| Summary: | 碩士 === 國立臺灣大學 === 微生物與生化學研究所 === 97 === 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.
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