| Summary: | 碩士 === 台北醫學院 === 生物醫學技術研究所 === 88 === Hepatitis C virus (HCV) is the major etiological agent of posttransfusion viral hepatitis C. Most HCV-infected individuals develop chronic disease which may progress to liver cirrhosis and eventually hepatocellular carcinoma. With an estimated more than 100 million carriers, HCV infection is one of the most important cause of liver disease worldwide. HCV is an enveloped virus with a single-strand, positive sense, RNA genome of approximately 9500 nucleotides and is classified in the family Flaviviridae. The HCV NS3 protein is an immunodominant antigen, possessing viral protease and helicase. Sequence analysis of the NS3 protein indicates that it is highly conserved, implying it is an important viral protein in its life cycle.
In this study, hepatitis C antibody libraries were established by Dr. Toshiaki Muruyama. The heavy chain genes with either kappa or lambda light chain genes resulted in 1.3 X 107 (HCK) and 2.1 x 106 (HCL) clones in size, respectively. After biopanning against the NS3 of HCV, we used Western blot analysis to randomly check 30 clones and found that 15 contained Fab fragment.The preliminary enzyme-linked immunoabsorbent assay (ELISA) data suggest that 2 clones containing lambda light chain (HCL4 and L7) may be specific for NS3. Furthermore, their NS3 binding activity was confirmed by AxSYM HCV 3.0 Microparticle Enzyme Immunoassay (MEIA). DNA sequence analysis indicated that HCK5 and YKns3a1 contained identical CDR3 region in kappa light chain gene, but differed in the heavy chain. Moreover HCL4 and YKns3b used the same heavy chain, but different light chain genes! Taken together, our results showed that the NS3 binding activity of those screened anti-NS3 Fab antibodies was mainly determined by the heavy chain. The conclusion is consistent with most of the previous studies which indicated that the heavy chain plays an important role in antigen binding activity!
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