Study On Expression Of Truncated EGFR In COS7 And CHO-K1 Cells

Object: To establish transient expression system of COS7 and stable expression system of CHO-K1, and to obtain high expression of soluble truncated Epidermal Growth Factor Receptor (sEGFR501).Methods: The transient expression system of COS7 was constructed by using pcDNA3.1-sEGFR501 expression vector and the process of Calcium-phosphate co-precipitation transfection was optimized with Enhanced Green Fluorescent Protein (EGFP) as a reporter. To study the conditions of large-scale mammalian cell culture, COS7 was cultured in a 500mL bioreactor BelloCell(?) -500 and the whole process was monitored by the biochemical analysis apparatus BIOPROFILE250. The stable expression system of CHO-K1 was constructed with pIRESneo-sEGFR501, the Cationic liposome transfection was also optimized with EGFP as a reporter, and the stable expression strains were selected via G418. The expression level of the two systems was evaluated by using RT-PCR and ELISA.Result: The expression level of sEGFR501 in transient expression system of COS7 is about 50ng/mL·day·10~6cells, whereas it raised to 150ng/mL·day·10~6cells and could keep being stable after 20 passages in stable expression system of CHO-K1. The cell density of COS7 cultured in the 500mL bioreactor reached to 4.67×10~8/500mL. by 6 days.Conclusion: Both of the transient expression system of pcDNA3.1-sEGFR501/COS7 and the stable expression system of pIRESneo-sEGFR501/CHO-Kl were established successfully, and it provided a basic to study the expression of other recombinant proteins in the two systems.