关键词: 重组骨桥蛋白, 结直肠癌, 非小细胞肺癌, HEK293E, 瞬时转染

Abstract: This work aims to transfect human embryonic kidney 293E（HEK293）,to secretly express and purify his-tagged recombinant human osteopontin（rhOPN）using mammalian expression vector with transient transfection technology,as well as to study how the rhOPN promotes the cell proliferation of colorectal cancer and non-small cell lung cancer（NSCLC）. The expression vector pcDNA3.1-OPN for OPN fusion 6×his-tagged was constructed and synthesized,and was transiently transfected into HEK293E cells with a polyetherimide（PEI）. The rhOPN was purified by a nickel affinity chromatography column. The mobility and purity of the purified rhOPN in gel was analyzed by SDS-PAGE electrophoresis and Western blot. In addition,the binding capacity between rhOPN and anti-OPN antibody was accessed by ELISA,and the effects of the rhOPN on the proliferation of colorectal cancer and NSCLC cells were analyzed with CCK-8（Cell Counting Kit-8）. The results demonstrated that rhOPN was successfully expressed by transiently transfecting pcDNA3.1-OPN into HEK293E cells and 95% purity was achieved. The rhOPN was well recognized by anti-OPN antibodies,and it significantly promoted the proliferation of HT29 cells in vitro at a concentration of 36 μg/mL. It also remarkably promoted the proliferation of NSCLC H1299 and HCC827 cells in vitro at a concentration of 20 μg/mL. In sum,the rhOPN is successfully expressed in HEK293E cells using mammalian cell transient transfection technology and purified to be in high purity. It is found that the rhOPN can significantly promote the proliferation of human colorectal cancer cells and NSCLC cells and has fine biological activity.

Key words: recombinant human osteopontin, colorectal cancer, non-small cell lung cancer, HEK293E, transient transfection