rhEPO-L-Fc融合蛋白的表达、生物活性和初步药动学分析

Expression of rhEPO-L-Fc Fusion Protein and Analysis of Its Bioactivity and Pharmacokinetics

摘要:

为了延长人促红细胞生成素(hEPO)体内半衰期以达到更好的药效, 制备通过柔性接头相连接的重组人红细胞生成素-IgG1 Fc融合蛋白(rhEPO-L-Fc), 并对其生物学活性和体内药动学进行初步研究。利用PCR技术构建rhEPO-L-Fc融合基因, 克隆至表达载体pOptiVEC-TOPO?, 在二氢叶酸还原酶缺陷型中国仓鼠卵巢细胞(CHO-dhfr-)表达。Protein A 亲合层析柱纯化融合蛋白, SDS-PAGE、质谱、Western blotting鉴定表达产物, 细胞增殖实验检测融合蛋白的体外活性, 动物实验检测融合蛋白的体内活性和半衰期。成功构建pOptiVEC-TOPO?-rhEPO-L-Fc重组子, 实现了在CHO细胞表达,纯化后的rhEPO-L-Fc融合蛋白经鉴定, 其分子量和特异性均与理论值相符, 能刺激体外培养的EPO依赖型细胞生长, ED50 为2 ng/mL, 且明显增加大鼠外周血网织红细胞数, 体内消除半衰期达到27 h。rhEPO-L-Fc融合蛋白能延长hEPO体内半衰期, 为其临床研究奠定了基础。

Abstract:

To prolong serum half-life of human Erythropoietin for better efficacy, a new form of recombinant human erythropoietin (rhEpo-L-Fc) was generated by fusion of a full length human erythropoietin gene and the Fc fragment of human IgG1 with flexible linker sequence. The fusion gene rhEPO-L-Fc was constructed by PCR, then inserted into expression vector pOptiVEC?-TOPO?, and expressed in Chinese Hamster Ovary cells deficient in the DHFR enzyme(CHO-dhfr-). The chimeric protein was purified by Protein A affinity chromatography, showed expected molecular weight and demonstrated a similar bioactivity compared to that of the native recombinant human erythropoietin (rhEPO) in an EPO-dependent cell-based assay. In vivo pharmacokinetic studies showed that the rhEPO-L-Fc had an elimination half-life of 27 h. In vivo efficacy studies showed that a single dose administration of rhEPO-L-Fc in rats increased the reticulocyte number in the peripheral blood significantly. These results demonstrated that the new engineered rhEPO-L-Fc may become alternative therapeutic approach to extend the half-time of rhEPO to treat anemia.