A Study Of Construction Of High Density OTA Mimotope Phage And Its Application In ELISA

Ochratoxin A (OTA) mimotope is a short peptide, its stucture is similar to the epitope structure of OTA molecular and it can specifically bind with the OTA antibody. OTA mimotope is expected to become a substitute for natural OTA in immunology studying with the non-toxic, low-cost characteristics. In this study, two synthetic complementary oligonucleotides which contain Enterokinase cleavage site at the 5' terminal of OTA mimotope (IRPMVDP) sequence were put to hybridizing by annealing, and ligated to EcoR I and BamHI digested phagemid pC89S4 with T4 DNA ligase. Then, through phage display technology, the OTA mimotope display on KM 13 phage surface with high density. Then, we study the recombinant phage in immunogenicity identification, preservation methods and appliance in ELISA. Its main contents are as follows:1. Construction phagemid vector contain Enterokinase cleavage site and OTA mimotope sequence.The Enterokinase cleavage site and OTA mimotope sequence have been cloned into phagemid vector pC89S4 and display right sequnence by DNA sequencing.2. Construction high-density OTA mimotope recombinant phage and establishment non-toxic OTA ELISA detection method.High-density OTA mimotope recombinant phage which have the Enterokinase cleavage site connecting the N-terminal of mimotope are expressed though filamentous phage pⅧdisplay system. We obtain a high titer recombinant phage by using Enterokinase cleavage. It indicate that the residues in N-terminal of pⅧmay prevent mimotope binding with antibody. Then, we established non-toxic OTA ELISA detection method by using digested recombinant phage.3. Expression optimization of the OTA mimotope and studying preservation methods of the digested recombinant phage.We optimized the expression of OTA mimotope by testing adding quantity of phage, induction time and dose of IPTG. We obtain a large number of high-density OTA mimotope recombinant at the optimization condition. We also studying the preservation methods of recombinant phage and find a good protective agent.