Mip gene of Legionella pneumophila and ctxB gene of Vibrio cholerae were PCR amplified respectively. The amplified fusion DNA was ligated to pcDNA3.1(+) vector. The recombinant plasmid, which was named pcDNA3.1-mip/ctxB, was identified by restriction analysis, PCR and further confirmed by sequence analysis. NIH3T3 cell was transfected by recombinant plasmid pcDNA3.1-mip or pcDNA3.1-mip/ctxB with Lipofection strategy. Transient and stable products of mip/ctxB fusion gene was detected by immunofluorescence and Western blot. The results showed that NIH3T3 cell was transfected successfully and stable products can be detected in the transfected cell. To evaluate immunonicity of pcDNA3.1-mip and pcDNA3.1-mip/ctxB, BALB/c female mice were immunized intramuscularly with them and antigen specific antibodies, lymphocyte proliferative response, IFN-γ production and cytotoxic T-lymphocyte response of immunized mice were detected. The results showed that immunogenicity of pcDNA3.1-mip or pcDNA3.1-mip/ctxB immunized mice were higher than control and immunogenicity of pcDNA3.1-mip/ctxB immunized mice were higher than pcDNA3.1-mip immuneized mice. Statistic analysis by one way ANOVA showed that there was significantly difference between groups (P＜0.01).The results provide experimental proof to study of mip/ctxB fusion gene DNA vaccine.