Preparation and characterization of a murine single chain antibody library against Aspergillus fumigatus

Objective To construct a murine single chain antibody(scFv)library against galactomannan(GM)of Aspergillus fumigatus with phage display technique and to enrich and select specific anti-GM single chain antibodies and to identify the antibody library.Methods The total RNA was extracted from the spleen of mice immunized with Aspergillus fumigatus GM.The antibody VH and VL genes were amplified with real-time reverse transcription PCR(RT-PCR),and the VH and VL fragments were linked by overlapping PCR.The linked scFv fragments were transformed into pCANTAB5E phage display system,and the phage display library of scFv was constructed with the assistance of the helper phage.Immunotubes coated with gradient GM antigens were used for the screening of the murine natural source scFv phage antibody library.After four rounds of "absorption-elution-amplification" panning,positive clones obtained were sequenced and binding activity of scFv clones were detected with phage-enzyme-linked immunosorbent assay(ELISA).Results The phage display library of scFv against Aspergillus GM was constructed,which was about 1.8×10⁷ clones in capacity.Using Aspergillus GM coated immune test tubes,the phage titers were increased after 4 rounds of panning.Twenty clones with different gene sequence of variable region were randomly selected after 4 rounds of panning.The diversity of VH and VL was confirmed in the single chain antibody library.Ninety clones were tested for binding specificity to Aspergillus GM.Phage-ELISA results showed that 6 clones have specific binding reactions only with Aspergillus GM,while no binding reaction with Candida albicans.Conclusion A murine scFv phage display library was constructed and identified preliminarily.The antibody library constructed presents a high connection rate of variable fragments,good diversity and large storage capacity.ScFv clones have specific binding activity with Aspergillus GM.The study provides a base for further screening of high specific antibody against galactomannan of Aspergillus fumigatus.