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金黄色葡萄球菌核酸酶在大肠杆菌中的表达及其活性分析 被引量:2

Expression and nuclease activity analysis of staphylococcus nuclease in E.coli
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摘要 目的:构建金黄色葡萄球菌核酸酶(SN)基因的原核表达载体,研究其在大肠杆菌中的表达,并制备兔抗SN抗体。方法:从质粒pPLCSN中扩增SN基因片段,插入表达载体pLEX后转化大肠杆菌GI724,以色氨酸诱导表达。以表达的重组蛋白免疫日本大耳白兔制备抗SN抗体,用Westernblot分析兔抗SN抗体的特异性。结果:重组表达载体pLEXSN在大肠杆菌中获得表达,表达的可溶性蛋白占菌体蛋白总量的37%,具有良好的生物学活性。制备的兔抗SN抗体能够特异识别SN。结论:成功地在大肠杆菌中表达具有生物学活性的SN,并制备了兔抗SN抗体,为进一步探讨其作为抗病毒因子应用于病毒性疾病的治疗奠定了基础。 AIM: To express staphylococcus nuclease (SN) in E.coli and prepare rabbit antisera against SN. METHODS: The SN gene was amplified by high-fidelity PCR from plasmid pPLC-SN and then subcloned into expression vector pLEX to obtain the recombinant plasmid pLEX-SN. The expression of recombinant protein was induced by tryptophan. The expressed SN was used to immunize a rabbit to prepare specific antibody. RESULTS: SDS-PAGE analysis showed that the relative molecular mass (M_r) of the expressed SN was about 17 000 and the expressed SN accounted for about 37% of total bacterial proteins. The prepared antisera were specific to react with recombinant SN. CONCLUSION: Expression vector of SN has been successfully constructed and rabbit antibody against SN was prepared. These results lay the foundation for developing SN as antiviral protein.
出处 《细胞与分子免疫学杂志》 CAS CSCD 北大核心 2005年第4期513-515,共3页 Chinese Journal of Cellular and Molecular Immunology
基金 国家自然科学基金资助项目(No.30100006)
关键词 葡萄球菌核酸酶 表达 抗体 staphylococcus nuclease expression antibody rabbit
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  • 1徐立东,童宇峰,王金凤.金黄色葡萄球菌核酸酶C末端去9肽对酶蛋白溶液构象的影响[J].生物物理学报,2003,19(1):7-12. 被引量:3
  • 2Tucker PW,Hazen EE,Cotton FA.Staphylococcal nuclease reviewed:a prototypie study in contemporary enzymology[J].Mol Cell Biochem,1979,23(2):67-86.
  • 3Boeke JD,Hahn B.Destroying retrovirus from within[J].Trends Microbiol,1996,4(11):421-426.
  • 4Beterams G,Bottcher B,Nassal M.Packaging of up to 240 subunits of a 17kDa nuclease into the interior of recombinant hepatitis B virus capsids[J].FEBS Lett,2000,481(2):169-176.
  • 5Lachica RV,Genigeorgis C,Hoeprich PD.Metachromatic agar-diffusion methods for detecting staphyloccal nuclease activity[J].Appl Microbiol,1971,21(4):585-587.
  • 6Schumann G,Hermankova M,Cannon K,et al.Therapeutic effect of a Gap-nuclease fusion protein against retroviral infection in vivo[J].J Virol,2001,75(15):7030-7041.
  • 7Beterams G,Nassal M.Signifieant interference with hepatitis B virus replication by a core-nuclease fusion protein[J].J Biol Chem,2001,276(12):8875-8883.
  • 8Natsoulis G,Boeke JD.New antiviral strategy using capsid-nuclease fusion proteins[J].Nature,1991,352(15):632-635.

二级参考文献7

  • 1Loll PJ, Lattman EE. The crystal structure of the ternary complex of staphylococcal nuclease, Ca2+, and the inhibitor pdTp, refined at 1.65A [J]. Proteins, 1989,5:183~201.
  • 2Wang JF, Truckses DM, Abildgaard F, et al. Solution structures of staphylococcal nuclease from multidimensional, multinuclear NMR: nuclease-H124L and its ternary complex with Ca2+ and thymidine-3',5'-bisphosphate [J]. J Biomol NMR,1997,10:143~164.
  • 3Shortle D, Meeker AK. Residual structure in large fragments of staphylococcal nuclease: effects of amino acid substitutions[J]. Biochemistry, 1989,28:936~944.
  • 4Yin J, Jing G. Tryptophan 140 is important, but serine 141 Is essential for the formation of the integrated conformation of staphylococcal nuclease[J]. J Biochem (Tokyo), 2000,128:113~119.
  • 5Wang JF, LeMaster DM, Markley JL. Two-dimensional NMR studies of staphylococcal nuclease. 1. Sequence- specific assignments of 1H signals and solution structure of the nuclease H124L-thymidine 3',5'-bisphosphate-Ca2+ ternary complex[J]. Biochemistry, 1990,29:88~101.
  • 6Cuatrecasas P, Fuchs S, Anfinsen CB. Catalytic properties and specificity of the extracellular nuclease of Staphylococcus aureus[J]. J Biol Chem, 1967,242:1541~1547.
  • 7Wang JF, Hinck AP, Loh SN, et al. Solution studies of staphylococcal nuclease H124L. 2. 1H, 13C, and 15N chemical shift assignments for the unligated enzyme and analysis of chemical shift changes that accompany formation of the nuclease-thymidine 3',5'-bisphosphate-calcium ternary complex[J]. Biochemistry, 1992,31:921~936.

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