ABSTRACT Botulinum neurotoxin (BoNT) is the most toxic biomolecule known to the world (⁓100 times toxic than cyanide) and has been listed as category ‘A’ biowarfare agent. In the present investigation, we cloned the catalytic domain of BoNT type ‘F’. Maximum recombinant BoNT/F protein expression was achieved at 21°C, 0.75 mM IPTG at 6 h post induction. The rBoNT/F protein was purified and confirmed subsequently by MS-MS analysis as well as Western blot. High titer polyclonal antibodies were generated and elevated level of IgG1 isotypes was observed. Plate ELISA was developed for the detection of BoNT/F with the sensitivity of 3.9 ng/mL. Limits of Detection (LOD) was established for different fruit juices through spiking studies using rBoNT/F Lc protein as antigen and achieved a detection limit of 15.62 ng/mL for apple juice followed by 31 ng/mL for litchi and 62 ng/mL for grape, orange, and mango juices. The developed system has the potential for the detection of BoNT/F in food as well as environmental samples.

中文翻译：

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肉毒杆菌神经毒素血清型'F'催化结构域的表达和纯化：免疫学表征及其在检测中的应用

摘要 肉毒杆菌神经毒素 (BoNT) 是世界上已知毒性最强的生物分子（比氰化物毒性 ⁓ 100 倍），已被列为“A”类生物战剂。在本研究中，我们克隆了“F”型 BoNT 的催化结构域。在 21°C、0.75 mM IPTG 诱导后 6 小时实现最大重组 BoNT/F 蛋白表达。rBoNT/F 蛋白被纯化并随后通过 MS-MS 分析以及蛋白质印迹进行确认。产生了高滴度的多克隆抗体，并观察到 ​​IgG1 同种型水平升高。开发板 ELISA 用于检测 BoNT/F，灵敏度为 3.9 ng/mL。通过使用 rBoNT/F Lc 蛋白作为抗原的加标研究，建立了不同果汁的检测限 (LOD)，检测限为 15。苹果汁为 62 ng/mL，荔枝汁为 31 ng/mL，葡萄汁、橙汁和芒果汁为 62 ng/mL。开发的系统具有检测食品和环境样品中 BoNT/F 的潜力。