以 Pichia pastoris 生產重組塵蟎過敏原 Der p 1, 2 融合蛋白

Abstract

家居塵蟎 (HDM) 是引起呼吸系統免疫症狀的主要元兇。塵蟎中 Der p 1及 Der p 2 為造成氣喘、異位性皮膚炎等免疫反應的過敏原。為了提供發展口服耐受性的材料，本實驗室先前於酵母菌 Pichia pastoris X-33，已建構以 pPICZαA 為表現載體，並以α-螺旋做為 linker 連接 Der p 1 與 Der p 2 的融合過敏原基因。本研究利用 site-directed mutagenesis ，將融合過敏原 Der p 1 序列上第52號天門冬醯胺置換成麩醯胺，以去除醣基化現象。此質體經電轉形進入 Pichia pastoris X-33 ，並利用針對 Der p 2 之三明治酵素免疫分析法篩選出一株表現量最高的 Mut+ 菌株進行表現研究。此重組融合過敏原蛋白於 Hinton 氏搖瓶中培養並經甲醇誘導 120 小時，表現量為 15 µg/mL 。在 Bioflo110 發酵槽中進行細胞高密度培養，其細胞濕重可達 325 g/L ，融合過敏原蛋白質的產量於培養 126 小時為 127 mg/L ，但隨著培養時間增加，融合過敏原表現量呈現下降之趨勢，至 162 小時為 58 mg/L。推測可能受到培養基中存在之蛋白酶之水解。因此，另以小量培養之上清液加入重組 Der p 2，評估重組蛋白質被水解程度。結果顯示，在不添加任何蛋白酶抑制劑下48小時 rDer p 2 幾乎被完全降解；若添加 1 % casamino acids，rDer p 2 殘留40%。所以我們將針對如何抑制培養基之蛋白酶活性，以避免重組融合過敏原蛋白被降解所造成表現量降低之問題。

House dust mite (HDM) is major source that elicit airway allergic symptoms. In HDM, allergens Der p 1 and Der p 2 show the most significant effect on allergic responses including asthma and atopic dermatisis. In our previous study, gene of Der p 1 and Der p 2 was linked by an α-helix linker in a secretable vector, pPICZαA, and was expressed by the system of Pichia pastoris X-33. In this study, in order to avoid incorrect of glycosylation of rDer p 1, we used site-directed mutagenesis technology to replace the glycosylation site, Asn52, with Gln on the polypeptide sequence of the fusion protein. With an ELISA assay specific to Der p 2, we selected a Mut+ strain which showed the highest productivity for further productivity research. The production yield of fusion protein was 15 µg/ml in Hinton’s flasks after 120 h of culture and methanol induction. A high cell density culture in the Bioflo110 fermentor was achieved with 325 g/L wet biomass, and 127 mg/L fusion allergen production at 126 hours. However, the fusion allergen in the medium dropped with the increase of culture time, and the productivity of fusion allergen was 58 mg/L at 162 h. In orer to reveal whether this phenomenon was caused by degradeation by protease in the culture medium, rDer p 2 was added to the supernatant of cultured medium of the transformed Pichia pastoris, to quantify the residual fusion protein in the medium. Compared to the group that rDer p 2 was almost degraded after 48 h of treatment, 40 % fusion protein remained in the medium was observed in the group which was added 1 % casamino acids. It shows fusion protein was degraded during fermentation process. Therefore, to overcome the problem caused by protease in fermentation is the most important issue in the next step.