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制备氨基末端定点PEG修饰的尿酸酶,并比较尿酸酶修饰前后的免疫原性差异。采用M_r 20k的mPEG-丙醛选择性修饰尿酸酶的氨基末端;利用source 30Q离子交换层析和sephacryl S-200分子排阻层析分离纯化修饰后尿酸酶;测定修饰前后尿酸酶的酶动力学参数和免疫原性。尿酸酶和N端PEG修饰尿酸酶的SDSPAGE检测显示单一条带,SE-HPLC测定尿酸酶修饰前后的保留时间分别为26.578 min和23.818 min;PEG修饰后尿酸酶的最大反应速度为尿酸酶的80%;PEG修饰后尿酸酶与抗体的结合能力和体内的免疫原性均明显降低。这种方法可用来制备N端PEG定点修饰尿酸酶,修饰后可明显降低尿酸酶的免疫原性。
Preparation of amino-terminal fixed-point PEG-modified uricase, and comparison of uricase before and after the modified immunogenicity differences. M_r 20k of mPEG-propionaldehyde selectively modified amino terminal uricase; using source 30Q ion exchange chromatography and sephacryl S-200 molecular exclusion chromatography purification purified uricase; determination of uricase before and after modification of enzyme kinetics Parameters and immunogenicity. Uricase and N-terminal PEG-modified uricase SDSPAGE detection showed a single band, SE-HPLC determination of uricase before and after modification of retention time was 26.578 min and 23.818 min; PEG modified uricase maximum response rate of uricase 80 %; PEG modified uricase and antibody binding capacity and immunogenicity were significantly reduced. This method can be used to prepare N-terminal PEG-site-modified uricase, modified significantly reduce the immunogenicity of uricase.