重组葡激酶突变体K35R纯化过程中宿主菌菌体蛋白残余量的免疫分析  

Monitoring Host Cell Protein Contaminant Clearance During Purification of a Recombinant Staphylokinase Variant K35R by Immunological Assay

作　　者：贺进田[1] 王改珍[2] 狄静芳[1] 徐瑞光[1] 宋后燕[3] 

机构地区：[1]河北师范大学生命科学学院,河北石家庄050016 [2]河北科技大学环境科学与工程学院,河北石家庄050018 [3]复旦大学教育部分子医学重点实验室,上海200032

出　　处：《河北师范大学学报（自然科学版）》2006年第6期709-712,共4页Journal of Hebei Normal University：Natural Science

基　　金：国家863生物高技术研究发展计划项目(863-2001AA215291)

摘　　要：葡激酶突变体K35R（DGR）是双功能葡激酶突变体，是正在开发的治疗血栓性疾病的新药．为检测DGR纯化过程中宿主菌菌体蛋白的残留，建立了宿主菌菌体蛋白的免疫分析方法．用所制备的宿主菌菌体蛋白免疫新西兰兔，得到菌体蛋白的抗血清。使用SDS—PAGE和基于抗血清的免疫印迹方法检测DGR纯化过程中菌体蛋白的去除．进一步建立了对宿主菌菌体蛋白灵敏的酶联免疫吸附测定（ELISA）方法，其检测线性范围是5-100μg/L，并用ELISA法定量测定了纯化过程中中间产物和纯化产品中菌体蛋白的含量．Staphylokinase variant K35R （DGR） is a bifunctional protein that possesses fibrinolytic and antiplatelet aggregation activities, and is a promising drug for thrombotic disorders. Immunological assay was developed to monitor the host cell protein （HCP） contaminants during purification of DGR. Antiserum was prepared against HCP antigens unique to E. coli strain JF1125. The clearance of HCPs during purification of DGR was confirmed using SDS-PAGE and Western blot developed by the same HCP antisera. A sensitive HCP assay in the ELISA format was developed. The assay is specific for purification process with a useful detection range from 5 to 100μg/L,and is not affected by product level.

关 键 词：葡激酶 宿主菌菌体蛋白 酶联免疫吸附测定 

分 类 号：TQ450.2[化学工程—农药化工]