| Summary: | 碩士 === 嘉南藥理科技大學 === 生物科技系 === 101 === Oligodeoxynucleotides (ODN) containing unmethylated Cytosine phosphorothioate guanine (CpG) motifs can activate innate immune responses in mammals. Unmethylated CpG motifs act as a danger signal in vertebrate. In invertebrate, the effects of unmethylated CpG motif are still not clear. Because NF-κB signal transduction pathways are highly conserved, Drosophila melanogaster provides a good model to study these cascades. Drosophila melanogaster cell line malignant blood neoplasm-2 (Mbn-2) was explored as a model system to study insect immune responses in vitro. Previous studies showed that innate immunity against bacteria and fungi is governed largely by two NF-κB signal transduction pathways, Toll and immune deficiency (IMD).
The aim of the study is to investigate whether the stimulation of CpG DNA in Drosophila goes through the Toll pathway.
In order to silence Toll, we designed a plasmid with two T7 promoters to product dsRNA (dsTOLL). The expression of Toll in Drosophila cell line Mbn-2 after dsRNA treatment was analyzed by real-time RT-PCR. Furthermore, the expression of antimicrobial peptide Attacin and Drosomycin were also examined to investigate the possible signal transduction pathway of LPS and E.coli DNA recognition.
Our results indicated that the transfection of dsTOLL can be used in Drosophila cell line Mbn-2 to disrupt function of endogenous genes in vitro. The dsTOLL decreased the expression of Toll and Drosomycin after treated with LPS. These results showed the stimulation signal from LPS get through the Toll pathway in Drosophila. The expression of Attacin and Drosomycin in dsTOLL treated cells has no significant different after E.coli DNA stimulation. However, there is no evidence to show the recognition of E.coli DNA in Mbn-2 cells signaling by Toll pathway.
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