Objective: To construct a mouse single-chain fragment variable (scFv) antibody library specific to human P53 by overlapping PCR method and screen the single chain antibodies against human P53 protein with the yeast two-hybrid (Y2H) system.
Methods: The bait vector pGBKT-p53 expressing P53 protein in yeast AH109 cells was constructed by means of genetic engineering method. The total RNA which was extracted from the P53-immunized mouse spleen tissue was used to synthesize the single chain V(H)-linker-V(L) fragment by reverse transcription-PCR and overlapping PCR. And then the V(H)-linker-V(L) fragment constructed on the vector pGADT7 was transformed into the AH109 cells containing the bait vector. The positive clones were screened on the nutrition auxotrophic media. The characteristics of scFv were verified by immunocytochemistry.
Results: The bait vector pGBKT7-p53 was constructed successfully and transformed into AH109 cells. It had no self-activation in the yeast cells and no toxicity to the host. The library of V(H)-linker-V(L) was successfully obtained. The captured vector harboring V(H)-linker-V(L) fragments was constructed. We screened out the human P53 scFvs by the Y2H system. And scFv1/2/3 was proved to have a good affinity for human P53 protein, so it could be used for the identification of P53 protein in cells and tissues.
Conclusion: We obtained human P53 scFvs with a good affinity for human P53 protein by Y2H system, which provided a new way for screening scFv.