Construction of expression vector for lvgA/ctxB-EGFP fusion gene and study on its immunogenicity

摘要

Legionella pneumophila is an intracellular bacteria, and its successful parasitism in host cells involves two reciprocal phases: transmission and intracellular replication. In this study, we try to construct expression vectors of pcDNA3.1 (+)-lvgA/ctxB-EGFP fused gene and to investigate its transient expressions in NIH3T3 cells. Then preliminary analysis the immunogenicity of lvgA/ctxB fusion gene. The recombinant plamids pcDNA3.1(+)-EGFP, pcDNA3.1(+)-lvgA-EGFP, pcDNA3.1 (+)-ctxB-EGFP, pcDNA3.1(+)-lvgA/ ctxB-EGFP were transfected into NIH3T3 cells respectively with Lipofection method; Transient products were detected by immunofluorescence assay (IFA) and Western blotting. Study on there immunogenicity through the detection of cytokines in mice immunization. Mice were immunized intramusculary with different plasmids DNA as vaccines for immunization by detection the production of IFN-γ, IL-8. It was found that there was green fluorescence on the cells membrane and inside the cells and showed that NIH3T3 cells were transfected by fused expression vectors successfully, the production of IFN-γ, IL-8 by blood of mice immunized with fused genes all higher than pcDNA3.1(+) group and the production of pcDNA3.1(+)-lvgA/ctxB immunized was the most highest. The fused expression vectors were successfully constructed and transfected in NIH3T3 and induced specific immune responses in mice, the group immunized by the pcDNA3.1(+)-lvgA/ctxB was most obviously, which provide the basis for the research on DNA vaccine of Legionella pneumophila.