Preparation And Identification Of Single-chain Fragment Vairable Antibody Against Patulin

Patulin is lactone metabolites from a variety of fungi Aspergillus, Penicillium,Byssochlamys spp, discoveried in rotten hawthorn, apples, grapes, tomatoes and otherfruits, as well as expired ham, cakes and other foods. Patulin has teratogenic,carcinogenic, mutagenicon for cells and tissues of animals, causes vomiting, stomachcramps and other symptoms after ingesting of patulin.In view that patulin exists in fruit widely, and its toxic effect of eaters, preventionand control of patulin pollution has become a global important question. Fast andaccurate detection of the toxin as diffusion becomes the effective means to controlpollution. For the development of rapid immunological detection method based onantibody. In the experiment we use the method of mixed anhydride joint active esteron the basis of previous studies, successfully prepare PAT-BSA immunogen andcoating antigen PAT-OVA, with two methods of footpad immunization andconventional immunity, immuning BALB/c mice. When immunization titer is1:4000or more, on the other hand, preparating monoclonal antibodies, on the other hand,extracting RNA from BALB/c mouse spleen cells, reversing transcription to obtain afull genome cDNA as a template, obtaining the VH and VL genes. The assemblingmethod of obtaining single-chain antibody (VH-Linker-VL) is SOE-PCR, The genelength of ScFv is759bp, containing378bp light chain variable region gene,399bpheavy chain variable region gene,15bp Linker (Gly4Ser)3genes, after IgBlastanalysis, it has typical antibody variable range domain structure, using prokaryoticexpression system for single antibody gene expression effectively, expression ofprotein molecular weight was29kDa,identifying the biological activity of expressedproduct, it can be combined with patulin,at the same time, building single anti-patulinchain antibody library through the use of phage display technology, and making itsinitial screening, its activity was measured after three panning by enzyme-linkedimmunosorbent assay. Positive strains have been obtained, and preliminary purification for protein expression. Immunological detection method based on singleantibody experimental basis lay the foundation for the further development.