Preparation Of Single Chain Antibody Variable Fragment Against Fumonisin B₁

Fumonisin, a kind of mycotoxin derived from Fusarium moniliforme, can pollute crops and their products widely. It has diverse toxicity that threatens the health of human beings and animals. This study intended to establish a method, which was based on recombinant single-chain antibody against fumonisin B1, to detect the contents of fumonisin B1 in food and feed quickly and efficiently.FB1 is a micromolecule and hapten, so it needs to couple with a macromolecule protein and the conjugate will become a complete antigen. In this research, fumonisin B1 (FB1) was purchased, and glutaraldehyde (GA) one-step method was used to prepare FB1-BSA conjugate and GA two-step method to prepare FB1-KLH conjugate.FB1-BSA was used as immunizing antigen and FB1-KLH was used for detection, and the both conjugates were proved by the following titer assay results of antiserum. Balb/c mice were immunized with FB1-BSA conjugate, and the titer of mice antiserum was assayed by ELISA after booster, which achieved the expectation. Then, the immunized mouse was killed and its spleen was separated. Total RNA was extracted from the spleen and the first strand of cDNA was obtained by reverse transcription. And the genes VH and VL, from cDNA template were used to construct gene scFv. Subsequently, phage antibody library was established after the gene scFv was cloned into phage plasmid pCANTAB 5E. ScFv panning from the library by phage display techniques was carried out. The soluble expression and identification of scFv were performed. Finally, we obtained one strain of phage antibody being able to produce scFv with high affinity.