可受特定酵素觸發降解之複合水膠之研究：製備、鑑定與降解測試

Abstract

一般而言，相較於天然高分子，合成高分子的機械性質與化學性質更具有彈性，能夠透過化學反應來控制，但天然高分子也具有一些合成高分子無法取代的特性，如：專一性配對、專一性酵素切位、自組裝結構等。在本研究中，我們希望能結合合成高分子與天然高分子，建立在合成高分子良好的物理性質上，賦予其天然高分子才具有的特性-特定酵素可切割性。本研究將質粒pET32b(+)轉型入大腸桿菌BL21(DE3)藉此產出具有凝血酶切位與腸激酶切位的目標蛋白質，將此蛋白質與聚電解質─聚烯丙胺鹽酸鹽聚合，製作出高吸水性複合水膠，我們希望能夠將凝血酶與水膠反應，藉由切割蛋白質上的凝血酶切位，讓水膠結構崩解。在蛋白質生產過程中，透過洋菜糖凝膠電泳與十二烷基硫酸鈉聚丙烯醯胺凝膠電泳(SDS-PAGE)，我們確認了由大腸桿菌產出的蛋白質確實為分子量2萬道爾頓的目標蛋白質。在蛋白質純化的部分，我們藉由蛋白質上的組氨酸標籤與鎳離子的親和力，透過將鎳離子固定在樹脂上將目標蛋白從雜蛋白溶液分離開，最後透過與不同濃度的凝血酶反應證實目標蛋白確實能被酵素切割。純化後的蛋白質與聚烯丙胺鹽酸鹽透過不同比例混合並利用戊二醛進行交聯形成水膠。在降解測試中，在某些特定比例，凝血酶的加入確實能使水膠有更大程度上的降解。而在澎潤測試中，由於聚烯丙胺鹽酸鹽為聚電解質，其製成的水膠具有超吸水性但會受環境因素，如：酸鹼值、離子濃度等影響。結合了降解特性與澎潤特性，我們設計了細管堵塞疏導實驗作為應用端的體外測試，在實驗結果中可看到，對10P15TP水膠而言，凝血酶的加入確實能使其結構變得鬆散而更易被沖開。本實驗為一合成高分子與天然高分子成功結合的案例，複合水膠保有原本聚烯丙胺鹽酸鹽水膠的澎潤能力又具有目標蛋白的可降解特性。相信這種賦予合成高分子新特性的材料在未來定能更加蓬勃發展。

Generally, synthetic polymers are more flexible to control the mechanical properties and chemical properties than natural polymers. However, some natural polymers have unique features, such as the ability of specific binding, specific cleavage, and self-assemble, which synthetic polymers do not have. Therefore, in this research, we try to combine the synthetic polymers and the natural polymers to create a composite polymer which has well physical properties as synthetic polymer and could be cleaved by enzyme as natural polymers. By transforming pET32b(+) plasmid into the E. coli strain of BL21(DE), the target protein with thrombin cleavage site and enterokinase cleavage site is producted. The target protein crosslinks to polyallylamine hydrochloride (PAH) to create a superabsorbent composite hydrogel. We hypothesize the target protein on the composite hydrogel could be cleaved by thrombin and trigger the structure of the composite hydrogel collapse. In the process of protein production, the characterization of the target protein is carried out by the electrophoresis of agarose gel and SDS-PAGE. In the purification of the target protein, the purification of the target protein is carried out using HisTrap column. Last, the cleavage test of the target protein is accomplished to verify the target protein is enzymatically cleavable. The pure target protein blends with PAH in different ratios and is crosslinked by glutaraldehyde. In the degradation test, the presence of thrombin indeed triggers a better degradation in some of the composite hydrogels. In the swelling test, the composite hydrogels still maintain great ability to swell and the swelling ratios are apt to influence by the condition of environment. Basing on the result of degradation test and swelling test, the blocked tube test is established as one of the application of the composite hydrogels. In the blocked tube study, the blocked pressure of the 10P15TP composite hydrogels truly decreases after the reaction of thrombin. This study illustrates a new pattern to combining synthetic polymers and natural polymers successfully. We believe it is promising to integrate the unique element of synthetic polymers and natural polymers to create new materials in the future.