Study of the Binding Domain of Hepatitis B Virus e Antigen on Neutrophils

• Main Authors: Chien-Chiao Huang, 黃建喬
• Other Authors: Cheng-Po Hu
• Format: Others
• Language: en_US
• Published: 2004
• Online Access: http://ndltd.ncl.edu.tw/handle/52224087785999531818

碩士 === 國立陽明大學 === 微生物及免疫學研究所 === 92 === Hepatitis B virus (HBV) causes acute and chronic infections in the liver. The acute infection may result in serious illness, and approximately 0.5% of the patients exhibit fulminant hepatitis. The chronic infection is strongly associated with the liver cirrhosis and the hepatocellular carcinoma. HBeAg, a secretory product of HBV, is neither a structural component of HBV, nor is required for the viral infectivity and replication. It is suggested that HBeAg plays an immunomodulatory role to help virus escape the host immune system. We previously found that HBeAg could bind to human monocytes, neutrophils, and B cells. In order to identify the binding domain(s) of HBeAg to human neutrophils, four N-terminal and four C-terminal fragments of HBeAg with S, His, and Trx tags were constructed. They were N128 (a.a. -10 ~ 128), N112 (a.a. -10 ~ 112), N78 (a.a. -10 ~ 78), N41 (a.a. -10 ~ 41), 41C (a.a. 41 ~ 149), 78C (a.a. 78 ~ 149), 112C (a.a. 112 ~ 149), and 128C (a.a. 128 ~ 149). To identify the binding of HBeAg fragment(s) on neutrophils, a panel of monoclonal antibodies (mAb) against HBeAg was first characterized using Western blotting and peptide array. We found that the epitope recognized by #1 mAb located in the sequence AYRPPNAPI (131 ~ 139 a.a.) at the C terminus of HBeAg, while that recognized by #3 mAb located in the sequence LWGMDIDPYKEF (-3 ~ 9 a.a.) at the N terminus of HBeAg. Thus, #1 mAb can be used for the detection of C-terminal fragments, while #3 mAb can be used for the detection of N-terminal fragments. Using a binding assay followed by flow cytometry, we found that all C-terminal fragments except 128C could bind to neutrophils. These results suggest that the region of a.a. 112 ~ 128 seems to be required for the HBeAg binding. However, all the N-terminal fragments also bind to neutrophils. Thus, other sequences within a.a. -10 to 112 may also participate in the binding. We further removed the tags from N78 and 78C fragments using enterokinase. We found that both the N78 and 78C fragments without tags bound to neutrophils. Thus, our preliminary results suggest that more than one region in the HBeAg may be involved in the binding on neutrophils. The exact sequences for the binding need further investigation.