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Recombinant adeno-associated virus vector as well as preparation method and application thereof

A viral vector, virus technology, applied in the field of biomedicine, to achieve broad market prospects, excellent infection effect, and the effect of reducing intraocular pressure

Inactive Publication Date: 2018-12-21
SHANGHAI FIRST PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are no reports about the adeno-associated virus vector expressing Nrf2 protein in the present invention and its preparation method and its application in the treatment of retinal ganglion cell degeneration in glaucoma.

Method used

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  • Recombinant adeno-associated virus vector as well as preparation method and application thereof
  • Recombinant adeno-associated virus vector as well as preparation method and application thereof
  • Recombinant adeno-associated virus vector as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1 Construction and separation and purification of recombinant adeno-associated virus vector

[0061] gene sequence

[0062] Shown in SEQ ID NO:1 is the Nrf2(CA) sequence after splicing,

[0063] Shown in SEQ ID NO: 2 is the amino acid sequence of human Nrf2 (CA) after splicing,

[0064] Shown in SEQ ID NO:3 is the Left ITR sequence,

[0065] Shown in SEQ ID NO:4 is the CMV sequence,

[0066] Shown in SEQ ID NO:5 is the humanβ-globin intron sequence,

[0067] Shown in SEQ ID NO:6 is the MCS sequence,

[0068] Shown in SEQ ID NO:7 is hGHpA sequence,

[0069] Shown in SEQ ID NO:8 is the Right ITR sequence,

[0070] Shown in SEQ ID NO: 9 is the Ampicillin Resistance sequence,

[0071] Shown in SEQ ID NO:10 is the full nucleotide sequence,

[0072] Shown in SEQ ID NO:11 is the f1ori sequence,

[0073] Shown in SEQ ID NO:12 is the recombinant sequence,

[0074] Shown in SEQ ID NO: 13 is the Nrf2(CA)' sequence.

[0075] The construction of plasmid AAV_Nrf2(C...

Embodiment 2

[0077] Embodiment 2 The treatment of recombinant adeno-associated virus vector to glaucoma model mice

[0078] 1 Experimental materials

[0079] 1.1 Experimental animals

[0080] Normal C57BL / 6J mice were purchased from Shanghai Slack Experimental Animal Co., Ltd., and the light cycle was 12h light-12h dark, with free access to food and water, and all animal research was strictly in accordance with the "Regulations on the Management of Experimental Animals" issued by the National Science and Technology Committee. "conduct.

[0081] 1.2 Experimental reagents and consumables

[0082] Normal saline (Zhejiang Tianrui Pharmaceutical Co., Ltd.), disposable injection needle (Becton Dickinson and Company, USA), Nrf2 antibody (Abcam), TUJ1 (Abcam), secondary antibody goat anti-rabbit IgG (JacksonImmuno), goat serum (Sigma), Polyethylene glycol octylphenyl ether (Triton X-100) (Sigma), Paraformaldehyde (Sigma).

[0083] 1.3 Experimental Instruments

[0084] Ophthalmology Experiment...

Embodiment 3

[0102] Example 3 Treatment of recombinant adeno-associated virus vectors to glaucoma model rats

[0103] 1 experimental drug

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PUM

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Abstract

The invention relates to a recombinant adeno-associated virus vector. The recombinant adeno-associated virus vector comprises constitutively active human Nrf2 (Nrf2 (CA)), a CMV promoter / enhancer, beta-globin intron for increasing gene expression, human growth hormone poly (A) tail sequence, etc. The invention also relates to a preparation method of the recombinant adeno-associated virus vector and the application of the recombinant adeno-associated virus vector or a composition containing the recombinant adeno-associated virus vector in preparing a medicine for treating glaucoma retinal ganglion cell denaturation. The recombinant adeno-associated virus vector provided by the invention can obviously reduce the intraocular pressure of a mouse, increase the survival rate of RGCs of the mouse, and effectively treat glaucoma retinal ganglion cell pathological changes.

Description

technical field [0001] The invention relates to the technical field of biomedicine, specifically, a recombinant adeno-associated virus vector and its preparation method and application. Background technique [0002] Glaucoma is a group of diseases characterized by optic atrophy and visual field defect, and pathological increased intraocular pressure is the main risk factor. It has been recognized that glaucoma is a neurodegenerative disease, and its pathological features are retinal ganglion cell (RGC) apoptosis and axonal degeneration, as well as the accompanying progressive loss of visual function. RGCs are responsible for outputting visual signals captured by the retina to the brain. Continuous degeneration or apoptosis of RGCs caused by high intraocular pressure, multiple genetic factors, or neurodegenerative diseases can lead to optic nerve head lesions, visual field loss, and then develop into glaucoma. . [0003] The pathological mechanism of glaucoma is complex, in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/864C12N15/66A61K48/00A61P27/06
CPCA61K48/0058A61P27/06C12N15/66C12N15/86C12N2750/14143
Inventor 罗学廷孙晓东高敏
Owner SHANGHAI FIRST PEOPLES HOSPITAL