摘要
目的获得转染人蛋白CcDNA的工程细胞株,实现重组蛋白C的表达。方法将构建好的重组表达载体pIRES-hPC用脂质体介导的方法转染HEK293细胞,经G418加压筛选、细胞有限稀释法等获得克隆细胞株,收集无血清培养上清,浓缩后进行鉴定。结果经G418筛选得到了21株克隆化细胞,SDS-PAGE和Westernblot鉴定表明,表达产物含有人蛋白C,且具有抗凝活性。结论在哺乳动物细胞中已成功表达重组人蛋白C,为进一步深入研究及产业化奠定了基础。
Objective To obtain a recombinant cell strain transfected with human protein C cDNA and e xpress the protein. Methods Transfect HEK293 cells with a constructed recombinant expression vector pIRES-h PC in the mediation of liposome.Select positive transfectants by G418 pressure s creening and clone the single cell colony by limiting dilution.Culture the clone d cells in a serum-free medium for 3 days,collect the culture supernatant and c oncentrate with 70% PEG8000.Identify the expressed product by SDS-PAGE and West ern blot. Results A total of 21 cloned cell strains were obtained by G418 pressure screening.S DS- PAGE and Western blot proved that the human protein C with anticoagulation activ ity was expressed. Conclusion Recombinant human protein C was successfully expressed in mammal animal cells.It laid a foundation of further study and industrialization of human protein C.
出处
《中国生物制品学杂志》
CAS
CSCD
2005年第4期294-296,共3页
Chinese Journal of Biologicals
基金
军事医学科学院创新基金资助项目(020901)
