Variations Of Immune Related Genes Expression In Flounder (Paralichthys Olivaceus) Injected And Immersed With Vaccine

Vaccination is an effective method for controlling diseases of fish. At present,researchers have paid more attention to variations of immune related genes expressionto evaluate the immune effect of vaccine, besides the changes of antibody level ofserum and mucus, enzyme activity, number of lymphocytes, respiratory burst,complement activity and so on. The toll-like receptor(TLR) Signal pathway members,TLR2, TLR5M, myeloid differentiation factor88(MyD88) and nuclear transcriptionfactor(NF)-κB, cytokines such as interleukin-1β (IL-1β), IL-6, Interferon(IFN)γ,tumor necrosis factor α (TNF α) and chemokines, Humoral factor, c-type lysozymeand heat shock protein(HSP)70are the key factors in inducing inflammatory andimmune cell migration, immune regulation and stimulating the proliferation of T cellsand B cells. Major histocompatibility complex class I (MHC Ⅰ), majorhistocompatibility complex class II (MHC II), T cell surface receptor CD4, T cellsurface receptor CD8and immunoglobulin M (IgM) can play key roles in differentstages such as presentation and recognition of antigens and immune response afterpathogens invasion. These factors finally to remove pathogens and maintain healthythrough a series of immune response. Numerous studies have showed that the vaccinecould cause rapid changes in many immune related genes.For purpose of studying the influence of immune methods of vaccine onexpression of immune related genes, Paralichthys olivaceus were intraperitonealinjected and immersed with inactivated Vibrio anguillarum vaccine respectively inthis paper. Healthy flounder were divided into three groups, each group had60tails.Two groups were experimental groups and the last group was blank control.5tailswere taken respectively at0h,4h,8h,12h,24h,48h,72h,96h,7d and14d afterimmunization, then total mRNA was extracted and reverse transcribed into cDNA from spleen, head kidney and gill. The expression of seventeen immune relatedgenes, TLR2, TLR5M, MyD88, NF-κ B, IL-1β, TNF α, IL-6, IFNγ, c-typelysozyme, HSP70, MHC Ⅰ, MHC II, CD4, CD8, IgM and C3in spleen, head kidneyand gills was determined by quantitative real-time polymerase chain reaction(qRT-PCR) with18sRNA as internal control.The results showed that, the expression of all the tested genes except TLR5Mvaried significantly. In the injection group, the expression of TLR2, MyD88, NF-κB,IL-1β, TNF α, IL-6, IFNγ, CXC, C3, c-type lysozyme and HSP70reached the peakbetween12-24h butTLR2, MyD88, NF-κB, CXC, and C3reached the peak between48-72h in gills. MHC Ⅰ, MHC Ⅱ, CD4, CD8occurred between48-72h and peak ofIgM appeared at7d, the gene maximum expression was2-70times higher than that ofcontrol group. By contrast, in immersion group, the expression of TLR2, MyD88, NF-κB, IL-1β, TNF α, IL-6, IFNγ, CXC, C3, c-type lysozyme and HSP70reached theirpeak between12-48h, MHCⅠ, MHCⅡ, CD4, CD8occurred within48-96h, IgMpeak appeared at96h in the gills, but after14d in spleen and head kidney, themaximum amount was2-20times higher than control. The results indicated that, thetranscriptional level of genes in injection group was higher than that of immersiongroup in spleen and head kidney. In addition, the highest value appeared earlier ininjection group than in immersion group. However, the gene expression peak in gillsappeared later than immersion group. Data are referring for the evaluation of vaccine.