Investigation On Mechanism Of Mutually Exclusive Splicing Of Drosophila MRP1Gene;Study Of The Silkworm-expressed Cholera Toxin B Subunit And Human Glutamic Acid Decarboxylase Fusion Protein Protecting Against Type Ⅰ Diabetes

Alternative splicing is an essential regulated mechanism in eukaryotic mRNA posttranscriptional processing.Mutually exclusive splicing is a special type of alternative splicing, also which is a strictly regulated means to increase proteomic diversity. In this form,only one variable exon out of a cluster of mutually exclusive exons is chosen in the mature mRNA isoform, thus generating protein isforms. Many works have been done to study the mechanism of the mutually exclusive splicing.The multidrug-resistance associated protein which is involved in multidrug resistance of tumor cells encoded by MRP is a member of the ATP-binding cassette transmembrane transporter superfamily. We find that MRP1gene of Drosophila melanogaster has seven mutually exclusive exons. In this study, we first obtain the gene sequences of MRP1in fourteen Drosophila species by bioinformatics methods. Then we analysis EST sequences and phylogenetic relationships of mutually exclusive exons of Drosophila melanogaster. According to the expression of mutually exclusive exon8of Drosophila melanogaster MRP1mini-gene in S2cells, exon8.4was mainly included. Through a series of deletion mutagenesis, we find that there exist regulatory elements in the108bp intron on the left side of the exon8.4, also in the244bp intron on the right side of the exon8.4. These experimental data will provide a basis for the mutually exclusive splicing regulation mechanism of MRP1gene.Type I diabetes(TED)is a spontaneous organ-specific autoimmune disease, which results from the specific destruction of β cells secreting insulin. The CTB serving as a mucosal carrier molecule can promote effective oral immune tolerance.Here we constructed the expression vector of the CTB and human GAD65fusion gene, which was transformed into DH10Bac competent cells for transpositional recombination by Bac-to-Bac baculovirus expression system, generating the recombinant Bacmid through three kinds of resistance and blue-white screening. Once we got recombinant Bacmid, it was used to transfect BmN cells of Bombyx mori to produce recombinant baculovirus. After the recombinant virus infecting BmN cells of Bombyx mori,we performed Western blot analysis to detect recombinant protein. When the fusion protein was boiled and produced in a monomeric form, we can detect a single band of21kDa on SDS-PAGE; conversely the unboiled recombinant protein was yielded a band of105kDa produced in a pentameric form. A GM1-ELISA was performed to detect the affinity of the recombinant protein for GM1-ganglioside, we find that the GM1binding ability of CTB-hGAD65was lower than that of the CTB.