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

Production of recombinant Der p 1 and 2 fusion house dust mite allergen by Pichia pastoris

蕭念杰(Nien-Chieh Hsiao)

指導教授 ： 李昆達

國立臺灣大學/生命科學院/微生物與生化學研究所/碩士(2009年)

https://doi.org/10.6342/NTU.2009.01914

摘要

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。

關鍵字

畢赤酵母 ； α-螺旋 ； α-訊息序列 ； 融合過敏原蛋白 ； Der p 1 ； Der p 2 ； N型醣基化

並列摘要

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.

並列關鍵字

Pichia pastoris ； α-helix ； α-factor singal sequence ； fusion allergnen ； Der p 1 ； Der p 2 ； N-glycosylation

參考文獻

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