Biopanning The Mimotopes Of Ochratoxin A And Their Immunogenicity

Ochratoxin A?OTA?is a toxic secondary metabolite produced by molds.It is widely distributed,highly toxic and poses a major threat to animals and human health.Because OTA can cause harm to a variety of foods and food raw materials,it is urgent to take effective measures to prevent and control OTA pollution.The artificial antigens prepared by coupling mycotoxins with carrier protein can be used to immunize animals,which can resist the invasion of mycotoxins.However,artificial antigens have potential toxicity to organisms,and mycotoxin standard materials are expensive.In comparison,using phage display technology to screen mimotopes to prepare epitope vaccines against mycotoxins has great potential and research significance in the prevention and control of mycotoxins poisoning in animals and humans.The main contents of this experiment include:1.Biopanning the mimotopes of OTA from a 7-mer phage peptide libraryUsing a monoclonal antibody against OTA as a ligand,mimotopes were selected from a 7-mer phage peptide library.After four rounds of biopanning,thirty blue phage plaques were randomly selected.They were identified by Phage-ELISA and indirect competitive ELISA.Among these phage clones,twenty-seven clones could bind to anti-OTA monoclonal antibodies and the twenty-seven positive phage clones could be effectively inhibited by OTA.After DNA sequencing,the amino acid sequences were deduced from their nucleotide sequences.Finally,twenty-seven phage clones were identified as six different OTA mimotopes.The inhibition curves of three mimotopes?P1,P4 and P12?that appeared most frequently during the screening process were plotted.The results showed that all three phages had high sensitivity to OTA.IC₅₀of P1,P4 and p12 were 0.347 ng/m L,0.142 ng/m L and 3.437ng/m L,respectively.Among them,P4 was the most sensitive to OTA.2.Study the immunogenicity of OTA mimotopes and artificial antigenThe screened phages P1,P4,P12(10¹²pfu)and OTA-BSA?50?g?were used as antigens to immunize Kunming mice,respectively.Seven days after the third immunization,mouse serum samples were collected to explore their immunogenicity.In the phage-immunized group,the coating antigens were phage P1,P4 and P12,respectively.In the OTA-BSA immunized group,OTA-OVA coated plates were used to detect titer of mouse immune serum.The indirect competitive ELISA results showed that the immune serum of OTA-BSA could induce mice to produce antibodies against OTA.The immune serum of the specific phages could also induce antibodies production and produced antibodies against the phages,but no antibodies against OTA were detected.In this study,six mimotopes of OTA were successfully obtained through biopanning and identified.The three mimotopes?P1,P4 and P12?and OTA-BSA were used to immunize mice,respectively.It was found OTA-BSA had immunogenicity.However,the serum of mice immunized with mimotopes did not produce antibodies against OTA.Whether the mimotopes of OTA could be used as a vaccine candidate still needs more in-depth and comprehensive research.