菸草懸浮細胞高密度培養生產重組塵蟎過敏原Der p 2

Abstract

為有效提供植物來源之重組塵蟎過敏原Der p 2，以發展用於減緩氣喘之可食用性疫苗，本實驗室先前已建立可生產重組Der p 2 (rDer p 2) 的菸草懸浮細胞株。但由於在生物反應器的培養中，菸草細胞有明顯的褐化現象，使得細胞生長受抑制，降低了rDer p 2的產量。本研究為了提升rDer p 2的產量，首先經由通氣量與細胞生長量的關係，確定可提供充足溶氧且不會使細胞褐化的通氣速率為0.06 vvm。接著在搖瓶培養，發現採用3% 蔗糖作為初始碳源進行批次培養 (batch culture) 時，具有最高的rDer p 2的生成率。以5 L生物反應器進行批次培養時，rDer p 2產量在第19天達最高，為4.85 mg/L，是搖瓶批次培養的2.3倍；而以批次饋料進行培養時，生物反應器內之rDer p 2在第14天達最高之 7.00 mg/L，為搖瓶批次饋料培養的1.4倍。此外，菸草懸浮細胞之蛋白質粗抽液，經硫酸銨分劃、Sephacryl S-100膠體過濾，與DEAE-sepharose離子交換處理後，可獲得以rDer p 2 為主要蛋白質之溶液，其純化倍數為2.1倍，回收率為8.1 %。

To efficiently provide recombinant protein of the major house dust mite allergen Der p 2 from a transgenic plant system for development of an edible vaccine to reduce asthma allergy, clones of recombinant Der p 2 (rDer p 2) producing tobacco suspension cell lines had been established in our previous study. However, the browning of tobacco cells caused the low cell growth as well as low rDer p 2 production yield in the culture period. In this study, by examining the relationship between aeration rate and the biomass accumulation, the aeration rate at 0.06 vvm was considered adequate for cell growth without browning of cells. Based on the data revealed from flask cultures, evaluating the ratio of rDer p 2 to carbon source, 3% sucrose was used as the initial carbon source. In batch cultures, the rDer p 2 production yield in the 5 L bioreactor reached 4.85 mg/L on the 19th day, which is 2.3 times the yield of flask batch cultures. In fed-batch cultures, the rDer p 2 production yield in the 5 L bioreactor reached 7.00 mg/L on the 14th day, which is 1.4 times the yield of fed-batch cultures in flasks. Furthermore, the rDer p 2 produced by tobacco suspension cells was purified approximately 2.1-fold by ammonium sulfate fractionation, gel filtration through Sephacryl S-100 and ion-exchange on DEAE-sepharose, with the recovery yield about 8.1%.