Construction and quality examination of murine naive T7 phage display antibody library.

摘要

A naive single-chain variable fragment (scFv) antibody library of BALB/c mouse was constructed using T7 phage and the quality was examined. Fifty-five primers were designed to clone variable regions of light (440 bp) and heavy (450 bp) chains of antibody genes from non-immune mice. After overlap extension PCR, scFv fragments about 820 bp were obtained. Primary antibody library which titer reached 1.74x107 pfu was constructed after package. After amplification the titer reached 1.78x1011 pfu/ml. Ten random clones were amplified and five clones were sequenced to determine the quality of the library. Random PCR indicated that 90% phages contained desired fragments. Distinct difference among the five sequenced scFv fragments indicated good quality of the library. Phosmet conjugated with carrier protein was used as ligand to screen the library and specific plaques could be isolated. These results demonstrated that the library could be used as an effective platform to screen specific antibodies against certain molecules.