Study on expression of the house dust mite allergen Der p 2 in Flammulina velutipes

• Main Authors: Chia-Chang Chung, 張家禎
• Other Authors: Ching-Tsan Huang
• Format: Others
• Language: zh-TW
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/48467094406231182795

碩士 === 國立臺灣大學 === 微生物與生化學研究所 === 94 === House dust mites are the most significant indoor allergens. Dermatophagoides pteronyssinus group 2 protein (Der p 2), originated from the digestive tract of host dust mites, plays a major role in induction of asthma, allergic rhinitis and allergic dermatitis. Up to 85% of asthma patients are sensitized to Der p 2 in Taiwan. There are 387 bp in the cDNA clone which encodes a 129-aa protein with a molecular mass of 14 kDa. Oral tolerance can induce a group of antigen-specific regulatory T cells to suppress antigen-specific immune response through the oral administration of protein antigens presented by intestinal lymphoid tissue. However, the application of oral tolerance for the treatment of allergey was limited due to complicated purification and high cost of candidate allergens. To solve this problem, this study would like to develop the Der p 2-transgenic mushrooms possessing advantages of conveniently oral delivery and inexpensive protein production process. In this study, four different strategies were used to improve the heterogenous gene expression in Flammulina velutipes. Plasmids with an intron at 5’ of der p 2, the endoplasmic reticulum retention signal HDEL at 3’ of der p 2, fusion gene of der p 2 and egfp (enhanced green fluorescent protein), and modified the codon of der p 2 (mder p 2) were constructed for F. velutipes. After transforming the target DNA to mycelium by eleporation, only transformants of mder p 2 with an obvious signal were observed in Western blot analysis. Using Der p 2 sandwich enzyme-linked immunosorbent assay (sandwich-ELISA), the transformant pAImDS-19 expressed the highest Der p 2 protein, c.a. 0.02% of the total soluble proteins. This study demonstrated the expression of mite allergen in mushroom and suggested that the codon usage or the G + C content are crucial for the expression of heterogenous protein in F. velutipes.