Preparation And Application Of Aflatoxin Fluorescence Labeled Mimic Peptide

Aflatoxin(AFT)is a natural pollutant with strong toxicity and carcinogenicity,and its physical and chemical properties are stable.AFT is easy to pollute agricultural products,which not only causes huge economic losses,but also seriously threatens the health of human,livestock and poultry.Efficient prevention of AFT pollution requires timely detection and monitoring,detection generally requires AFT standard,but AFT standard is expensive,and AFT is a highly toxic chemical,which has hidden dangers to experimental analysts and the environment.Therefore,it is of great significance to study and prepare substitutes for AFT and to develop non-toxic and low-toxicity detection methods.The technology of phage random peptide library is to insert a large number of randomly encoded peptide sequences into the phage vector to form a phage display library.There is only one foreign peptide chain sequence on the surface of each phage particle,and has its relative spatial conformation and biological activity.In this study,anti-AFB₁3C7 monoclonal antibody was selected as the target molecule,phage random 7-peptide library was selected,the mimic epitopes of antibody AFB₁ 3C7 were screened,and the synthetic peptides were selected to replace the AFB₁ standard,and the properties of the peptides were studied and identified.The main research contents are as follows:1.Screening of mimic epitope peptides of aflatoxin.After three rounds of screening,39 phage clones were selected and verified by adding anti-AFB₁ 3C7 monoclonal antibody to the phage random heptapeptide library.36 of them could specifically bind to anti-AFB₁ 3C7 monoclonal antibody,of which30 could be inhibited by AFB₁.DNA was extracted and sequenced.The results showed that the repetitive sequences of 30 positive phage particles were actually 17phage clones,and their common sequence was histidine(H)-preserved(P)-tryptophan(W).Abbreviated as XXXXHPW,XXXHPWX,XXHPWXX,XHPWXXX,X are arbitrary amino acids.The competition curve was established with 17 positive phage particles.The linear range,detection limit and semi-inhibitory concentration(IC₅₀)were similar,and the linear range was between 1 and 2000 pg/m L.2.Synthesis and Verification of mimic Peptide.Seventeen peptides were chemically synthesized according to the foreign sequence inserted by A1-A17 bacteriophage.An indirect competitive ELISA method for the analysis of peptides was established by optimizing antigen coating concentration,antibody dilution rate,peptide solution,competitive reaction time,pH and ionic strength.The best reaction system of indirect competitive ELISA for synthetic peptides is as follows:the concentration of antigen coating is 0.5?g/m L,the dilution ratio of antibody is 1/32000,the solution of polypeptide is water,the PBS buffer with competitive binding time of 10 min,pH is 7.4 diluted 3C7 antibody,and the competitive inhibition curve is established under this condition.The IC₅₀ of XXXXHPW mimic peptide is between 28.18?g/m L and 74.13?g/m L.The IC₅₀ of XXXHPWX mimic peptide is between 4.37?g/m L and 9.908?g/m L,the IC₅₀ of XXHPWXX mimic peptide is between 3.28?g/m L and 5.956?g/m L,and the IC₅₀ of XHPWXXX mimic peptide is between 3.24?g/m L and 14.12?g/m L3.Fluorescence labeling and verification of mimic peptides.7-amino-4-methylcoumarin(AMC)was used to label the C-terminal of mimic peptide SMFHPWS and GAWHPWS,and an indirect competitive ELISA assay for fluorescent labeling of mimic peptide was established.The results showed that there was a good linear relationship in the range of 0.078125-100?g/m L.The lowest detection limits(IC₁₀)of IC₅₀ were 1.62?g/m L and 0.845?g/m L,respectively,and the affinity constants were 3.5214×10⁴mol^-1 and 2.6316×10⁴mol^-1,respectively.IAC-HPLC internal standard analysis was established with two fluorescent labeled mimic peptides.Among them,the concentration of fluorescence-labeled mimic peptide SMFHPWS has a good linear relationship at 2-100 ng/m L(R²=0.9994).However,when the fluorescence-labeled mimic peptide in the sample solution is sampled,eluted and eluted,the recovery rate of fluorescent mimic peptide is low,which may be due to the low affinity of fluorescence-labeled mimic peptide SMFHPWS,which can not be used to establish IAC-HPLC internal standard analysis.High affinity clones need to be further screened.