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构建了 SARS冠状病毒主要蛋白酶3CLpro 及其缺失N 端七肽即N-finger的突变体3CLpro(?1–7)融合表达质粒,并于大肠杆菌表达系统中表达。得到的融合蛋白经肠激酶酶切,亲和层析,最终得到纯化的3CLpro及其突变体 3CLpro(?1–7)。酶活性实验显示,去除 N-finger多肽的突变体3CLpro(?1–7)丧失了3CLpro所具有的对含有其自动水解位点的荧光底物的水解活性,表明处于非酶活性中心的N-finger 多肽是酶活性所必需的。利用固相合成法,进一步合成了N-finger七肽。酶抑制实验表明N-finger七肽表现了对3CLpro 部分竞争抑制活性。
The 3CLpro (1-7) fusion expression mutant of SARS-CoV major protease 3CLpro and its deletion N-terminal heptapeptide was constructed and expressed in E. coli expression system. The resulting fusion protein was digested with enterokinase and affinity chromatographed to finally obtain purified 3CLpro and its mutant 3CLpro (? 1-7). Enzyme activity experiments showed that 3CLpro (1-7), a mutant that removes the N-finger polypeptide, loses the hydrolytic activity of 3CLpro with a fluorescent substrate containing its autohydrolysis site, indicating that N- Finger polypeptides are required for enzymatic activity. N-finger heptapeptide was further synthesized by solid-phase method. Enzyme inhibition experiments showed that N-finger heptapetide exhibited partial competitive inhibition of 3CLpro.