| Fumonisin B1, a water-soluble secondary fungi metabolite, is mainly producedby Fusarium moniliforme, Fusarium proliferatum and especially contaminates maize,wheat and other cereals. FB1can cause equine leukoencephalomalacia in horses,porcince pulmonary edema in pigs and primary liver cancer, oesophageal cancer inhumans and so on. To accurately analyze mycotoxins, many kinds of methods havebeen developed, such as Gas chromatography(GC), High-performance liquidchromatography (HPLC) and antigen-antibody based immunoassays. Compared withchromatography methods, immunoassays have advantages of simple, rapid, cheapand very suitable for large-scale, high throughput screening of the samples. Smallmolecules including mycotoxins cannot provide two epitopes for simultaneouslybonding by two antibodies, So far immunoassay small molecules mainly arecompetitive immunoassay format. However, the competitive immunoassay must usemycotoxin standards to prepare coating antigen or competitive antigen by chemicalsynthesis. But the mycotoxin standards are very toxicity and expensive, in addition,the process of chemical synthesis mycotoxin complete antigen is facing complexsynthesis steps, pollute environment.In this study, the specific peptides that mimic FB1were selected fromPh.D.-12Phage Display Peptide Library and biosynthesis of FB1detection antigenby molecular cloning technique. The biosynthesis of FB1detection antigen-basedimmunoassay was established for determination of FB1in corn samples. In addition,the peptides that recognize anti-FB1antigen-antibody complex were screened fromPh.D.-12Phage Display Peptide Library by subtractive panning, which could bedeveloped noncompetitive immunoassay for detection of small moleculars. The mainresearch results are as follows:1. By phage display and biopanning technology against3F11,7types of peptidethat mimic FB1which can competive binding3F11were obtained and named F1, F3,F4, F7, F15, F17and F22.The standard curves of F1, F3, F4, F7, F15, F17, F22. were developed by indirect competitive phage-ELISA, the IC50value was0.422±0.021ng/mL,0.534±0.024ng/mL,0.464±0.031ng/mL,0.351±0.023ng/mL,0.875±0.059ng/mL,0.405±0.022ng/mL and0.312±0.025ng/mL, respectively. Apolyvinylidene fluoride (PVDF) membrane-based test device was performed with F1,the cut-off value was2.5ng/mL and the result can be obtained just by the naked eyes2. The7types of peptide that mimic FB1genes were cloned into PMAL-PIII byPCR and constructed the expression vector of pMAL-F1-MBP，pMAL-F3-MBP，pMAL-F4-MBP， pMAL-F7-MBP， pMAL-F15-MBP， pMAL-F17-MBP andpMAL-F22-MBP. The recombinant expression vectors were transformed into TB1and induced by0.3mM IPTG. The bioactivity of biosynthesis of FB1detectionantigens were analyzed by indirect ELISA, the result showed that only F1-MBP andF15-MBP can competive binding3F11with FB1, the IC50value was2.15±0.13ng/mL,1.26±0.08ng/mL.3. Taking F1-MBP as research object, compared with F1-MBP and FB1-BSA atdifferent pH value, ionic strength, methanol concentration and heat treatmentinfluence of ELISA assay，the result showed that immunology performance ofF1-MBP and FB1-BSA was no difference. The sensitive detection FB1wasdeveloped by biosynthesis FB1antigen F1-MBP. The IC50value of F1-MBP was2.15±0.13ng/mL and the cutoff value of membrane-based dot was2.5ng/mL. Thesensitivity was10-fold higher than that of FB1-BSA with the same antibody.30naturally contaminated maize samples were analyzed by F1-MBP ELISA, F1-MBPdot-immunoassay and converntional ELISA. The results measured by F1-MBPELISA were compared with conventional ELISA analysis by a correlation test，respectively. The coefficients of correlation R2was0.98. It demonstrated that theF1-MBP could be used as substitute of FB1-BSA for immunoassay.4. The peptides that recognize anti-FB1antigen-antibody complex werescreened from Ph.D.-12Phage Display Peptide Library by subtractive panning,23positive clones obtained by Phage-ELISA.However, the insert sequences showedthat multi-insert segment. |
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