| Summary: | 碩士 === 國立中興大學 === 農業生物科技學研究所 === 85 === The goal of my research is to find an optimal method for the
production of Hepatitis B surface antigen (HBsAg) and Hepatocyte
growth factor (HGF). The firstchapter of my thesis mainly
described the effect of stirring speeds, concentrations of
serum, working volumes and the modes of culture methods on M-BmN
cell growth. M-BmN cells cloned from Bombyx mori, could be
cultured in suspensionin a shaking flask at the speed of 80 rpm
or below. If 0.2% of Pluronic F-68was added in the IPL-41
medium, the cells could grow even the stirring speed
wasincreased to 250 rpm . As for the effect of serum
concentration on cell growth, our study showed that cells grew
better in the medium supplemented with 10%of fetal bovine serum
(FBS) than that with 5% FBS. We also found when the working
volume was 60 ml or more in a 250 ml of shaking flask, the pH of
culturemedium kept dropping and finally inhibited the cell
growth. In contrast, the change of pH values in the culture
medium,when the cells were growing in a flaskwith a working
volume of 30 ml, first dropped and then increased again. The
increase of pH was used as a sign to indicate the timing to feed
glucose and glutamine. The most important contribution of my
study was to develop this fed-batch method based on the pH value
of culture medium. We were able to increase the maximum cell
density of M-BmN cells from 38 x 10^5 cells/ml to 48.6 x 10^5
cells/ml and HBsAg production from 230 to 560 ng/ml. The second
chapter of mythesis was to describe the production of HGF by
using a recombinant AcMNPV baculovirus. First of all, we
screened four cell lines, including SF-9, SF-21-AE, High-5 and
NTUSL-1 and found High-5 cells could highly secret HGF into the
culture medium. Second, the production level of HGF was still
low even we picked the new plaques from a high passage number of
virus solution to infect cells. In contrast, if we did the new
cotransfection and obtained a new virus solution to infect
cells, we could have high production of HGF. Finally, we also
applied the fed-batch method developed here to increase the
production of HGF.
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