Abstract:

Objective: To construct a novel vector with the nucleotide sequence of ochratoxin A (OTA) mimotope based on M13 phage with pⅧ gene, which can give a highly efficient amplification of high-density OTA mimotope so as to provide a basis for preparing an alternative to OTA competitive antigen. Methods: The mimotope with the highest affinity was selected out of OTA mimotopes from the random peptide library of phase pⅢ. Two nucleotide sequences containing the nucleotide sequence of this mimotope and endonuclease sites were chemically synthesized, formed into a double-chain sequence by annealing, inserted into the gap between the genes of pⅧ leader peptides and mature amino acids for connection with vector pC89S4 that had been doubly digested with EcoRⅠ and BamHⅠand transformed into XL1-Blue competent cells. The transformed XL1-Blue competent cells were incubated in liquid SOC medium with shaking and then spreaded on solid LB medium in order to provide single colonies for cell amplification before plasmid extraction. The extracted plasmids were separately digested with XbaⅠ, EcoR Ⅰ and BamHⅠ, preliminarily screened and identified by sequencing. Results: The results of plasmid sequencing were in good accordance with the synthetic sequence. Conclusion: A phase display vector that can efficiently amplify high-density OTA mimotope has been successfully constructed.

Key words: ochratoxin A, phage display, p Ⅷ, mimotope