Expression Of TNFR1-IgG4 And EGFP-IgG Eukaryotic Expression Vector

Objective: To express Tumor Necrosis Factor Receptor1-IgG4, (TNFR1-IgG4) in Chinese Hamster Ovary(CHO) cells with enhanced green fluorescent protein(EGFP) as a positive control, and lay a foundation for function study and the clinical application of TNFR1-IgG4.Methods: pMM-TNFR1-IgG4/WG and pMM-EGFP-IgG4/WG were Identified with NheI or XbaI single enzyme digestion and Nhe and XbaI double enzyme digestion. The plasmid pMMGR was linearizedwith Ndeâ… and the plasmids pMM-TNFR1-IgG4 and pMM-EGFP-IgG4 were linearized with Notâ… . Recombined plasmids were transfected into CHO cell with lipid transferring reagent respectively. After selecting with G418 and dialysed free-serum medium, positive clones were obtained, then cultured under the pressure of MTX. The expression of enhanced green fluorescent protein gene was observed with fluorescence micoroscope, and the expression of TNFR1-IgG4 was detected with SDS-PAGE and Western Blot.Results: The eukaryotic expression vectors pMM-TNFR1-IgG4/WG,pMM-EGFPIgG4/WG and pMMGR transfected into CHO cells successfully, EGFP positive clones were observed to be green fluorescence with fluorescent microscope. TNFR1-IgG4 protein was purified with protein A agarose resin successfully.Conclusion: It is demonstrated that EGFP gene is successfully expressed in CHO cells transfected with eukaryotic expression vector pMM-EGFP-IgG4/WG, and positive clones can emit green fluorescence. Therefore, EGFP gene is a good positive control of producing secreted fusion protein. The eukaryotic expression vector TNFR1-IgG4 gene expressed successfully in CHO cells.