以批次饋料培養方法於菸草細胞中生產 重組塵蟎過敏原蛋白 Der p 2

Abstract

為有效提供轉殖植物來源之Dermatophagoides pteronyssinus 2 (Der p 2) 做為發展口服耐受性治療以用於緩和過敏性氣喘，本實驗室先前已進行了表現重組Der p 2菸草轉殖細胞之生物反應器批次培養研究。本研究中，我們進一步嘗試以批次饋料培養方法培養轉殖細胞，以提高重組Der p 2之產量。根據搖瓶培養的數據，並且避免造成細胞滲透壓之逆境，我們決定以3% 最終濃度之蔗糖進行多次饋料之策略。與搖瓶培養相較，生物反應器批次饋料培養之單位細胞rDer p 2 含量為其8倍 (87.5 μg/g)，而產量在第12天達到8.2倍之多 (1002.6 μg/L)。同時，為了建立植物來源重組Der p 2之另一生產方法，我們構築了可表現帶有外泌訊息序列之重組Der p 2的菸草毛狀根 (S組)，轉植毛狀根經培養並與本實驗室先前所建構表現未帶有外泌訊息序列Der p 2之毛狀根 (R組) 做比較。經20天的培養，S組之27株轉型毛狀根中，最高表現量為358.4 μg/L，較R組中最高值175.3 μg/L為高；而S組外泌的比例佔總表現量最高者可達49.0%，R組則是22.0%。結果顯示，在毛狀根中加上訊息序列於Der p 2基因前之設計，有助於重組Der p 2的外泌 (p < 0.05)。

To efficiently afford recombinant protein of Dermatophagoides pteronyssinus 2 (Der p 2) from transgenic plant system for development of oral tolerance to reduce asthma allergy, the transgenic Der p 2 expressing tobacco cells and batch cultivation in bioreactors had been established, in our former study. For improving the yield of recombinant Der p 2, fed-batch culture method is applied in this study. Based on the basal data reveled in flask cultures and avoid to induce the osmosis stress of plant cells, the strategy of multiply fed the cultures with 3% sucrose (concentration after fed) was decided. Compared with the results of batch culture in flasks, the fed-batch culture in bioreactors achieved the 8-fold of the rDer p 2 content (87.5 μg/g) and 8.2-fold Der p 2 yield at the 12th day (1002.6 μg/L). Meanwhile, for an alternative route for rDer p 2 production from plant systems, we established tobacco hairy roots which can express signal peptide-recombinant Der p 2 (S group). These transgenic hairy roots were cultivated and compared with transgenic roots previously established that could express Der p 2 without signal peptide (R group). After 20 d cultivation, the maximum Der p 2 yield among 27 S transgenic clones was 358.4 μg/L, higher than the best R transgenic clone (175.3 μg/L); maximum released Der p 2 of S group was 49.0% of total Der p 2, also higher than the maximum value (22.0%) of R group. It demonstrated that fusion of the signal peptide sequence with Der p 2 gene improved the secretion of recombinant Der p 2 efficiently (p < 0.05).