摘要
目的 构建含有Fc片段的绒毛膜促性腺激素（human chorionic gonadotropin,hCG）新型长效重组hCG-Fc融合蛋白,可显著延长其在血液中的半衰期,有利于提高患者用药依从性.方法 首次将Fc DNA片段有序连接到hCG基因3＇端,构建hCG-Fc表达载体并将其稳定表达在哺乳动物细胞,通过无血清悬浮细胞培养和基于亲和层析的蛋白纯化,制备了新型重组hCG-Fc融合蛋白.检测其分子量、ELISA免疫活性和体内生物活性,并测定其在大鼠体内血循环半衰期.结果 重组融合蛋白分子量与理论推断值相当,ELISA活性为2 965 IU/mg,体内生物活性为2 288 IU/mg,消除半衰期为174 h.结论 hCG-Fc融合蛋白构建表达成功,且具有显著的体内外活性,药代动力学分析显示hCG-Fc重组融合蛋白半衰期是重组hCG的近6倍.这种新型长效融合蛋白在临床上可大大减少注射次数,提高患者用药依从性,具有显著的社会和经济效益.
Objective To make a novel long-acting hCG-Fc fusion protein that could prolong hCG＇s serum half-life and improve the patients＇ compliance. Methods The expression vector was constructed by the gene engineering that contains the gene encoding hCG-Fc which Fc fragment was fused into the 3＇ end of hCG gene. The expression vector was transfected into CHO cells and stable clones were selected to express hCG-Fc fusion protein specifically. The serum free culture medium of cells grown was collected for protein purification using affinity based chromatography. The analysis of purified protein was performed as follow： molecular weight by SDS-PAGE, protein identity by Western blotting, activity in vitro by ELISA, activity in vivo by seminal vesicle weight gain assay, and serum half-life by single-subcutaneous injection into rats. Results The molecular weight of hCG-Fc fusion protein is similar to the theoretical size. The ELISA activity of hCG-Fc protein is 2 965 IU/mg and in vivo activity is 2 288 IU/mg. The serum half-life in rats is 174 h. Conclusions hCG-Fc fusion protein is active and functional based on its in vitro activity and in vivo activity. Pharmacokinetic study shows that its serum half-life is almost six times of the recombinant hCG＇s. This novel long-acting fusion protein can signifi- cantly reduce the times of injection and improve the patients＇ compliance of medicine, and also could have huge social and economic benefits.
出处
《实验动物与比较医学》
CAS
2014年第2期113-119,共7页
Laboratory Animal and Comparative Medicine
基金
广州市创业领军人才创业启动基金项目（LCY201305）
