- 學位論文
角質蛋白-1與C反應蛋白及氧化低密度脂蛋白之交互作用
Keratin-1 Interaction with C-reactive Protein and Oxidized Low Density Lipoprotein
摘要
動脈硬化被認為是慢性發炎疾病，而C反應蛋白(C-reactive protein；CRP)是一個非常靈敏的發炎性指標，並且CRP在動脈硬化的形成扮演重要的角色。氧化低密度脂蛋白(oxLDL)引起內皮細胞活化及功能障礙、泡沫細胞的形成和平滑肌細胞的增生，導致動脈硬化斑塊的形成。 先前實驗發現角質蛋白-1 (keratin-1)會和CRP產生結合，並且會抑制CRP引起的NO釋放量下降。為了更進一步闡明KRT1與CRP還有oxLDL之間的交互作用，我們將實驗室先前建構的質體(pcDNA3.1(+)-KRT1)利用PCR方式將KRT1放大，並與在C端含有6X His-tag的pET29c進行接合，形成pET29-KRT1，再轉型入大腸桿菌中。利用IPTG誘導KRT1的表現，使用Ni2+ column方式純化KRT1蛋白。接著，利用酵素免疫分析(Enzyme-Linked ImmunoSorbent Assay；ELISA)測試KRT1與CRP和oxLDL之間的交互作用。 實驗結果顯示KRT1與CRP在含鈣離子的環境下，會與KRT1產生交互作用，在不含鈣離子的環境卻不會產生交互作用。因此判斷KRT1是透過CRP的B face與CRP產生結合。另外，實驗結果也發現KRT1會與oxLDL結合，並且隨著oxLDL氧化的程度影響與KRT1結合的親和性。 由KRT1與CRP及KRT1與oxLDL的交互作用，可以證明KRT1與動脈硬化的相關性。
並列摘要
Atherosclerosis is considered as a chronic inflammatory disease. C-reactive protein (CRP) is a sensitive indicator of inflammation and plays an important role in atherosclerosis. Oxidized low density lipoprotein (oxLDL) contributes to the atherosclerotic plaque formation by induction of endothelial cell activation and dysfunction, foam cell formation, and smooth muscle cell proliferation. Previously, we found that keratin-1 (KRT1) binds to CRP and interfered the CRP effect on reduction of nitric oxide release. To further elucidate the interaction of KRT1 with CRP and oxLDL, KRT1 gene was amplified from pcDNA3.1(+)-KRT1 using polymerase chain reaction (PCR) and inserted into pET29c with a 6X His-tag at the C-terminus, forming plasmid pET29-KRT1. pET29-KRT1 was transformed into E. coli. The KRT1 expression was induced by IsoPropyl-Beta-D-ThioGalactoside (IPTG) and purified using Ni2+-column. The interaction of KRT1 with CRP and oxLDL was assayed by the enzyme-linked immunosorbent assay (ELISA). Our results showed that KRT1 bound to CRP in the presence but not absence of Ca2+, suggesting that the binding of CRP to KRT1 may be through the B-face of CRP. We also confirmed that KRT1 bound to oxLDL and the binding affinity was dependent on the oxidation degree of oxLDL. The binding of KRT1 to CRP and oxLDL strongly suggests the involvement of KRT1 in the process of atherosclerosis.