The Immunogenicity And Protective Immunity Of Mip Gene DNA Vaccine Against Legionella Pneumophila

Legionnaires' disease is the infectious disease of respiratory tract caused by Legionella.Humans become infected with Legionella pneumophila by inha ling aerosols from L.pneumophila-contaminated water sources and caused dis ease such as untypical pneumonia and Pontiac fever.If patients were theated incorrectly, death ratio of infection was very high.The older and immunodeficiency people are the susceptible people. Legionella is an inhabitant of soil, natural and man-made aquatic environments.With the popularity of air-condi tion and humidifier in modern life, Legionella infection will increase and will be more and more severe.China emphases prevention and incure Legionnaires' disease as a new emerging infectious disease. But human haven't find any good way to deal with it. This study choose the subject of L. pneumophila DNA vaccine.According to the results of indexing the domesitic and overseas reference, this study choose microphage infectivity potentiator gene (mip gene) of L. pneumophila as candidate vaccine gene.This study firstly combine mip gene with adjuvant cholera toxin subunit B gene (ctxB gene). We hope to provide experimental basis and proof for study of safe and effect vaccine.This study includes four parts.In the first part, 828bp L. pneumophila mip gene and 376bp ctxB gene were PCR amplified from a template of L. pneumophila serogroup 1 and Vibrio cholerae 569B genosome DNA. The amplified DNA was cloned into pUC 18 vector respectly and the recombinant plasmid pLpmip and pctxB were constructed. 24kDa protein of pLpmip and llkDa protein of pctxB were induced to express in prokaryotic cell successfully.In the second part, PCR product of pLpmip and pctxB were cloned into pcDNA3.1(+) vector and recombinant plasmid pcDNA3.1-mip, pcDNA3.1-ctxB and pcDNA3.1-mip/ctxB were constructed. NIH3T3 cells were transfected by recombinant plasmid pcDNA3.1-mip, pcDNA3.1-ctxB and pcDNA3.1-mip/ctxB, pcDNA3.1-mip/CTB with Lipofection strategy. Transient products were detected in cell membrane and inside the cell by immunofluorescence.24kDa protein of pcDNA3.1-mip, 24kDa and llkDa protein of pcDNA3.1-ctxB, 35 kDa protein of pcDNA3.1-mip/ctxB were detected in stable transfercted cells with Western-blot.In the third part, BALB/c female mice were immunized intramuscularly with the DNA vaccine. Immunogenicity and protective immunity were evaluated through antigen specific antibodies, lymphocyte proliferative response, IFN-y production and cytotoxic T-lymphocyte response of immunized mice. The results showed that immunogenicity of DNA vaccine immunized mice were higher than control. Humoral immunity and cellular immunity of pcDNA3.1-mip and pcDNA3.1-ctxB, pcDNA3.1-mip/ctxB immunized mice were higher than pcDNA3.1-mip immuneized mice. Antigen specific antibodies of pcDNA3.1-mip and pcDNA3.1-ctxB immuneized mice were higher than pcDNA3.1-mip/ctxB immuneized mice. Cell mediatedimmune responses of pcDNA3.1-mip/ctxB immunized mice were higher than pcDNA3.1-mip and pcDNA3.1-ctxB immunized mice. Statistic analysis by one way ANOVA showed that there was significantly difference between groups (PO.01).In the fourth part, BALB/c female mice were immunized intramuscularly with the DNA vaccine. After the last immunization, the immunized mice were challenged with L. pneumophila serogroup 1. 28 days after changllege, the bacterial counts and the pathologyical changes of lung were detected to evaluated the protective immunity. The results showed that the bacterial counts of DNA vaccine immunized mice were lower than control, whereas no significantly difference between the three DNA vaccine immunized groups. Exudative pathological changes of were observed in control. On the contrary, similar changes were not observed in DNA vaccine immunized groups. It can be concluded that the three DNA vaccine have protective immunity. The protective afficacy of pcDNA3.1-mip and pcDNA3.1-ctxB, pcDNA3.1-mip/ctxB were higher than pcDNA3.1-mip. Comparing pcDNA3.1-mip and pcDNA3.1-ctxB with pcDNA3.1-mip/ctxB, there was no significantly difference.This study showed that the three DNA vaccine have higher immugenecity and some protective immunity, immugenecity and protective immunity combining with adjvnant CTB were higher than mip gene alone. Higher cell mediated immunity was induced by mip/ctxB fusion gene. On the other hand, higher humoral immunity was induced by mip gene combining with ctxB gene. Because L. pneumophila is a facultative intracellular parasite, cell-mediated immunity against L. pneumophila palys an important role in inhibition ofbacterial growth. It is meaningful to construct fusion gene of candidate geneand adjvnant gene. Untill today, such study is not reported domestic andoverseas. This study provided experimental proof to study L. pneumophilavaccine.