Construction and Structure Prediction of Murine Single Chain Fv Gene against Carbofuran

The gene of single chain Fv fragment (scFv) was constructed against carbofuran and the physical and chemical characteristics, secondary structure and tertiary structure of the scFv was predicted using computer assisted modeling. The variable region gene of the heavy and light chains were amplified respectively from 5D1 hybridoma cells that can secret monoclonal antibodies with high activity and specificity against carbofuran. These two fragments were then spliced together through a flexible linker to scFv by using splicing overlap extension (SOE) PCR. The scFv gene was named cbf and the sequence of scFv gene was measured. The physical and chemical characteristics, secondary and tertiary structure were predicted using Internet and corresponding software. The whole scFv gene contained 750 bp was cloned successfully and encoded 228 amino acids. Theoretically, the scFv was relative molecular masses 26315.1, the isoelectric point (pI) was 5.66 and the antibody belongs to acid protein. The predictied secondary structure showed the protein contained 18 α-helix, 108 β-sheet, 24 β-turn and 93 random coli. The mimic tertiary structure model of scFv indicted the linker was isolated from VH and VL. The VL, as well as the VH, was involved in composing the hydrophobic"pocket" which was beneficial to the antigen binding. The construction and analysis of scFv against carbofuran laid the foundation for the further research into expression, purification and bioactivity of the genetic engineering antibody.