pCANTAB-5E

The ras gene plays an important role in the occurrence and development of human tumors. Neutralizing Ras proteins in the cytoplasm may be an effective way to block ras signaling. In this study, the researchers prepared anti-p21Ras single-chain fragment variable antibodies (scFv) and studied their immunoreactivity with human tumors. Studies suggest that scFvs may contribute to ras signaling blockade and may be potential therapeutic antibodies for ras-derived tumors.

In this study, the scFv gene consists of a heavy chain variable region (VH) and a light chain variable region (VL), connected by a neutral linker (Figure 1a). VH (340 bp) and VL (320 bp) were amplified from cDNA of KGH-R1 hybridoma. Then, the two were connected to the DNA linker by overlap extension PCR to construct a 750 bp scFv gene (Figure 1b). The scFv gene library was ligated to the phagemid pCANTAB-5E, and then competent TG1 cells were transformed and grown in plates to obtain 1 × 10⁷ TG1 colonies transformed with the recombinant phage plasmid (Figure 1c). All TG1 colonies were collected and co-cultured with M13KO7 helper phage to establish a scFv phage display library in which the scFv-g3p fusion protein was expressed at the phage tip. To obtain phage expressing anti-p21Ras scFv antibodies from the library, the library was selected and panned sequentially using H-p21Ras, K-p21Ras, and N-p21Ras proteins. After three rounds of panning, the eluted phages were maintained at 1 × 106 pFU/mL, and the number of input phage pools was maintained at 1 × 10⁹ pFU/mL (Figure 1d).

Figure 1. Preparation of anti p21Ras scFv. (Yang, Ju-Lun, et al. 2016)