Construction And Screening Of A ScFv Antibody Library Against Organophosphorus Peesticides

Traditional analytical techniques, which are time-consuming due to the complicatedsample preparations, need professional operators and are expensive and inconvenient for theapplication in the field of quick and instant multitudinous samples screening. Phage displayantibody library technology, which is as important as monoclonal antibody technology in therealm of producing specific antibodies and detection of pesticides, with its advantages such ashigh-performance, time saving and so on, gives a bright prospect in the development ofimmunoassay for pesticides. In this study, a phage display antibody library against Ops wassuccessfully constructed, for the objective to use single chain variant fragment (ScFv) toquickly and effectively detect the residues of Ops in the food.On the basis of the work that has been done, the mouse with the highest antiserum titerswas used to extract the total RNA from its spleen cells. After the RT-PCR，immunoglobulinvariable heavy chain region (V_H) genes and variable light chain region (V_L) genes，which wasabout340bp and325bp respectively，were first amplified. After being purified, equal mol V_Hand V_Lgenes were jointed together through splicing by overlap extension(SOE)，by the use offragments containing parts of the linker. The ScFv genes were about750bp through the processof PCR, which applied the primers that contain the slice sites of restriction endonuclease.Following Sfi I and Not I’ digesting，the ScFv gene fragments were ligated into the phagevector PCANTAB5E that had been digested using the same enzymes and were transformedinto competent E.coli TGI cells. Plasmid electrophoresis showed that ScFv genes were insertedand the aimed DNA fragments were attained after PCR. With the help of helper phage M13K07,the transformed bacterium were then reinfected. After overnight incubation in specific mediumand sedimenttion by polyethylene glycol(PEG), the supernant was collected. That is the originalphage display antibody library.The unspecific phages were eluted after panning by the use of Poly-L-Lysine artificialantigen. Then log phase E.coli TG1was added. Reinfected by M13KO7and sedimented byPEG, the first round of panning was over. Repeating for six times, the capacity of the phagedisplay antibody library was2.22×106pfu/mL.HRP/anti-M13monoclonal was the second antibody in the indirect ELISA while M13KO7 was the negative control and one strain of phage antibody being able to produce ScFv with highaffinity was obtained. Used it to infect log phase E.coli HB2151to produce the soluble antibodyby the inducible expression of IPTG. Detected by ELISA, the result was positive, indicating thatthe progress was successful.