建構高效能的BHK疫苗生產細胞株與細胞代謝特性分析

Establish high performance vaccine production BHK cell strains and analyze their metabolic characteristics

李俊毅(Chun-Yi Lee)

指導教授 ： 陳裕星

長榮大學/健康科學學院/生物科技學系(所)/碩士(2011年)

https://doi.org/10.6833/CJCU.2011.00179

摘要

利用基因工程技術轉染哺乳動物細胞以製備重組蛋白質、疫苗、單株抗體等，是生物科技產業中一大應用領域，透過基因工程的操作，使得細胞可以合成自身所需的生長因子、可調控細胞週期，改變關鍵代謝物質流量以提升細胞活性等。在調控細胞代謝所使用之重組基因中，透明顫菌之血紅蛋白(Vitreoscilla hemoglobin, VHb) 相當受到矚目，此蛋白在雙體構型時對氧氣有高度親和性可截取氧氣以提供代謝所需，在低氧的環境下可增加大腸桿菌細胞內氧氣量，增強終
端氧化酶的活性以促進NADH 的利用，使細胞產生較高的TCA 循環酵素活性、較高的ATP 合成量。有鑑於動物細胞在培養時多依賴糖解作用提供生長代謝所需能量，TCA 循環活性不彰，本研究探討表達VHb 基因對BHK 細胞生長之影響，在轉染VHb 基因並獲得穩定轉染細胞株之後分別進行生長曲線、植板效率與代謝物質分析。結果發現在低密度與細胞平板培養時，細胞生長劣於對照，但在高密度與滾瓶培養條件下，細胞可維持高的生長速率，有效利用培養基並減少乳酸堆積，推測在低細胞密度培養條件下，氧氣並非對生長之限制因子，但在高
細胞密度時，可維持細胞增殖速率達到高密度細胞培養，在生化工程之運用上仍有極高潛力。

關鍵字

透明顫菌血紅蛋白 ； 倉鼠腎臟細胞 ； 生長曲線

並列摘要

The use of genetic engineering technology in mammalian cells transfected to prepare recombinant proteins, vaccines, monoclonal antibodies etc., is a major biotechnology industry applications, through genetic engineering operations, making cells can synthesize.Their required growth factors can regulate the cell cycle, changes in key metabolic cell activity to enhance the flow of material. And so on. In the regulation of cell metabolism by the use of recombinant gene in Vitreoscilla of hemoglobin (Vitreoscilla hemoglobin, VHb) rather receive much attention, this protein when the catamaran configuration has high affinity for oxygen can be cut. Of oxygen to provide the metabolic needs in low-oxygen environment of E. coli cells can increase the amount of oxygen, enhance the end Terminal oxidase activity in order to promote the use of NADH, causes cells to produce a higher TCA cycle enzyme activity, Higher ATP synthesis. In view of cultured animal cells more dependent on metabolism of sugar solution by providing role need energy, TCA cycle activity failed, this study, BHK cells expressing VHb gene on the growth of the shadow Ring, VHb gene in transfected and stable transfected cell lines were carried out after the growth curve, plating efficiency And metabolites analysis. Found in low-density cell culture plate, the cells inferior to the control, but And roller bottles in high-density culture conditions, cells can maintain high growth rates, and reduce the effective use of media Lactic acid accumulation, suggesting that at low cell density culture conditions, oxygen is not the limiting factor for growth, but high Cell density, cell proliferation rate can be maintained to achieve high-density cell culture, biochemical engineering in the use of still Very high potential.

並列關鍵字

Vitreoscilla hemoglobin (VHb) ； baby hamster kidney fibroblast (BHK) ； growth curve

參考文獻

Aberg, A., S. Hahne, et al. (1989). "Evidence for two different classes of redox-active cysteines in ribonucleotide reductase of Escherichia coli." J Biol Chem 264(21): 12249-52.

Altamirano C. et al. (2006). "Specific nutrient supplementation of defined serum-free medium for the improvement of CHO cells growth and t-PA production." Electron. J. Biotechnol. 9, 61–67.

Ahsan, M. K., I. Lekli, et al. (2009). "Redox regulation of cell survival by the thioredoxin superfamily: an implication of redox gene therapy in the heart." Antioxid Redox Signal 11(11): 2741-58.

Al-Rubeai, M., R. P. Singh, et al. (1995). "Death mechanisms of animal cells in conditions of intensive agitation." Biotechnol Bioeng 45(6): 463-72.

Borth, N., D. Mattanovich, et al. (2005). "Effect of increased expression of protein disulfide isomerase and heavy chain binding protein on antibody secretion in a recombinant CHO cell line." Biotechnol Prog 21(1): 106-11.