Abstract
The envelope protein is the major surface antigen of Japanese encephalitis virus that is believed to induce protective immunity and neutralizing antibody response in hosts. The neutralization of virus by antibody is an important mechanism to protect host free from virus infection. It is demonstrated that neutralizing antibody to the E protein play a dominant role in the protective immunity against JEV infection. To improve the affinity of JEV neutralizing antibody 2H2 and to investigate the direction and possibility of 2H2 evolution, DNA shuffling technique and phage display system were performed in this study. At first, we mutagenized recombinant Fab by shuffling 2H2 VH alone, and then the library was established, resulting in 8.2 x 104 clones in size. After three times panning and phage ELISA, 2H2 reveals the highest affinity. By DNA sequencing of 10 clones with higher affinity in phage ELISA assay, shuffled 2H2 mutants indicated that all point mutations occurred in FR of VH. Our results suggested that these mutations have little effect in antigen–antibody interaction, and 2H2 is a highly maturated antibody. Next, we shuffled both neutralizing antibody 2H2 and E3.3 simultaneously to generate VH chimera. Established library results in 8.8 x 104 clones in size. We analyzed VH sequences of 10 clones possessing higher affinity after selection, and they showed more mutation in CDR2, CDR3 and FR3 resulted from homologous recombination between 2H2 and E3.3. No dominant influence in affinity of antigen binding was found in the sequence of these mutants that have no insertion and deletion. In conclusion, our data suggested that 2H2 and E 3.3 show high consensus in the evolution of immune system, and they exhibite highly maturated architecture.