The Effects Of Ganoderma Lucidum Spores On The Growth Of Chinese Hamster Ovary Cells And Establishment Of Cell-based Screening For Serum-free Growth Factors

Chinese hamster ovary cell(CHO) is an immortal and unlimited proliferation cell line, which is the preferred expression system for recombinant glycosylated protein production. Although several biocompatible medium and bioreactors have been developed for lowering down the cost and enhancing the production. Ganodermalucidum spore(Gls), which is a traditional Chinese medicine, can also promote cell mitosis, improve T lymphocyte cell proliferation, enhance immune activity, inhibit DNA polymerase activity and Ras oncogene protein post-translational modifications, promote cytokine production and improve cell proliferation with low cell toxicity. Recently,its new application in mammalian cell culture attracted much attention.In this work, Gls extract was used as the candidate of the serum analogues and the optimal concentration of Gls extract was achieved. A stably expressing green fluorescent protein(GFP) cell line was established for high throughout screening. From spinner flask dynamic culture, Gls extract not noly promoted cell proliferation, but also the protein expression was kept at the same level with 10% FBS culture. Using this noninvasive and high-throughput screening method for serum analogues for, three kinds of cell growth including soya peptone, Vc and GSH( abbreviation for SVG) were chosen for composition of serum free medium, and it was confirmed that the SVG medium is a broad-spectrum medium for mammalian cell lines.0.01 %(w/v)Gls with, low serum concentration(1% FBS) can maintain the normal growth and proliferation just like 10% FBS medium. It was demonstrated that Gls extract can inhibit the apoptosis which was induced by serum starvation. In addition, the standard beta glucan polysaccharide has same effect on promoting cell proliferation compared with Gls extract. In spinner flask, cell number of CHO cells in 10% FBS medium increased by 11.41 folds, while those in the 1% FBS plus 0.01% Gls medium and Serum Free medium(SF) were 10.29 and(6.19 fold, seperately. Compared with 10%FBS medium, it has nearly the same levels of the cell number, specific growth rate and protein expression in the 1% FBS w 0.01% Gls.In this study, an optical method was established for serum analogues screen,the main steps were as follow: fisrtly, p EGFP-N2 was transformed into Escherichia coli DH5α and the endotoxin free plasmid was extracted; secondly, pEGFP-N2 was transfected into CHO cells by using lipofectamine 3000 regent; thirdly, geneticin G418 was used for GFP cell line screen; lastly, fluorescence intensity of GFP and cell number showed a linear relationship between the judgment based on CHO cell proliferation which has very good market prospect.