融合蛋白Epo-Tpo(C)的表达及初步活性检测

Eukaryotic expression,purification and analysis of the fusion protein of Epo-Tpo(C)

目的 进一步研究促血小板生成素Tpo的功能 ,探讨新型Epo的可能性。 方法 构建了融合蛋白Epo Tpo(C)的真核表达载体 ,在CHO细胞中表达融合蛋白 ,染料亲合柱Blue SepharoseCL 6B初步纯化 ,网织红细胞计数法体内测活并测定了体内半衰期和融合蛋白对大鼠肾衰模型的疗效。结果 初步纯化的融合蛋白体内活性比ELISA结果高 2 0 % ,半衰期为 12h ,比标准Epo长近5 0 % ,对大鼠肾衰模型有良好的疗效。结论 Tpo富糖基化的C端结构域能显著延长Epo的半衰期 ,融合蛋白Epo Tpo(C)

Objective To further elucidate the function of Tpo(C) and create a new type of Epo.Methods Tpo(C) was linked to Epo to produce a fusion protein\_\_Epo\|Tpo(C).The fusion gene was expressed in CHO cell.After selection with G418,Epo\|Tpo(C)\|expressing cell lines was obtained.The supernatant had 15 U/ml Epo activity.The results of PCR and Southern Blotting indicated the fusion gene was fused into the genome.RT\|PCR and RNA Dot Blotting analysis found the Epo\|Tpo(C) mRNA.The supernatant was purified with Blue Sepharose CL\|6B.Results The rate of recovery was 94%.The \%in vivo\% activity tested by reticulocytes was 20% higher than ELISA.The half\|life was about 12 hours while the Epo standard sample had half\|life of 8 hours tested with ELISA in rat.Conclusion The results proved that Tpo(C) could protect Epo and improve its function.The fusion protein had satisfactory effect in the model of rat renal failure.