分子生物學中，質體DNA（pDNA）的純化為至關重要的一環。過去的幾十年間，pDNA純化方法透過不同方法進行改良，獲取更好的產量及純度以用於後續應用。本研究，利用以下方法建立一新系統以進行質體DNA的純化：鋅指蛋白（Zinc finger protein）與具特異性的DNA序列、SpyTag-SpyCatcher接合系統以及纖維素與纖維素結合域（CBD）；此系統使用改造之鋅指蛋白與抗生素抗性基因（例如：卡那黴素或氨芐青黴素）具特異性的 9 bp 區域進行結合。
本研究成功建構出可分別表達融合蛋白zinc finger-Spytag (ZFP-Spytag) 和 CBD-Spycatcher之大腸桿菌菌株。而 Spytag/Spycatcher 系統中自然發生之異肽鍵與其複合物固定於微晶纖維素上，可使ZFP-Spytag 融合蛋白與 CBD-Spycatcher 融合蛋白進行接合。由接合測試中可得知Spytag/Spycatcher接合所需時間為 5 分鐘，並以3:1的比例接合ZFP-Spytag 和 CBD-Spycatcher。最後，為了探討以本研究所建之新系統進行細胞裂解物中純化目標質體DNA其穩健性，藉由瓊脂凝膠電泳檢查目標質體與本研究之蛋白質複合體以完成初步測試，並藉由Cross-binding確認其特異性。截至目前，我們成功建立以簡單的系統以進行目標 pDNA 的純化，未來需清除裂解物以評估靈敏度與純度。

Purification of plasmid DNA (pDNA) is a very important step in molecular biology. Over the past few decades, pDNA purification methods have been improved with a different approach to obtain better yield and purity for downstream applications. In this study, a new system to purify plasmid DNA utilizing the interaction between zinc finger protein and specific DNA sequence; Spytag and Spycatcher; and cellulose-binding domain (CBD) with cellulose was proposed. The system uses the engineered zinc finger protein to bind specifically to a 9 bp region of the antibiotic resistance gene of interest (e.g., Kanamycin or Ampicillin). Here, two fusion proteins namely zinc finger-Spytag (ZFP-Spytag) and CBD-Spycatcher were constructed and expressed in Escherichia coli. The ZFP-Spytag fusion protein was conjugated to CBD-Spycatcher fusion protein based on the naturally occurring isopeptide linkages of the Spytag/Spycatcher system and the complex was immobilized on microcrystalline cellulose. The conjugation test of Spytag/Spycatcher was found to be 5 minutes and the ratio of ZFP-Spytag and CBD-Spycatcher was 3:1, respectively. Taken together, to discover the robustness of our system in purifying target plasmid DNA from cell lysates, a preliminary test was done by examining the binding of targeted plasmid after miniprep with our complex protein on agarose gel electrophoresis. Furthermore, the specificity was also investigated by doing cross-binding. Up to now, our newly established simple system could be an alternative for targeted pDNA purification, and yet, the sensitivity and the purification from cleared lysate need to be assessed in the future.

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