Mala J, Puthong S, Maekawa H, Kaneko Y, Palaga T, Komolpis K, Sooksai S
Program in Biotechnology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand.
The Institute of Biotechnology and Genetic Engineering, Chulalongkorn University, Bangkok 10330, Thailand.
J Genet Eng Biotechnol. 2017 Jun;15(1):69-76. doi: 10.1016/j.jgeb.2017.02.008. Epub 2017 Mar 17.
Norfloxacin belongs to the group of fluoroquinolone antibiotics which has been approved for treatment in animals. However, its residues in animal products can pose adverse side effects to consumer. Therefore, detection of the residue in different food matrices must be concerned. In this study, a single chain variable fragment (scFv) that recognizes norfloxacin antibiotic was constructed. The cDNA was synthesized from total RNA of hybridoma cells against norfloxacin. Genes encoding V and V regions of monoclonal antibody against norfloxacin (Nor155) were amplified and size of V and V fragments was 402 bp and 363 bp, respectively. The scFv of Nor155 was constructed by an addition of (GlySer) as a linker between V and V regions and subcloned into pPICZαA, an expression vector of . The sequence of scFv Nor155 (GenBank No. AJG06891.1) was confirmed by sequencing analysis. The complementarity determining regions (CDR) I, II, and III of V and V were specified by Kabat method. The obtained recombinant plasmid will be useful for production of scFv antibody against norfloxacin in and further engineer scFv antibody against fluoroquinolone antibiotics.
诺氟沙星属于已被批准用于动物治疗的氟喹诺酮类抗生素。然而，其在动物产品中的残留可能会对消费者产生不良副作用。因此，必须关注不同食品基质中该残留物的检测。在本研究中，构建了一种识别诺氟沙星抗生素的单链可变片段（scFv）。从抗诺氟沙星杂交瘤细胞的总RNA合成cDNA。扩增编码抗诺氟沙星单克隆抗体（Nor155）的V和V区的基因，V和V片段的大小分别为402 bp和363 bp。通过在V和V区之间添加（GlySer）作为接头构建Nor155的scFv，并亚克隆到pPICZαA（一种表达载体）中。通过测序分析确认了scFv Nor155（GenBank编号AJG06891.1）的序列。V和V的互补决定区（CDR）I、II和III通过Kabat方法确定。所获得的重组质粒将有助于在[具体表达系统]中生产抗诺氟沙星的scFv抗体，并进一步构建抗氟喹诺酮类抗生素的scFv抗体。
