重组人骨桥蛋白在哺乳动物细胞中的表达、纯化和活性研究  

Study on Expression,Purification and Activity of Recombinant Human Osteopontin in Mammalian Cells

作　　者：周美琪 肖欣怡 杨卓一 白思益 陈会 袁运生 ZHOU Mei-qi;XIAO Xin-yi;YANG Zhuo-yi;BAI Si-yi;CHEN Hui;YUAN Yun-sheng(Engineering Research Center of Cell and Therapeutic Antibody,School of Pharmacy,Shanghai Jiao Tong University,Shanghai 200240)

机构地区：[1]上海交通大学药学院细胞工程及抗体药物教育部研究工程中心,上海200240

出　　处：《生物技术通报》2020年第8期129-135,共7页Biotechnology Bulletin

基　　金：国家科技重大专项重大新药创制课题(2019ZX09201001);国家自然科学基金项目(31671388);上海交通大学医工交叉项目(YG2019QNA50)。

摘　　要：旨在运用哺乳动物表达载体的瞬时转染技术,转染人类胚胎肾细胞(Human Embryonic Kidney 293E,HEK293E),分泌性表达和纯化带His标签的重组人骨桥蛋白(Recombinant Human Osteopontin,rhOPN),并研究其促结肠癌以及非小细胞肺癌细胞增殖功能。合成和构建OPN融合6×His标签重组蛋白的表达载体pcDNA3.1-OPN,利用聚醚酰亚胺(Polyetherimide,PEI)瞬时转染法将pcDNA3.1-OPN转染到HEK293E细胞中,并用镍亲和层析柱对rhOPN进行纯化。SDS-PAGE电泳和Western Blot被用来检测纯化后的rhOPN蛋白在凝胶上的迁移率和纯度。此外,ELISA方法被用于测定rhOPN与抗OPN抗体间的结合能力,并用CCK-8(Cell Counting Kit-8)方法初步研究rhOPN蛋白对结肠癌及非小细胞肺癌细胞增殖的影响。结果显示,通过将pcDNA3.1-OPN瞬时转染HEK293E细胞,成功表达和纯化出纯度达95%的rhOPN,且rhOPN可以被抗OPN抗体很好的识别。rhOPN被证实在36μg/ml浓度时即可显著促进体外培养的HT29细胞增殖,在20μg/mL时能够促进体外培养的非小细胞肺癌H1299及HCC827细胞增殖。运用哺乳动物细胞瞬时转染技术,在HEK293E细胞中成功表达并纯化出高纯度的rhOPN蛋白,并发现rhOPN可以显著促进结肠癌及非小细胞肺癌细胞增殖,具有良好的生物学活性。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 SDSPAGE 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 36μg/mL.It also remarkably promoted the proliferation of NSCLC H1299 and HCC827 cells in vitro at a concentration of 20μg/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.

关 键 词：重组骨桥蛋白 结直肠癌 非小细胞肺癌 HEK293E 瞬时转染 

分 类 号：Q952[生物学—动物学]