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丙型肝炎病毒丝氨酸蛋白酶在病毒复制和包装中的重要作用使其成为特异性抗病毒药物研究的首选靶标。根据丝氨酸蛋白酶晶体结构特点 ,用柔性连接子连接NS3丝氨酸蛋白酶结构域和NS4A的核心序列 ,构建成单链丝氨酸蛋白酶基因并且在大肠杆菌中获得高水平的可溶性表达 ,纯化后的目的蛋白能够切割重组蛋白底物NS5ab。随后 ,以单链丝氨酸蛋白酶为靶分子对噬菌体展示的随机十二肽库进行了三轮淘筛 ,挑选的 44个克隆中有 3 7个克隆能够特异性地结合丝氨酸蛋白酶 ,并且这种结合作用为竞争性ELISA试验结果所支持。对 1 3个克隆进行序列测定 ,得到 6种序列 ,它们在氨基酸组成上存在明显偏性 ,富含组氨酸和色氨酸 ,缺乏酸性氨基酸 ;6种序列存在一个共有序列
The important role of the hepatitis C virus serine protease in viral replication and packaging makes it the preferred target for the study of specific antiviral drugs. According to the characteristics of the serine protease crystal structure, the NS3 serine protease domain and NS4A core sequence are connected by a flexible linker to construct a single-stranded serine protease gene and acquire a high level of soluble expression in E. coli. The purified target protein can be cut and recombined Protein substrate NS5ab. Subsequently, phage-displayed random dodecapeptide libraries were screen-screened with single-stranded serine protease for three rounds, and 37 out of 44 clones selected specifically bound serine proteases, and this binding Supported by competitive ELISA test results. The sequences of 13 clones were sequenced and 6 sequences were obtained. They had obvious deviation in amino acid composition, rich in histidine and tryptophan, and lacked acidic amino acids. There was one consensus sequence in 6 kinds of sequences