Expression And Activity Analysis Of Engineering Antibodies Against Bt Cry2Aa Toxins

Bacillus thuringiensis has been widely used in agriculture production for pests control as biological pesticides or genetically modified organism(GMO).It brings huge economic and social benefits,meanwhile,the potential risk of ecological effect by Bt toxins has been raised.Thereby,on-site test technologies certainly need to ensure the agri-product safety and evaluate the risk of ecological environment effect.With the genetic engineering technology development,noval method based on recombinant antibody becomes a realistic solution for Bt toxins detection.In our study,phage display,peptide and single-chain fragment antibody have been developed for the Bt Cry2Aa toxin detection.Phage display technology is a efficiency way for the engineered antibody screening,preparation and expression,also is a most of convient and fast way for anti-idiotype antibody panning.Furthermore,a humanized single chain antibody(single-chain antibody fragment,Tomlinson scFvs)library displayed by phage has been establishted by the MRC Centre of the University of Cambridge in 2002.This Library was made with the heavy chain variable region of humanized antibody(VH)and light chain variable region(VL)connected by several amino acids as the linker,the molecular weight of the scFv was about 30 kD.Another successful phage display library is made by random peptide,such as 8,12,or 15-mer peptide phage display library and so on.In this library peptide gene is fused to the gene of M13 capsid protein(p?),thereby,rodam peptides displayed in the N terminal of p?.The main results of our study are as follows:1.Development of an Indirect Competitive ELISA method based on peptide for the Cry2Aa detection via peptide library screening.A phage displayed random 12-mer peptide library(Ph.D.-12TM Phage Display Peptide Library)is screened by Cry2Aa toxin.After 4 rounds of panning,20 single colonies is picked randomly,and the positive clones are identified by monoclonal phage competitive enzyme-linked immunosorbent assay(ELISA),and PCR,DNA electrophoresis and sequencing.Totally 8 peptides,which could recognized Cry2Aa specifically have been confirmed.The peptide(amino acid sequence:GTPWHHHRHLIV)which showed better binding ability than others was employed to develop an indirect competitive ELISA for the Cry2Aa detection.The peptide based indirect competitive ELISA shows that the IC50 is 1.139?g/mL,and the minimum detection limit is 0.0211?g/mL.The linear of detection ranged from 0.0478 to 0.555?g/mL.With this new detection mode,Cry2Aa can be detected rapidly and accurately.2.Development of sandwich ELISA for the Cry2Aa Detection based on scFv antibodyBy optimizing solid phase screening strategy,humanized phage antibody library(Tomlinson ?)is used for screening single chain antibody against Cry2Aa toxin.After 4 rounds of "adsorption-elution-amplification",200 single colonies are randomly selected from the last round of elution,and identified by monoclonal ELISA,PCR amplification,DNA electrophoresis and sequencing.12 positive phage scFvs are successfully obtained.The positive clones scFv-A3 which has better binding activity is transformed into the host bacteria HB2151,then expressed and purified.Coating purified scFv-A3 as "the capture antibody",rabbit polyclonal antibody against Anti-Cry2Aa as "the detection antibody for Cry2Aa",A double antibody sandwich ELISA method is established for detecting Cry2Aa.The results showed that,the linear detection range of this method between 7.1ng/mL-3.8?g/mL,linear regression equation is y=167.96x + 0.4513(R2 = 0.9909),the detection limit is 1.08 ng/mL.3.Preparation,Expression and Activity Study on Anti-idiotypic antibody of Cry2AaPolyclonal antibody is obtained by immunizing New Zealand rabbits,and the purified polyclonal antibody sera and Cry2Aa negative antibody sera used as coating antigen alternatively,deduction of screening(subtractive panning)strategy is conducted to screen anti-idiotypic antibody of Cry2Aa toxic in the humanized phage displayed antibody library(Tomlinson ?).For screening,two different ways are employed:only one round of screening is conducted,then washing 40 times;the other is four round of screening(same as screening anti Cry2Aa single chain antibody).200 clones are picked from both screening way for the activity analysis.The results showed that:38 positive clones are obtained after one round of screening,however,125 positive clones are obtained after four rounds of screening.We finally confirmed two kinds of anti-idiotypic scFvs were "Ab2?" by binding with the receptor(diamondback moth BBMV).The positive clones scFv-B10 and scFv-F2 which had better binding activity provide the preliminary foundation for the study of insecticidal activity.The reactive value(OD450)of B10 was 0.806,and the reactive value(OD450)of F2 was 0.584.