Construction Of Two Vectors To Express Deoxynivalenol Mimotope And Application Study

Deoxynivalenol (DON) is a mycotoxin produced by Fusarium. It has been a widespread contamination of cereals such as wheat and corn. Foods such as beer, bread, breakfast cereals and feed processed by these contaminated raw materials will pose a serious threat to human and animal safty. DON hazards include animal and human toxicity, teratogenicity, and effects to reproductive toxicity and cells and immune system.Commonly used analysis methods for the detection of DON including high-performance liquid chromatography, thin layer chromatography and liquid-mass spectrometry. However, there are shortcomings like time-consuming, high cost and complex operation for these methods. Enzyme-linked immunosorbent assay (ELISA) is an immunochemical detection method. Those features like high sensitivity, fast and easy operation make it a significant method in detecting mycotoxin. But conventional ELISA methods need to use artificial antigen conjugating toxin as a competitive antigen. The high cost to purchase DON mycotoxin limit its application, and the most important defect is artificial antigen conjugating toxin still do harm to researchers. So it is urgent to seek a nontoxic substance to replace the toxic artificial antigen in ELISA. DON mimotope (CMRPWLQ), designated as CDON, is a cheap and nontoxic7peptide which can bind with anti-DON antibody. Based on these characteristics, DON mimotope provides a new way to solve the above problems. We constructed two expression vectors to express the CDON fusion protein, and deteted the reactogenicity of the two fusion proteins with anti-DON antibody by ELISA. At last, we established nontoxic ELISA method for detecting DON without toxin. The main results are as follows:1. Oligonucleotide T11T12containing DON mimotope and BamH Ⅰ+Xho Ⅰ restriction sites was cloned into the double digested plasmid pGEX-4T-1, then we constructed a recombinant plasmid pGEX-4T-1-CDON. In the same way, another recombinant phagemid pC89S4-CDON was constructed by inserting oligonucleotide T13T14containing DON mimotope and BamH Ⅰ+EcoR Ⅰ restriction sites into the double digested phagemid pC89S4.2. Engineered bacteria pGEX-4T-1-cdon was obtained by transforming vector pGEX-4T-1-CDON to host bacteria BL21(DE3). In the expression process, the induction temperature and time, the idensity of bacteria when IPTG was added as well as the final concentration of IPTG in medium were optimized. The optimal condition is as fellows:when the idensity of bacteria reached0.6,1mM IPTG was added in the medium and expressed at25℃for6h. GST-CDON fusion protein was analysed by SDS-PAGE and purified by GST-resin.3. Engineered bacteria pGEX-4T-1-cdon was obtained by transforming vector pC89S4-CDON to host bacteria XL1-Blue. In the expression process, the induction temperature and time, the idensity of bacteria when helper phage KM13was added, the idensity of bacteria when IPTG was added as well as the final concentration of IPTG in medium were optimized. The opotimal condition is as fellows:when the idensity of bacteria reached0.3, KM13was added to superinfect bacteria, and when the idensity of bacteria reached0.6,1mM IPTG was added in the medium and expressed at25℃for8h. Recombinant phage displaying pⅧ-CDON fusion protein was purified by PEG/NaCl and analysed by ELISA.4. The binding effect of the two fusion proteins with anti-DON antibody was compared by ELISA. Indirect competitive ELISA was used to detect the DON in corn samples spiked with the toxin. The results demonstrate pⅧ-CDON fusion protein presents a better reactogenicity and specificity than GST-CDON when bind with antibody.