重组犬促红细胞生成素Fc融合蛋白的真核表达及生物学活性分析OA

The canine erythropoietin Fc fusion protein(cEPO-Fc)was expressed in Chinese hamster ovary(CHO)cells and its biological activity was analyzed.The cEPO-Fc fusion gene was designed and synthesized using the cEPO gene coding sequence and the canine IgG Fc coding sequence.A eukaryotic expression plasmid was constructed.Stable cell pools were obtained through electroporation,homologous recombination and pressure screening methods.Three high-expression stable cell lines were selected through flow cytometric single-cell sorting and double-antibody sandwich ELISA methods.The cEPO-Fc fusion protein was purified by protein G affinity chromatography.After SDS-PAGE and Western blot analysis,functional activity of the cEPO-Fc fusion protein was analyzed in vitro and in vivo.The results showed that the size of the reduced monomer of the expressed cEPO-Fc fusion protein was 65 kDa,and the size of the non-reduced dimer was 130 kDa,which was consistent with the predicted values.The TF-1 cell proliferation test results showed that the cEPO-Fc fusion protein had the activity of promoting TF-1 cell proliferation,with an ED50 of 9.25～10.27 ng/mL.A single subcutaneous injection of 10～80 μg/kg dose of the cEPO-Fc fusion protein could effectively promote the increase in the number and proportion of reticulocytes in the peripheral blood of dogs.The group with a dose of 40 μg/kg had the largest increase.No adverse reactions were observed in clinical observations.This study obtained a canine-derived long-acting EPO drug candidate molecule,providing a reference for the development of therapeutic drugs for canine renal anemia.