Optimization Of GS Selection Markers For The Construction Of High Yield And Stable CHO Cell Lines

Antibody drugs have been successfully used in the diagnosis and treatment of many diseases due to their strong targeting specificity.At present,more than 70%of antibody-related drugs on the market are produced by CHO cells,so improving the expression level of CHO is of great significance in the development and industrialization of antibody-related drugsGlutamine synthase(GS)selection system,with GS gene as the selection marker,can effectively reduce the concentration of NH4+in cells,and at the same time use the metabolic waste to synthesize the nutrients needed by themselves,which has been successfully applied in the commercial production of a variety of antibodies and recombinant protein drugs.In order to further improve the expression level of exogenous proteins in CHO cells,this study took CHO cells as host cells and GS genes from three different species(Human,Dog,CHO)as selection markers to construct cell lines.Batch,overgrowth,fed-batch and other culture methods were used to monitor the cell density,cell activity and expression level of foreign proteins,and analyze the influence of GS on the yield and construction efficiency of cell lines.On this basis,the concentration of glucose and lactic acid in the culture medium,the GS activity in the cell line,the transcription level of GS and target genes,and the binding mode of GS from different species and their substrates were analyzed.The experimental results showed that GS selection markers of different species caused significant differences in the expression levels of exogenous genes.Among them,the cell lines constructed with huamn-gs selection markers had the highest expression of foreign proteins,followed by cho-gs selection markers,while the cell lines constructed with dog-gs as selection markers were significantly worse than the first two in terms of expression of foreign proteins and cell growth.Cell lines constructed with huamn-gs selection markers had significantly higher glucose uptake in single cells than the other two selection markers.There was no significant difference in the activity of glutamine synthase labeled by GS.There was no significant difference in the transcription level of the target gene or the selection marker.Thro ugh the structural simulation of GS,it was found that the types and number of amino acids combined with MSX in human-gs were more than cho-gs and dog-gs,which indicated that the binding force between human-gs and its substrates was stronger than cho-gs or dog-gs.To sum up,this study found that compared with cho-gs and dog-gs,cell lines constructed with huamn-gs as the selection marker had better expression level and better selection efficiency,possibly because Human-gs can enhance CHO glucose uptake and has stronger binding ability with the reaction substrate.This study will provide a new reference for further optimization of GS expression system.