A型肉毒毒素轻链的原核表达、纯化及活性鉴定  

Prokaryotic Expression and Purification of BoNT/A LC and its Identification

作　　者：罗森[1] 齐芬芳[1] 宫路路[1] 刘昊[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》2015年第5期632-635,共4页Letters in Biotechnology

基　　金：国家自然科学基金(31201352)

摘　　要：目的:在大肠杆菌中表达、纯化A型肉毒毒素轻链(Bo NT/A LC),研究其生物学活性。方法:根据Gen Bank中报道的Bo NT/A LC基因序列设计特异引物,从肉毒梭菌中扩增Bo NT/A LC基因片段,构建重组大肠杆菌p ET-32a Bo NT/A LC/BL21(DE3)Rosetta,IPTG诱导目的蛋白高效表达,表达产物经Ni螯合亲和层析纯化,SDS-PAGE鉴定目的蛋白,并利用相应底物对纯化产物进行生物活性分析和酶活动力学测定。结果与结论:构建了重组大肠杆菌p ET-32a Bo NT/A LC/BL21(DE3)Rosetta,原核表达获得高水平可溶性重组Bo NT/A LC,纯化得到纯度较高的蛋白质,重组Bo NT/A LC的酶活略高于A型肉毒毒素全毒素,可作为试剂用于Bo NT/A LC抑制剂高通量体外检测方法研究。Objective： To prokaryotic express, purify and identify the type A botulinum neurotoxin light chain （BoNT/A LC）. Methods： The sequence of BoNT/A LC gene was amplificated from Clostridium botulinum and in- serted into pET-32a to create plasmid pET-32a-BoNT/A LC. The recombinant plasmid was transformed into E.coli BL21（DE3）Rosetta and induced by IPTG at 20~C. BoNT/A LC protein with His-tag was purified using Ni2＊-NTA agarose. After purification, The activity of the protein was also analyzed by SNAP-25. Results ＆ Conclusion： The expression plasmid pET-32a-BoNT/A LC was successfully constructed and the recombinant pET-32aBoNT/A LC/BL21 （DE3）Rosetta was induced by IPTG to express the recombinant BoNT/A LC. The purified recombinant BoNT/A LC retained proteolytic activity was examined by SNAP-25. BoNT/A LC gene has been successfully cloned and expressed, with its product has been purified and identified.

关 键 词：A型肉毒毒素轻链 金属内肽酶 原核表达 

分 类 号：Q78[生物学—分子生物学]