Analysis Of Pyrethroid Metabolites And Molecular Mimicry Of Cry2Aa Toxin Based On Antibodies

Antibodies are multifunctional molecules with high affinity and specificity to antigens produced in vertebrates.In plant protection,antibodies can be used not only as bioaffinity reagents of pesticide analysis,but also as molecular mimics of pesticides,which can bind to receptors of target insect,and even trigger their biological effects.Here,we established immunoassys for the detection of 3-phenoxybenzoic acid(3-PBA),which is a common metabolite of pyrethroids.At the same time,anti-idiotypic antibodies was used to mimic Bacillus thuringiensis(Bt)Cry2Aa toxin,and its biological activity was also studied along with its parental toxin.(1)Establishment of a colloidal gold based lateral flow immunoassay for 3-phenoxybenzoic acidMonoclonal antibodies(mAb)against 3-PBA were produced by inducing ascites in vivo and purified by Protein G column.By indirect competitive ELISA,The I50 and I10 of purified mAbs were determined to be 0.63 and 0.13 ?g/mL,with a dynamic range between 0.19-2.04 ?g/mL.The 15 nm colloidal gold particles were prepared by sodium citrate reduction method and used to label purified antibodies.The optimum label concentration of antibody was 48 ?g/mL.Immunochromatographic test strip was assembled with cellulose nitrate membrane as a carrier.The optimized concentration of coating antigen and colloidal gold labeled antibody were 2 mg/mL and 5 ?L/cm,respectively.The cut-off value for the assay was 1 ?g/mL and the detection time was within 10 min.Forty river water samples were spiked with 3-PBA at different levels and determined by the lateral flow immunoassay without any sample pretreatments.The negative false rate was 2.5%and no positive false results were observed.This method is potential to be an on-site screening method for monitoring 3-PBA in water or the other environmental samples.(2)Construction of a single chain variable fragment(scFv)against 3-PBA and its bioinformatics analysisIn this study,a mouse hybridoma which can secret mAbs againt 3-PBA was used as a template to extract its total RNA,and then to be reverse transcribed into the first strand of cDNA.Degenerate primers were used to amplify heavy chain variable region(VH)and kappa light chain variable region(V?)genes of mouse antibody by PCR.Then overlap extension PCR was used to connect VH and V? genes with a 45 bp linker.The scFv gene was cloned into pET26b(+),and then transformed to E.coli BL21(DE3).After antibody expression,the binding activity of 3-PBA scFv was confirmed by ELISA and Western blot.Subsequently,the physicochemical properties,secondary structure,alleles and functional regions of scFvs were predicted and analyzed by ExPASy and IMGT.The results showed that the molecular weight of the 3-PBA scFv was 29905.61 Da and the theoretical isoelectric point was 8.95.It was a hydrophilic and unstable protein.The full length of scFv gene was 840 bp,with the VH and V? gene were 370 bp and 319 bp,respectively.The most similar alleles for variable frament of VH and V? gene were Musmus IGHV1-18*01 F or Musmus IGHV1-22*01 F and Musmus IGKV4-72*01 F.The lengths of the three complementarity-determing region(CDR)of VH and V? are 8-8-16 and 5-3-9.Comparing with the germline genes,the total amino acid mutation rate of scFv was 20%.In addition,the three-dimensional structure of the scFv was bulit by SWISS-MODEL.3-PBA was docking into its scFv by using the Autodock Vina.It was found that there were hydrogen bonds,hydrophobic interactions,CH-? interaction and cation-? interaction between scFvs and 3-PBA.The key amino acid sites involved in antigen recognition were GLY-74,ASN-76,GLU-81,VAL-123,LEU-130,TRP-252 and ARG-83.These amino acids were all located in the CDR and their adjacent regions of antibodies by IMGT.In this work,a scFv against 3-PBA was firstly constructed,which laid a foundation for the antibody expression and its application in 3-PBA detection.In addition,the key amino acid and the interaction between scFv and 3-PBA were analyzed.The binding mode between scFvs and 3-PBA was reasonably explained,which provided important information for the further modification and production of antibodies.(3)Expression,function analysis and application of a single chain variable fragment against 3-PBAThe temperature,induction time and concentration of inducer on the expression of 3-PBA scFv were optimized in this study.The results showed that 16?,8 h and 0.25 mM IPTG were the optimal inducing conditions.The inclusion body of scFv was purified by Ni based immobilized metal ion affinity chromatography and eluted by 200 mM imidazole.The electrophoresis pure protein was obtained with a yield of 2.68 mg/L,and then be dissolved in guandinine and renatured by dialysis.After optimizing the working concenrtration of the coating antigen,scFvs and methanol,an indirect competitive ELISA was established The half maximal inhibitory concentration(I50)of scFv was calculated to be 0.55 ?g/mL,the linear detection range(I20-I80)was between 0.15-2.64 ?g/mL,and the detection limit(I10)was 0.05 ?g/mL.The sensitivity of 3-PBA scFv is slightly higher than its parental monoclonal antibodies.The cross reactivities of scFv with fenpropathrin,beta-cypermethrin and fen valerate were between 0.56-1.15%,for 3-phenoxybenzaldehyde and 3-phenoxybenzyl alcohol were 93.22%and 87.30%.3-PBA scFv could also be used to fast screening of these two compounds.The half-life of scFvs stored at 4? was between 14-21 days,scFvs showed no decrease in binding signal under-20? and-80? for 3 months.In tap water recovery test,the average recoveries of ELISA method was between 83%and 96%,the average intra and inter coefficient of variation were between 1.78%-8.54%and 4.65%-10.56%respectively.The compared results of ELISA and HPLC showed that they had good relativity(R2=0.9892).This method could be used as an alternative screening method for 3-PBA in water and other environmental samples.(4)Construction and isolation of Cry2Aa anti-idiotypic single chain variable fragments by chain shufflingUsually,none or seldom anti-idiotypic antibodies could be directly isolated from non-immunized antibody libraries.In our previous study,two anti-idiotypic scFvs of Bacillus thuringiensis(Bt)Cry2Aa toxin(B10 and F2)were isolated from a human semi-synthetic antibody library(Tomlinson I library).In order to get more scFvs,heavy chain and light chain shuffled libraries with the capacity of 1.4×106 and 1.2×106 were constructed by chain shuffling based on these two clones.The whole affinity of light chain shufffed library was determined to be 4.2 times higher than the heavy one by polyclonal ELISA.The two library were selected by using anti-Cry2Aa polyclonal antibody as the antigen.The positive rates for the two libraries were 13.6%and 57.4%.After sequencing,1 heavy chain shuffled clone and 6 light chain shuffled clones were obtained.Subsequently,competitive phage ELISA was used to determine the apparent affinity between the mutant and the antigen.The best clone(MUT10)had an apparent affinity of 1.42×106 M-1 to anti-Cry2Aa polyclonal antibody.The molecular mimicry function of MUT10 to Cry2Aa was also tested by inhibition test.100 ?g/mL Cry2Aa could inhibit 30.1%binding of MUT10 to anti-Cry2Aa polyclonal antibodies and 50?L supernatant of MUT10 could inhibit 46.7%binding of Cry2Aa to its polyclonal antibodies.MUT10 could mimic partial stucture of Cry2Aa.This study provided basic materials for the analysis of bioactivity of Cry2Aa anti-idiotypic scFvs.(5)Preliminary analysis of the bioactivity of Cry2Aa and its anti-idiotypic scFvs against Plutella xylostellaThe bioactivity of Cry2Aa and its anti-idiotypic scFvs against a target insect of Lepidoptera Plutella xylostella was tested and compared on the basis of our previous study.The binding curve of Cry2Aa to P.xylostella BBMV was determined by ELISA.The apparent binding affinity was 266.6 nmol/L by using Scatchard analysis.The binding ability of nine Cry2Aa anti-idiotypic scFv with P.xylostella BBMV was also determined by ELISA.The results showed that MUT10 has the highest specific binding.Subsequently,the LC50 of Cry2Aa against P.xylostella was determined to be 27.90?g/mL by leaf soaking method.While the ajusted mortality of 107 cfu/mL MUT10 against P.xylostella was 12.8%.The binding proteins of Cry2Aa and MUT10 with midgut protein were analyzed by ligand blot,and a common binding band above 245 kDa was analyzed by LC-MS/MS.The results showed that ATYSEGPNGSVR fragment was obtained by enzymatic hydrolysis.After searching in protein database,an uncharacterized protein of P.xylostella with molecular weight of 332 kDa was matched with the fragment and its function was annotated as lipocalin family.This study provides useful data for explaining the action mechanism of Cry2Aa and its anti-idiotypic scFv against P.xylostella.In this study,a novel bioaffinity reagent and immunoassay for metabolites of pyrethroids(3-PB A)were constructed.The bioinformatics data of the 3-PB A scFv were obtained and the binding pattern was clarified.In addition,anti-idiotypic scFvs which can mimic the structure of Bt Cry2Aa toxin were isolated.We also investigate the biological activity of Cry2Aa and its anti-idiotypic scFvs against P.xylostella.This could be a new strategies to explore insecticidal protein resources.