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從噬菌體抗體基因庫篩選對 C 型肝炎病毒 NS5A 抗原具有特異性之抗體片段與分析

從噬菌體抗體基因庫篩選對 C 型肝炎病毒 NS5A 抗原具有特異性之抗體片段與分析.

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從噬菌體抗體基因庫篩選對 C 型肝炎病毒 NS5A 抗原具有特異性之抗體片段與分析

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  1. 從噬菌體抗體基因庫篩選對C型肝炎病毒NS5A抗原具有特異性之抗體片段與分析從噬菌體抗體基因庫篩選對C型肝炎病毒NS5A抗原具有特異性之抗體片段與分析 • C型肝炎病毒是主要造成人類輸血後非A非B型肝炎的病毒，它不只會造成暫時性感染的急性肝炎，亦會造成慢性肝炎、肝硬化、肝細胞癌。C型肝炎的非結構蛋白5具有RNA聚合酵素之活性，且在病毒複製過程中扮演重要角色。在本實驗中，我們利用兩個噬菌體抗體基因庫篩選與分析對NS5A有專一性的抗體。包含重鏈與kappa或lambda輕鏈的基因庫大小分別為：1.3×107和2.1×106 pfu。隨機挑選二十個菌株進行DNA序列分析，結果指出：這些抗體片段有四種組合，分別為NS5L6、NS5L13、NS5L14、與NS5K8。在這四種組合當中，其中六個屬於NS5L6這群且含有相同重鏈與輕鏈基因。甚至NS5L6的輕鏈與NS5L13的輕鏈幾乎相同，這可能暗示輕鏈是決定抗體具有對NS5A抗原專一性之關鍵。任意選擇的菌株表現蛋白後，用西方墨點法、抗人類k或l輕鏈抗體分析後，證實50 kDa的Fab之蛋白存在。酵素連結免疫分析法結果顯示，NS5L6、13、14與C型肝炎病人血清相較下，皆具有NS5A結合之專一性。綜合以上所述，我們的結果說明：噬菌體表現抗體的技術，也許可取代傳統製造人類專一性單株抗體方法。在未來，這些抗體將可被用來發展成抗感染性疾病的藥物。

  2. The selection and characterization of Fab fragments for hepatitis C virus NS5A from phage display antibody libraries • Hepatitis C virus (HCV) is the major source of non-A, non-B hepatitis, which causes not only acute hepatitis by transient infection, but also chronic hepatitis, liver cirrhosis and hepatocellular carcinoma in human. Nonstructural protein 5 (NS5) of HCV possesses RNA polymerase activity and plays a key role in viral replication. In this study, we utilized two phage display antibody libraries to generate and analyze antibodies specific for NS5A. The sizes of antibody libraries containing kappa and lambda light chain are 1.3×107 and 2.1×106 pfu (plaque forming unit), respectively. Sequence analysis of 20 randomly selected clones indicated that these Fab fragments consisted of four groups, represented by NS5L6, NS5L13, NS5L14, and NS5K8. Of which, six NS5L6 clones contain identical heavy and light chain genes. Moreover, the light chain gene used by NS5L13 is almost identical to that of NS5L6 clones, suggesting that this light chain might be crucial for the NS5A-binding activity. The Fab expression of these chosen clones was verified as a 50 kDa protein on western blot using anti-human k or l light chain antibodies. ELISA results revealed that NS5L6, 13, and 14 all have NS5A-binding specificity comparable to that of sera from HCV-infected subjects. Viewed as a whole, our results suggested that the phage display antibody technology might provide an alternative way for the generation of human specific monoclonal antibodies. These generated antibodies could be useful for the development of therapeutic agents against infectious diseases in the future.

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