Cloning and expression of recombinant TNFR - FC fusion protein

Abstract

Recombinant TNFR-Fc fusion protein (rhTNFR-Fc) has been wide to reduce inflammatory response, which is especially useful to treat arthritis. Thus, rhTNFR-Fc needs stable expression process in mammalian cell. In this study, the coding gene TNFR-Fc was amplified and then cloned into plasmid pOptVEC-TOPO to create pOptVEC-TOPO (rhTNFR-Fc). The plasmid pOptVEC-TOPO (rhTNFR-Fc) contained the right direction gene was isolated to transfect stably into Chinese hamster ovary (CHO) DG44 mammalian cell line using lipofectamine 2000. The expression capability of transfected CHO DG44 cell line was checked by Western blotting. To enhance the expression capability of transfected CHO DG44, this cell line was treated with 11700nM MTX – the highest MTX concentration the transfected cells can survive. We will use this concentration in the next experiments to obtain high yields of purified secreted proteins from stably transfected CHO DG44 cell lines. Key words: TNFR-Fc, CHO GD44, MTX.