TM-FactorXa-EPO融合蛋白在CHO-K1细胞中的表达研究

Study on TM-FactorXa-EPO Fusion Protein Expression in CHO-K1 Cell

为了构建一种能快速表达人EPO蛋白的真核表达系统,本研究运用的方法是构建基于TNF-α跨膜段(transmembrane,TM)的pcDNA3.1-TM-FactorXa-EPO真核表达载体。把构建好的载体瞬时转染CHO-K1细胞,用FactorXa消化TM-FactorXa-EPO融合蛋白,rhEPO从FactorXa位点处切割下来,达到高效表达和可控酶切。结果本研究构建的表达系统能高效快速表达人EPO。ELISA检测到转染24 h的细胞:经FactorXa酶消化总蛋白,rhEPO浓度为285.21μg/mL;经FactorXa酶消化胞外rhEPO,rhEPO浓度为298.65μg/mL。

The eukaryotic expression system was constructed which can quickly expression human EPO protein. Construction of eukaryotic expression vector pcDNA3.1 - TM - FactorXa - EPO had TNF - α transmembrane Fragment （transmembrane, TM）. Constructed vectors were transiently transfected CHO- K1 cells and the transfected cells rapidly and efficiently expressed the EPO protein which was similar to natural EPO protein. Using FactorXa to digest the fusion protein expressed by the recombinant vector, the rhEPO in the extracellular was cut down at the point of FactorXa site from the fusion protein. The results of this study constructed expression system can quickly and efficiently express human EPO. The concentration of rhEPO was 285.21 μg/mL in total cellular protein by ELISA after 24 h transfection, and in the FactorXa enzyme digestive juice the rhEPO concentration was 298.65 μg/mL by ELISA