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以邻氨基苯甲酸(Abz)为荧光发射基团、2,4-二硝基苯基乙二胺(Eddnp)为荧光猝灭基团,设计合成了SARS-CoⅤ3CL蛋白酶的新型荧光多肽底物:H2N-E(Eddnp)STLQSGLK(Abz)-CONH2.用液相色谱-质谱(LC-MS)联用技术进行了表征,表明该多肽底物能被SARS-CoⅤ3CL蛋白酶识别,并在QS之间被专一性酶解.另外,利用该多肽底物的荧光共振能量转移(FRET)特性,对SARS-CoⅤ3CL蛋白酶的酶解动力学性质进行了研究,结果表明,此荧光多肽底物可以作为荧光探针,应用于SARS-CoⅤ3CL蛋白酶活性的测定及其抑制剂的筛选.
A novel fluorescent peptide substrate of SARS-CoⅤ3CL protease was designed and synthesized by using anthranilic acid (Abz) as a fluorescent emitting group and Eddnp as a fluorescent quenching group. H2N-E (Eddnp) STLQSGLK (Abz) -CONH2 was characterized by liquid chromatography-mass spectrometry (LC-MS) technique, indicating that the polypeptide substrate is recognized by the SARS-Co V3CL protease and is Specific enzymolysis.In addition, the enzymatic hydrolysis kinetics of SARS-CoV3CL protease was studied by the fluorescence resonance energy transfer (FRET) of the substrate. The results showed that the fluorescent peptide substrate could be used as a fluorescent probe Needle for determination of protease activity of SARS-Co Ⅴ CL and screening of its inhibitors.