期刊文献+

蛋白质多肽文库构建中限制性显示技术的接头设计

The Flexible Adapter Design of Restriction Display Technique in Constructing Peptide Library
下载PDF
导出
摘要 为了探讨限制性显示(RD)技术在构建蛋白质多肽文库中灵活的接头设计,分别根据原核表达载体pET22b以及酵母表达载体pNMT-TOPO设计了三套接头,三套接头依次增加一个碱基以保证与之连接的片段总有可能表达正确的开放阅读框.然后以HIV-1B亚型代表株U26942全基因质粒DNA为对象,利用RD技术分别建立了相应的蛋白质多肽文库.从每个库中各随机挑选12个克隆进行测序分析并进行蛋白质表达预测.结果从原核表达文库中获得了一个可以表达HIVPol多肽的克隆,SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)结果显示该克隆在细菌BL21(DE3)中有较高的表达,蛋白质印迹为阳性,与理论预测相符.这些结果提示,RD技术是一种建立基因组随机多肽文库的新方法,该方法灵活的接头设计可以满足不同的表达载体需求. To show the flexible adapter design of restriction display (RD) technique in constructing peptide library, two series of adapters were designed according to vector pET22b in E.coli and vector pNMT-TOPO in S.pombe. Each series of adapter has three in turn one-base-increment adapters, which allowed the inserted DNA fragment probably expressed in the correct reading flame. HIV-1 subtype B whole gene was used as an example, and flagments expression libraries with two different adapters were constructed by RD technique, randomly 12 clones from each library were sequenced for translation analysis. As a result, a clone from the prokaryotes library was obtained, which could encode HIV Pol peptide. Then the positive plasmid was induced to express protein in E.coli BL21 (DE3). SDS-PAGE and Western blot showed positive result, which is consistent with expected. It can be concluded that RD technique is a new approach for constructing genome random peptide library, and its flexible adapters design can meet with kinds of expression vectors.
出处 《生物化学与生物物理进展》 SCIE CAS CSCD 北大核心 2005年第8期758-764,共7页 Progress In Biochemistry and Biophysics
基金 广州市重大科技攻关项目(2001-Z-005-01)~~
关键词 限制性显示 接头设计 多肽文库 restriction display, flexible adapter design, peptide library
  • 相关文献

参考文献10

  • 1李凌,马文丽,祝骥,朱利娜,宋艳斌,吴清华,郭秋野,郑文岭.应用RD-PCR技术制备HIV基因芯片探针[J].中国生物化学与分子生物学报,2002,18(1):105-109. 被引量:13
  • 2马文丽 郑文岭 Jame FB 等.限制性显示(RD-PCR):一种新的差异显示技术[A].孙志贤主编.全军生物化学与分子生物学研究进展[C].北京:军事医学科学出版社,1998.113-4.
  • 3毛向明,马文丽,姜立,郑文岭.应用RD-PCR方法制备K562细胞表达谱芯片基因探针[J].第一军医大学学报,2002,22(6):548-550. 被引量:8
  • 4Jiang L, Ma W L, Zheng W L, et al. Preliminary study of the differentiation-related genes from K562 cells induced by Hemin with the method of RD-PCR. Acta Anat Sini, 2001, 32:35-360.
  • 5Li L, Ma W L, Zhu J, et d. A modified restriction display PCR methods in sample-labelling of DNA microarray. J Virol Methods,2003,114 (l): 71 -75.
  • 6Davis C A, Benzer S. Generation of cDNA expression libraries enriched for in-flame sequences. Proc Natl Acad Sci USA, 1997, 94(6): 2128-2132.
  • 7Farabaugh P J. Programmed translational Frameshifting.Microbiological Review, 1996, 60 (1): 103-134.
  • 8zur Megede J, Otten G R, Doe B, et al. Expression and immunogenicity of sequence-modified human immunodeficieney virus type 1 subtype B pol and gagpol DNA vaccines. J Virol, 2003,77 (11): 6197-6207.
  • 9Sambrook J, Fritsch E F, Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd. New York: Cold Spring Harbor Laboratory Press,1989. 16-572.
  • 10Bao Z, Wenli M, Qinghua W, et al. Construction of a cDNA fragment library from SH-SY5Y cells using restriction display PCR.Br J Biomed Sci, 2002, 59 (1): 35-37.

二级参考文献6

共引文献31

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