A型肉毒毒素轻链多步纯化及其金属蛋白酶活性研究  

Multi-step purifications of botulinum neurotoxin A light chain and identification of its metalloproteases activity

作　　者：卢晓雪 赵缓[2] 黄彦杰[2] 李涛[2] 王慧[1,2] 

机构地区：[1]广西医科大学,南宁530021 [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：《军事医学》2016年第4期319-321,共3页Military Medical Sciences

基　　金：国家自然科学基金资助项目(31201352)

摘　　要：目的利用基因工程方法原核表达N-His标签的A型肉毒毒素轻链(Bo NT-ALC)蛋白,多步纯化获得高纯度蛋白,并对其金属蛋白酶活性进行鉴定。方法以A型肉毒毒素为模板,构建重组表达载体p ET22b-ALC,转化BL21(DE3),诱导蛋白表达,采用镍柱亲和纯化、阴离子交换以及分子筛纯化获取高纯度目的蛋白;通过体外切割特异性底物SNAP-25实验进行酶活性研究。结果与结论获得高纯度的Bo NT-ALC蛋白,对金属蛋白酶活性进行了验证。该纯化方法获得的蛋白纯度高、均一性好,且具有很好的酶活性,为后续研究奠定了基础。Objective To obtain highly purified botulinum neurotoxin A light chain（ Bo NT-ALC） protein in E. coli by genetic engineering and multi-step purifications,and identify its metalloproteases activity. Methods The full-length of Bo NT-ALC was cloned from Bo NT A by PCR and inserted into plasmid p ET-22 b. Then p ET-22b-ALC was transformed into E. coli BL21（ DE3） strains and induced by IPTG. The protein was purified by Ni-NTA sepharose,anion exchange column and gel filtration. The enzymatic activity of the protein was identified by SNAP-25. Results and Conclusion A highly purified and homogeneous protein is obtained,which shows good enzymatic activity.

关 键 词：A型肉毒毒素 蛋白纯化 金属蛋白酶类 

分 类 号：R3411[医药卫生—基础医学]