hK1-L-Fc融合蛋白在CHO细胞中的表达及其活性研究  

Expression of hK1-L-Fc Fusion Protein in CHO Cells and Analysis of its Bioactivity

作　　者：邓春平[1] 陶建军[2] 侯永敏[2] 钟翎[1] 

机构地区：[1]中山大学药学院,广州510006 [2]广东天普生化医药股份有限公司,广州510520

出　　处：《中国生物工程杂志》2011年第3期23-28,共6页China Biotechnology

基　　金：国家自然科学基金(30873457);广东省科技计划(2008A060202010)资助项目

摘　　要：为进一步改造重组人激肽释放酶1(hK1),以期提高其生物活性,制备了通过柔性接头相连接的重组人激肽释放酶1-L-IgG1 Fc融合蛋白(hK1-L-Fc)。采用重叠延伸PCR技术构建hK1-L-Fc融合基因,克隆至表达载体pcDNA3.1,在中国仓鼠卵巢细胞(CHO-S)中表达。利用Protein A亲和层析柱纯化融合蛋白,SDS-PAGE、Western blotting、飞行时间质谱(MALDI-TOF-MS)、HPLC检测表达产物,底物法检测融合蛋白的体外活性。结果显示:成功构建pcDNA3.1-hK1-L-Fc重组表达载体;获得稳定表达融合蛋白的细胞株;无血清悬浮批式培养的表达量在0.7 mg/L以上;纯化的蛋白其纯度在95%以上,分子量约60 kDa;活性检测显示其比活性在9.2 U/mg以上,较hK1-Fc蛋白提高了18%以上。To further modify the recombinant human kallikrein 1（hK1） for higher activity,new form of recombinant human kallikrein 1（hK1-L-Fc） was constructed by fusion of the human kallikrein1 gene and the coding sequences for Fc fragment of human IgG1 with a flexible linker peptide.The fusion gene hK1-L-Fc was constructed by PCR,then inserted into expression vector pcDNA3.1,and expressed in Chinese Hamster Ovary cells.The chimeric protein was purified by Protein A affinity chromatography,and further analyzed by SDS-PAGE、Western blotting、MALDI-TOF-MS and HPLC.The bioactivity of fusion protein in vitro was determined by the reaction with its substrate.The results showed that recombinant expression vector pcDNA3.1-hK1-L-Fc was constructed successfully and the cell lines stably secreting fusion protein were obtained;The expression output of the fusion protein was higher than 0.7 mg/L in batch culture;The purity of the protein with about 60 kDa molecular weight was higher than 95%,and the biological activity of the fusion protein was more than 9.2 U/mg,which increased more than 18% than that of hK1-Fc fusion protein.

关 键 词：激肽释放酶1 融合蛋白 CHO细胞 

分 类 号：Q78[生物学—分子生物学]