| It is proved that bacterial DNA has potent immunological properties because of its content of immunostimulatory sequences centering on CpG motifs.In part one, to investigate whether DNA from various bacterial species differ in these properties, the activity of a panel of DNA was assessed in in vitro, cultures of Carassius auratus macrophages. This panel varied in base composition and included DNA form Escherichia coli, Agrobacterium tumefaciens, Micrococcuslysodeikticus, Aeromonas Hydrophila (A.Hydrophila), Bifidobacterium adolescentis, Streptococcus thermophilus, Enterococcus faceium, Bacillus Licheniformis, Bacillu subtzlis, Saccharomyces cerevisiae. In assays of superoxide anion and Bactericidal activity, these DNA showed marked differences in their immunostimulatory activity. Then, the difference between groups were analysis with Tukey-Kramer multiple comparison procedure. Of all, A.Hydrophila and E.coli demonstrated the highest activity while Saccharomyces had the lowest activity. And later, the genomes of bacteria were analyzed for the frequency of unmethylated cytosine-guanosine ([CG]) dinucleotides with visual C++. We found the [CG] dinucleotide content of bacterial DNA correlates with the immunostimulatory activities.ln next part, E.coli DNA, was used as the immunostimulant for its clear research backgroud.In part two, the genomic DNA of Escherichia coli (E.coli), which was demonstrated one of the best bacterial with immunostimulating effect, was used to evaluate the immunostimulating effect of bacterial DNA on innate immune responses in bivalve mussel. The results showed that the genomic DNA of E.coli had no significant effect on the production of superoxide anion (O2-) or acid phosphatase (AP) by haemocytes of bivalve mollusks (Hyriopsis cumingii Lea) in vitro. However, the bactericidal activity of the haemocytes was significantly increased when the cells were incubated with 50 or 100μg/ml bacterial DNA for 12 and 24 h. Antibacterial activity, lysozyme activity, and prophenoloxidase (proPO) production of haemolymph were also increased, when the bivalve mollusks were injected with 50 or lOOug/ml of bacterial DNA for 12 and 24 h. These activities returned to the control level after 48 h. This work showed the bacterial DNA with unmethylated CpG motif could also activate some parameters of the immune system of bivalve mollusks in vivo and in vitro.In part three, we studied the effects of E.coli genomic DNA on immune responses of fish in vitro and in vivo, as well as the expression of some CKs inclding alpha-TNF, alpha-IFN, Mx, MHC-II, MHC-I, pre-B-cell enhancing factor (PBEF), natural killer cell enhancement factor (NKEF) on Carassius auratus, the Carassius auratus were injected with 50μg/ml E.coli DNA for 2, 12, 24, 48 and 72 hours, and the total RNA of head kidney was isolated. And then the CKs were amplified. The results The results shown the expressions of TNF- α , IFN- α , NKEF, PBEF and Mx had upregulated by the injection of bDNA after 12 or 24 hours, however, the expressions of MHC-I and MHC-II were stable in all time, which shown that the bDNA could stimulate the monocyte and macrophage to secrete CKs such as TNF, IFN to enhance the...
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