自剪切重组麦芽糖结合蛋白-人鼻病毒3C蛋白酶融合蛋白在大肠杆菌表达系统中表达、纯化及活性检测  

Expression,purification and cleavage activity analysis of self-processed recombinant MBP-HRV 3C fusion protease in E.coli expression system

作　　者：董哲君[1,2] 赵海舰[2] 许小毛[3] 方保民[3] 郭健[4] 肖飞[4] 

机构地区：[1]北京协和医学院研究生院,北京100730 [2]北京医院卫生部临床检验中心,北京100730 [3]北京医院呼吸与危重症医学,北京100730 [4]卫生部老年医学研究所,北京100730

出　　处：《国际检验医学杂志》2014年第20期2721-2722,2725,共3页International Journal of Laboratory Medicine

基　　金：国家自然科学基金资助项目(81170050)

摘　　要：目的利用大肠杆菌表达系统高效表达一种可以自剪切的重组的麦芽糖结合蛋白-人鼻病毒3C蛋白酶融合蛋白酶,获得一种可溶性好、酶切特异性强,且在低温下仍保持酶活性的新型基因工程工具酶。方法将编码人鼻病毒3C蛋白酶的遗传微粒克隆入表达载体pRSF-duet,转化大肠杆菌BL21(DE3),经镍柱亲和层析纯化得到人鼻病毒3C蛋白酶。通过自身体内酶切实验进行活性检测。结果人鼻病毒3C蛋白酶在大肠杆菌表达系统中得到了高效的表达,并可特异性识别及剪切人鼻病毒3C蛋白酶酶切位点。结论获得了新型的基因工程工具酶。Objective To obtain a novel tool-enzyme for genetic engineering with good solubility,strong specificity of enzyme digestion and maintaining the enzyme activity at low temperature by using E.coli expression system to express self-processed re-combinant MBP-HRV 3C fusion protease.Methods The cDNA encoding HRV 3C protease was cloned into pRSF-Duet vector.The recombinant plasmid was transferred into E.coli BL21 （DE3）for expression.HRV 3C protease was obtained through Nichol col-umn affinity purification.The cleavage activity of HRV 3C protease was determined by in vivo experiment.Results HRV 3C prote-ase was highly expressed in E.coli expression system,and the obtained HRV 3C protease could recognize and digest HRV 3C site. Conclusion A novel tool-enzyme for genetic engineering is obtained.

关 键 词：人鼻病毒3C蛋白酶 表达 工具酶 

分 类 号：R346[医药卫生—基础医学]