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变形链球菌葡聚糖结合蛋白D基因的分子克隆及其原核表达和初步纯化 被引量：1

Cloning,expression and purification of streptococcus mutans glucan-binding protein D
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摘要 目的:克隆变形链球菌葡聚糖结合蛋白D(gbpD)基因,在大肠杆菌中表达并对重组蛋白进行初步纯化,为进一步的蛋白功能研究奠定基础。方法:体外培养变形链球菌UA159菌株并以其基因组为模板,对gbpD基因编码区进行PCR扩增,连接pMD-18T载体并测序。随后将该片段克隆入原核表达载体pPROEX HTb中,构建表达质粒pPROEX/gbpD,转化大肠杆菌DH5α并用IPTG诱导表达。表达产物经镍-次氮基三乙酸(Ni-NTA)柱进行亲和层析纯化。结果:成功克隆变形链球菌gbpD基因并在大肠杆菌中得到可溶性表达,经Ni-NTA柱纯化后获得纯度大于95%、相对分子量(Mr)为76×103的变链菌GbpD蛋白。结论:获得Mr为76×103的变形链球菌GbpD蛋白。对进一步确定GbpD的葡聚糖结合能力,探讨其在变链菌致龋过程中的作用,以及在龋病预防领域的应用前景都具有重要的意义。 Objective：To clone, express and purify the recombinant S. mutans glucan-binding protein D. Method：The gbpD gene was amplified from the S. mutans chromosome by PCR and cloned into pMD-18T Simple vector and sequenced. Then the segment was inserted into expression vector pPRoEX HTb. The recombined plasmid was transformed into E. coil DHSa and induced by IPTG to express the fusion protein. The 6x his-tagged fusion protein was purified by affinity chromatography on Ni-NTAresin. Result：The gbpD gene was successfully cloned and expressed in E. coll. The purification rate of the recombinant protein was more than 95 percent. Conclusion：The gbpD gene was cloned and the 6× his-tagged fusion protein of GbpD obtained, which will be helpful for the further study .
作者 赵红萍 吴补领 苏凌云 孙叶芳 王秦芳 刘艳丽 逄键梁
机构地区 第四军医大学口腔医学院
出处 《临床口腔医学杂志》 2005年第10期604-607,共4页 Journal of Clinical Stomatology
基金 国家"十五"科技攻关项目(2004BA720A24)
关键词 变形链球菌 葡聚糖结合蛋白D 蛋白表达 streptococcus mutans gluccm-binding protein D proten expression
分类号 R378 [医药卫生—病原生物学]
  • 相关文献

参考文献7

  • 1Matsumura M, Izumi T, Matsumoto M, et al. The role of glucanbinding proteins in the cariogenicity of streptococcus mutans[J]. Microbiol Immunol, 2003,47: 213 - 215.
  • 2Banas JA, Vickerman MM. Glucan-binding proteins of the oral streptococci[J]. Crit Rev Oral Biol Med, 2003,14: 89 - 99.
  • 3Banas JA. Virulence properties of streptococcus mutans[J]. Frontiers in Bioscience, 2004,9:1267 - 77.
  • 4Ajdic P, McShan W, McLanghlin R, et al. Genome sequence of Streptococcus mutans UA159, a cariogenic dental pathogen[J]. Proc Natl Acad Sci, 2002,99:14434 - 9.
  • 5Shah DSH, Russell RRB. A novel glucan-binding protein with lipase activityfrom the oral pathogen Streptococcus mutans[J]. Microbiology, 2004,150: 1947 - 56.
  • 6苏凌云,吴补领,李福洋,卫克文.变形链球菌gbpA的葡聚糖结合区GBD的分子克隆及其在大肠杆菌中的表达[J].牙体牙髓牙周病学杂志,2002,12(4):197-200. 被引量：1
  • 7Haast W, Banas JA. Ligand-binding properties of the carboxyl-terminal repeat domain of streptococcus mutans glucan-binding protein A [J ]. J Bacteriology, 2000,182: 728 - 733.

二级参考文献6

  • 1[1]Banas JA, Russell RRB, Ferretti JJ. Sequence analysis of the gene for the glucan- binding protein of streptococcus mutans ingbritt [J]. Infect Immun, 1990,58:667
  • 2[2]Douglas CWI, Russell RRB. Effect of specific antisera on adherence properties of the oral bacterium streptococcus mutans [J]. Arch Oral Biol,1982,27:1039
  • 3[3]Russel RRB, Donald AC, Douglas WI. Fructosyltransferase activity of a glucan-binding protein from streptococcus mutans.J [J]. Gen Microbiol,1983,129:3243
  • 4[4]Smith DJ, Akita H, King WF, et al. Purification and antigenicity of a novel glucan-binding protein of streptococcus mutans [J]. Infect Immun, 1994,62:2545
  • 5[5]Sato Y, Yamamoto Y, Kizaki H. Cloning and sequence analysis of the gbpC gene encoding a novel glucan-binding protein of streptococcus mutans [J]. Infect Immun,1997,65(2):668
  • 6[6]Haast W, Banas JA. Ligand-binding properties of the carboxyl-terminal repeat domain of streptococcus mutans glucan-binding protein A [J]. J Bacteriology,2000,182(3):728

同被引文献10

  • 1Banas JA.Virulence properties of Streptococcus mutans.Frontiers in Bioscience,2004,9:1267-1277.
  • 2Shah DSH,Russell RRB.A novel glucan-binding protein with lipase activity from the oral pathogen Streptococcus mutans.Microbio,2004,150:1947-1956.
  • 3Ajdic P,McShan W,McLaughlin R,et al.Genome sequence of Streptococcus mutans UA159,a cariogenic dental pathogen.Proc Natl Acad Sci,2002,99:14434-14439.
  • 4Hinds J,Mahenthiralingam E,Kempsell KE,et al.Enhanced gene replacement in mycobacteria.Microbio,1999,145:519-527.
  • 5Nelson DL,Cox MM.Lehninger principles of biochemistry.3th ed.New York:Wortb Publishers,2000,931-977.
  • 6Tao L.Streptococcal integration vectors for gene inactivation and cloning.Methods Cell Sci,1998,20:59-64.
  • 7Yoshida A,Kuramitsu HK.Multiple Streptococcus mutans genes are involved in biofilm formation.Appl Environ Microbiol,2002,68:6283-6291.
  • 8Banas JA,Vickerman MM.Gluc an-binding proteins of the oral streptococci.Crit Rev Oral Biol Med,2003,14:89-99.
  • 9Yim HH,Rubens CE.Site-specific homologous recombination mutagenesis in group B streptococci.Methods Cell Sci,1998,20:13-20.
  • 10陈效友,李传友,马玙,刘冲,王敬慧,张雪峰,昌增益.卡介苗菌MDP1基因敲除技术的研究[J].中华结核和呼吸杂志,2004,27(3):183-187. 被引量：4

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临床口腔医学杂志
2005年 第10期

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