| Background and ObjectiveInitially, Mints gene was cloned as a candidate of Friedreich ataxia (FRDA), and named xll. However, in succedent research it was found that Mints can directly interact with APP which play an important role in Alzheimer's disease (AD). Mints interact with APP through their PTB domain and prolong the half-life of APP in neuron and reduce the secretion of neurotoxin A P which can form senile plaque of AD. Subsequently, Mints were found and dentified by yeast two-hybridization assay, which could interact with Muncl8-l, so named Muncl8-l interacting protein (Mintl and Mint2). Muncl8-l is a neuronal protein that interacts with sytaxin land is required for synaptic vesicle exocytosis. After sequence analysis, we found part sequence of Mintl and Mint2 are Xll proteins that are expression production of xll gene, which was a candidate gene for FRDA. Masaya Okamoto attested that human Xll proteins and rat Xll proteins are homologous isoforms, and they are conservative in evolution.Till now, Mints protein family has three members: Mintl, Mint2 and Mint3. Mints are neuron-specific (Mintl and Mint2) and ubiquitous (Mint3) adaptor protein composed of isoform-specific N-terminal sequence and common C-terminal pHospHotyrosine-binding (PTB) and two PDZ domains. Mints proteins are the only one protein family we know, which contains both PTB and PDZ domains. Mints proteins can widely join in signal transduction in neurons, play an important role in synaptic vesicle exocytosis and receptor located to target position, interact withAPP and regulate APP metabolism and interact with other PDZ proteins to form a protein network to participate in many cell metabolism events by it's domains. AD will be understood better than before through the study on Mints proteins, and this will provide new idea for the therapy. At the same time research on Mints proteins will supply new complementarity to signal transduction between neurons. Therefore Yatsuka Nakajima et al exerted in situ hybridization and immunohistochemistry to localize Mintl and Mint2 in mouse brain and got an elaborate morpHological result. On this base they studied the distribution of Mintl protein on both presynaptic and postsynaptic terminals to know how Mints proteins participate in synaptic vesicle exocytosis.For the sake of further elucidation on expression and distribution of Mints proteins in nerve system, in our lab we use Mint2 antibody made by ourselves through Western blot, in situ hybridization and fluorescent immunohistochemistry combining confocal microscope to detect Mint2 mRNA and Mint2 protein in normal rat spinal cord. We conform Mint2 expresses in spinal cord, and this will provide morpHological proof for further functional study. Methods1. Construct expressional plasmid: using molecular clone methods clone Mint2 411 -656 residues into pGEX4T3, and then we identified the new plasmid.2. Protein expression and purification: BL21 was transformed by the new pGEX4T3 plasmid. GST fusion protein was induced with IPTG Then we analysised the total protein by 15% SDS-PAGE electropHoresis. The production was purified by Glutathione-SepHarose 4B.3. Test Mint2 self-made antibody: through Western blot we compare the antibodyspecificity with commercial antibody, which we purchased from Santa Cruz company4. Using Western blot to detect Mint2 protein in spinal cord of rat5. Construct transcriptive plasmid: The primer was designed according to the cDNA sequence of rat Mint2 in Genebank with assistant tool DNAstar, we clone 1 -780residues in pSK plasmid and then linearized by BamH I .6. Transcriptived Digoxigenin-linked NTP into cRNA probe by T3 transcriptase.7. Detect the expression of Mint2 mRNA in different sections spinal cord of rat.8. Detect Mint2 protein distribution in rat spinal cord of rat and use confocal microscope scan the slice.Result1. SDS-PAGE electropHoresis shows E. coli expresses 37 kD GST- Mint2 protein, which is coincident with design.2. Western blot shows that the self-made Mint2 an... |
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