摘要
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。
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.
出处
《食品科学》
EI
CAS
CSCD
北大核心
2010年第21期271-274,共4页
Food Science
基金
广东省科技攻关计划项目(2007A020300007-8)
广东药学院科研启动基金项目(2006SMK06)
关键词
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
chloramphenicol
disulfide-stablized single chain variable fragment
over-expression
β-cyclodextrin companion
renaturation
