Abstract:

To construct a disulfide-stablized single chain variable fragment directed against chloramphenicol ( sdsFvCAP), both cloned V genes were introduced a cystine code by site-specific mutagenesis, then assembled into sdsFvCAP gene by overlap extension PCR. Under the control of strong promoter T7, the sdsFvCAP gene was failed to be individually expressed in Escherichia coli, but over-expressed as inclusion when GC content in the terminal 5 sequence was decreased by synonymous mutation. The inclusion protein was refolded with β -cyclodextrin companion system, and showed affinity similar to its parent monoclonal antibody (MAb). The sdsFvCAP prepared in this study could be potentially used instead of conventional antisera or MAb for development of a rapid and affordable immunoassay for the detection of residual CAP in foods.

Key words: chloramphenicol, disulfide-stablized single chain variable fragment, over-expression, &beta, -cyclodextrin companion, renaturation