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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-DC) 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-DC was compared with that of pUB-DC plasmid using Green fluorescent protein (GFP) and Erythropoietin (EPO) as reporter proteins. Our results showed that pUB-PyOri-DC-EPO can help development of high expressing clone in one round of selection/amplification as compared to multiple rounds of selection/amplification with pUB-DC-EPO plasmid. CHO-DG44/EPO clone generated using pUB-PyOri-DC-EPO gave a productivity of 119 mg/L in shake flask.