The Consruction And Application Of The Animal Expression Vector Based On GS Screening System

Currently many monoclonal antibodys (mAb) used in clinical trials and approved for the treatment are increasing significantly in recent years. They are used for the treatment of cancer, autoimmune diseases, allergic diseases, transplantation, and anti-idiotype vaccine. With the technology in cell culture, particularly in the development of mammalian cell culture, it improvs the production of monoclonal antibodies and other recombinant proteins. The process of monoclonal antibodies involves that cloning the transfected gene, the adaption of different culture conditions, culturing in a bioreactor and the industrial level. In fact, due to the high demands of monoclonal antibodies, building a large-scale production process meets the needs of the market.The optimization of the production is not only in the bioreactor, but also in an early step which play a role in the final production. Indeed, it is known that several parameters such as the cell type, the transfection of the vector, the vector promoter, the selection, post-transcriptional regulation and the medium which are inportant. Therefore, the optimization of the production is not only in the bioreactor during the process but in the first few steps.The topics of the optimistical production are the vector screening system, promoter elements, the adaptation period, the optimization of serum-free medium and the optimistical condition of fed-batch culture and other aspects of research. The vector of pHGS1.0was affiliated GS selection marker and replaced the hCMV promoter successfully. After inserting the gene, transfected cells successfully express, and the experiment of Western Blot confirms the target protein of approx90kD. With G418and MSX screening, the highest protein expression level is acquired.OD450and IgG2quantitative detection kit select20monoclones. Between20monoclones,2、6、8、9、10、12and13are negative, and the other are positive. The protein expression of15reachs4ug/mL.In the adherent culture the maximum cell density is4×106cell/mL and the recombinant protein expression reachs3μg/mL. The maximum specific growth rate is0.35d-1. The maximum expression rate is3.5μg/mL/cell/d. The glucose consumption rate increases first, then decreases and the lactic acid rate decreases first, then increases, decreases last.In adaptation phase the maximum cell density is2.5×106cell/mL, and the recombinant protein expression reached3.8μg/mL by adding the F-68into the medium. When the concentration of dextran sulfate reachs25ug/mL, the optative condution of the protein expression can be achieved. After the adaptation period, the cell growth rate reached0.34d-1and the expression of the recombinant protein to6μg/mL. The consumption rate of the glucose and the generation rate of the lactose remains virtually changless over the yield.The method of Plackett Burman screens the factors of the medium. The result is that insulin, transferrin and putrescine play key roles. The results of response surface experiment showed that when the concentration of the insulin is2.78mg/L, the transferrin to0.78mg/L, the putrescine to8.6mg/L, the maximum cell density reachs4.2×106cell/mL. Through the optimization of cell culture medium, the level of recombinant protein expression can be up to5μg/mL. By using MATLAB software in serum-free batch culture, the dynamics model which gives an account the consumption of glucose consumption and the production of lactate establishs a good foundation.After stable cell lines were passaged in16times in a row, the protein expression of serum-free batch culture is higher than that of serum batch culture, and the density maintains stable.In fed-batch culture conditions the maximum density and protein expression of different initial seeding density is not different. The cell density is8×106cell/mL, and the level of protein expression is about4.5μg/mL. The temperature of33℃has almost no influence of the cell growth and maximum density, but the level reachs9μg/mL. It is nearly two times higher than the other two incubation temperature. When the pH at5.5, the cell density maintains about6X106cell/mL, and protein expression is the highest. It finds that by adjusting the pH, the cell density can be improved at24h.