The production of hepatitis B virus surface antigen(HBsAg) and hepatocyte growth factor(HGF) by using baculovirus expression system

本論文先探討M-BmN家蠶細胞株在懸浮狀況下培養之情形，嘗試了解有關 攪拌速率，血清濃度，起始接種密度，操作體積等因子對細胞生長之影響 。我們已發現0.2%之Pluronic F-68可有效地保護細胞在高轉速(高至250 rpm)下生長，同時高轉速確實有助於提高細胞密度。在血清濃度方面，顯 示M-BmN細胞在10% 血清中的狀況比在5%血清為好。而操作體積方面，則 發現過大的體積會導致溶氧量下降，使pH值過度偏低而不利於細胞生長。 而在起始接種密度部份，發現較高的接種密度，細胞將長得更好，達到最 大細胞密度的時間也較短。本研究較重大的貢獻是發展出利用pH值來當做 饋料批次培養的指標，並發現其中以當pH值上升時一次加13.5 mM glucose和6.8 mM glutamine最佳，再者是分次加4 mM glucose則次 之，而一開始培養即加入13.5 mM glucose則最差，這三種饋料批次培養 所能達到之最高密度分別為48.6 x 10^5、43.8 x 10^5、38 x 10^5 cells/ml。此法亦可應用於增加異源重組蛋白之表現量，目前B型肝炎表 面抗原之產量已可由批次培養所得之230 ng/ml提高到利用饋料批次培養 所得之560 ng/ml。其次我們利用另一種桿狀病毒AcMNPV表現系統來生產 HGF，我們嘗試去篩選高HGF產量之細胞株和利用上述所發展之方法，做高 細胞密度之饋料感染，結果發現Sf-9，Sf-21-AE，High-5，SL這四種細胞 株在相同條件下的HGF產量，以High-5這個細胞株最高為7.15 ng/ml；而 利用饋料批次方法，將SL細胞株培養至高密度再感染，發現其HGF產量也 比低細胞密度感染時高。顯然利用我們所發展之新饋料批次感染方法來生 產重組蛋白時，不管是在AcMNPV或BmNPV桿狀病毒表現系統，均能有效提 高異源蛋白之產量。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