B型肉毒毒素轻链的高效制备及活性鉴定  

High-Level Expression and Purification of BoNT/B LC and its Identification

作　　者：罗森[1] 陈芳红[2] 李涛[2] 王慧[2] 

机构地区：[1]遵义医药高等专科学校,贵州遵义563002 [2]军事医学科学院微生物流行病研究所病原微生物生物安全国家重点实验室,北京100071

出　　处：《生物技术通讯》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)

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.

关 键 词：肉毒毒素 金属内肽酶 原核表达 

分 类 号：Q78[生物学—分子生物学]