Humanized Anti-Bacillus Thuringiensis Bt (CrylB) Single-chain Antibody Screening Toxin Protein, Expression And Biological Activity Assay

In this study,considering the ecological impact of the transgenic crops (trans-Bt,GOM) planting,and the health effect to human and other mammals,Cry1B toxin was chosen as the research target,aim at the rapid detection methods development.A large synthetic phage displayed human library (Tomlinson J) was employed to produce single-chain antibodies (scFvs) against Cry1B toxin by affinity panning,then scFv antibody was expressed via several different cell systems,thereafter,two scfv antibody based immunological methods (ELISA,TRFIA) were developed for the determination of Cry1B toxin.After four rounds screening of "adsorption-elution-amplification" through the amplified naive phage-displayed human single fold scFv Tomlinson J library,8positive clones were picked up,and confirmed to be specific for the Cry1B recognition,after sequencing,for scFv antibodies,the different of amino acid was found in CDR area.Clone No.1E2,which shows highest antigen binding ability than the others,was chosen to infest cell of E.coli HB2151for the host replacement,then secretory expressed scFv antibodies were purified for the antigen recognizing ability identification.The results demonstrated that the antibody encoding genes inserted correctly,and the expressed antibody got it’s antigen binding ability improved,in the optimized cell culture condition of30℃、1mmol/L IPTG、and12h culture, the soluble scFv antibody was expressed successfully,and the concentration of purified scFv reached to132μg/mL.ScFv genes of1E2was conducted into pFastBac MHT-A vector to introduce the competent cells of DH10BacTM E.coli,and the positive recombinant clones were identified by "white-blue plaque selection".Furthermore,Bacmid-sc Fv recombined vector in positive clone was extracted to infect insect cell of sf9for eukaryotic expression.The results showed that pFastBacrMHT-A-scFv recombinant plasmid was constructed successfully,and complete and correct scFv sequence was transferred into cell of sf9,and the significant pathological changes of insect cells was noticed,and the concentration of purified scFv reached to103μg/mL.ScFv antibody based ID-ELISA and TRFIA assay were developed for Cry1B toxin Detection.For phage display scFv based ID-ELISA,the results indicated that the50%inhibition of control (IC50) is1.0732μg/mL,and the detection limit (IC10) is13.4ng/mL for the determination of CrylB in the range from0.5μg/mL to4.0μg/mL;For prokaryotic expressed scFv,the50%inhibition of control (IC50) is1.398μg/mL, and the detection limit (IC10) is25.7ng/mL for the determination of CrylB in the range from0.5μg/mL to4.0μg/mL;For eukaryotic expressed scFv,the50%inhibition of control (IC50) is1.010μg/mL,and the detection limit (IC10) is11.3ng/mL for the determination of CrylB in the range from0.5μg/mL to10.0μg/mL.TRFIA method was developed for the determination of Cry1B,based on the scFv expressed by eukaryotic expression system,the detection limit was0.17ng/ml in the range of CrylB from1.00to800.00ng/mL.The cross-reactivity of CrylB analogues was negative.Conclusions:Several phage display scFv antibodies were isolated from Tomlinson J phage displayed library,and expression the scFv proteins with prokaryotic expression system and eukaryotic expression system was successfully achieved.The competitive indirect ELISA by the soluble scFv from eukaryotic expression system and the sandwich time-resolved fluoroimmunoassay (TRFIA) for CrylB toxin determination was established with high sensitivity,which has the potential quality to be commercial marketed.