The selection and characterization of Fab fragments for hepatitis C virus NS5A from phage display antibody libraries

• Main Authors: Yi-Ning Shih, 施憶寧
• Other Authors: Yi-Yuan Yang
• Format: Others
• Language: zh-TW
• Published: 2002
• Online Access: http://ndltd.ncl.edu.tw/handle/19304418441007595951

碩士 === 台北醫學院 === 細胞及分子生物研究所 === 90 === 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.