Preparation Of Anti-idiotypic Antibodies And Phage Display Study Of Cry1 Toxins

The creation and application of efficient and safe insect-resistant materials is to improve the toxicity to target pests and environmental safety.At present,in the field of pest control,the wide use of chemical pesticides poses a great threat to the ecological environment,and easily leads to pest resistance.In order to effectively delay the problem of pest resistance,it is a research hot spot to actively develop the creation and use of biological pesticides.Bacillus thuringiensis（Bt）is the most successful and widely used biological pesticide in the world.According to previous studies,the variable regions of heavy and light chains of antibody have structural similarity with Domain II-III of Cry toxin with 3-D structure in spatial structure,and have the potential to simulate the structure and biological function of Cry toxin.Therefore,in this study,the mimic material of Bt Cry1F toxin was prepared and identified by combining the‘anti-idiotypic antibody’theory and phage display technology.At the same time,with the help of phage display technology,the toxin on its surface were explored and studied by using phage display technology.Through phage display technology,we studied the screening and preparation of toxin mimic materials,and the efficient fusion expression of toxin on its surface,in order to provide new technical means for biological control of pests.The research mainly includes the following three aspects:1.Amplification and titer determination of helper phage M13K07.Helper phage M13K07(NEB,titer:1×10¹¹pfu/m L)with known titer was used to infect TG1(OD₆₀₀=0.5)in logarithmic phase.After coating solid medium（kanamycin resistance）overnight,the isolated single colonies were picked and transferred into2×TY liquid for overnight culture.The helper phage contained in the supernatant was enriched and purified by PEG/Na Cl,and then resuspended with PBS and centrifuged to remove cell debris to prepare helper phage M13K07.The ELISA standard curve method was used to replace the traditional plate counting method to determine the titer of the amplified helper phage.The M13K07 helper phage was used as the standard substance.The M13K07 helper phage was diluted and coated according to the double gradient dilution method.The HRP labeled anti-M13 monoclonal antibody was used to identify and determine.The standard curve was established to calculate the titer of the amplified helper phage.Compared with the traditional plate counting method,the ELISA standard curve method can not only achieve the same accuracy,but also has the unique advantages of simple,rapid and independent of TG1.2.Screening and identification of anti-idiotypic antibodies against Cry1F toxin.RNA was extracted from the spleen cells of Rat immunized with Cry1F and reverse transcribed into c DNA.V_H and V_L were amplified by degenerate primers,respectively.The single-chain variable antibody fragments（sc Fvs）were connected by the linker peptide（Gly₄Ser）₃ and cloned into the phagemid vector to construct the immune library.F（ab）’₂ fragment of mouse anti-Cry1F polyclonal antibody was used as the screening target molecule to screen and enrich the constructed mouse-derived immune library.Finally,three positive clones with high binding value to BBMV receptor of Ostrinia furnacalis were obtained by monoclonal ELISA（with the binding characteristics of mimic toxin and receptor）.The amino acid sequence of a strain with the highest binding value and Cry1F toxin sequence were selected for the comparative analysis of the physico-chemical properties and secondary structure composition of amino acids,and the 3D structures of toxins and mimics were further constructed.The spatial structure distribution of amino acids in the receptor recognition region was compared and analyzed,providing a theoretical reference for the subsequent study of mimics and the characteristics of toxin and receptor recognition.3.Preliminary study on phage display and insecticidal activity of Cry1Ac.In this study,the recombinant phagemid p IT2-Cry1Ac was constructed.Sta rting from the replacement of terminator TAG between Cry1Ac and g III genes and the selection of helper phage for rescue package,the effects of terminator mutation and helper phage rescue with different functions on the display effici ency of Cry1Ac were explored and studied.The results confirmed that terminat or mutation and M13K07 helper phage rescue could make Cry1Ac display on t he phage surface efficiently.PBS（blank control）,M13K07（negative control）,p I T2-Cry1Ac-wt and p IT2-Cry1Ac-mt rescued by helper phages M13K07 were us ed as experimental groups,and P.xylostella was fed respectively to determine the insecticidal activity against P.xylostella.The results showed that the correc ted mortality of p IT2-Cry1Ac-wt before mutation was only 26.47%after rescue d by M13K07,while the corrected mortality of p IT2-Cry1Ac-mt after mutation was 63.19%after rescued by M13K07,which confirmed that the mutation ter minator was an important factor affecting the efficient display of Cry toxin on the phage surface,and provided technical support for the display of Bt toxin with insecticidal activity by phage in the future.