期刊文献+

大肠杆菌表面CS3菌毛展示随机肽库的构建

Construction of a CS3 fimbria-displayed random peptide library on Escherichia coli cell surface
下载PDF
导出
摘要 目的:在肠毒素性大肠杆菌CS3菌毛表面构建 10肽随机肽库.方法:首先将原有的单酶切CS3菌毛呈现载体改造为双酶切载体,并证实改造后的载体能正确形成CS3菌毛.同时设计合成2条寡核苷酸序列,链1含有1个10肽随机编码序列(NNS)10, 链2可与链1的3′端互补.两条链经过退火、延伸、酶切和回收与经过同样酶切的呈现载体连接,连接产物纯化后分多次电击转化,获得随机肽库.随机挑选10个克隆进行测序并对测序结果进行分析.结果:获得1个库容量为1.8×106大小的随机肽库.测序结果显示,所构建肽库的基本框架与预期设计相符,4个寡核苷酸出现的频率也与理论值相接近.结论:在肠毒素性大肠杆菌CS3菌毛表面成功构建库容量为1.8×106大小的10肽随机肽库.为下一步利用肽库进行筛选奠定了基础. AIM: To construct a CS3 fimbria-displayed random peptide library on the Escherichia coli cell surface. METHODS: Firstly, a new display vector with double restriction sites was reconstructed, and the ability of the new vector to form CS3 fimbriae on E. coli surface was confirmed by Western blot and transmission electron microscopy. Then two oligonucleotides were synthesized, one of which contained a random oligonucleotide encoding region (NNK)10 and the other was its complement. The two synthesized oligonucleotides was annealed and then extended by Klenow Fragment. The double-stranded oligonucleotides were digested by Xho Ⅰ and BamH Ⅰ and purified by PAGE, then inserted into the digested display vector. The ligation product was purified and electroporated into XL1-Blue. Ten randomly selected clones were sequenced and the sequences were analyzed.RESULTS: A library with diversity of 1.8 × 10^6 was obtained. The sequencing results confirmed the basic frame of the constructed library to be correct and the bases of A, T, G, and C were distributed randomly.CONCLUSION: A random ten-peptide library is displayed on the Escherichia coli cell surface using CS3 fimbriae display system.
出处 《世界华人消化杂志》 CAS 北大核心 2006年第2期158-162,共5页 World Chinese Journal of Digestology
基金 国家高技术研究发展863计划项目.No.21004AA215212
关键词 CS3菌毛 细菌表面展示 随机肽库 CS3 fimbria Bacterial surface display Random peptide library
  • 相关文献

参考文献14

  • 1Georgiou G, Stathopoulos C, Daugherty PS, Nayak AR, Iverson BL, Curtiss R 3rd. Display of heterologous proteins on the surface of microorganisms:from the screening of combinatorial libraries to live recombinant vaccines. Nat Biotechnol 1997, 15:29-34.
  • 2Lu Z, Murray KS, Van Cleave V, LaVallie ER,Stahl ML, McCoy JM. Expression of thioredoxin random peptide libraries on the Escherichia coli cell surface as functional fusions to flagellin: a system designed for exploring protein-protein interactions.Biotechnology 1995, 13:366-372.
  • 3Zhao S, Lee EY. A protein phosphatase-l-binding motif identified by the panning of a random peptide display library. J Biol Chem 1997, 272:28368-28372.
  • 4Herrmann A, Pieper M, Schrader J. Selection of cell specific peptides in a rat carotid injury model using a random peptide-presenting bacterial library. Biochim Biophys Acta 1999,1472:529-536.
  • 5Brown CK, Modzelewski RA, Johnson CS, Wong MK.A novel approach for the identification of uni-que tumor vasculature binding peptides using an E. coli peptide display library. Ann Surg Oncol 2000,7:743-749.
  • 6Kjaergaard K, Schembri MA, Klemm P. Novel Zn(2+)-chelating peptides selected from a fimbriadisplayed random peptide library. Appl Environ Microbiol 2001, 67: 5467-5473.
  • 7Jalajakumari MB, Thomas CJ, Halter R, Manning PA. Genes for biosynthesis and assembly of CS3 pili of CFA/II enterotoxigenic Escherichia coli: novel regulation of pilus production by bypassing an amber codon. Mol Microbiol 1989, 3:1685-1695.
  • 8高荣凯,张兆山,李淑琴,黄翠芬.外源抗原表位在大肠杆菌CS3菌毛上的呈现[J].高技术通讯,2000,10(5):16-20. 被引量:3
  • 9Bakker D, van Zijderveld FG, van der Veen S,Oudega B, de Graaf FK. K88 fimbriae as carriers of heterologous antigenic determinants. Microb Pathog 1990, 8:343-352.
  • 10Der Vartanian M, Girardeau JP, Martin C, Rousset E,Chavarot M, Laude H, Contrepois M. An Escherichia coli CS31A fibrillum chimera capable of inducing memory antibodies in outbred mice following booster immunization with the entero-pathogenic coronavirus transmissible gastroenteritis virus.Vaccine 1997, 15:111-120.

二级参考文献1

共引文献2

相关作者

内容加载中请稍等...

相关机构

内容加载中请稍等...

相关主题

内容加载中请稍等...

浏览历史

内容加载中请稍等...
;
使用帮助 返回顶部