- 作者: Sun-Young Lee Seon Hee Kim Vic Narry Kim Jeong Ho Hwang Mirim Jin Jaegwan Lee Sun-Young Kim
- 作者服務機構: Institute for Molecular Biology and Gretics, Seoul National University, Seoul, Research Laboratories, II-Dong Pharmaceutical Company, Kyung-Ki-Do, and Department of Biotechnology, Chung Ang University, Kyunggido, Korea
- 中文摘要: --
- 英文摘要: We have explored the possibility of using avian cells forthe expression of human proteins. We found that variousavian cells including quail fibrosarcoma cells(QF), duckembryo cells(DE)and primary chicken embryo fibro-blasts(CE)could efficiently be transfected with DNA bycalcium phosphate coprecipitation, and that promoterswhich are transcriptionally active in mammalian cellsalso functioned well in these avian cells. Among the pro-moters we tested, the major immediate early promoterof human cytomegalovirus drove the highest level ofchloramphenicol acetyl transferase(CAT) expression,outperforming the SV40 early promoter and the RSVLTR. Using the bacterial CAT gene as a reporter, wefound that levels of CAT activity were higher in QF andDE cells than in mammalian cells such as CHO, HeLa,Vero and 293T cells. We further cloned a sequenceencoding human erythropoietin(EPO) and compared itsexpression in QF and mammalian cells. Consistent withthe CAT data, in transient transfection assays, OF cellsproduced higher levels of EPO than the mammalian celllines tested. QF cells which can be passaged permanent-1y were stably transfected with an EPO expression vec-tor. The subcloned QF line was able to produce up to1,700 U/ml EPO from 3 × 10 cells in 72 h. Purified QF-produced EPO showed a broad but discrete proteinband, ranging from 33 to 41 kD and was as biologicallyactive as CHO-produced EPO. Although a number of fac-tors still remain to be optimized, our results demonstratethe potential of avian cells such as QF as producers ofheterologous proteins.
- 中文關鍵字: --
- 英文關鍵字: Heterologous gene expression. Avian cells. Erythropoietin. Quail fibrosarcoma cells. Duck embryo cells