篩選具辨識環單磷酸腺苷之抗體片段並探討其結合專一性

Abstract

本研究利用噬菌體展示庫 (phage display library)，以環單磷酸腺苷 (adenosine 3' :5'-cyclic monophosphate，cAMP) 為抗原，利用噬菌體展示技術篩選出具辨識cAMP能力的抗體片段蛋白 (ScFv)，經六輪篩選後，成功得到多株噬菌體 (1D、 2E與5H) 具有辨識 cAMP 之能力。其中以2E之反應最佳，故選擇2E進行後續研究。2E經基因定序後證實所得之抗體片段蛋白僅為輕鏈可變區之片段，其對應蛋白質命名為cAMP-VL，該蛋白質可以大腸菌表現系統生合成再經Ni-column純化而得，以電噴灑離子化法測得其分子量為14052.8，與理論值吻合。酵素連結免疫吸附分析系統 (Enzyme-linked immunosorbent assay, ELISA) 將cAMP以化學方式修飾於BSA後固定化於96孔盤上對不同濃度的2E噬菌體與cAMP-VL進行辨識，推算出偵測極限分別為200 phage particle/mL及0.66 µg/mL；另以石英晶體微天平 (quartz crystal microbalance, QCM) 分析含有cAMP-VL之噬菌體與cAMP之結合能力，以已固含胺基之cAMP衍生物之晶片測試含cAMP-VL噬菌體，求得KD = 74900 phage particle/mL。為釐清cAMP-VL對cAMP之辨識特性，亦針對cAMP之結構相似物如Adenine、Adenosine、ATP、ADP及AMP等進行專一性測試，結果顯示，cAMP-VL對於結構中含有嘌呤分子具有較高之專一性。此外，為了提升cAMP-VL結合能力，透過蛋白質模擬技術以研判脯氨酸 (P59)，精氨酸 (R61)，穀氨酸 (E81)等胺基酸殘基可能位於結合區內，經由突變或可改進cAMP-VL之結合能力，我們逐一以蛋白質突變技術完成P59R、R61K與E81Q等單點突變蛋白並進一步測試突變蛋白對cAMP之辨識能力，發現對於cAMP辨識能力降低為野生型的20 % - 50 %，表示突變點為辨識決定點，未來可針對脯氨酸，精氨酸，穀氨酸等胺基酸殘基進行其他氨基酸突變改善對於cAMP的辨識能力。

The study is aimed to screen a single chain antibody fragment (scFv) from phage display library for cAMP recognition. The cAMP derivative (containing alkyl amine moiety) was conjugated on BSA and employed for scFv screening. After 6 rounds of panning, 3 clones (designated as 1D, 2E and 5H) with high titers were obtained. Since 2E exhibited higher titer than the other two clones, 2E clone, virtually composed of only the light chain variable region (VL), was mainly used in this study. The corresponding protein, named as cAMP-VL, was overexpressed and purified. The molecular weight was analyzed (14052.8 Da) by electrospray ionization mass spectrometry and confirmed to be consistent with the theoretical value (14054.3 Da). The binding features of cAMP-VL as free protein or on the surface of phage were further analyzed by ELISA or quartz crystal microbalance (QCM). The experiments were conducted using cAMP as probe for interacting with 2E phage and cAMP-VL. The detection limits of 2E phage and cAMP-VL are 200 phage particle/mL and 0.66 µg/mL, repectively. In the case of QCM analysis, cAMP derivative was immobilized on QCM chip, the dissociation constant (Kd) was estimated to be 74,900 phage particle/mL. In order to investigate the binding specificity of cAMP-VL, several nucleotides (adenosine,ATP,ADP and AMP) and nucleosides were used for competitive binding assessment. Results showed that cAMP-VL possesses high specificity to purine moiety. Further study on structure simulation attempted to sketch the binding domain of cAMP. P59, R61, and E81 were found to be the possible candidates for binding. P59R, R61K and E81Q mutants were constructed and purified for binding study. The outcome showed all three mutants moderately damaged the interaction with cAMP indicating these residues are somewhat important for cAMP binding. More studies will be proposed to improve the binding affinity of cAMP-VL toward cAMP based on the structure simulation or the complex of 3D protein structure.