Abstract: Japanese Encephalitis Virus (JEV) was isolated from a porcines intumescent testis. After specimens were made into homogenate and filtrated，it was inoculated to passage cells and injected into rat brain to isolate JEV, then JEV was identified by RTPCR. According to published cDNA sequence of E gene in GenBank of JEV SA14 and SA14142 strain, the synthesized specific primers were designed. E gene fragments of JEV Guizhou strain were amplified by using RTPCR. The PCR products were sequenced and comparatively analyzed. The products of RTPCR were cloned into pMD19T vector to form recombinants pMD19TE, then were subcloned into Prokaryotic Expressing Vector pET32a (+) after double enzyme digestion and sequence analysis. The pET32aE was transformed into the E. coli strain BL21 (DE3) and the expression of the E protein was induced. After analysis by SDSPAGE electrophoresis of the induced expressed protein and Western blot, we got about 59 kDa expressed protein in line with the expected size. Through further extracting and purifying the target protein, the expressed protein was used to immunize mice by adding adjuvant, and then the antibody titers of mice were detected. The results showed that the target protein induced antiJE antibody in mice. This study laid the foundation of the research of subunit vaccine of Japanese Encephalitis.