Construction And Screening Of Phage Display Antibody Library Specific For Fenvalerate

Wide application of various types of pesticides has greatly improved the yield of vegetables, fruits, tea and crops. Otherwise, pesticide residues onvegetables, fruits, tea and crops lead to a great deal of human health problems. Each year, it is reported that there are many food safety accidents caused by pesticide residues. All countries have made a law to limit pesticide residues in agricultural products. For human health and Chinese food export, it is quite necessary to establish a quick and easy method to detect pesticide residues. In this study, we constructed fenvalerate genetically engineered antibody library using phage display technique, and screened the primary library to pick out fenvalerate specific scFv.Total RNA was extracted from spleen of Balb/c mouse that was immunized with FAAE-KLH. Then mRNA was purified from total RNA and cDNA was obtained by reverse transcription. The cDNA was used as template to amplify the variable regions of heavy chain and light chain of antibody genes using degenerate primers. The size variable regions of heavy chain and light chain were 350bp and 325bp, respectively. The light chain variable regions and heavy chain variable regions were linked into scFv fragments by overlap extension PCR. The size of scFv fragments were about 750bp. scFv fragments and phagemid were digested by Sfi I and Not I simutaniously. The digested products were ligated and then transformed into TG1 competent cells. Primary antibody library was successfully constructed with capacity of 5.16×106 clones. After activation and amplification, the helper phage titer was reached 1.3×1013 cfu/mL. After being infected with helper phage, in the TG1 cells which containing recombinant phagemid vector, scFv would be displayed on phage surface. Based on the principle of antigen-antibody specific interaction, the primary antibody library was panned. After five rounds of panning, the specific phage clones were enriched. The recombinant phage clones were tested using PCR method and the result indicated that the positive rate reached 100% since second round panning. Fifteen positive clones were picked out according to phage ELISA experiment including 21,22,23,24,25,26,27,28,41,42,43,51,52,54, and 58. The five clones 22, 26,27,51,52 were sequenced and the results suggested that heavy chain belonged to IGHV1 family and encoded 119 animo acid, light chain belonged to IGKV4 family encoded 108 animo acid.These results could be the foundation for preparation of fenvalerate genetically engineered antibodies and development of correspinding ELISA detection methods.