pCDisplay-4 vector

CD133, also known as Prominin-1, is expressed on stem cells in many tissues and tumors. In this work, researchers identified and characterized a single-chain variable fragment (scFv) for efficient and specific recognition of CD133.

Here, researchers used the VL and VH fragments of clone 7 anti-CD133 antibody to construct recombinant anti-CD133 scFv. VL and VH fragments were amplified by RT-PCR. Using these fragments and a DNA linker encoding Ser7Gly10Arg, scFv was constructed by performing overlap extension PCR. The resulting PCR products were analyzed by gel electrophoresis (Figure 1a). The scFvs were then cloned into the pCDisplay-4 vector, transformed into E. coli XL-1 BLUE, and subsequently infected with VCSM13 helper phage to release intact phage particles displaying the scFv library on their coat surface. The phage library was then screened for reactivity to CD133 by a standard panning process that involves incubating phage particles with CD133 protein-coated plates. Only a small fraction of the phage library was found to bind to the plate. These conjugates are then recovered, amplified in bacteria and screened by subsequent panning. After 3 such cycles, the phage conjugates were enriched 394-fold (Figure 1b). At this stage, 30 phage clones were randomly picked and their reactivity toward human CD133 was confirmed by phage ELISA (Figure 1c). The results show that CD133 scFv can recognize and bind to human CD133 protein, but the binding affinity is lower than its parent monoclonal antibody.

Figure 1. Cloning of anti-CD133 scFv by pCDisplay-4 vector. (Swaminathan S K, et al. 2013)