A simpler and more cost-effective peptide biosynthetic method using the truncated GST as carrier for epitope mapping.
A simpler and more cost-effective peptide biosynthetic method using the truncated GST as carrier for epitope mapping.
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使用截短的 GST 作为表位作图载体的更简单且更具成本效益的肽生物合成方法
DOI：
10.1371/journal.pone.0186097
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发表时间：
2017
期刊：
影响因子：
3.7
通讯作者：
Xie Y
中科院分区：
文献类型：
--
作者：
Xu WX;Wang J;Tang HP;Chen LH;Lian WB;Zhan JM;Gupta SK;Ji CN;Gu SH;Xie Y
需要开发更好的方法来绘制表位图和/或识别抗体识别基序。在此，我们介绍了一种改进的生物合成肽(BSP)方法，该方法以新开发的pXXGST-3为载体，在pXXGST-1的克隆位点上含有病毒E7基因。使用pXXGST-3而不是pXXGST-1至关重要，因为它使BSP方法的使用更简单、更容易执行，并且更具成本效益地进行表位映射。这些优点体现在两个方面：1)在BamH I和Sal I酶切位点之间插入315个碱基，便于回收双酶切产物，大大减少了自连接克隆的产生；2)用聚丙烯酰胺凝胶电泳法筛选表达8/18肽的重组(r-)克隆时，不再需要控制蛋白。该方法包括以下核心步骤：(I)设计编码重叠的8/18肽的正链和负链DNA片段；(Ii)设计的DNA片段的化学合成；(Iii)利用表达每个8/18肽的pXXGST-3载体与截短的GST188蛋白融合，开发r-克隆；(Iv)通过在SDS-PAGE凝胶上运行每个诱导克隆的细胞球，然后对每个验证的r-克隆的插入DNA片段进行测序，筛选r-克隆；以及(V)用单抗或多克隆抗体进行Western blotting。这种改进的GST188-BSP方法为表位定位提供了一个强有力的替代工具。
There is a need to develop better methods for epitope mapping and/or identification of antibody-recognizing motifs. Here, we describe improved biosynthetic peptide (BSP) method using a newly developed plasmid pXXGST-3 as vector, which has a viral E7 gene in the cloning sites of pXXGST-1. It is crucial to employ pXXGST-3 instead of pXXGST-1, since it makes use of the BSP method simpler and easier to perform, and more cost-effective for epitope mapping. These merits are embodied in two aspects: i) convenient recovery of double enzyme-digested product due to the existence of 315 bp inserted between BamH I and Sal I sites, and thus greatly reducing the production of self-ligation clones, and ii) no longer requiring control protein when screening recombinant (r-) clones expressing 8/18mer peptides by running polyacrylamide gel electrophoresis. The protocol involves the following core steps: (i) design of plus and minus strands of DNA fragments encoding overlapping 8/18mer peptides; (ii) chemical synthesis of the designed DNA fragments; (iii) development of r-clones using pXXGST-3 vector expressing each 8/18mer peptide fused with truncated GST188 protein; (iv) screening r-clones by running the cell pellets from each induced clone on SDS-PAGE gel followed by sequencing of inserted DNA fragments for each verified r-clone; and (v) Western blotting with either monoclonal antibodies or polyclonal antibodies. This improved GST188-BSP method provides a powerful alternative tool for epitope mapping.
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影响因子：
2.7
作者：
Lenstra JA;Kusters JG;van der Zeijst BA
通讯作者：
van der Zeijst BA
影响因子：
4.1
作者：
He, Ya-ping;Xu, Wan-xiang;Xie, Yi
通讯作者：
Xie, Yi
DOI：
10.1016/j.bbrc.2004.05.066
发表时间：
2004-07-02
影响因子：
3.1
作者：
Hua R;Zhou Y;Wang Y;Hua Y;Tong G
通讯作者：
Tong G
影响因子：
3.7
作者：
Christensen, ND;Dillner, J;Galloway, DA
通讯作者：
Galloway, DA
影响因子：
5.5
作者：
Slupetzky K;Gambhira R;Culp TD;Shafti-Keramat S;Schellenbacher C;Christensen ND;Roden RB;Kirnbauer R
通讯作者：
Kirnbauer R