卵形鲳鲹Hepcidin-5的原核表达、纯化及多克隆抗体的制备
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摘要
为了制备高效的卵形鲳鲹hepcidin-5蛋白多克隆抗体和对其特异性进行鉴定,本研究通过PCR扩增的方法获得了卵形鲳鲹抗菌肽hepcidin-5(Tro H5)的成熟肽cDNA序列,并构建了原核表达载体pET-32a(+)/TroH5,然后将其转入大肠杆菌表达菌株BL21(DE3)中进行原核表达.经IPTG诱导,成功表达了相对分子质量约为35kDa的融合重组蛋白.经不同诱导条件的优化,最终确定在IPTG终浓度为0. 1mmol/L和温度为20℃时,其表达量最大.可溶性分析表明:该融合蛋白主要存在于上清中.经镍柱亲和层析纯化获得了纯度较高的融合蛋白,然后以免疫小鼠制备多克隆抗体,经ELISA法检测,其效价达到1∶105.蛋白免疫印迹(Western blot)检测表明:所制备的多克隆抗体特异性强.本研究为进一步探究hepcidin-5的生物学功能奠定了基础.
In order to prepare the efficient anti-hepcidin-5 polyclonal antibody against golden pompano and identify its specificity,in the study,the sequence that encoded the mature peptide of hepcidin-5 in golden pompano( Trachinotus ovatus) was cloned. Then,the recombinant vectors of pET-32 a( +)/TroH5 was constructed and transformed into Escherichia coli BL21( DE3) for expression. The results indicated that the fusion recombinant protein was successfully induced by IPTG and the putative molecular weight was 35 kDa; the fusion recombinant protein was mainly expressed in the supernatant. After being purified by Ni SepharoseTM affinity chromatography column,the fusion recombinant protein with high purity was obtained. The purified fusion recombinant protein was used to immunize mouse to prepare polyclonal antibody. The polyclonal antibody titer was determined to be1: 105 by ELISA. Western blot analysis showed that the polyclonal antibody was specific. The study provide the foundation for further exploring the biological functions of hepcidin-5.
In order to prepare the efficient anti-hepcidin-5 polyclonal antibody against golden pompano and identify its specificity,in the study,the sequence that encoded the mature peptide of hepcidin-5 in golden pompano( Trachinotus ovatus) was cloned. Then,the recombinant vectors of pET-32 a( +)/TroH5 was constructed and transformed into Escherichia coli BL21( DE3) for expression. The results indicated that the fusion recombinant protein was successfully induced by IPTG and the putative molecular weight was 35 kDa; the fusion recombinant protein was mainly expressed in the supernatant. After being purified by Ni SepharoseTM affinity chromatography column,the fusion recombinant protein with high purity was obtained. The purified fusion recombinant protein was used to immunize mouse to prepare polyclonal antibody. The polyclonal antibody titer was determined to be1: 105 by ELISA. Western blot analysis showed that the polyclonal antibody was specific. The study provide the foundation for further exploring the biological functions of hepcidin-5.
引文
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[15]Chernysh S,Kim S I,Bekker G,et al. Antiviral and antitumor peptides from insects[J]. Proceedings of the National Academy of Sciences USA,2002,99(20):12628-12632.
[16]Pigeon C,Ilyin B,Courselaud B,et al. A new mouse liver specific gene,encoding a protein homologous to human antimicrobial peptide hepcidin,is overexpressed during iron overload[J]. Journal of Biological Chemistry,2001,276:7811-7819.
[17]Chen J Y,Lin W J,Lin T L. A fish antimicrobial peptide,tilapia hepcidin TH 2-3,shows potent antitumor activity against human fibrosarcoma cells[J]. Peptides,2009,30(9):1636-1642.
[18]Engvall E,Perlmann P. Enzyme-linked immunosorbent assay(ELISA):Quantitative assay of immunoglobulin G.[J]. Immunochemistry,1971,8(9):871-874.
[19]Sow F B,Florence W C,Satoskar A R,et al. Expression and localization of hepcidin in macrophages:a role in host defense against tuberculosis[J]. Journal of Leukocyte Biology,2007,82(4):934-945.
[20]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,2007,44:1922-1934.
[21]Zhang H,Yuan Q P,Zhu Y P,et al. Expression and preparation of recombinant hepcidin in Escherichia coli[J]. Protein Expression&Purification,2005,41:409-416.
[22]Sorensen H P,Mortensen K K. Soluble expression of recombinant proteins in the cytoplasm of Escherichia coli[J]. Microbial Cell Factories,2005,4:1-8.
[2]Douglas S E,Gallant J W,Leibscher R S,et al. Identification and expression analysis of hepcidin like antimicrobial peptides in bony fish[J]. Developmental&Comparative Immunology,2003,27:589-601.
[3]Reddy K V R,Yedery R D,Aranha C. Antimicrobial peptides:premises and promises[J]. International Journal of Antimicrobial Agents,2004,24(6):536-547.
[4]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,2001,276:7806-7810.
[5]Krause A,Neitz S,Magert H J,et al. Leap-1,a novel highly disulfide-bonded human peptide,exhibits antimicrobial activity[J]. Febs Letters,2000,480:147-150.
[6]Hilton K B,Lambert L A. Molecular evolution and characterization of hepcidin gene products in vertebrates[J]. Gene,2008,415(1-2):40-48.
[7]Kim Y O,Hong S,Nam B H,et al. Molecular cloning and expression analysis of two hepcidin genes from olive flounder Paralichthys olivaceus[J]. Bioscience,Biotechnology,and Biochemistry,2005,69(7):1411-1414.
[8]Hirono I,Hwang J Y,Ono Y,et al. Two different types of hepcidins from the Japanese flounder Paralichtys olivaceus[J].Febs Journal,2005,272(20):5257-5264.
[9]Falzacappa M V V,Muckenthaler M U. Hepcidin:Iron-hormone and anti-microbial peptide[J]. Gene,2005,364(1):37-44.
[10]Wang Y D,Kung C W,Chi S C,et al. Inactivation of nervous necrosis virus infecting grouper(Epinephelus coioides)by epinecidin-1 and hepcidin-5 antimicrobial peptides and down regulation of Mx2 and Mx3 gene expressions[J]. Fish&Shellfish Immunology,2010,28(1):113-120.
[11]Hu X Y,Camus A C,Aono S,et al. Channel catfish hepcidin expression in infection and anemia[J]. Comparative Immunology,Microbiology&Infectious Diseases,2007,30:55-69.
[12]Courselaud B,Pigeon C,Inoue Y,et al. C/EBPαRegulates hepatic transcription of hepcidin,an antimicrobial peptide and regulator of iron metabolism[J]. Journal of Biological Chemistry,2002,277(43):41163-41170.
[13]Weinstein D A,Roy C N,Fleming M D,et al. Inappropriate expression of hepcidin is associated with iron refractory anemia:implications for the anemia of chronic disease[J]. Blood,2002,100:3776-3781.
[14]Vizioli J,Salzet M. Antimicrobial peptides:new weapons to control parasitic infections[J]. Trends in Parasitology,2003,6(3):89-91.
[15]Chernysh S,Kim S I,Bekker G,et al. Antiviral and antitumor peptides from insects[J]. Proceedings of the National Academy of Sciences USA,2002,99(20):12628-12632.
[16]Pigeon C,Ilyin B,Courselaud B,et al. A new mouse liver specific gene,encoding a protein homologous to human antimicrobial peptide hepcidin,is overexpressed during iron overload[J]. Journal of Biological Chemistry,2001,276:7811-7819.
[17]Chen J Y,Lin W J,Lin T L. A fish antimicrobial peptide,tilapia hepcidin TH 2-3,shows potent antitumor activity against human fibrosarcoma cells[J]. Peptides,2009,30(9):1636-1642.
[18]Engvall E,Perlmann P. Enzyme-linked immunosorbent assay(ELISA):Quantitative assay of immunoglobulin G.[J]. Immunochemistry,1971,8(9):871-874.
[19]Sow F B,Florence W C,Satoskar A R,et al. Expression and localization of hepcidin in macrophages:a role in host defense against tuberculosis[J]. Journal of Leukocyte Biology,2007,82(4):934-945.
[20]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,2007,44:1922-1934.
[21]Zhang H,Yuan Q P,Zhu Y P,et al. Expression and preparation of recombinant hepcidin in Escherichia coli[J]. Protein Expression&Purification,2005,41:409-416.
[22]Sorensen H P,Mortensen K K. Soluble expression of recombinant proteins in the cytoplasm of Escherichia coli[J]. Microbial Cell Factories,2005,4:1-8.
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