中国生物工程杂志

Objective: To clone the genes encoding human anti-DNA-PKcs antibodies from large phage antibody library for the use of cancer therapy or diagnosis in future. Method: Human DNA-PKcs protein fragments with high antigenicity and low homologous to other proteins were defined through antigenicity analysis and BLAST searching. After prokaryotic expression and purification, the protein segments were coated onto immuno-tube. Panning of large scale phage library against antigen was conducted to select specific antibodies against DNA-PKcs. The binding activity and specificity were tested by ELISA method. Soluble scFv was prepared through transfecting HB2151 with the selected phage antibody genes. Result: 250 AA of DPK3 and 257 AA of DPK4 segments were defined as high antigenicity and low homology to other protein through bioinformatic analysis. After 4 rounds of panning, 26 clones were identified with the specific binding ability with DPK3, and 31 clones with DPK4. DNA fingerprinting revealed 5 individual positive clones to DPK3 and 21 individual positive clones to DPK4. Sequencing analysis showed that variable regions of these ScFvs belong to different subgroups. Conclusion: Human DNA-PKcs antibody genes were successfully obtained from large scale phage antibody library.