Hatfield C, Duus K M, Chen J, Jones D H, Grose C
University of Iowa College of Medicine, Iowa City, USA.
Biotechniques. 1997 Feb;22(2):332-7. doi: 10.2144/97222rr02.
We describe a rapid PCR method that directly inserts an epitope tag into an open reading frame (ORF) to facilitate protein detection. This project was performed within a varicella-zoster virus (VZV) system. In earlier work, we produced a monoclonal antibody (MAb 3B3) to one VZV ORF called gE. MAb 3B3 bound to its epitope under extreme denaturing conditions. To further characterize the epitope, we devised a technique that identified the epitope by its insertion into another protein of interest. The 3B3 epitope was mapped to 11 residues (residues 151-161; QRQYGDVFKGD) in the gE ectodomain by using the technique of recombination PCR. At the same time, the 3B3 epitope was inserted in-frame into another VZV protein for which no MAb was available. The end result, VZV gL3B3.11, was a unique construct possessing a 33-bp insertion that expresses gL-3B3 protein recognized by the MAb 3B3. The 3B3 epitope was verified to be both highly functional and stable. An important advantage of this recombination PCR method of epitope mapping and tagging is that the epitope sequence can be inserted anywhere along the nucleotide sequence of an ORF, regardless of existing restriction sites.
我们描述了一种快速聚合酶链反应（PCR）方法，该方法可将表位标签直接插入开放阅读框（ORF）以促进蛋白质检测。本项目是在水痘-带状疱疹病毒（VZV）系统中进行的。在早期工作中，我们针对一种名为gE的VZV ORF制备了单克隆抗体（MAb 3B3）。MAb 3B3在极端变性条件下与其表位结合。为了进一步表征该表位，我们设计了一种技术，通过将其插入另一种感兴趣的蛋白质中来鉴定表位。通过重组PCR技术，3B3表位被定位到gE胞外域的11个残基（残基151 - 161；QRQYGDVFKGD）上。同时，3B3表位被读框内插入到另一种没有可用MAb的VZV蛋白中。最终产物VZV gL3B3.11是一种独特的构建体，具有一个33 bp的插入片段，表达可被MAb 3B3识别的gL - 3B3蛋白。3B3表位被证实具有高度功能性和稳定性。这种用于表位定位和标记的重组PCR方法的一个重要优点是，表位序列可以沿着ORF的核苷酸序列插入到任何位置，而无需考虑现有的限制性酶切位点。
