Screening And Expression Of The Single Chain Variable Fragments And Development Of Immunoassay Of Two Bt Endotoxins

As a Gram-positive, spore-forming bacterium, Bacillus thuringiensis(Bt) was initially characterized by its production of insecticidal parasporal crystals during sporulation, which has become a dominating bio-insecticide and genetic material of genetically modified (GM) crops worldwide. The Cry toxins are specifically toxic to the following insect:Lepidoptera, Coleoptera, Hymenoptera and Diptera. The toxinology study has conformed that the formation of ionic pores in the membrane of insect epithelial midgut cells after Bt toxin combining, leads to insect death. However, some reports showed that Bt toxins could infect human health and soil eco-system. For example, Bt transgenic rice could made a negative effect on bacteria and yeasts, while an active effect on actinomyces. In addition, crisis assessment need to be verified for GM crops, such as their potential allergenicity and environment residual persistence. Therefore, it is necessary to establish a convenient method for Cry toxins determination.Currently, the detection of Bt-GM crops and Bt residue are mainly performed by instrument analysis, DNA-based and protein-based methods. The reversed phased high-performance liquid chromatography can distinguish the Bt-transgenic and non-transgenic corps. However because of its high cost and professional requirement for its operators, this method is not suited for most testing agency. The DNA-based method like polymerase chain reaction (PCR) focuses on the Bt DNA whether inserted into crops. Although PCR assay is highly sensitive, the complicated process, high cost and difficulty for a high throughput analysis restrict its application. Especially the technology can’t evaluate the level of expression or residue. On the other hand, the immunoassay has been recognized as the most successful method, such as ELISA and colloidal gold strip. Nonetheless, it requires specific antibodies for binding targets.Antibody phage display technology is an attractive alternative to hybridoma technology. It displays recombinant antibodies on the surface of phage particles that can be screened by enabling the phage to interact with immobilized ligands. It reduces batch to batch variation and the single chain variable fragments(scFvs) can be produced using prokaryotic or eukaryotic expression system.Under such background, this dissertation surrounding the topic of development of novel Bt toxin detection methods and has carried out the following research work.1. Construction and application of a naive mouse scFv phage display libraryThe total RNA was extracted from spleen cells of non-immunized Balb/c mice. VH and VL were amplified using universal primers. To facilitate the display of antibody molecules in phages, the VH and VL fragments are assembled as single-chain molecules with a (Gly4Ser)3linker peptide resulting in the construction of scFv genes fused to the pⅢ gene in phagemid (pCANTAB5E) by digesting with sfi I and Not I, then transformed into competent E. coli cells via electroporation. The naive single chain antibody library contained2.04×107independent clones. Then the scFv phage library was used to pan scFv against Bt toxin. But the Kaff of functional scFv were too low to detect. So we choose the commercial human single fold scFv library for the next research.2. Isolation of scFv specific for Bt toxin from human single fold scFv librariesIn this study, scFvs, which could specifically recognize and detect Cry1C, were isolated from naive phage displayed human antibody libraries (Tomlinson I+J) by iterative affinity selection procedure instead of immunization process. Following the competitive eluted phages were treated by trypsin, which disable the phages without scFvs. Meanwhile, with increasing selection pressure, after four rounds of panning,1C6individual scFv was obtained and sequenced. Thereafter, conformed novel anti-Cry1C scFv, was expressed in E. coli HB2151and purified by Ni metal ion affinity chromatography. The Kaff was (5.8±0.5)×106M-1.Tomlinson J phage antibody library was panned for four rounds of "adhesion-elution-amplification". The specificity of the antigen binding activity of the selected clones was identified by ELISA and the variable genes were analyzed and identified. The1AC6positive clone was obtained, which could bind Cry1Ac specifically. The1AC6was expressed in HB2151and purified by Ni metal ion affinity chromatography. The experimental results showed that the best induced express condition is culturing overnight with0.8mM IPTG, at25℃. The immuno-activities of1AC6was confirmed by competitive inhibition ELISA. The Kaff was (3.33±0.05)×107M-1. The3-D model of scFv-1AC6was homologically built with SWISS server. Then the interaction between scFv-lAC6and Cry1Ac was docked by GRAMM sever. The result showed that CDR involved in combination with antigen, which further implied that the1AC6could specificity bind with CrylAc.3. Establish the novel immuno-detection methods for Cry toxinImmunoassay is widely used in the area of rapid toxin detection. The research established several novel detection methods for Cry toxin based on polyclonal antibodies and scFv.An indirect competitive ELISA assay of1C6was developed for the determination of CrylC toxin in the range from0.023to4.35μg/mL, and50%inhibition concentration (IC50) was0.39μg/mL. This approach showed ignorable cross-reactivity with toxin CrylAc and Cry1B. The ic-ELISA approach was exploited for the determination of CrylC in spiked ground rice samples with a mean recovery rate of92.5%and coefficient of variation less than5.0%. This study proves that phage display libraries provide a valuable system for the low-cost, rapid and continuous generation of specific antibody fragments directed against Cry1C toxin target and develop a simple detection method. The results show that1C6scFv could be a valuable tool for detection of CrylC in food and agricultural samples.To establish a rapid detection of CrylAc residue in soil, the time-resolved fluorescence immunoassay (TRFIA) using goat anti-rabbit IgG conjugated with a Eu-N1as a tracer was developed to quantify CrylAc in five types of soil (black soil, yellow soil, red soil, purplish soil, fluvo-aquic soil). The results showed that the limit of detection of Cry1Ac achieved to0.3ng/mL by TRFIA, the detection range ranged from0.3to500.0ng/mL, and the coefficient of variations were both below10.0%. The mean recovery of CrylAc in soil was75.03%. The recovery of Cry1Ac in black soil with the highest organic matter content was the lowest, and the recoveries of CrylAc were reduction with the increase of the organic matter content in soil. The Cry1Ac residue detected by TRFIA and ELISA was a high correlation, and the coefficient of correlation was0.9967. Therefore, TRFIA was a simple, specific, highly sensitive, quantitative assay for Cry1Ac in soil.Phage display is a very powerful technique since the selected phages maintain a physical link between the displayed scFv (phenotype) and the encoding gene (genotype). The novel immuno-PCR detection method was set up based on the character. The immuno-PCR assay showed high sensitivity with minimum detection limit of0.2ng/mL and found to be200times more sensitive than ic-ELISA. Under the optimized assay conditions, Cry1Ac protein can be determined in the concentration ranged from0.2to100ng/mL. As results showed this assay could be a powerful tool for the detection of trace amounts of Cry1Ac protein.The double-antibody sandwich ELISA for detection of insecticidal protein Cry1Ac was developed. The1AC6was used as coating antibody and Cry1Ac-IgG as detecting antibody. The best working conditions were optimized for double-antibody sandwich ELISA. The results showed that the concentration of1AC6were2.5μg/mL and the dilution of IgG-HRP was1:1600are the best conditions. The sandwich ELISA has a detection limit of14ng/mL.With the colloidal gold labeled1AC6, immnnochromatography assay for detection of insecticidal protein CrylAc were developed. IgG coated on the nitrocellulose membrane for the test line, and goat anti-rabbit antibody was blotted on the nitrocellulose membrane for the control line. The detection can be completed within10min and has a detection limit of58ng/mL for CrylAc. These results suggest the possibility for detecting transgenic Bt-crop with simply and reliably characters.