EH-L-Fc在CHO细胞中的表达、纯化及活性分析  

Expression, Purification and Function Analysis of EH-L-Fc Fusion Protein

作　　者：庞玉红 刘玉斌[2] 于爱平[2] 吕英涛[1] 

机构地区：[1]青岛科技大学,山东青岛266042 [2]军事医学科学院放射与辐射医学研究所,北京100850

出　　处：《生物技术通讯》2017年第4期422-428,共7页Letters in Biotechnology

基　　金：国家科技重大专项"重大新药创制"(2012ZX09102301-008)

摘　　要：目的:为延长重组新蛭素(EH)的半衰期,制备通过连接肽连接的重组新蛭素与IgG1Fc的融合蛋白EH-LFc,并对其进行功能分析。方法:采用重叠PCR技术构建Eh-L-Fc融合基因,克隆至表达载体pcDNA3.1,用脂质体将重组表达载体转染至中国仓鼠卵巢细胞(CHO)中,G418抗性筛选稳定克隆株;Western印迹检测培养上清中EH-LFc蛋白的表达,用有限稀释法对G418抗性筛选出的混合克隆单克隆化,通过Protein A亲和层析柱纯化融合蛋白,Lowry法检测蛋白浓度,SDS-PAGE、HPLC法检测目的蛋白纯度,质谱法分析相对分子质量,凝血因子Ⅹa裂解融合蛋白后采用纤维蛋白凝块法测定其抗凝活性。结果:构建了重组表达载体pcDNA3.1-Eh-L-Fc,并获得稳定表达EHL-Fc的细胞株。表达产物的相对分子质量为72 168,HPLC检测亲和层析获得的EH-L-Fc纯度达93.9%。完整的EH-L-Fc无抗凝活性,经凝血因子Ⅹa裂解后其抗凝比活性为96.6 ATU/mg。结论:获得稳定表达EH-L-Fc的CHO细胞株和较高纯度的重组融合蛋白,且该重组融合蛋白经凝血因子Ⅹa裂解后可释放抗凝活性。EH-L-Fc融合蛋白的获得为研究新蛭素的长效剂型奠定了重要基础。Objective： To prolong the half-fife of recombinant neorudin（EH）, a new form of recombinant neorudin（EH-L-Fc） was constructed by fusion of the neorudin gene and the coding sequence for fragment of human IgG1 with a rigid linker peptide, and the function of EH-L-Fe was analyzed. Methods： The fusion gene was obtained by SOE-PCR, and constructed into expression vector pcDNA3.1. The recombinant pcDNA3.1-Eh-L-Fc was transfected into CHO cells by liposomes. Stable gene expression cell lines were selected by geneticin（G418） resis- tance screening. The expression of EH-L-Fc protein in the cell culture supernatant was measured by Western blot- ting. The monoclone was selected from the resistance screening polyclone by limiting dilution analysis. The fusion protein was purified by Protein A affinity chromatography, and then the purity of the EH-L-Fc protein was analyzed by SDS-PAGE and RP-HPLC. The fusion protein was cleaved by coagulation factor X a and the anticoagulant activity was determined in vitro by fibrin clot method. Results： The results showed that the recombinant expression vector pcDNA3.1-Eh-L-Fc was successfully constructed, and the stable cell clone CHO-pcDNA3.1-Eh-L- Fc was obtained. The expression product is a dimer with a molecular weight of 72 168 Da. The purity of the recombinant protein was 93.9% with a 96.9 ATU/mg anticoagulant specific activity after cleaved by factor X a. The complete recombinant EH-L-Fc has no anticoagulant activity. Conclusion： The stable CHO cell line harboring the recombinant Eh-L-Fc was successfully obtained. The EH-L-Fc fusion protein with a high purity and bioactivity after cleavage by factor X a provides an important foundation for the further research of long acting EH-L-Fc.

关 键 词：新蛭素 抗凝 FC融合蛋白 

分 类 号：Q78[生物学—分子生物学]