Understanding Molecular Mechanism Of DON Antigen Mimicry By Nanobody N-28 And Its Modification By Site Saturation Mutagenesis

Deoxynivalenol (DON) is a secondary metabolite produced by some Fusarium fungi and is commonly found in cereal grains and their byproducts. The contamination of DON in food is a potential hazard for humans and animals. DON displays acute and chronic toxicity, which can cause anorexia, vomiting, diarrhea, fever, and growth retardation. In addition, DON can induce cytotoxicity and immunotoxicity. Various methods have been developed to detect the toxic effects and high occurrence of DON. Among them, immunoassay was widely used for DON detection due to its simplicity, specificity, rapidity and suitable for high-throughput screening.Immunoassay is based on antibody-antigen recognition, for improving the immunoassay sensitivity, one approach is the development of natural or artificial high-affinity antibodies. However, production of high-affinity antibodies is difficult and complicated; it is usually a bottleneck problem in the improvement of immunoassay sensitivity. Another approach to enhance sensitivity is antigen modification. As for small molecules, the heterologous immunoassay system presents a good alternative to homologous assays in achieving higher sensitivity in competitive immunoassay format. The conventional chemical synthetic method for antigen modification was usually peroformed by "trial and error", involving not only the handling of toxic mycotoxin which pose a threat to the environment and human health, but also complicated.In this study, nanobodies that mimic DON were selected form a naive alpaca nanobody library. The nanobodies were used to substitute the conventional chemical synthesized DON-BSA conjugates, which could reduce the DON contamination. Besidies, the nanobody based heterologous immunoassay was more sensitive than the DON-BSA conjugates based homologous immunoassay. Moreover, the 3D structure of "nanobody (N-28)-anti-DON ScFv" complex was presented and verified by molecular modeling and alanine-scanning mutagenesis, which provide evidence that nanobody can mimic an antigen at the molecular level. Furthermore, site saturation mutagenesis was used to modify nanobody N-28 and to improve the sensitivity of N-28-based immunoassay. The main research results are as follows:1. Biopanning of DON antigen mimeticsA combined strategy including 1 round of Glycine-HCl elution and 3 rounds of DON competitive elution was carried out in the biopanning, after four cycles of panning, two phage clones (P-28 and P-31) were identified to be specific binding to anti-DON MAb. The IC50 of the immunoassay developed with P-28 and P-31 was 78.89±1.77 ng/mL and 68.18±1.15 ng/mL, respectively, the linear range was 21.24-316.52 ng/mL and 16.36-262.05 ng/mL.2. Expression and application of DON antigen mimeticsGene fragments of P-28 and P-31 were subclone to pET-25(+) vector, respectively. Then the recombinant vectors was transformed into E.coli Rosetta (DE3), after IPTG induced expression and purification, nanobody N-28 and N-31 was obtained. The IC50 of the immunoassay developed with N-28 and N-31 was 8.77 ± 0.41 ng/mL and 19.97±0.84 ng/mL, respectively, which were 18-and 8-fold more sensitive than the DON-BSA based immunoassay. A total of 70 samples of cereals and feedstuffs were analyzed using N-28 based ELISA and commercial ELISA kit, respectively. The results obtained from the two methods were in good agreement with each other.3. The mechanism of N-28 mimic DON antigenThe 3D structures of "N-28-ScFv" and "DON-ScFv" complex were generated by using molecule modeling. Comparing the two structures, N-28 and DON was dock into the same binding site of ScFv, the CDR3 amino acids (Thr102-Ser106) of N-28 could mimic the DON and interact with ScFv. Then the molecular modeling predicted structure was confirmed by alanine-scanning mutagenesis.4. Modification of N-28 by site saturation mutagenesisSite saturation mutagenesis was performed on five residues of N-28 to alter its binding activity. After mutagenesis, mutants were selected by phage-ELISA and sequencing. A total of 77 mutants were obtained, three of them (N-28-T102Y、 N-28-V103L�'�N-28-Y105F) exhibited higher sensitivity (3.2-,1.5-and 2.2-fold) compare to wild-type. Thirty samples of cereals and feedstuffs with unknown concentrations of DON were analyzed using N-28-T102Y based phage-ELISA and commercial ELISA kit. The results obtained from the two methods were in agreement with each other (R2=0.981).