Cloning and expression of envelope protein of Thai genotype I strain KE-093 of Japanese encephalitis virus.

The purpose of this study was to clone and express envelope (E) gene of Japanese encephalitis virus (JEV) genotype I, Thai strain KE-093. The E gene was amplified by PCR and cloned using the expression vector, pET-15b. Analysis of the insert sequence revealed a point mutation, which was corrected by site directed mutagenesis. The envelope 53 kDa protein expression was generated by in vitro coupled transcription translation system. Heterologous expression in Escherichia coli Rosetta 2 strain, but not in E. coli BL21 (DE3) resulted in 2 immunoreactive bands (13 and 53 kDa) using anti-JEV E protein antibodies, and an additional band (35 kDa) using anti-His antibodies, suggesting that E protein antigenicity is located at the carboxy-terminal region. This is the first report of a successful cloning and heterologous expression of an E gene of JEV genotype I. This should prove useful in the application for diagnostics and vaccine development of JEV genotype I strains.