Preparation And Activity Analysis Of Monoclonal Antibody And Single-Chain Antibody Of Bt Cry1 Toxins

The Cry1 family toxins,mainly including Cry1Aa,Cry1Ab,Cry1Ac,Cry1B,Cry1C,Cry1E,and Cry1F,are produced by the Gram-positive bacterium Bacillus thuringiensis(Bt)during sporulation.These proteins have insecticidal activity against Lepidopterous larvae.However,Cry toxins are considered harmless to human and farm animals,for there are no receptors in the small intestine of mammals.Thus,Bt genes have been widely introduced in the creation of genetically modified(GM)crops such as com,soy,and potato,which have been planted to a large extent.However,as such GM crops have been commercialized worldwide and provided as food or animal feed in the past decade,public health and environmental safety concerns have been raised.Thus,a sensitive and convenient detection method for Cryl toxins in plants and food products is particularly urgent.Rely on maturely monoclonal antibody technology,genetic engineering technology and phage display technology,this paper was mainly study on preparing monoclonal antibody and scFvs,apply detection Bt Cryl toxins and analyze the activity.This research work mainly includes the following aspects:1.Production of monoclonal antibody broadly recognizing Cryl toxins and establishment of the assay method for simultaneous determination of seven Bt Cryl toxins.First,by comparing the three-dimensional structures of seven Cryl toxins(Cry1Aa,CrylAb,CrylAc,CrylB,CrylC,CrylE,and CrylF),analyzing the conserved sequences,and considering the antigenicity and hydrophilicity,three polypeptides(T1,T2,and T3)have been chosen and coupled to keyhole limpet hemocyanin(KLH)as immunogens for the generic monoclonal antibody(Mab)generation.Thereafter,a double antibody sandwich enzyme-linked immunosorbent assay method(DAS-ELISA)was developed for simultaneous determination of seven Cryl toxins.The results revealed that the haptens T1,T2,and T3 had different effects in the production of antibodies.Among them,the obtained Mab(strain 2D3)generated by T2 can recognize seven Cryl toxins simultaneously.Equilibrium dissociation constant(KD)values for seven Cryl toxins with Mab 2D3 were 1.198×10-8 M,2.197×10-8 M,1.367×10-8 M,2.092×10-8 M,5.177×10-8 M,4.016×10-8 M,and 3.497×10-8 M,respectively.For 2D3-based DAS-ELISA,the limits of detection(LOD)and limits of quantification(LOQ)can reach 15 and 30 ng·mL-1 for each Cryl toxin,respectively.Our study is the first report of a broadly specific immunoassay for multidetermination of seven major Cryl toxins,and it will provide a new idea and technical routes for development of multidetermination immunoassays.2.Construction,identification and activity analysis of the single-chain variable fragment(scFv)for Bt Cryl toxins.Using the hybridoma cell line 2D3,the variable region genes of heavy chain(VH)and light chain(VL)were amplified using the universal primer sets by PCR.The genes of VH and 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.The scFv gene was then constructed into prokaryotic expression system E.coli BL21 after combined with vector PET-26b.The expression level of scFv was identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE)and western blot,and its activity was identified by indirect ELISA.As a result,VH and VL gene was successfully amplified and scFv was successfully constructed and expressed.The key position of scFv for recognition were studied by homologous modeling and molecular docking.VH and VL were separately expressed and assayed by indirect ELISA.The results showed that VL was the main combination domain.The VH and VL protein were both able to specifically recognize Cryl toxins and the activities of VL protein was higher,which validated the result of molecular docking.Afterwards,VL-VL tandem was designed and the activities of the VL-VL antibody was assayed.The results indicated that the VL-VL had higher activity then scFv.3.Selection of scFv antibodies from an immunized mouse library by phage display and established a DAS-ELISA for the detection of Bt Cryl toxins.In this study,scFvs against Cryl toxins were selected from an immunized mouse phage antibody displayed library which was successfully constructed at about 6.25×107 CFU/mL.After four times of affinity screening,seven positive phage-scFvs against Cryl toxins were selected,sequenced and characterized by ELISA.Among them,clone scFv-3H9 showing relative stable and high binding ability to six Cryl toxins was selected for expression and purification.SDS-PAGE indicated that the soluble scFv-3H9 fragments approximately 27 kDa were successfully expressed in E.coli HB2151 strain.The purified soluble scFv-3H9 were used to establish the DAS-ELISA for the detection of the six Cryl toxins,with the LOD and LOQ of 3.14-11.07 ng mL-1 and 8.22-39.44 ng mL-1,respectively,and the correlation coefficient was higher than 0.997.The measured average recoveries of Cryl toxins from spiked rice leaf samples were ranged from 84.2%to 94.8%,with coefficient of variation(CV)less than 8.2%,showing good accuracy for the quantitative detection of Cryl toxins in agricultural samples.These results showed promising applications of scfv-3H9 for the detection of six Cryl toxins with the new established DAS-ELISA.4.Production of monoclonal antibody specific recognizing Bt CrylAb toxin and establishment of the assay methods.In this study,based on molecular design,a novel hapten polypeptide was synthesized and conjugated to KLH.Then,through animal immunization with this antigen,a monoclonal antibody named 2C12,showing high affinity to CrylAb and having no cross reaction with Cry1Ac,was produced.The KD value of Cry1Ab toxin with Mab 2C12 was 1.947×10-8 M.Based on this specific monoclonal antibody,a DAS-ELISA and nanogold immunochromatographic test strip were developed for the specific determination of CrylAb toxin.In ELISA determination,the LOD and LOQ values were determined as 0.47±0.11 and 2.43±0.19 ng mL-1,respectively.The average recoveries of CrylAb from spiked rice leaf and rice flour samples ranged from 75.3%to 115.2%,with CV less than 8.6%within the quantitation.range(2.5-100 ng mL-1),showing good accuracy for the quantitative detection of CrylAb toxin in agricultural samples.The assay was based on double antibody sandwich format with the visual detection limits(vLOD)of 0.1 μg mL-1,which is similar to commercial test strips for CrylAb/CrylAc toxin.The test results of immunochromatographic assay were all positive validated against the DAS-ELISA in the analysis of CrylAb toxin in spiked rice leaf samples between 0.5-10 μg g-11.In addition,10%,5%and 0%error probability results were found in 20 times repeated tests for Cry1Ab concentration of 0.1,0.2 and 0.5-1 μg mL-1,respectively,demonstrating the reproducibility of the immunochromatographic test strip.Furthermore,the test(T)and control(C)red lines on the strip stored for 3 months were also visible with the concentration of CrylAb toxin 0.1 μg mL-1,suggesting the test strip could be stored for 3 months without significant loss of sensitivity.To our knowledge,this is the first report on qualitative determination of CrylAb toxin by immunochromatographic assay without interference from Cry1Ac toxin.In conclusion,this study provides a new approach for the production of high specific antibody.