Persic L, Righi M, Roberts A, Hoogenboom H R, Cattaneo A, Bradbury A
Societa Italiana per la Ricerca Scientifica, Roma, Italy.
Gene. 1997 Mar 10;187(1):1-8. doi: 10.1016/s0378-1119(96)00627-0.
We define intracellular immunization as the inhibition or inactivation of the function of a molecule by the ectopic intracellular expression of antibody binding domains which recognise the molecule. Such recombinant antibodies can be directed to different compartments of eukaryotic cells by means of previously defined targeting signals, thus permiting the study of any molecule in any cellular compartment for which an antibody is available. For this purpose, we have created a set of vectors based on the VHExpress vector described [Persic, L., Roberts, A., Wilton, J., Cattaneo, A., Bradbury, A. and Hoogenboom, H.R. (1997) An integrated vector system for the eukaryotic expression of antibodies or their fragments after selection from phage display libraries. Gene 187, 000-000], which has been modified to express scFvs (single chain fragments) linked to specific targeting signals. These permit the localisation of scFvs to different intracellular compartments: the endoplasmic reticulum (scFvE-er), the nucleus (scFvE-nuclear), the mitochondria (scFvE-mit), the cytoplasm (scFvE-cyto), and as secreted proteins (scFvE-sec). The function of these vectors has been assessed by the immunofluorescence of COS cells transiently transfected with constructs containing the alphaD11 scFv.
我们将细胞内免疫定义为通过识别该分子的抗体结合域的异位细胞内表达来抑制或失活分子的功能。此类重组抗体可借助先前确定的靶向信号被导向真核细胞的不同区室，从而能够研究任何可获得抗体的细胞区室中的任何分子。为此，我们基于已描述的VHExpress载体[Persic, L., Roberts, A., Wilton, J., Cattaneo, A., Bradbury, A.和Hoogenboom, H.R. (1997) 一种用于从噬菌体展示文库中筛选后在真核细胞中表达抗体或其片段的整合载体系统。基因187, 000 - 000]构建了一组载体，该载体已被修饰以表达与特定靶向信号相连的单链抗体片段（scFvs）。这些载体可使单链抗体片段定位于不同的细胞内区室：内质网（scFvE - er）、细胞核（scFvE - nuclear）、线粒体（scFvE - mit）、细胞质（scFvE - cyto）以及作为分泌蛋白（scFvE - sec）。这些载体的功能已通过用含有αD11单链抗体片段的构建体瞬时转染的COS细胞的免疫荧光进行了评估。
