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siRNA and recombinant vector for restraining MAG gene expression
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A technology of gene expression and recombinant vector, applied in the direction of DNA/RNA fragment, recombinant DNA technology, introduction of foreign genetic material using vector, etc.
Inactive Publication Date: 2013-05-29
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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[0005] At present, the use of RNA interference technology to specifically inhibit the gene expression of Nogo A, MAG and OMgp in oligodendrocytes and then down-regulate the signal transduction to NgR, and creatively Three high-efficiency siRNAs that inhibit different genes are connected in series on the same mammalian expression vector to achieve simultaneous high-efficiency inhibition of the three major axon growth-related inhibitors Nogo A, MAG, and Omgp.
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Embodiment 1
[0039] Synthesis and functional verification of siRNAs that inhibit the expression of NogoA, MAG and OMgp genes, respectively
[0040] 1 Materials and methods
[0041] 1.1 Materials
[0042] Experimental animals: Sprague-Dawley (SD) healthy rats 1-3 days after birth, male or female, with an average body weight of 8 g, purchased from the Experimental Animal Center of Second Military Medical University.
[0043] Main instruments: ultra-clean bench, carbon dioxide (cch) incubator, 37 ℃ constant temperature water bath oscillator, constant temperature air bath vibration shaker. Mouse anti-04 IgM, antibody Rabbit- Anti -Neurite Outgrowth Inhibitor Protein A, Mouse-Anti-myelin associated-glycoprotein, Mouse-Anti-Oligodendrocyte-Myelin-glycoprotein, secondary antibody Goat anti-Rabbit IgG, Goat anti-Mouse IgG ( Both were purchased from R&D Company). Inverted phase-contrast microscope, 74μm cell strainer, microdissection instruments, etc.
[0044] Main reagents and their preparatio...
Embodiment 2
[0071] Construction and functional verification of multigene-specific siRNA vectors targeting the expression of NogoA, MAG and OMgp genes
[0072] 1 Materials and methods
[0073] 1.1 Materials
[0074] Experimental animals: Sprague-Dawley (SD) healthy rats 1-3 days after birth, male or female, with an average body weight of 8 g, purchased from the Experimental Animal Center of Second Military Medical University.
[0075] Main instruments: ultra-clean bench, carbon dioxide (cch) incubator, 37 ℃ constant temperature water bath oscillator, constant temperature air bath vibration shaker. Mouse anti-04 IgM (purchased from R&D Company), inverted phase-contrast microscope, 74 μm cell strainer, microdissection instruments, etc.
[0076] Main reagents and their preparation: DMEM medium: containing 10% fetal bovine serum, 0.6% glucose, 4mmol / L glutamine, 5mmol / L sodium pyruvate, 50μg / ml penicillin, 50μg / ml streptomycin. Orientation medium: DMEM medium: containing 0.5% fetal bovine...
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Abstract
The invention provides a siRNA for restraining expression of myelin related glycoprotein (MAG). The nucleotide sequence of the siRNA is selected from: a) the nucleotide sequence shown in SEQIDNO.2; or b) the nucleotide sequence complementary to SEQIDNO.2. The invention further provides a recombinant vector capable of simultaneously restraining the expression of myelin related axon growth inhibitor (NogoA), the MAG and oligodendroglia cell myelin glycoprotein (Omgp). The siRNA disclosed by the invention plays a foundation for specifically restraining the expression of the three axon inhibitors NogoA, MAG and OMgp in injured nerve cells by a sequent single-chain antibody connected in series with a specific siRNA molecule in a non-covalent combination manner, stopping the inhibition effect of the myelin related inhibitors on regeneration of the axon, observing the effect of the siRNA for promoting regeneration and recovery of neuron axon after spinal cord injury in a mouse model body and researching the action mechanism of the siRNA.
Description
[0001] This application is a divisional application for the application number: 201110322415.7, application date: October 21, 2011, and the title of invention: siRNA and recombinant vector for inhibiting the expression of NogoA, MAG and OMgp genes and their applications. technical field [0002] The present invention relates to siRNA and recombinant vectors for inhibiting gene expression and their application, specifically, for inhibiting Nogo A (myelin-associated axon growth inhibitor), MAG (myelin-associated glycoprotein) and Omgp (oligodendrocyte Myelin glycoprotein) expression siRNA and recombinant vector and its application. Background technique [0003] At present, the research on spinal cord injury mainly focuses on how to break through the difficult problem of axon regeneration, including how to induce and strengthen the regeneration and elongation of axons, guide the reconnection of regenerated axons with target organs, and rebuild neural pathways. The reconstructi...
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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/85C12N15/66
Inventor 严望军刘铁龙肖建如袁文贾连顺
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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