Abstract
Japanese encephalitis is the major cause of epidemic encephalitis worldwide especially in eastern and southern Asia. It is spreading geographically and becomes a more concerned issue in recent years. Utilization of antiviral neutralizing antibody to treat disease for human disease control is of great interest. The 2H2, a neutralizing antibody (TCID50=0.053 nM) against domain III of the Japanese encephalitis virus (JEV), was utilized in this study. The possible neutralization mechanism for 2H2 is that it blocks the virus binding to the host cell (Wu et al., 2004). To improve the binding affinity of 2H2, random mutagenesis and phage display were performed. The mutated 2H2 libraries were created by introducing random nucleotides into the five and four residues of the CDR2 and CDR3 region of heavy chain, respectively. The recombinant phage libraries (2.51x108 and 2.05x108 combinations in the CDR2 and CDR3 libraries, respectively) were enriched by four rounds of successful biopanning and from which, five high-affinity clones were screened out by phage ELISA. Two high-affinity variants (CDR2-26 and CDR3-30) were selected out after nine times of phage ELISA assay. Residues 54 and 100 of heavy chain (TyrH54 and GlyH100) might play important roles in the maintenance of high affinity of 2H2. LysH63 was very constant and very likely made the most contribution in the keeping of high affinity.