Study On The Effect Of Cpg Dna On The Activation Of Immunological Cells In The Carassius Auratus Gibelio

In mice and humans, B cells, antigen-presenting cells including monocytes, macrophages and dendritic cells and natural killer cells can be stimulated directly or indirectly by the bacterial DNA and oligodeoxynucleotides containing the CpG dinucleotides (CpG DNA).In the essay, we used headkidney macrophages of carassius auratus gibelio as an invitro model and investigated the effects of six CPG DNA, on fish immunocytes for the first time. The CPG-DNA included the optimal motifs : the 1826 (GACGTT) and 2006 (GTCGTT) for the mice and humans cells, the 1670 (AACGTT) used in Atlantic salmon, the 1670-N containing the same motif of those in 1670 , but near the 5' end the 1670-D containing two repeats motif of those in 1670 and the 1670-R with an inverted CPG . The respiratory burst activity of oxygen was detected by the NBT method, the nitrogen content was measured to detect the respiratory burst activity and the proliferation of peripheral blood leukocytes was determinated by the MTT method, we used headkidney macrophages of carassius auratus gibelio as an invitro objects and investigated the effects of six CPG DNA, on expression of two important cytokines including IL-1βand IL-12 on carassius auratus gibelio for the first time . After 24h of incubation with the six sequences of CpG DNA, total RNA of head kidney macrophages was isolated. The qualitative analysis of the expression of these two kinds of immune factors was be measured by General RT-PCR. Using the fluorescence quantitative RT-PCR, the expression of these two kinds of immune factors was detected by quantitative analysis. The results are as follows:1. The activation of the macrophages and peripheral blood leukocytes can be caused by the other five kinds of CPG DNA, in addition to 1670-R, mainly expressed in the respiratory burst activity of oxygen and nitrogen, and the improvement of cell proliferation. After RT-PCR amplification, the cDNAs of IL-1β, IL-12 were observed in CpG DNA stimulated macrophages. Using quantitative RT-PCR, the upregulated expression of IL-1βand IL-12 in CpG DNA-induced head kidney macrophages was observed, while no amplification of the corresponding cDNAs were found in control group. Using quantitative RT-PCR, the upregulated expression of IL-1βand IL-12 in CpG DNA-induced head kidney macrophages was observed,compared with the control group, there were significant (P<0.05) or very significantly differences (P<0.01).1670-R compared with the control group, there is no obvious enhancement.2. Among which, 1670-D was the most effective ODNs.3. Dose-response effects of CpG DNA on the immunomodulation was observed, with 10μg·mL^-1 being the most effective concentration(P<0.01).The results suggests that CpG DNA could be used as a novel immunostimulant in fish which promote the cell-and hurmoral-mediated immune responses. And it may have potential application in the minimizing the impact of fish diseases and enhance the efficiency of vaccines.