• 中国中文核心期刊
  • 中国科学引文数据库核心期刊
  • 中国科技核心期刊
  • 中国高校百佳科技期刊
高级检索

葡萄球菌核酸酶的制备及性质研究

洪流, 郎君超, 贺冬梅, 刘坤锋, 吴洁

洪流, 郎君超, 贺冬梅, 刘坤锋, 吴洁. 葡萄球菌核酸酶的制备及性质研究[J]. 中国药科大学学报, 2017, 48(2): 214-219. DOI: 10.11665/j.issn.1000-5048.20170213
引用本文: 洪流, 郎君超, 贺冬梅, 刘坤锋, 吴洁. 葡萄球菌核酸酶的制备及性质研究[J]. 中国药科大学学报, 2017, 48(2): 214-219. DOI: 10.11665/j.issn.1000-5048.20170213
HONG Liu, LANG Junchao, HE Dongmei, LIU Kunfeng, WU Jie. Studies on preparation and properties of Staphylococcus nuclease[J]. Journal of China Pharmaceutical University, 2017, 48(2): 214-219. DOI: 10.11665/j.issn.1000-5048.20170213
Citation: HONG Liu, LANG Junchao, HE Dongmei, LIU Kunfeng, WU Jie. Studies on preparation and properties of Staphylococcus nuclease[J]. Journal of China Pharmaceutical University, 2017, 48(2): 214-219. DOI: 10.11665/j.issn.1000-5048.20170213

葡萄球菌核酸酶的制备及性质研究

基金项目: 国家自然科学基金资助项目(No.81673340,No.31270985);江苏省基础研究计划资助项目(No.BK20161462)

Studies on preparation and properties of Staphylococcus nuclease

  • 摘要: 为获得纯度较高的葡萄球菌核酸酶(SNase)以研究其与糖尿病的关系,构建基因工程表达菌E.coli BL21/pET28a-His-SNase,诱导其表达可溶性胞外蛋白,并对纯化的SNase进行初步性质研究。采用分子生物学方法设计和构建含有SNase基因的表达载体pET28a-His-SNase,再转入E.coli BL21(DE3)感受态细胞。经过乳糖诱导表达、超声破碎及镍柱亲和色谱等纯化步骤后,以SDS-PAGE和Western blot鉴定His-SNase,并对其性质进行了初步研究。结果表明,乳糖诱导后His-SNase能高效表达,亲和色谱后纯度高达85%以上,且其具备较高的核酸酶活性,作用pH范围较广,有很好的耐热性。本研究为进一步探究葡萄球菌核酸酶与糖尿病的关系奠定了基础。
    Abstract: In order to study the relationship between Staphylococcal nuclease(SNase)and diabetes mellitus, genetic engineering bacteria E. coli BL21/pET28a-His-SNase was constructed, the expression of soluble extracellular protein SNase was induced and the a preliminary research was made on it. An expression vector pET28a-His-SNase plasmid containing the His-SNase gene was constructed and transformed into E. coli BL21(DE3)competent cells. The protein was induced by lactose and purified by ultrasound destruction and Ni-affinity chromatography, respectively. It was then analyzed by SDS-PAGE and Western blot. The enzymatic properties for SNase has been preliminary studied as well. Results indicated that the purity of the correctly expressed fusion protein His-SNase was over 85%. SNase showed good activity within a wide range of pH and good heat resistance. This experiment might be a foundation work for the further study on the relationship between SNase and with diabetes.
  • [1] Haskins K,Portas M,Bergman B,et al.Pancreatic islet-specific T-cell clones from nonobese diabetic mice[J].Proc Nat Acad Sci U S A,1989,86(20):8000-8004.
    [2] Panina-Bordignon P,Lang R,van Endert PM,et al.Cytotoxic T cells specific for glutamic acid decarboxylase in autoimmune diabetes[J].J Exp Med,1995,181(5):1923-1927.
    [3] Bekris LM,Kavanagh TJ,Lernmark A.Targeting type 1 diabetes before and at the clinical onset of disease[J].Endocr Metab Immune Disord Drug Targets,2006,6(1):103-124.
    [4] White SA,Shaw JA,Sutherland DER.Pancreas transplantation[J].Lancet,2009,373(9677):1808-1817.
    [5] Wen Y,Chen B,Ildsrad ST.Stem cell-based strategies for the treatment of type 1 diabetes mellitus[J].Expert Opin Biol Ther,2011,11(1):41-53.
    [6] Mócsai A. Diverse novel functions of neutrophils in immunity,inflammation,and beyond[J].J Exp Med,2013,210(7):1283-1299.
    [7] Segal AW.How neutrophils kill microbes[J].Annurev Immunol,2005,23:197-223.
    [8] Brinkmann V,R eichard U,Goosmann C,et al.Neutrophil extracellular traps kill bacteria[J].Science,2004,303(5663):1532-1535.
    [9] Darrah E,Andrade F.NETs:the missing link between cell death and systemic autoimmune diseases[J].J Front Immunol,2012,3:428.
    [10] Villanueva E,Yalavarthi S,Berthier CC,et al.Netting neutrophils induce endothelial damage,infiltrate tissues,and expose immunostimulatory molecules in systemic lupus erythematosus[J].J Immunol,2011,187(1):538-552.
    [11] Kessenbrock K,Krumbholz M,Schonermarck U,et al.Netting neutrophils in autoimmune small-vessel vasculitis[J].Nat Med,2009,15(6):623-625.
    [12] Khandpur R,Carmona-Rivera C,Vivekanandan-Giri A,et al.NETs are a source of citrullinated autoantigens and stimulate inflammatory responses in rheumatoid arthritis[J].Sci Transl Med,2013,5(178):178ra40.
    [13] Wang Y,Xiao Y,Zhong L,et al.Increased neutrophil elastase and proteinase 3 and augmented NETosis are closely associated with β-cell autoimmunity in patients with type 1 diabetes[J].Diabetes,2014,63(12):4239-4248.
    [14] Diana J,Simoni Y,Furio L,et al.Crosstalk between neutrophils,B-1a cells and plasmacytoid dendritic cells initiates autoimmune diabetes[J].Nat Med,2013,19(1):65-73.
    [15] Dwivedi N,Upadhyay J,Neeli I,et al.Felty′s syndrome autoantibodies bind to deiminated histones and neutrophil extracellular chromatin traps[J].Arthritis Rheum,2012,64(4):982-992.
    [16] Nakashima K,Hagiwara T,Yamada M.Nuclear localization of peptidylarginine deiminase V and histone deimination in granulocytes[J].J Biol Chem,2002,277(51):49562-49568.
    [17] Simon D,Simon HU,Yousefi S.Extracellular DNA traps in allergic,infectious,and autoimmune diseases[J].Allergy,2013,68:409-416.
    [18] Taniuchi H,Anfinsen CB.Simultaneous formation of two alternative enzymology active structures by complementation of two overlapping fragments of staphylococcal nuclease[J].J Biol Chem,1971,246(7):2291-2301.
    [19] Shortle D,Meeker AK.Residual structure in large fragments of staphylococcal nuclease:effects of amino acid substitutions[J].Biochemistry,1989,28(3):936-944.
    [20] Singh PK,Chan PF,Hibbs MJ,et al.High-yield production and characterization of biologically active GST-tagged human topoisomeraseIIα protein in insect cells for the development of a high-throughput assay[J].Protein Expr Purif,2011,76(2):165-172.
    [21] Vorackova I,Suchanova S,Ulbrich P,et al.Purification of proteins containing zinc finger domains using immobilized metal ion affinity chromatography[J].Protein Expr Purif,2011,79(1):88-95.
    [22] Zakalskiy AE,Zakalska OM,Rzhepetskyy YA,et al.Overexpression of(His)6 tagged human arginase I in Saccharomyces cerevisiae and enzyme purification using metal affinity chromatography[J].Protein Expr Purif,2012,81(1):63-68.
    [23] Shiokawa D,Tanuma S.Characteration of human DNase1 family endonucleases and activation of DNase gamma during apoptosis[J].Bioehemistry,2001,40:143-52.
计量
  • 文章访问数:  881
  • HTML全文浏览量:  1
  • PDF下载量:  1457
  • 被引次数: 0
出版历程
  • 刊出日期:  2017-04-24

目录

    /

    返回文章
    返回
    x 关闭 永久关闭