Cyclic Heptapeptide-based Biosynthesis Of F-2 Complete Antigen Mimics And Its Application In Immunoassay

As F-2 mycotoxin widely exist in grains or feeds,like wheat and grains,humans and animals may cause potential harm after eating contaminated food with mycotoxins.Therefore,effective monitoring and rapid detection of F-2 toxin in argo-food is an important means to prevent it from harming human health.The development and application of F-2 toxin immunoassay detection methods are mainly based on competitive immunoassay models.However,the synthesis and preparation of complete antigens mainly depend on chemical synthesis,in the process of which the standards and toxic reagents are potentially harmful to the operator’s health.More and more researchers have begun to improve the preparation methods of artificial antigens,develop alternatives to mycotoxins,and develop green immunoassay methods based on mycotoxin mimics.Phage display technology was used for panning mycotoxin mimic epitopes,and further using genetic engineering to biosynthesize complete antigens,this method can obtain chemical synthesis antigen substitutes with lowcost,stable performance,simple,green and non-toxic.The purpose of this study is to use phage display peptide technology,biopanning the cyclic heptapeptide and linear dodecapeptide phage mimic epitopes of F-2 toxin,and biosynthesizing the complete antigen of F-2 toxin as an alternative to the traditional chemical synthesis antigen,for comparative analysis the difference between the cyclic heptapeptide and linear dodecapeptide mimetic epitopes in the format of phage and biosynthetic complete antigen in immunoassay performance,and then explore the application of biosynthetic complete antigen mimetics in immunoassay and analysis methods.The main research results are as follows:1.Using anti-F-2 monoclonal antibody as a target molecule,the phage displayed mimotope peptides were panned from phage display cyclopeptide and dodecapeptide libraries,and the phage display-based cyclic heptapeptide and dodecapeptide ic-ELISA standard curves were established.A total number of 9 cyclic heptapeptides colones and 7 linear dodecapeptides colones with different amino acid sequences were obtained,and the half-maximum inhibition concentration(IC50)of the established standard curves were 0.07-0.82 ng/mL and 0.33-0.87 ng/mL,respectively.Furthennore,the sensitivity of immunoassay based on the mimic epitope of cyclic heptapeptide is generally higher than that of linear Dodecapeptide.2.Using molecular cloning technology,the phage-displayed F-2 toxin epitope cycloheptapeptide(Z7-1/6/9)and dodecapeptide(Z12-1/2/6)genes were inserted into pMAL-pⅢ vectors,respectively.The peptide-MBP fusion expression vector was successfully constructed and introduced into E.coli TB1 to induce expression,and 4 cyclic heptapeptide-based F-2 biosynthetic antigens(Z7-1A-MBP,Z7-1B-MBP,Z7-6-MBP,and Z7-9-MBP)were expressed and purified.The indirect competition ELISA(ic-ELISA)established by using it as a coating antigen and showed a typical competition inhibition"S" curve.The IC50 values were 0.36±0.01,0.39±0.02,0.43±0.02,and 2.13±0.10 ng/mL,respectively.In addition,the ic-ELISA standard curves based on biosynthetic antigens(Z12-1-MBP,Z12-2-MBP,and Z12-6-MBP)were established with IC50 values of 0.57±0.01,3.66±0.09,and 5.44±0.10 ng/mL.3.The peptide Z7-4 and Z12-2 displayed by phage clones were cloned into pET-22b-Nluc vector and then constructed a luciferase fusion expression vector based on cyclic heptapeptide and dodecapeptide.After induced expression by IPTG,Z7-4-Nluc and Z12-2-Nluc fusion proteins were obtained.When it was used as the coating antigen,the indirect competition ELISA standard curve was established,and the IC50 was 0.47 ± 0.02 and 2.10±0.11 ng/mL,respectively.4.On the basis of obtaining the biosynthetic Peptide-MBP antigens of FB1,F-2 and OTA,using them as substitutes for artificial antigens and using QDs/QBs as signal output elements,a triple fluorescence immunochromatographic strip analysis method based on biosynthetic antigen was established.And the minimum detection limits for FB1,F-2,and OTA detection under naked eye conditions were 0.25 ng/mL,3.0 ng/mL,and 0.5 ng/mL,respectively.Experimental results such as specificity analysis and spike experiments show that the fluorescent immunochromatographic test strip has good specificity,accuracy and stability.In addition,in order to evaluate the practicability,10 samples of corn,wheat and rice were selected for real sample detection.The results showed that six actual samples were positive,and the results of mFICA real samples detection were consistent with those of UPLC-MS/MS.