establish a basis for further study the biological function and clinical application of Lateolabrax japonica Hepcidin.
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Keywords:
- Lateolabrax japonica /
- Hepcidin /
- Cloning /
- Expression /
- Biological function
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[1] [3] Shike H, Lauth X, Westerman M E, et al. Bass hepcidin is a novel antimicrobial peptide induced by bacterial challenge [J]. European Journal of Biochemistry, 2002, 269(8): 2232-2237
[1] Krause A, Neitz S, M?gert H J, et al. LEAP-1, a novel highly disulfide-bonded human peptide, exhibits antimicrobial activity [J]. FEBS letters, 2000, 480(2-3): 147-150
[2] [4] Douglas S E, Gallant J W, Liebscher R S, et al. Identification and expression analysis of hepcidin-like antimicrobial peptides in bony fish [J]. Developmental and Comparative Immunology, 2003, 27(6-7): 589-601
[2] Park C H, Valore E V, Waring A J, et al. Hepcidin, a urinary antimicrobial peptide synthesized in the liver [J]. Journal of Biological Chemistry, 2001, 276(11): 7806-7810
[3] [5] Zhang Y A, Zou J, Chang C I, et al. Discovery and characterization of two types of liver-expressed antimicrobial peptide 2 (LEAP-2) genes in rainbow trout [J]. Veterinary Immunology and Immunopathology, 2004, 101(3-4): 259-269
[4] [6] Shike H, Shimizu C, Lauth X, et al. Organization and expression analysis of the zebrafish hepcidin gene, an antimicrobial peptide gene conserved among vertebrates [J]. Developmental and Comparative Immunology, 2004, 28(7-8): 747-754
[5] [7] Wang K J, Zhou H L, Yang M. A Novel hepcidin-like Antimicrobial Peptide Isolated from the Japanese Bass (Lateolabrax japonicus Cuvier et Valenciennes) [J]. Journal of Xiamen University (Natural Science), 2004, 43(3): 237 [王克 坚, 周红玲, 杨明. 海水养殖鲈鱼分离出一种hecidin 抗菌 肽新基因. 厦门大学学报(自然科学版), 2004, 43(3): 237]
[6] [8] Ren H L, Wang K J, Zhou H L, et al. Cloning and organisation analysis of a hepcidin-like gene and cDNA from Japan sea bass, Lateolabrax japonicus [J]. Fish and Shellfish Immunology, 2006, 21(3): 221-227
[7] [9] Chen S L, Xu M Y, Ji X S, et al. Cloning, characterization, and expression analysis of hepcidin gene from red sea bream (Chrysophrys major) [J]. Antimicrobial Agents and Chemotherapy, 2005, 49(4): 1608-1612
[8] [10] Hirono I, Hwang J Y, Ono Y, et al. Two different types of hepcidins from the Japanese flounder Paralichthys olivaceus [J]. FEBS Journal, 2005, 272(20): 5257-5264
[9] [11] Bao B, Peatman E, Li P, et al. Catfish hepcidin gene is expressed in a wide range of tissues and exhibits tissue-specific upregulation after bacterial infection [J]. Developmental and Comparative Immunology, 2005, 29(11): 939-950
[10] [12] Bao B, Peatman E, Xu P, et al. The catfish liver-expressed antimicrobial peptide 2 (LEAP-2) gene is expressed in a wide range of tissues and developmentally regulated [J]. Molecular Immunology, 2006, 43(4): 367-377
[11] [13] Zheng W, Liu G, Ao J, et al. Expression analysis of immune- relevant genes in the spleen of large yellow croaker (Pseudosciaena crocea) stimulated with poly I:C [J]. Fish and Shellfish Immunology, 2006, 21(4): 414-430
[12] [14] Rossi F, Chini V, Cattaneo A G, et al. EST-based identification of genes expressed in perch (Perca fluviatilis, L.) [J]. Gene Expression, 2007, 14(2): 117-127
[13] [15] Huang P H, Chen J Y, Kuo C M. Three different hepcidins from tilapia, Oreochromis mossambicus: analysis of their expressions and biological functions [J]. Molecular Immunology, 2007, 44(8): 1922-1934
[14] [16] Huang W S, Li S J, Cai L, et al. Cloning, sequence analysis and gene organization of antimicrobial peptide hepcidin gene from Nile Tilapia (Oreochromis niloticus) [J]. Journal of Xiamen University (Natural Science), 2007, 46(3): 390-395 [黄文树, 李少菁, 蔡灵, 等. 尼罗罗非鱼Hepcidin 基因结 构与序列分析. 厦门大学学报(自然科学版), 2007, 46(3): 390-395]
[15] [17] Chen S L, Li W, Meng L, et al. Molecular cloning and expression analysis of a hepcidin antimicrobial peptide gene from turbot (Scophthalmus maximus) [J]. Fish and Shellfish Immunology, 2007, 22(3): 172-181
[16] [18] Yang M, Wang K J, Chen J H, et al. Genomic organization and tissue-specific expression analysis of hepcidin-like genes from black porgy (Acanthopagrus schlegelii B) [J]. Fish and Shellfish Immunology, 2007, 23(5): 1060-1071
[17] [19] Cho Y S, Lee S Y, Kim K H, et al. Gene structure and differential modulation of multiple rockbream (Oplegnathus fasciatus) hepcidin isoforms resulting from different biological stimulations [J]. Developmental and Comparative Immunology, 2009, 33(1): 46-58
[18] [20] Lauth X, Babon J J, Stannard J A, et al. Bass hepcidin synthesis, solution structure, antimicrobial activities and synergism, and in vivo hepatic response to bacterial infections [J]. The Journal of Biological Chemistry, 2005, 280(10): 9272-9282
[19] [21] Cuesta A, Meseguer J, Esteban M A. The antimicrobial peptide hepcidin exerts an important role in the innate immunity against bacteria in the bony fish gilthead seabream [J]. Molecular Immunology, 2008, 45(8): 2333-2342
[20] [22] Wang G L, Xue L Y, Jin S, et al. Epidemiological studies on the skin ulcer disease of Lateolabrax japonicus in marine cage culture [J]. Acta Hydrobiol Sin, 2000, 24(4): 392-395 [王国 良, 薛良义, 金珊, 等. 海水网箱养殖鲈鱼皮肤溃疡病的流 行病学研究. 水生生物学报, 2000, 24(4): 392-395]
[21] [23] Li W, Chen S L. Fusion expression of hepcidin gene from Paralichthys olivaceus in Escherichia coli [J]. Chinese High Technology Letters, 2007, 17(7): 765-770 [李伟, 陈松林. 牙鲆抗菌肽hepcidin 基因在大肠杆菌中的融合表达. 高技 术通讯, 2007, 17(7): 765-770]
[22] [24] Zhou J Y, Chen J G, Wang J Y, et al. cDNA cloning and functional analysis of goose interleukin-2 [J]. Cytokine, 2005, 30(6): 328-338
[23] [25] Qi J, Chen J G, Wang J Y, et al. Expression of Goose Interleukin- 2 gene in Escherichia coli and Isolation of Its Soluble Monomer [J]. Chinese Journal of Biotechnology, 2008, 24(2): 183-187 [齐静, 陈吉刚, 王金勇, 等. 鹅IL-2 基因在大 肠杆菌中的表达及其可溶性单体的分离. 生物工程学报, 2008, 24(2): 183-187]
[24] [26] Hunter H N, Fulton D B, Ganz T, et al. The solution structure of human hepcidin, a peptide hormone with antimicrobial activity that is involved in iron uptake and hereditary hemochromatosis [J]. The Journal of Biological Chemistry, 2002, 277(40): 37597-37603
[25] [27] Ai Q H, Mai K S. Advance on nutritional immunity of fish [J]. Acta Hydrobiologica Sinica, 2007, 31(3): 425-430 [艾 庆辉, 麦康森. 鱼类营养免疫研究进展. 水生生物学报, 2007, 31(3): 425-430]
[26] [28] Srinivasulu B, Syvitski R, Seo J K, et al. Expression, purification and structural characterization of recombinant hepcidin, an antimicrobial peptide identified in Japanese flounder, Paralichthys olivaceus [J]. Protein Expression and Purification, 2008, 61(1): 36-44
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