重组人βNGF在大肠杆菌中的可溶表达、纯化及活性鉴定  

Soluble Expression,Purification and Activity Assay of Recombinant Human βNGF Expressed in Escherichia coli

作　　者：白羊 章永垒 邹有土 黄奋飞 陈胜亮 阮卡 葛平辉 马燕玲 王明灶 陈星 

机构地区：[1]未名生物医药有限公司,厦门361009

出　　处：《生物技术通报》2016年第11期202-207,共6页Biotechnology Bulletin

基　　金：国家"重大新药创制"科技重大专项(2011ZX09401-017)

摘　　要：旨在大肠杆菌中可溶表达重组人神经生长因子（Recombinant humanβnerve growth factor,rhβNGF）,并对表达产物进行分离纯化和生物学活性鉴定。成功扩增h NGFβ亚基基因,将其克隆入pMAL-c2X表达载体,构建了hβNGF-MBP的大肠杆菌表达体系并进行诱导表达,表达产物经纯化后以Factor Xa酶切去除麦牙糖结合蛋白（MBP）,Western blot鉴定后以TF-1细胞法检测生物学活性。结果显示,pMAL-c2X-hβNGF经酶切和测序证实构建正确,25℃、180 r/min、0.5 mmol/L IPTG诱导下可溶表达hβNGF-MBP融合蛋白。hβNGF-MBP经Factor Xa酶切后可去除MBP标签,SDS-PAGE分析纯化的hβNGF位于13 k D左右,纯度可达95%。Western blot鉴定为hβNGF,结果表明,比活约为1×10~6 U/mg。在大肠杆菌中成功可溶表达hβNGF,并具有较高的生物学活性。The goal of this work is to express the recombinant human nerve growth factor（rhβNGF）in Escherichia coli in soluble form,to separate and purify the expressed products,and to determine the biological activity. First,hβNGF gene was amplified and then inserted intoexpression vector p MAL-c2 X,then E. coli expression system of hβNGF-MBP was constructed and induced for expression. Further,the MBP inpurified expressed products was cleaved by Factor Xa enzyme,after identified by Western blot,the biological activity was examined by TF-1method. The results showed that enzyme digestion and sequencing confirmed that the recombinant plasmid p MAL-c2X-hβNGF was constructedcorrectly,hβNGF-MBP was secretory expressed under 2℃,180 r/min and 0.5 mmol/L IPTG induction. MBP tag in hβNGF-MBP was removedby Factor Xa digestion,and the purified hβNGF via SDS-PAGE was about 13 k D with the purity over 95%. The protein was identified ashβNGF by Western blot. The biological activity test showed that the specific activity was about 1×106 U/mg. Overall,recombinant hβNGF wassuccessfully expressed in E. coli in soluble form with high biological activity.

关 键 词：人神经生长因子 大肠杆菌 可溶表达 生物学活性 

分 类 号：Q78[生物学—分子生物学]