Molecular Design,Expression And Recognition Mechanism Of Engineering Antibodies Against Pesticides

To prepare engineering antibodies against pesticides,organophosphorous insecticides triazophos and parathion were chosen as model compounds.Firstly,a water-soluble adjuvant named QuickAntibody(QA)was introduced into the procedure of mouse immunization for the development of target-specific monoclonal antibodies(mAb).QA is a ready-to-use water-compatible solution that can be easily mixed with immunogen before immunization through the simple operation of intra-muscular injection.QA treatments gave little adverse effects to the experimental animals,and were preferable in harvesting splenocytes during the steps of cell fusion.The efficiency of gaining immune-positive hybridomas was satisfactory(>30%),and the resultant mAbs showed similar sensitivities to those previously reported mAbs which were prepared by Freund's Ajuvants.The new-developed mAbs showed high-affinity to triazophos(IC50 of 0.91 ng/mL),and to parathion(IC50 of 8.18 ng/mL),respectively.Using the hybidoma cell lines that secreted anti-triazophos mAb or anti-parathion mAb with high affinity and specificity,the variable region genes of these mAbs were amplified with isotype-specific universal primer sets by RT-PCR.The variable regions of heavy chain(VH)and light chain(VL)were then assembled as single-chain variable fragment(scFv)via splicing by overlap extension polymerase chain reaction(SOE-PCR)with a(Gly4Ser)3 linker in VH-VL orientation.After sequencing identification,the scFv gene was respectively constructed into prokaryotic expression system,Bac-to-Bac baculovirus expression system,and Pichia pastoris expression system.The soluble scFv was identified by western blot and indirect competitive enzyme linked immunosorbent assay(ic-ELISA).Finally,triazophos-speicific and parathion-specific scFvs with similar sensitivities to their corresponding mAbs were successfully constructed and expressed in P.pastoris economically and effectively.The effect of orientation of VH and VL to scFv was studied.The sensitivities (IC50)of two domain order of scFvs were not changed,scFvs against triazophos were around 0.4?0.5 ng/mL,scFvs against parathion were around 8.5?10.5 ng/mL,which were similar to those of the parent mAbs.The VL-VH orientation of anti-triazophos scFv and anti-parathion scFv were both expressed in a high level,with the expression amount around 700 mg/L.Afterwards,taken anti-triazophos scFv as model,several bi-valent tandem scFvs were designed and expressed in different domain order and linker length.The activities of bi-valent tandem scFvs were compared and found that it was not related to the linker length and the mono-valent scFv,but it was related to the domain order.VL gene in the N-terminus was conducive to obtain the high-affinity product in P.pastoris,with the high affinity(the IC50 value around 0.8?0.9 ng/mL)and selectivity to triazophos.On the basis of the study above,different bi-specific tandem scFvs against triazophos(T)and parathion(P)were further designed,expressed,and identified by ic-ELISA.VL gene in the N-terminus was proved to be the important factor for obtaining the high-activity antibody.Bi-specific tandem scFv in the model of VLT-VH-T-(Gly4Ser)-VLp-VHp showed good recognition to coating antigens of triazophos and parathion.However,its sensitivity to triazophos and parathion were both reduced around seven times compared with their own mono-valent scFv.Moreover,its selectivity to parathion was also changed.As a result,VL gene in the N-terminus might be a good choice to design scFv,bi-valent tandem scFv and bi-speicific tandem scFv antibody.Besides,the interference and unexpected binding between variable regions from different antibody should be taken into account when designing the bi-speicific tandem scFv antibody.According to the amino acid sequences of the triazophos-specific scFv and the parathion-specific scFv,their three dimensional structures were further constructed through homologous modeling by Discovery StudioTM software(Accelrys).The molecular docking study of the interaction between scFv and the target analyte was performed and the key amino acid residues for antibody recognition were found.Besides,the binding affinity and kinetics between scFv and the target analyte were investigated by a surface plasmon resonance(SPR)biosensor to confirm the strong immunoreactions.These results would be useful to explore the mechanism of antibody binding with the target pesticide and to further design or reform engineering antibody with high affinity and stability.