Bacteroides coprocola菌β-葡萄糖苷酶基因的表达纯化及活性测定  

Expression and purification of Bacteroides coprocola β-glucosidase gene and its enzymic activity

作　　者：韩洋 路迪 钱露 邢雅玲 应晓敏 HAN Yang;LU Di;QIAN Lu;XING Ya-ling;YING Xiao-min(Institute of Military Cognition and Brain Sciences, Academy of Military Medical Sciences, Academy of MilitarySciences, Beijing 100850, China;Institute of Radiation Medicine, Academy of Military Medical Sciences, Academy ofMilitary Sciences, Beijing 100850, China)

机构地区：[1]军事科学院军事医学研究院军事认知与脑科学研究所,北京100850 [2]军事科学院军事医学研究院辐射医学研究所,北京100850

出　　处：《军事医学》2018年第10期753-756,共4页Military Medical Sciences

基　　金：国家重点研发计划资助项目(2017YFC0908300);国家科技重大专项资助项目(2018ZX10201-001)

摘　　要：目的构建一种拟杆菌Bacteroides coprocola(BC)β-葡萄糖苷酶基因的原核表达载体,并纯化该β-葡萄糖苷酶,检测其水解不同底物的能力。方法以NCBI数据库中提供的BC菌β-葡萄糖苷酶基因序列为参考,合成出该基因的全长序列,测序正确后插入到含GST基因的原核表达载体p GEX-4T-1中;以IPTG诱导表达,并经谷胱甘肽琼脂糖珠纯化融合蛋白; SDS-PAGE和Western印迹检测β-葡萄糖苷酶基因的表达,并通过不同底物检测其活性及比较各法的优劣。结果 PCR扩增结果与双酶切实验表明,原核表达载体构建成功; Western印迹结果显示,β-葡萄糖苷酶基因在大肠杆菌中特异表达;酶活性实验表明,该菌β-葡萄糖苷酶在对硝基苯-β-D-葡萄糖苷(4'-nitrophenyl-beta-D-glucopyranoside,p NPG)和京尼平苷(geniposide)中均有活性,且用p NPG作为底物测定的活性结果更可靠。结论用两种底物成功检测出纯化的BC菌β-葡萄糖苷酶的活性,为进一步研究其功能奠定了基础。Objective To construct a prokaryotic expression vector of Bacteroides coprocola( BC) β-glucosidase gene,purify the protein and test its ability to hydrolyze different substrates. Methods Full length of BC β-glucosidase gene was synthesized according to reference sequence provided in NCBI,the gene was inserted into the vector p GEX-4 T-1 containing GST gene and expressed in E. coli,and GST fusion protein was purified using Glutathione Sepharose 4 B. The expression ofβ-glucosidase gene was detected by SDS-PAGE and Western blotting,the activity was determined by using different substrates and the two methods were compared. Results The prokaryotic expression vector expressing β-glucosidase gene was successfully constructed and checked by PCR and double digestion. The specificity was identified by Western blotting.BC β-glucosidase was active in both p-nitrophenyl-β-D-glucoside( p NPG) and geniposide,and the result could be more reliable by using p NPG as substrate. Conclusion BC β-Glucosidase is able to hydrolyze p NPG and geniposide,which can shed light on the function of BC β-glucosidase.

关 键 词：拟杆菌属 Β-葡萄糖苷酶基因 原核表达 蛋白纯化 活性检测 

分 类 号：R378.2[医药卫生—病原生物学]