Construction Of ScFv Phage Antibody Library Against Milbemycin Oxime

Phage display antibody library technology, which has been widely used in the diagnosis and treatment of disease, and functional protein expression, was a high-performance, time saving, and effective technique for producing specific antibodies. Several high affinity antibodies against micro-molecular substances, such as pesticides or veterinarian, have been reported to be successfully obtained by phage display antibody technology, which gave it a bright prospect in the development of immunoassay for pesticide. In this study, a phage display antibody library against milbemycin oxime was successfully constructed, the objective was to develop a specific phage display library for screening antibodies against compounds with 16-membered macrocyclic backbone.1. Immunogen synthesizing and animal immunizing. The hapten 5-o-Succinoylmilbemycin-oxime were bound to BSA by the carbodiimide activation. Four Balb/c mice were immunized with MILO-BSA, after the titers of antiserum reached 1:25600. A MILO-OVA -based CI-ELISA was conducted with the antiserum, the inhibition curve show range for detection milbemycin oxime was between 20% and 70%.2. The obtained of immunoglobulin variable genes and splicing. The total RNA was extracted from spleen cells of mouse with the highest antiserum titers. Using the RT-PCR, immunoglobulin variable heavy chain region genes (V_h) and variable light chain region genes (V-l), which were about 360bp and 340bp respectively, are first amplified. The approximately mol V_H and V_L genes which have been purified were jointed together through splicing by overlap extension (SOE), using the fragments contain parts of the linker. The ScFv genes were about 750bp generated by PCR, using the primers that contain the slice sites of restriction endonuclease. 3. The construction of ScFv phage display antibody library. After digesting with SfiI and NotI, the ScFv gene fragments have been ligated into the phage vector pCANTAB5E that have been digested using the same enzymes and were transformed into competent E.coli TG1 cells. Plasmid electrophoresis showed that there were inserting fragments and the aimed fragments were amplified using PCR, with primers R1 and R2. The transformed bacterium were infected with helper phage M13K07, sedimented the cell and collected the supernant. Then a phage display antibody library containing 2.4×10 p.f.u/mL bacteriophage plaque has been successfully constructed .The reseach are help for establishing new screen methods of antibody library and antibody panning.