摘要
目的:在大肠杆菌中高效表达B型肉毒毒素轻链(BoNT/BLC)并纯化,研究其生物学活性。方法:根据报道的BoNT/B LC基因序列设计引物,从肉毒梭菌中扩增BoNT/BLC基因片段,将其克隆至原核表达载体pET-22b中,重组质粒转化大肠杆菌BL21(DE3)Rosetta感受态细胞,构建重组大肠杆菌pET-22b BoNT/BLC/BL21(DE3)Rosetta,在20℃条件下用IPTG诱导目的蛋白表达,表达产物经His Trap FF柱纯化,用SDS-PAGE对目的蛋白进行鉴定,并利用相应底物对纯化产物进行生物活性分析。结果与结论:构建了重组大肠杆菌pET-22b BoNT/BLC/BL21(DE3)Rosetta,BoNT/BLC表达量达到了细菌总蛋白的30%左右,通过一步亲和纯化目的蛋白后经SDS-PAGE检测其纯度在95%以上,制备的重组LC的酶活略高于B型肉毒毒素全毒素,可作为试剂用于BoNT/BLC抑制剂高通量体外检测方法的研究。
Objective: To prokaryotic express, purify and identify the light chain of botulinum neurotoxin serotype B(BoNT/B LC). Methods: The sequence of BoNT LC gene was amplified from Clostridium botulinum and inserted into pET-22 b vector for construction of the plasmid pET-22 b-BoNT LC. The recombinant plasmid was transformed into E.coli BL21(DE3)Rosetta and induced by IPTG at 20℃. BoNT LC protein with His-tag was purified using a His Trap FF column. After purification, the activity of the protein was also analyzed by its substrate.Results & Conclusion: BoNT LC gene has been cloned and expressed with the level of 30% of total bacterial protein. The purity of purified protein was determined to be more than 95% by SDS-PAGE. The recombinant BoNT LC proteolytic activity was slightly higher than botulinum toxin type B toxin, suggesting it can be used as a reagent for high throughput screening of BoNT LC inhibitors in vitro.
出处
《生物技术通讯》
CAS
2018年第1期59-63,共5页
Letters in Biotechnology
基金
国家自然科学基金(31201352)
遵义市科技计划(遵市科合社字(2014)06号)
关键词
肉毒毒素
金属内肽酶
原核表达
botulinum neurotoxin
endopeptidase
prokaryotic expression
