Chinestra Patrick, Lajoie-Mazenc Isabelle, Faye Jean-Charles, Favre Gilles
INSERM UMR 1037, Cancer Research Centre of Toulouse, Claudius Regaud Cancer Institute, University of Toulouse, Toulouse, France.
Methods Mol Biol. 2012;827:283-303. doi: 10.1007/978-1-61779-442-1_19.
We describe a phage display approach to select active Rho-specific scFv sensors. This in vitro technique allows preserving the antigen conformation stability all along the selection process. We used the GTP locked RhoBQ63L mutant as antigen against the Griffin.1 library composed of a human synthetic V(H) + V(L) scFv cloned in the pHEN2 phagemid vector. The method described here has permitted to identify an scFv that discriminates between the activated and the inactivated form of the Rho subfamily.
我们描述了一种用于筛选活性Rho特异性单链抗体片段(scFv)传感器的噬菌体展示方法。这种体外技术能够在整个筛选过程中保持抗原构象的稳定性。我们使用GTP锁定的RhoBQ63L突变体作为抗原,针对克隆于pHEN2噬菌粒载体中的人源合成V(H)+V(L) scFv组成的Griffin.1文库进行筛选。本文所述方法已成功鉴定出一种能够区分Rho亚家族激活形式和失活形式的scFv。