A型肉毒毒素轻链多步纯化及其金属蛋白酶活性研究

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（ BoNT－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pET22b－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BoNT－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(BoNT-ALC) protein in E.coli by genetic engineering and multi-step purifications, and identify its metalloproteases activity.Methods The full-length of BoNT-ALC was cloned from BoNT A by PCR and inserted into plasmid pET-22b.Then pET-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.