Abstract Orjective:To obtain recombinant CHO-K1 with expressing sPDGFRα and to identify the biological activities of sPDGFRα secreted in non-serum medium. Methods:Recombinant human sPDGFRα expression vector pIRES-Neo3-sPDGFRα-Fc was constructed and then transfected into CHO-K1 cells by using LipofectamineTM 2000. After screened with G418 in 8 weeks, some monoclone cells were selected randomly to amplify in 96-well-plate to 24-well-plates, and then to identify positive cell clones by RT-PCR. Furthermore, the candidate cell clones were test by Real-Time PCR and Western blot assays. Finally, anti-proliferation activities of the expressed sPDGFRα were analyzed by MTT. Results: sPDGFRα-Fc was cloned into pIRES-Neo3 correctly. The sPDGFRα-Fc expression level in recombinant CHO-K1 cell clones were concordant in between Realtime PCR and Western blot assay. sPDGFRα-Fc obtained from cultured non-serum medium of positive CHO-K1 could significantly inhibit proliferation of vascular endothelial cell.Conclusion:Successed to select recombinant CHO-K1 cell lines with high expressed sPDGFRα-Fc. The sPDGFRα-Fc can inhibit the cell proliferation significantly and it means sPDGFRα-Fc might be a new anti-cancer drug in the future.
Abstract 13C metabolic flux analysis (13CMFA) have been the research hotspots of metabolic engineering internationally due to its accuracy and applicability.It is vital that the measurement of 13C labelling pattern of proteinogenic amino acids for 13C metabolic flux analysis.To acquire 13C-labelling proteinogenic amino acids,Pseudomonas denitrifican which products Vitamin B12 was firstly fed with mimimal culture medium contained 20% U-13C and 80% natural glucose，after the culture reached a steady state, then about 20 mg biomass was hydrolyzed by 1 ml of 6 mol/L hydrochloric acid for 24h at 110℃.Then amino acids was separated,concentrated, evaporated in a vacuum, and derivatized with MBDSTFA, TBDMS-derivatized amino acids can be observed by GC-MS last we get 13C labelling pattern of fifteen aminio acids through mass spectrum.The experimental methods and sample preparation offers referential value for the development of 13C metabolic flux analysis in our courtry.
