Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/97572
標題: 應用Ssp dnaB mini-intein系統生產基因重組抗菌胜肽cecropinB2
Construction and production of recombinant cecropinB2 via Ssp dnaB mini-intein system
作者: 方㛄婷
Yi-Ting Fang
關鍵字: cecropinB2
抗菌胜肽
Ssp dnaB mini-intein
大腸桿菌
幾丁質結合結構域
cecropinB2
antibacterial peptides
Ssp dnaB mini-intein
E. coli
chitin binding domain
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摘要: Cecropin是一種陽離子抗菌肽,由35-39個殘基組成。此抗菌胜肽被鑑定具有較強的抗菌活性能力,對真核細胞毒性較低,某些種類的抗菌胜肽更具有殺滅癌細胞的能力。 在本研究中,利用基因工程技術應用於生產抗菌胜肽cecropinB2。因此,我們構築抗菌胜肽的內含肽表達質體,並在大腸桿菌ER2566表現系統中表達融合蛋白。選擇CecropinB2作為靶蛋白,以幾丁質結合結構域 (chitin binding domain)親和標籤與可自身切割Ssp DnaB內含肽 (intein)做為純化方法。透過融合蛋白的親和吸附將它們應用於幾丁質樹脂柱。內含肽切割活性強烈受到內含肽相鄰的胺基酸的影響。選擇三種胺基酸片段:CRA或SRA或GRA作為內含肽後的輔助插入片段,用於幫助Ssp DnaB內含肽的切割。經由pH值和溫度調節,內含肽以自身切割並且目標純化蛋白被洗脫並收集。目標胜肽以SDS-PAGE分析確認。經由幾丁質結合結構域親和管柱純化後能夠生產出約58.73 mg/L之抗菌胜肽cecropinB2。純化之cecropinB2對革蘭氏陰性菌株大腸桿菌均表現出抗菌活性。這項研究表明,透過CBD-intein系統可產生大量且具有強抗菌活性的重組cecropinB2胜肽。
Cecropin is a cationic antibacterial peptide and composed of 35-39 residues. This antibacterial peptide was identified to possess strong antibacterial activity and low toxicity against eukaryotic cell and some antibacterial peptides have the capability to kill cancer cell. In this study, gene engineering was applied to produce the antibacterial peptide cecropinB2. We constructed an intein-based expression vector for an antimicrobial peptide and expressed the fusion peptide in Escherichia coli ER2566 expression system. CecropinB2 was selected as the target protein and the purification method of chitin binding domain (CBD) affinity tag with auto-cleavage Ssp DnaB intein was selected. The adsorption of the fusion protein was carried out by applying them to the chitin resin column. The intein cleavage activity is also strongly affected by the amino acids adjacent to the intein. Three amino acid segments: CRA or SRA or GRA were selected as the helper insert after intein for accelerating cleavage of the Ssp intein. Through the pH value and temperature adjustment, the intein was auto-cleaved and the target peptide was eluted and collected. The target peptide was confirmed by the SDS-PAGE. After purified by CBD tag column, about 58.73 mg/L of antibacterial peptide cecropinB2 can be produced. The purified cecropinB2 showed antibacterial activity against the gram-negative strains E. coil. This study demonstrated that large amounts of recombinant cecropinB2 peptide with strong antibacterial activity can be produced via the CBD-intein system.
URI: http://hdl.handle.net/11455/97572
文章公開時間: 2021-08-13
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