葡萄球菌S. aureus及S. epidermidis是經常造成醫院內及傷口感染之主要細菌之一，其易形成生物膜於人體醫療置入裝置表面，為引起臨床各類急性及慢性感染之主因。細菌形成生物膜後將自身包埋在由蛋白質、多醣和細胞外DNA（eDNA）所組成的細胞外基質(extracellular polymeric substances, EPS)中，保護細菌抵抗外在不利生長的環境及外物攻擊如抗生素等。本論文將利用Dispersin B（DspB），其具有糖苷糖水解酶(glycosyl hydrolases)的功能，能夠將EPS中的多醣水解，有效水解形成表皮葡萄球菌(S. epidermidis)生物膜之多醣類Poly--1,6-N-acetyl-D-glucosamine（PNAG），使包埋在生物膜中的細菌能夠釋放出來，受天然貽貝足蛋白(mussel foot protein, mfp)其蛋白富含多巴(dihydroxy phenylalanine, DOPA）能夠輕易黏附於各類表面的啟發，本論文設計帶有6xTyr-tag之DspB融合蛋白，利用酪氨酸酶催化序列中酪胺酸形成多巴，將材料浸泡於溶液中，利用6xDopa基團於玻片表面上固定化DspB，可實際應用於抑制生物膜之形成。
融合6xTyr-tag後之DspB於表達時會增加DspB之不可溶性，而將6xTyr-tag融合於DspB序列之C端時會降低DspB的活性，帶有銀離子結合胜肽AgBP2之DspB融合蛋白分散S. epidermidis生物膜效果較佳，因此也使用AgBP2-DspB來探討6xTyr融合對其表面固定化之效果，再經酪氨酸酶催化後6xTyr會形成6xDopa能將DspB融合蛋白固定化於表面，其中AgBP2-DspB、6Y-AgBP2-DspB及DspB-6Y擁有較高固定化量，與未經處理之表面生物膜厚度相比可分別減少43%、34%及24%，而總螢光強度則減少65.3%、53.3%及51.9%，顯示固定化後帶有DspB之表面擁有抑制S. epidermidis形成生物膜之能力。

Bacterial strain Staphylococci frequently causes biofilm-associated infections in human body. In the biofilm formed by Staphylococcal, bacterial cells are embedded in an extracellular matrix composed of proteins, polysaccharides and extracellular DNA (eDNA) that protect the cells from killing by most of antimicrobial agents employed. Poly--1,6-N-acetyl-D-glucosamine (PNAG) was found to be the major polysaccharide in the matrix of S. epidermidis biofilm. The biofilm can be effectively eradicated, if PNAG structure were hydrolyzed to expose the cells to antimicrobial agents. Dispersin B (DspB), a family 20 -hexosaminidase, is known for its high activity to cleave PNAG. Mussel foot proteins (mfps), containing many dihydroxy phenylalanine (DOPA) residues in its structure was found can adhere strongly on various surfaces.
In this work, oligoTyr residues were fused to DspB and transformed into oligoDOPA by tyrosinase for the purpose of facile immobilization of DspB on various surface to prevent biofilm formation. A synthetic gene encoding DspB of Aggregatibacter actinomycetemcomitans with 6xTyr fusion was expressed in Escherichia coli. The 6xTyr-tag fusion, however, was found to induce the inclusion body formation of the target protein. The activity of DspB against PNAG was enhanced when a silver binding peptide was fused to the N-terminal of DspB (AgBP2-DspB、6Y-AgBP2-DspB). 6xTyr fused DspB demonstrated a better surface immobilization only after tyrosinase was employed to transform 6xTyr into 6xDopa. The DspB fusion protein AgBP2-DspB along with 6xDopa modified DspB 6Y-AgBP2-DspB and DspB-6Y could be easily immobilized on glass slide and able to reduce 43%, 34%, and 24% of S. epidermidis biofilm thickness respectively.

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