以基因重組人類二氫葉酸還原酶建立抗癌藥之篩選系統

Abstract

由混合物中，直接篩選出有作用的藥物，是非常具有經濟效益的方法，因此建立可行的篩選系統是本研究最終目的。本研究主要是利用人類的二氫葉酸還原酶結合表面電漿子共振 (surface plasmon resonance，SPR) 之光學偵測方法，建立抗癌藥物之篩選系統。 首先以聚合酶鏈鎖反應成功地合成558 鹼基對之人類二氫葉酸還原酶基因，並建構於載體pET22b(+)，轉型進入BL21 (DE3) 宿主大量表現。選殖菌株產生之二氫葉酸還原酶為胞內酵素，以陰離子交換樹脂層析管柱純化，得純度90 %以上的二氫葉酸還原酶 (DHFR)。 為便於與金表面鍵結，本研究利用基因突變技術於野生種DHFR之C-端加入一個半胱胺酸 (cystein)，命名為DHFR-cys，以進行篩選系統之建立。系統溫度設定為25 ℃，選擇pH 7.2 HBS-EP緩衝液為流洗溶液，pH 5.2 NaOAc作為再生試劑 (regeneration buffer)。實驗已成功將DHFR-cys固定化於感應晶片之金箔表面，並以DHFR之抑制劑MTX評估系統可行性。然而由於MTX為一小分子，其使得表面電漿之光學偵測方法靈敏度不高，導致偵測之困難。即使如此，所建立之系統未來或許可用於篩選分子量較大的化合物或月生月太。

Construction of a screening system for drug discovery possesses economic benefit. This study is investigated in the combination of human dihydrofolate reductase and SPR optical detection to develop an anticancer drug screening system. A Homo sapiens dihydrofolate reductase (DHFR) gene fragment was amplified by PCR (Polymerase Chain Reaction). The 588-bp DHFR gene fragment was further constructed into pET22b(+) vector and expressed in Escherichia coli BL21 (DE3) cells. The intracellular DHFR was purified over 90 % homogeneity by anion-exchange chromatography. For DHFR, the optimal pH is 9.2 and the optimal temperature is 60 ℃. Since DHFR is necessarily immobilized on the gold nano-film for further screening application, wild type DHFR was mutated to become DHFR-cys, which has an extra cystein on its C-terminal domain. In this study, we clearly demonstrated that DHFR-cys could be immobilized on sensor chip. Besides Methotrexate (MTX), a potent inhibitor of DHFR, was used to evaluate the feasibility of this system. However, owing to the low molecular weight of MTX, the recognition of DHFR-cys toward MTX cannot be unequivocally identified by SPR optical detection system. Further studies are suggested to focus on improving the sensitivity of SPR by using different type of senor chip, changing the source of analytes or the way of its preparations.