Screening of special sc Fv antibody against human p53 protein by yeast two-hybrid system
ZHENG Meiyun;LI Miaojun;SHEN Guoying;LIN Suying;LI Huibo;MENG Chun;WANG Hang;College of Biological Science and Engineering,Fuzhou University;
Objective To construct a mouse single-chain fragment variable( sc Fv) 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 p GBKT-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 VH-linker-VLfragment by reverse transcription-PCR and overlapping PCR. And then the VH-linker-VLfragment constructed on the vector p GADT7 was transformed into the AH109 cells containing the bait vector. The positive clones were screened on the nutrition auxotrophic media. The characteristics of sc Fv were verified by immunocytochemistry. Results The bait vector p GBKT7-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 VH-linker-VLwas successfully obtained. The captured vector harboring VH-linker-VLfragments was constructed. We screened out the human P53 sc Fvs by the Y2 H system. And sc Fv1 /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 sc Fvs with a good affinity for human P53 protein by Y2 H system,which provided a new way for screening sc Fv.