关键词: 呋喃它酮代谢物, 单链抗体, 噬菌体展示, 序列分析

Abstract:

Objective: To construct a phage single chain Fv (scFv) library against derivatives of furaltadone metabolites (AMOZ). Methods: The total RNA extracted from a hybridoma cell line (BC3-E8) secreting monoclonal antibodies against AMOZ was reverse-transcribed into cDNA by RT-PCR. The heavy chain (VH) and the light chain (VL) variable region genes were amplified respectively by PCR with the previously designed degenerate primer. The VH and VL genes were spliced into scFv fragment with a DNA linker encoding (G1y4Ser)3 by splicing overlap extension (SOE). Then the scFv fragment was cloned into the phagemid pCANTAB5E and after the cloning, the phagemid was transformed into the competent Escherichia coli TG1. With the rescue of helper phage M13K07, a phage scFv library was constructed. Ten positive clones were randomly selected and identified by PCR and double enzymatic digestion. Furthermore, the sequences of these positive clones were sent for sequencing and analyzed by the DNAMAN software. Results: VH, VL and scFv DNA fragments were amplified successfully. The constructed phage scFv library had a capacity of 1.2 × 106 and the titre was about 2.0 × 1010 PFU. PCR and double enzymatic digestion identification showed that the ratio of positive insert was high. Multiple sequence alignment showed that the difference of scFv sequences was 8.38%, and those of VH and VL sequences were 3.68% and 14.34%, respectively, and the discrepancy were mostly concentrated in corresponding nucleic acid sequences of the CDR antigen region. Conclusion: a scFv phage antibody library against derivaties of furaltadone metabolites has been constructed successfully. This will lay a foundation for the further enrichment and expression of scFv.

Key words: derivatives of furaltadone metabolites, single chain Fv (scFv), phage display, sequencing