Abstract: Objective To express protein NS1 of Japanese encephalitis virus(JEV) in Escherichia coli and to evaluate its immunogenicity.Methods JEV NS1 gene was cloned into the pET30a vector.After being purified with Ni-NTA chromatography, the expression products were analyzed with sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) and Western blot.Rabbits were immunized with the purified protein and their serum was assayed with enzyme-linked immunosorbent assay(ELISA) and immunifluorescence assay(IFA).Results Recombinant NS1 protein was expressed and purified successfully.Western blot results showed that the purified product could reacted with the ascites of mice immunized with JEV.Indirect ELISA results showed that the serum titer reached above 1:51200.IFA showed that when the serum was diluted to 1：50, 1：100, and 1：200, the florescent light could be detected in Escherichia coli, and its immunogenicity was verified.The result was observed in baby hamster kidney(BHK) cells infected with JEV.Conclusion Recombinant JEV NS1 protein was expressed in Escherichia coli and the purified protein was of immunogenicity, which could be used for developing diagnostic reagent of JEV.