Biosynthesis Of Zearalenone Complete Antigen Through Mimotope And Its Application In Immunoassay

Mycotoxins are toxic secondary metabolites of fungal origin; most of mycotoxins are hazardous to the health of both humans and animals and now recognised as a major cause of food safety issue in China. Detection and control of mycotoxins contamination play an important role in food safety system. Currently, many mycotoxin detection methods have been developed, the competitive immunoassay is now the most applied and representative method for the rapid screening analysis of mycotoxins.The preparation of antibody and complete antigen is the precondition and core of setting up competitive immunoassay for small analytes. However, at present, the complete antigen preparation of small analytes including mycotoxins is mainly dependent on the chemical synthesis approach, while the process of chemical synthesis is time-consuming, costly and might be toxic to manufacturers and users, which often hinders its extensive application.In this study, we combine the technology of phage displayed peptide library, molecular simulation and exogenous gene expression to develop the method of biosynthesis of complete antigen for model small molecules mycotoxin, zearalenone(ZEN) and achieve its application in immunoassay. The main contents of research are:(i) the biopanning of ZEN mimotope by phage displayed peptide library (12-mer) and its identification;(ii) the research of the immunological detection performance of ZEN mimotope;(iii) construction and expression of ZEN mimotope-based vectors and the research of immunological detection performance of expression fusion proteins;(iv) elucidating the structure-activity relationship of the ZEN mimotopes. The main research results are as follows:1. Monoclonal antibody (7G5), which recognizes the ZEN, was used to select for peptides that mimic ZEN by employing a library of filamentous phages that have12-mer peptides on their surfaces. After three rounds of biopanning, five mimotope peptides were obtained to be able to mimic ZEN in binding with the7G5and designated Z5, Z8, Z14, Z15, and Z18. The theoretical isoelectric point, heat stability index, extinction coefficient, average hydrophilic physicochemical properties and secondary structure of ZEN mimotope were analyzed by ExPasy and Swiss-pdbviewer softwares. The results showed that five mimotope have the same secondary structure, which were predominantly in radom coil and helix, and the same hydrophilic, heat stable properties.2. The inhibition curves of ZEN by indirect competitive ELISA were established with ZEN mimotopes, the IC50value was23.5±2.0,2.6±0.4,7.0±0.8,1.2±0.3,14±1.6ng/mL for Z5, Z8, Z14, Z15, and Z18, respectively. Furthermore, the peptides which have the same amino acids as ZEN mimotope were synthesized and conjugated with HRP (Z5-, Z8-, Z14-, Z15-, and Z18-HRP) by maleimide method; the inhibition curves of ZEN by direct competitive ELISA were established with peptide-HRP conjugates, and the IC50value was15±2.4,2.0±0.6,7.2±1.5,0.7±0.1,11±3.1ng/mL for Z5-, Z8-, Z14-, Z15-, and Z18-HRP, respectively.3. The Z5(12-mer), P4(7-mer), and ZEN-BSA conjugates were immobilized on sensor chip, the reaction kinetics between mimotope/conjugates and7G5were detected by surface plasmon resonance (SPR) analysis. The results showed that sensorgram curves obtained were well-behaved and the SPR response unit (RU) caused by7G5binding increased as the concentration of antibody increased, which is in accordance with the indirect immunoassay principle. The equilibrium dissociation constant (KD) measured for P4:7G5(KD=38.7nM) and Z5:7G5(KD=39.8nM) was-100-fold higher than that measured for ZEN-BSA:7G5(KD=0.31nM).4. A polyvinylidene fluoride (PVDF) membrane-based test device was constructed to develop a non-instrumental assay performed with ZEN mimotope. The cut-off level for this method of detecting ZEN, assessed visually, was50μg kg-1and the final results can be obtained within10min. Furthermore, a PVDF membrane-based dot immunoassay for rapid and simultaneous detection of multi-mycotoxins in cereal samples is also developed. The cut-off level for this method, assessed visually, were20,60,1000,20, and250μg kg-1for aflatoxin B1, zearalenone, deoxynivalenol, ochratoxin A, and fumonisins B1respectively, the final results can be obtained within10min.5. Five ZEN mimotope-pMal-pⅢ expression vector (Z5-, Z5-, Z8-, Z14-, Z15-, and Z18-pMal-pⅢ) were constructed to express mimotope-MBP fusion proteins which mime ZEN complete antigen. The mimotope-MBP fusion proteins were coated in microplates, and the inhibition curves of ZEN by indirect competitive ELISA were established, the IC50value was1.5±0.2,1.6±0.3,3.5±0.5,7.2±0.8,1.8±0.4ng/mL for Z5-, Z5-, Z8-, Z14-, Z15-MBP fusion protein coated microplates, respectively. Z15-pRX-AP expresssion vector was also constructed to express Z15-AP fusion proteins, whilie the binding ability between Z15-AP and7G5is very low.6. The structure-activity relationship of the ZEN mimotopes was studied, the results showed that the linker peptides and multi-copies structure were not key factors which effect the immunological detection performance of ZEN mimotope; while the cyclized Z15based direct immunoassay is more sensitive than Z15based.