二硫键稳定的抗氯霉素单链抗体的构建 - 食品科学.pdfVIP

※生物工程 食品科学 2010, Vol. 31, No. 21 271 李黄金，陈 伟，赵 林，柴伟佳，唐 伟 (广东药学院生命科学与生物制药学院，广东 广州 510006) 为构建一种食品残留检测用二硫键稳定的抗氯霉素单链抗体(sdsFvCAP) 。以抗氯霉素抗体可变区基因为模板， 通过引物突变 PCR 在轻链和重链的可变区分别引入一个半胱氨酸定点突变，以重叠延伸 PCR 组装成 sdsFvCAP 。在 大肠杆菌系统强启动子 T7 驱动下，sdsFvCAP 基因不能单独表达，而通过同义突变降低其 5 ＇端序列 GC 含量则可实 现高效表达。表达产物主要以包涵体形式存在，经 β- 环糊精分子伴侣系统复性可获得亲和力与母本抗体相似的 sdsFvCAP 。所构建的sdsFvCAP 具有取代单克隆抗体用于氯霉素残留检测的潜力。 ：氯霉素；二硫键稳定的单链抗体；高效表达； β- 环糊精分子伴侣；复性 Construction of Disulfide-Stablized Single Chain Variable Fragment Directed against Chloramphenicol LI Huang-jin ，CHEN Wei ，ZHAO Lin ，CHAI Wei-jia ，TANG Wei (College of Life Science and Biopharmaceuticals, Guangdong Pharmaceuticals University, Guangzhou 510006, China ) 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 ；β-cyclodextrin companion ；renaturation 中图分类号：Q81 文献标识码：A 文章编号：1002-6630(2010)21-0271-04 氯霉