Preparation And Homology Modeling Of Anti-triazophos Engineering Antibody

To prepare the engineering antibody,hybridoma cells secreting antibody with high affinity and specificity were screened by hybridoma technology,from which variation region genes of the antibody would be cloned.Based on the anti-triazophos antibody produced by the selected cells,a direct competitive enzyme-linked immunosorbent assay(ELISA) for triazophos was developed.The influence of several physicochemical factors on the immunoassay was studied.The optimized ELISA has been used to quantify triazophos in foods and environmental samples spiked at different amounts.The excellent recoveries achieved not only confirmed the potential of the immunoassay for monitoring of triazophos in food and environmental samples, but also proved the hybridoma cell was good enough to prepare the engineering antibodies.From the hybidoma cell lines,which can secrete anti-triazophos McAb with high affinity and specificity,the variable region genes of the McAb were amplified with universal primers by RT-PCR.To construct single-chain Fv antibody(ScFv) gene, methods splicing by overlap extension(SOE) was used for stringing variable region genes of heavy chain and light chain with a(G₄S)₃ linker.After sequencing identification,the ScFv gene was cloned into the expression vector PET-29a(+) with the direction of V_H-linker-V_L,which was constructed by endonuclease digestion and ligation.Then the recombinant vector was transformed into E.coli BL21(DE3) strain. The identified positive strain can express the anti-pesticide ScFv protein under the induction of IPTG.The collected ScFv protein was denaturalized with urea,purified with immunoaffinity column and renaturalized by dialysis with renaturalizing solution.The ScFv' immuno-activities were confirmed by competitive inhibition ELISA.On the basis of the variable region genes of the McAb above,the amino acid sequences of the variable region were obtained.Then the three dimensional structure of the ScFv was constructed through homologous modeling by series of software of Discovery Studio^TM(Accelrys).The heavy variable region(V_H) and light variable region(V_L) domains were respectively modeled through recognition of target model, alignment between query sequence and target sequence,modeling,evaluation of structure rationality.Then the V_H and V_L model were superimposed upon the target model of antibody variable region by sequence alignment and the linker was added between V_H and V_L,thus the three-dimensional structure of ScFv was constructed.It was proved the modeling structure was reasonable after structure optimization by the methods of dynamics and energy minimization.Also the molecular modeling study of the interaction between ScFv and triazophos was performed by software.The docking showed that triazophos was deeply located within the binding pocket of ScFv.The result of the docking analysis indicated that triazophos mainly bear hydrogen-bond and Van(?)er Waals interaction with ScFv whose 13 residues can involve in the stable binding interactions.The results from the binding mode analysis may be useful in rational lapten design,developing engineering antibody with high affinity and specificitr,and studying the theory of interaction between antibody and pesticide.Finally,we carried out the initial research for construction the dsFv(disulfide stabilized Fv fragments).Site-specific mutagenesis in the light and heavy chain DNA fragment had been created by PCR using mutagenic primers.Thus a pair of inter-chain disulfide bond would be introduced between the light and heavy chain variable region at the 44^th site of the heavy chain and the 100^th site of the light chain. The binding mode between dsFv and triazophos was virtually the same as that of ScFv and triazophos.Hydrogen-bond and Van der Waals were the main interactive force.Residues involved in the binding interaction were at similar position.However, dsFv had more residues involved in the docking.There seemed to be affinity differencr between dsFv and ScFv.