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