Metta M K, Kunaparaju R K, Tantravahi S
GIS, GITAM UNIVERSITY Department of Biotechnology Visakhapatnam India
GIS, GITAM UNIVERSITY Department of Biotechnology Visakhapatnam India.
Cell Mol Biol (Noisy-le-grand). 2016 Feb 29;62(2):101-6.
Recombinant therapeutic proteins have changed the face of modern medicine in the present trend and they continue to provide innovative therapies for deadly diseases. This study describes the development of a novel stable expression system for rapid amplification of genes in Chinese Hamster Ovary (CHO) cells. The expression system consists of a host CHO cell line and an expression vector (pUB-PyOri-D-C) which encodes for Polyomavirus (Py) Origin of Replication (PyOri) for amplification of integrated genes in the presence of Py Large T Antigen (PyLT) and Dihydrofolate Reductase (DHFR) selectable marker gene for selection in the presence of Methotrexate (MTX). Use of both PyOri/PyLT and DHFR can reduce the number of rounds of selection and amplification required for isolation of high producing clones. The efficiency of pUB-PyOri-D-C was compared with that of pUB-D-C plasmid using Green fluorescent protein (GFP) and Erythropoietin (EPO) as reporter proteins. Our results showed that pUB-PyOri-D-C-EPO can help development of high expressing clone in one round of selection/amplification as compared to multiple rounds of selection/amplification with pUB-D-C-EPO plasmid. CHO-DG44/EPO clone generated using pUB-PyOri-D-C-EPO gave a productivity of 119 mg/L in shake flask.
在当前趋势下，重组治疗性蛋白质改变了现代医学的面貌，并且它们继续为致命疾病提供创新疗法。本研究描述了一种新型稳定表达系统的开发，该系统用于在中国仓鼠卵巢（CHO）细胞中快速扩增基因。该表达系统由宿主CHO细胞系和一个表达载体（pUB-PyOri-D-C）组成，该载体编码多瘤病毒（Py）复制起点（PyOri），用于在Py大T抗原（PyLT）存在的情况下扩增整合基因，以及二氢叶酸还原酶（DHFR）选择标记基因，用于在甲氨蝶呤（MTX）存在的情况下进行选择。同时使用PyOri/PyLT和DHFR可以减少分离高产克隆所需的选择和扩增轮数。使用绿色荧光蛋白（GFP）和促红细胞生成素（EPO）作为报告蛋白，将pUB-PyOri-D-C的效率与pUB-D-C质粒的效率进行了比较。我们的结果表明，与使用pUB-D-C-EPO质粒进行多轮选择/扩增相比，pUB-PyOri-D-C-EPO可以在一轮选择/扩增中帮助开发高表达克隆。使用pUB-PyOri-D-C-EPO产生的CHO-DG44/EPO克隆在摇瓶中的产量为119 mg/L。
