Finer mapping of neutralizing epitope(s) on the C-terminal of Japanese encephalitis virus E-protein expressed in recombinant Escherichia coli system.

In order to localize denaturation-resistant neutralizing epitope(s) in the C-terminal 180 amino acids of Japanese encephalitis (JE) virus E-protein, four recombinant clones encoding different or overlapping nucleotide sequences were constructed by PCR from a recombinant plasmid pS22. The amplified fragments were cloned into PCR 1000 vector, and then transferred into Escherichia coli expression vector pRIT2T. The inserted genes were expressed as fusion proteins with protein-A and examined for their antigenicity and immunogenicity by Western blotting and mouse immunization, respectively. Among the four recombinant fusion proteins, the highest neutralizing antibody titre was obtained by the one expressed by the recombinant clone pRIT2T-B3, which carried the coding sequence of amino acid number 373-399 of JE virus E protein. The results indicated that this short region of 27 amino acids sequence near the C-terminal of JE virus E protein possesses neutralizing epitope(s). These data should assist in the design of an efficient subunit vaccine against JE virus infection in future.