臺灣博碩士論文加值系統

為了有效地提供歐洲塵蟎的重組過敏蛋白進行過敏原專一性的口服免疫治療，我們將第一類 (Der p 1) 及第二類 (Der p 2) 這兩個最主要的歐洲塵蟎過敏原蛋白連接成融合蛋白，並透過 Pichia pastoris 加以表現與生產。因 pPICZaA 質體在融合蛋白的 N 端前方具有 a-factor 外泌訊息序列，因此我們選取此質體來建構外泌蛋白的表現載體。連接兩個蛋白間的 linker 序列是設計成在轉譯成胺基酸序列後會形成 a-螺旋，a-螺旋被認為可以避免融合蛋白質在折疊成正常構型時互相干擾。成功的建構了 pPICZaA-Der p 1-linker-Der p 2 質體後，此質體經電轉形進入Pichia pastoris X33 (野生型) 中。我們利用針對 Der p 2 之三明治酵素連結免疫分析法篩選出一株表現量最高的 Mut+ 菌株。此融合過敏原蛋白在Hinton 氏搖瓶中培養與甲醇誘導 72 小時後產量為35.5 ug/mL。同時，我們利用針對 Der p 2 的西方墨點法分析樣品；西方墨點法的結果顯示出模糊拖尾且在較高的分子量位置的蛋白質條帶，這與我們所預測由於Der p 1的胺基酸序列中有一個N-糖基化的位置會造成糖基化之結果相符合。在 Bioflo110 發酵槽中進行細胞高密度培養，其細胞濕重可達 427 mg/mL ，目標融合過敏原蛋白的產量則為 203 mg/L。三明治酵素連結免疫分析法及西方墨點法的結果顯示，我們成功地在Pichia pastoris系統建構了一株融合過敏原蛋白的表現菌株並可透過細胞高密度培養生產此融合過敏原蛋白。

To efficiently generate recombinant allergens of Dermatophagoides pteronyssinus for allergen-specific immunotherapy such as the oral tolerance, we linked its major allergens, Der p 1 and Der p 2, into a fusion protein and expressed it via the system of Pichia pastoris. pPICZaA vector was chosen to construct a protein-secretable system because of the existence of a-factor signal sequence in front of the N-terminal of the fusion protein. Sequence of the linker linking the two proteins was designed to form a a-helix conformation after translated into amino acid sequence. The a-helix was expected to prevent the fusion protein from folding interference of the two recombinant allergens. After pPICZaA-Der p 1-linker-Der p 2 plasmid was constructed and transformed into the Pichia pastoris X33 (wild type) via electroporation, we selected a Mut+ strain which showed the highest productivity with an ELISA specific to Der p 2. The productivity of the fusion allergen in Hinton’s flask was 35.5 ug/mL after 72 h of methanol induction. We also analyzed the samples with Western blotting specific to Der p 2. The Western blotting showed some smear and higher molecular weight bands, which were coincided with our prediction that the existence of an N-glycosylation within Der p 1 amino acid sequence would leads to glycosylation. A high cell density culture in the Bioflo110 fermentor was achieved with 427 mg/mL wet biomass, and 203 mg/L fusion allergen production. According to the Western blotting and ELISA results, it concludes that we successfully constructed a fusion allergen expression strain of P. pastoris and produced the fusion allergen via a high cell density culture.

口試委員會審定書…………………………………………………………………..…i
Abstract...……………………………………………………………………..………iii
中文摘要………………………………………………………………………………iv
Abbreviation……………………………………………………………….……………v
專有名詞 中英文照表…………………………………………………..……………..vi
Contents…………………………………………………….………………………….vii
Contents of figures and tables………………………………………………………....x
Chapter I Introduction 1
1.1 House dust mite allergens 2
1.1.1 Allergen Der p 1 3
1.1.2 Allergen Der p 2 4
1.2 Immunotherapy of oral tolerance and edible vaccine 6
1.3 Expression of recombinant Der p 1 and Der p 2 in different hosts 9
1.3.1 Expression of recombinant Der p 1 10
1.3.2 Expression of recombinant Der p 2 13
1.4 Fusion proteins and the protein linker 15
1.5 Pichia pastoris 15
1.6 Aim of this study 17
Chapter II Materials and Methods 19
2.1 Microorganisms and vectors 20
2.2 Construction of plasmids and P. pastoris expression host 20
2.2.1 Construction of fusion allergen gene in yT＆A vector and then pPICZaA vector 21
2.2.2 Construction of P. pastoris expression host 26
2.3 Selection of a proper expression strain 28
2.3.1 Selection of a Mut+ expression strain 28
2.3.2 Selection of a strain with high copy number 29
2.3.3 Small scale cultures ELISA selection for higher productivity 29
2.4 Cultures in the Hinton''s flasks 30
2.5 Cultures in fermentors 31
2.6 Total soluble protein quantification 32
2.7 Preparation of recombinant Der p 2 protein as the standard in ELISA and Western blotting 32
2.8 Sandwich-ELISA 33
2.9 Western blotting 34
Chapter III Results 35
3.1 Construction of fusion allergen gene 36
3.2 Selection of a higher productivity strain 37
3.3 Western blot of strain 33 38
3.4 Strain 33 cultured in Hinton''s flask and fermentor 39
Chapter IV Discussion and Conclusion 41
4.1 Discussion 42
4.2 Conclusion 46
References 63
Tables and Figures 47

Appendix 76

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