CN116286837A - Nucleic acid molecule encoding human nuclear factor E2-related factor 2, expression vector and application thereof - Google Patents

Abstract

The invention relates to the field of biological medicine, in particular to a nucleic acid molecule for encoding a human nuclear factor E2 related factor 2, an expression vector and application thereof. The invention discloses a nucleotide sequence for encoding a human nuclear factor E2 related factor 2 (Nrf 2) protein, which comprises nucleic acid for encoding the human nuclear factor E2 related factor 2 (Nrf 2) protein. Further disclosed is a recombinant expression vector comprising the above nucleic acid. The nucleic acid for encoding the human nuclear factor E2 related factor 2 (Nrf 2) protein has the advantages of faster expression time and higher expression level, so that the nucleic acid can better treat the optic nerve related diseases.

Description

Nucleic acid molecule encoding human nuclear factor E2-related factor 2, expression vector and application thereof

technical field

The invention relates to the field of biomedicine, in particular to nucleic acid molecules encoding human nuclear factor E2-related factor 2, expression vectors and applications thereof.

Background technique

The optic nerve originates from the ganglion cell layer of the retina and is a part of the central nervous system. The visual information obtained by the retina is transmitted to the brain through the optic nerve. There are many diseases related to optic nerve damage, including glaucoma, ischemic optic neuropathy, Leber's hereditary optic neuropathy (LHON), autosomal dominant optic atrophy (Autosomaldominant optic atrophy, DOA) and so on. The damage of ganglion cells and their axons is irreversible, and is the main cause of vision loss or even blindness in patients. There are currently no effective drugs on the market for optic nerve damage. Drug research on optic nerve protection mainly focuses on neurotrophic factor drugs, such as BDNF, CNTF, etc., whose mechanism of action is to promote the growth of neurons and repair damaged optic nerves, but the therapeutic effect is not ideal.

Studies have found that the pathogenesis of various optic nerve injury diseases is due to the dependence of retinal ganglion cells and their axons on energy and their sensitivity to free radicals, which lead to damage or even apoptosis first, resulting in blindness. Therefore, the optic nerve can be protected by preventing or delaying the apoptosis of ganglion cells (RGCs) by eliminating free radicals of diseased cells and improving the antioxidant capacity of cells.

Oxidative stress is caused by the excessive production of reactive oxygen species (ROS) and electrophiles in cells, and excessive ROS can induce free radical chain reactions and damage cellular biomacromolecules such as proteins, lipids, and DNA. This in turn leads to cell death and tissue damage. At the same time, ROS is also involved in other pathological processes such as inflammation and fibrosis. The body forms a complex oxidative stress response system. When exposed to electrophile or active oxygen stimulation, it can induce a series of Protective proteins to alleviate cell damage.

Oxidative stress, especially within the mitochondria, can lead to a vicious cycle that directly damages cells. Oxidative stress, inflammation, and neuronal degeneration caused by the continuous exposure of the retina to reactive oxygen species are also associated with retinal diseases, and increasing evidence suggests that oxidative stress plays an important role in the accumulation process in retinal diseases.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a redox-sensitive transcription factor observed in many tissues, especially those exposed to the external environment (skin, lungs, and gastrointestinal tract) and associated with detoxification Associated tissues (liver and kidney). At rest, Nrf2 is trapped in the cytosol by the adapter protein Keap1, where it is rapidly degraded by the proteasomal pathway. Under oxidative stress, Nrf2 can enter the nucleus, bind to the antioxidant response element ARE, and initiate the transcription and expression of genes encoding many antioxidant enzymes and phase II detoxification enzymes. Nrf2 targets more than 250 genes, including NAD(P)H:quinone oxidoreductase-1 (NQO1), heme oxygenase-1 (HO-1), glutamic acid cysteine ligase, glutathione Glycerin S-transferase, glutathione peroxidase, catalase, superoxide dismutase and thioredoxin UDP-glucuronosyltransferase, etc. The expression of these downstream genes will be activated through Keap1-Nrf2 signaling, so as to respond to various stimuli (such as active poisons, pro-inflammatory factors, apoptosis and carcinogenesis), rescue tissue cells, and reduce damage. Activation of Nrf2 function is one of the key defense mechanisms against oxidative stress in many species. The well-known Nrf2 activator methyl Bardoxolone and its derivatives have been the subject of numerous clinical trials, including those aimed at the treatment of chronic kidney disease, pulmonary hypertension and mitochondrial myopathy. Recent studies have shown that Nrf2 activation protects the retina from retinal diseases. In particular, this finding is supported by the finding that Nrf2 knockout mice display age-related retinal degeneration.

Nrf2 contains seven highly conserved structural domains, Neh1-Neh6 (Nrf2-ECH homology). The Neh1 region contains a bZIP structure. When Nrf2 translocates to the nucleus, bZIP forms a heterodimer with small Maf proteins in the nucleus, enabling Nrf2 to recognize and bind to ARE, and then initiate the transcription of downstream related genes. The Neh2 domain is combined with Keap1 protein (Kelch-like ECH-associated protein-1) and then degraded by ubiquitination in the cytoplasm. Neh3 is located at the C-terminus and can combine with CHD6 (a chromo-ATPase/helicase DNA binding protein) to promote the regulation of antioxidant response element (ARE) on the transcription of related genes. Neh4 and Neh5 are structural domains involved in the initiation of downstream gene transcription. When Nrf2 translocates into the nucleus, it binds to ARE in the form of Nrf2-Maf, but it cannot start the transcription process at this time. It needs the combination of Neh4, Neh5 and CREB. Activate the transcription process. The Neh6 region is rich in serine and is a regulatory region for KEAP1-independent Nrf2 degradation. Neh7 binds to the RXRa nuclear receptor factor to inhibit the transcriptional activity of Nrf2. We deleted the domains that inhibit Nrf2 activity and responsible for Nrf2 degradation, retained its functional domains that activate downstream genes, and constructed them on double-stranded AAV vectors. The scAAV-Nrf2 mutant (miniNrf2) is more efficient than single-stranded infection, and it can activate the transcription and expression of downstream antioxidant and anti-inflammatory genes more strongly, and can be used to treat dry-AMD and other chronic ophthalmic diseases related to antioxidant and anti-inflammation disease.

Adeno-associated virus (AAV) belongs to the family Parvoviridae and is a type of single-stranded DNA-defective virus. Its genomic DNA is less than 5kb, without envelope, and its shape is a naked icosahedral particle. Adeno-associated virus can infect humans and some other primates. AAV vector is a kind of artificially transgenic vector produced by genetic engineering using certain characteristics of naturally occurring adeno-associated virus. It has no potential pathogenicity and is suitable for skeletal muscle, retina, liver cells, cardiac smooth muscle cells, and neurons. Cells, pancreatic islet B cells, joint synoviocytes, etc. have good infection effects, and the infection efficiency in vivo is extremely high. There are at least a dozen serotypes of AAV that have been discovered so far, and they mainly differ in the capsid protein Cap, thus leading to different infection efficiencies of various serotypes of AAV to different tissues and cells.

Recombinant adeno-associated virus (rAAV), as a widely used tool for gene transduction and gene therapy, can transduce foreign genes into dividing cells and terminally differentiated cells, and maintain relatively long-term expression of genes. Recombinant adeno-associated virus (rAAV) is favored by people because of its safety, low immunogenicity and non-pathogenicity, and has been widely used as an ideal carrier for in vivo research and gene therapy. Adeno-associated virus is a single-stranded DNA virus (ssAAV), which must first complete the conversion of the single-stranded genome to a transcriptionally active double-stranded form before expression can begin, a process that limits AAV vector-mediated gene transduction and directly affect the efficiency of virus infection.

Contents of the invention

In view of this, the present invention provides a nucleotide sequence encoding human nuclear factor E2-related factor 2 (Nrf2) protein capable of efficiently treating ophthalmic diseases.

In order to achieve the above-mentioned purpose of the invention, the present invention provides the following technical solutions:

The invention provides nucleic acid molecules having:

(I), a nucleotide sequence as shown in any of SEQ ID No.1, 2 or 3; or

(II), as the complementary nucleotide sequence of the nucleotide sequence shown in any of SEQ ID No.1, 2 or 3; or

(III), a nucleotide sequence having at least 95% sequence identity with the nucleotide sequence described in (I) or (II).

In some specific embodiments of the present invention, the nucleic acid molecule has a nucleotide sequence having at least 98% sequence identity with the nucleotide sequence described in (I) or (II).

The present invention also provides the polypeptide or protein encoded by the nucleic acid molecule.

The present invention also provides expression vectors, including said nucleic acid molecules.

In some specific embodiments of the present invention, the expression vector is a viral vector, and the viral vector includes a double-stranded adeno-associated viral vector.

In some specific embodiments of the present invention, the serotype of the viral vector is selected from AAV2, AAV5, AAV7 or AAV8 or a combination thereof.

In some specific embodiments of the present invention, the double-stranded adeno-associated viral vector further includes a promoter.

In some specific embodiments of the invention, said promoter comprises one or more of SYN, CMV or CAG.

Based on the above studies, the present invention also provides the application of the nucleic acid molecule, the polypeptide or protein, and the expression vector in the preparation of drugs for preventing and/or treating diseases related to optic nerve damage.

In some embodiments of the present invention, the diseases related to optic nerve damage include but are not limited to autosomal dominant optic atrophy (DOA), Leber's hereditary optic neuropathy (LHON), ischemic optic neuropathy or glaucoma.

In some specific embodiments of the present invention, the optic nerve injury-related disease is acute optic nerve injury or chronic optic nerve injury.

In some specific embodiments of the present invention, the activities of the nucleic acid molecule, the polypeptide or protein, and the expression vector include but are not limited to any of the following:

(1), causing stable high expression of Nrf2 gene in retinal ganglion cells; and/or

(2), long-term treatment of diseases related to optic nerve damage; and/or

(3), activation of cellular anti-inflammatory or antioxidant responses; and/or

(4), increasing the survival rate of ganglion cells; and/or

(5), preventing or delaying the apoptosis of ganglion cells; and/or

(6) Remove free radicals from diseased cells.

More importantly, the present invention also provides a drug, including the nucleic acid molecule, the polypeptide or protein, the expression vector, and pharmaceutically acceptable auxiliary materials.

The present invention also provides a pharmaceutical combination, including the nucleic acid molecule, the polypeptide or protein, the expression vector, and any other active ingredients. Those skilled in the art combine the medicines provided by the invention with any other active ingredients on the basis of the medicines provided by the invention, all of which fall within the protection scope of the invention.

The present invention also provides a pharmaceutical preparation, including the nucleic acid molecule, the polypeptide or protein, the expression vector, and a pharmaceutically acceptable carrier or excipient.

In some embodiments of the invention, the pharmaceutical formulation comprises a liquid formulation.

In addition, the present invention also provides a drug delivery method, wherein the drug, the drug combination, and the drug preparation are administered to the eye, and the administration method is vitreous cavity administration.

The present invention also provides a treatment method for optic nerve-related diseases, using the drug, the drug combination, and the drug preparation.

In some embodiments of the present invention, the diseases related to optic nerve damage include but are not limited to autosomal dominant optic atrophy (DOA), Leber's hereditary optic neuropathy (LHON), ischemic optic neuropathy or glaucoma.

In some specific embodiments of the present invention, the diseases related to optic nerve damage include acute optic nerve damage or chronic optic nerve damage.

In some specific embodiments of the present invention, the activity of the drug, the drug combination, and the drug preparation includes but is not limited to any of the following:

(1), causing stable high expression of Nrf2 gene in retinal ganglion cells; and/or

(2), long-term treatment of diseases related to optic nerve damage; and/or

(3), activation of cellular anti-inflammatory or antioxidant responses; and/or

(4), increasing the survival rate of ganglion cells; and/or

(5), preventing or delaying the apoptosis of ganglion cells; and/or

(6) Remove free radicals from diseased cells.

Adeno-associated virus is a single-stranded DNA virus (ssAAV), which must first complete the conversion of the single-stranded genome to a transcriptionally active double-stranded form before expression can begin, a process that limits AAV vector-mediated gene transduction and directly affect the efficiency of virus infection. Adeno-associated virus (scAAV) with self-complementary double-stranded DNA can overcome this limitation and does not need to go through a process of changing from single-stranded to double-stranded. That is to say, after double-stranded AAV virus enters cells, it can be directly expressed and expressed Time is faster and the level of expression is higher.

The invention discloses an expression vector of human nuclear factor E2-related factor 2 protein and its application. Specifically, the invention discloses a nucleotide sequence encoding human nuclear factor E2-related factor 2 (Nrf2) protein, which comprises The nucleic acid encoding human nuclear factor E2-related factor 2 (Nrf2) protein. Further disclosed is a recombinant expression vector comprising the above nucleic acid. The nucleic acid encoding human nuclear factor E2-related factor 2 (Nrf2) protein described in the present invention has faster expression time and higher expression level, so it can better treat optic nerve-related diseases.

Description of drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings that are required in the description of the embodiments or the prior art.

Figure 1 shows the plasmid electrophoresis diagram, in which numbers 1-5 represent pscAAV-mini Nrf2-1, numbers 6-10 represent pscAAV-mini Nrf2-2, and numbers 11-15 represent pscAAV-mini Nrf2-3;

Figure 2 shows the plasmid digestion map, and the electrophoresis shows that lanes 1, 2, and 3 are pscAAV-mini Nrf2-1 after enzyme digestion, and lanes 4, 5, and 6 are pscAAV-mini Nrf2-2, 7, and 8 after enzyme digestion , 9 is pscAAV-mini Nrf2-3 after enzyme digestion; from top to bottom, respectively, the uncut circular plasmid, the linear plasmid after enzyme digestion, and the target band after cutting;

Fig. 3 (a) the characteristic map of psAAV-CMV-miniNrf2-1 plasmid; Fig. 3 (b) shows the characteristic map of psAAV-CMV-miniNrf2-2 plasmid; Fig. 3 (c) shows the characteristic map of psAAV-CMV-miniNrf2-3 plasmid feature map;

Plasmid map and start and stop sites of each element: including promoter, gene sequence, virus packaging ITR sequence;

Figure 4 shows the detection of protein expression after constructing vector transfection HEK293 cells, lanes 1-5 are blank cells, pscAAV-GFP, pscAAV-mini Nrf2-1, pscAAV-mini Nrf2-2, pscAAV-mini Nrf2-3 plasmid transfection Protein detection after transfection of HEK293 cells (40X);

Figure 5 shows a map of subcellular localization;

Figure 6a shows that the expression level of the downstream gene HO-1 is regulated after plasmid transfection, and Figure 6b shows that the expression of the downstream gene NQO1 is regulated after plasmid transfection;

Figure 7 shows the expression of mini-Nrf21-3 in mouse retinal RGC cells; wherein, Figure 7a shows the expression of mini-Nrf2-1 in mouse retinal RGC cells; Figure 7b shows the expression of mini-Nrf2-2 in mouse retina Expression in RGC cells; Figure 7c shows the expression of mini-Nrf2-3 in mouse retinal RGC cells;

Figure 8(a) shows that the transcription of the downstream target gene NQO1 in mice is activated after virus infection; Figure 8(b) shows that the transcription of the downstream target gene HO-1 in mice is activated after virus infection.

Detailed ways

The invention discloses a nucleic acid molecule encoding human nuclear factor E2-related factor 2, an expression vector and its application. Those skilled in the art can learn from the content of this article and appropriately improve the process parameters to realize it. In particular, it should be pointed out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and the relevant personnel can obviously make changes or appropriate changes and combinations to the method and application described herein without departing from the content, spirit and scope of the present invention to realize and Apply the technology of the present invention.

Aiming at the deficiencies of the prior art, the first aspect of the present invention provides a nucleotide sequence encoding human nuclear factor E2-related factor 2 (Nrf2) protein capable of efficiently treating ophthalmic diseases.

Preferably, the nucleotide sequence encoding Nrf2 protein is one of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.

More preferably, the sequence is selected from sequences having ≥95% homology to SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.

More preferably, the sequence is selected from sequences having ≥98% homology to SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.

More preferably, the sequence is selected from sequences having ≥99% homology to SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.

In the present invention:

Sequence 1.mini Nrf2-1 (as shown in SEQ ID No.1)

GGATCCGCCACCATGGATGCTTTGTACTTTGATGACTGCATGCAGCTTTTGGCGCAGACATTCCCGTTTGTAGATGACAATGAGGTTTCTTCGGCTACGTTTCAGTCACTTGTTCCTGATATTCCCGGTCACATCGAGAGCCCAGTCTTCATTGCTACTAATCAGGCTCAGTCACCTGAAACTTCTGTTGCTCAGGTAGCCCCTGTTGTTTAGACGGTATG CAACAGGACATTGAGCAAGTTTGGGAGGAGCTATTATCCATTCCTGAGTTACAGTGTCTTAATATTGAAAATGACAAGCTGGTTGAGACTACCATGGTTCCAAGTCCAGAAGCCAAACTGACAGAAGTTGACAATTATCATTTACTCATCTATACCCTCAATGGAAAAAGAAGTAGGTAACTGTAGTCACATTTTCTAATGCTTTTGAGGATTCCTTCAGCAGCATCC TCTCCACAGAAGACCCCAACCAGTTGACAGTGAACTCATTAAATTCAGATGCCACAGTCAACACAGATTTTGGTGATGAATTTTATTCTGCTTTCATAGCTGAGCCCAGTATCAGCAACAGCATGCCCTCACCTGCTACTTTAAGCCATTCACTCTCTGAACTTCTAAATGGGCCCATTGATGTCTATCACTTTGCAAAGCTTTCAACCAAAACCACCCTGAAAGCAC AGCAGAATTCAATGATTCTGACTCCGGCATTTCACTAAACACAAGTCCCAGTGTGGCATCACCAGAACACTCAGTGGAATCTTCCAGCTATGGAGACACACTACTTGGCCTCAGTGATTCTGAAGTGGAAGAGCTAGATAGTGCCCCTGGAAGTGTCAAAACAGAATGGTCCTAAAACACCAGTACATTCTTCTGGGGATATGGTACAACCCTTGTCACCATCTCAG GGGCAGAGCACTCACGTGCATGATGCCCAATGTGAGAACACACCAGAGAAAGAATTGCCTGTAAGTCCTGGTCATCGGAAAACCCATTCACAAAAAGACAAACATTCAAGCCGCTTGGAGGCTCATCTCACAAGAGATGAACTTAGGCAAAAGCTCTCCATATCCCATTCCCCTGTAGAAAAATCATTAACCTCCCTGTTGTTGACTTCAACGAAATGATGTCCAA AGAGCAGTTCAATGAAGCTCAACTTGCATTAATTCGGGATATACGTAGGAGGGGTAAGAATAAAGTGGCTGCTCAGAATTGCAGAAAAAAAAAACTGGAAAATATAGTAGAACTAGAGCAAGATTTAGATCATTTGAAAGATGAAAAAGAAAAATTGCTCAAAGAAAAAGGAGAAAATGAAAAAAGCCTCACCTACTGAAAAAACCAACTCAGCACCTTATATCTCGAAGTTTT CAGCATGCTACGTGATGAAGATGGAAAACCTTATCTCCTAGTGAATACTCCCTGCAGCAAACAAGAGATGGCAATGTTTTCCTTGTTCCCAAAAGTAAGAAGCCAGATGTTAAGAAAAACCTCGAG

Sequence 2.mini Nrf2-2 (as shown in SEQ ID No.2)

GGATCCGCCACCATGGCCCAGCACATCCAGTCAGAAACCAGTGGATCTGCCAACTACTCCCAGGTTGCCCACATTCCCAAATCAGATGCTTTGTACTTTGATGACTGCATGCAGCTTTTGGCGCAGACATTCCCGTTTGTAGATGACAATGAGGTTTCTTCGGCTACGTTTCAGTCACTTGTTCCTGATATCCCGGTCACATCGAGAGCCCAGTCTTCATTGC TACTAATCAGGCTCAGTCACCTGAAACTTCTGTTGCTCAGGTAGCCCCTGTTGATTTAGACGGTATGCAACAGGACATTGAGCAAGTTTGGGAGGAGCTATTATCCATTCCTGAGTTACAGTGTCTTAATATTGAAAATGACAAGCTGGTTGAGACTACCATGGTTCCAAGTCCAGAAGCCAAACTGACAGAAGTTGACAATTATCATTTTTACTCATCTATACCCTCA ATGGAAAAAGAAGTAGGTAACTGTAGTCCACATTTCTTAATGCTTTTGAGGATTCCTTCAGCAGCATCCTCTCCACAGAAGACCCCAACCAGTTGACAGTGAACTCATTAAATTCAGATGCCACAGTCAACACAGATTTTGGTGATGAATTTTATTCTGCTTTCATAGCTGAGCCCAGTATCAGCAACAGCATGCCCTCACCTGCTACTTTAAGCCATTCACTCTCTGAACTTCT AAATGGGCCCAAAACAGAATGGTCCTAAAACACCAGTACATTCTTCTGGGGATATGGTACAACCCTTGTCACCATCTCAGGGGCAGAGCACTCACGTGCATGATGCCCAATGTGAGAACACACCAGAGAAAGAATTGCCTGTAAGTCCTGGTCATCGGAAAACCCATTCCAAAAGACAAACATTCAAGCCGCTTGGAGGCTCATCTCACAAGAGATGAACTT AGGGCAAAAAGCTCTCCATATCCCATTCCCCTGTAGAAAAAAATCATTAACCTCCCTGTTGTTGACTTCAACGAAATGATGTCCAAAGAGCAGTTCAATGAAGCTCAACTTGCATTAATTCGGGATATACGTAGGAGGGGTAAGAATAAAGTGGCTGCTCAGAATTGCAGAAAAAAAAACTGGAAAATATAGTAGAACTAGCAAGAATTTAGATCATTTGAAAGATGA AAAAGAAAAATTGCTCAAAGAAAAAGGAGAAAATGACAAAAGCCTTCACCTACTGAAAAAACAACTCAGCACCTTATCTCGAAGTTTTCAGCATGCTACGTGATGAAGATGGAAAACCTTATTCTCCTAGTGAATACTCCCTGCAGCAAACAAGAGATGGCAATGTTTTCCTTGTTCCCAAAAGTAAAGCCAGATGTTAAAAAAACTCGAGSeq3.mini Nrf2-3 (as shown in SEQ ID No.3)

GGATCCGCCACCATGGCCCAGCACATCCAGTCAGAAACCAGTGGATCTGCCAACTACTCCCAGGTTGCCCACATTCCCAAATCAGATGCTTTGTACTTTGATGACTGCATGCAGCTTTTGGCGCAGACATTCCCGTTTGTAGATGACAATGAGGTTTCTTCGGCTACGTTTCAGTCACTTGTTCCTGATATCCCGGTCACATCGAGAGCCCAGTCTTCATTGC TACTAATCAGGCTCAGTCACCTGAAACTTCTGTTGCTCAGGTAGCCCCTGTTGATTTAGACGGTATGCAACAGGACATTGAGCAAGTTTGGGAGGAGCTATTATCCATTCCTGAGTTACAGTGTCTTAATATTGAAAATGACAAGCTGGTTAAACAGAATGGTCCTAAACACCAGTACATTCTTCTGGGGATATGGTACAACCCTTGTCACCATCTCAGGGG CAGAGCACTCACGTGCATGATGCCCAATGTGAGAACACACCAGAGAAAGAATTGCCTGTAAGTCCTGGTCATCGGAAAACCCATTCACAAAAAGACAAACATTCAAGCCGCTTGGAGGCTCATCTCACAAGAGATGAACTTAGGGCAAAAGCTCTCCATATCCCATTCCCCTGTAGAAAAAATTAACCTCCCTGTTGTTGACTTCAACGAAATGATGTCCAAAAGAGCA GTTCAATGAAGCTCAACTTGCATTAATTCGGGATATACGTAGGAGGGGTAAGAATAAAGTGGCTGCTCAGAATTGCAGAAAAAAAAAACTGGAAAATAGTAGAACTAGAGCAAGATTTAGATCATTTGAAAAGATGAAAAAGAAAAATTGCTCAAAGAAAAAGGAGAAAATGACAAAAGCCTCTCACCTACTGAAAAAACCAACTCAGCACCTTATCTCGAAGTTTTCAGCAT GCTACGTGATGAAGATGGAAAACCTTATCTCCTAGTGAATACTCCCTGCAGCAAACAAGAGATGGCAATGTTTTCCTTGTTCCAAAAAGTAAGAAGCCAGATGTTAAAGAAAAACCTCGAG

The second aspect of the present invention provides an adeno-associated virus vector encoding human nuclear factor E2-related factor 2 (Nrf2) protein.

Specifically, the present invention provides an adeno-associated virus vector capable of long-term treatment of optic nerve injury-related diseases, and the adeno-associated virus vector includes a sequence for expressing Nrf2 protein from a promoter.

Preferably, the promoter is one of CAG, SYN or CMV.

More preferably, the promoter is selected from CMV.

More preferably, the plasmid map of the vector is shown in FIG. 3 .

The third aspect of the present invention provides the use of any of the nucleotides, adeno-associated virus vectors, and plasmids described above in the treatment of acute or chronic optic nerve-related diseases; preferably, the diseases are DOA, LHON, ischemic optic neuropathy and glaucoma.

The fourth aspect of the present invention provides a pharmaceutical preparation, which contains the above-mentioned viral vector, and a pharmaceutically acceptable carrier or excipient; preferably, the pharmaceutical preparation is a liquid preparation.

The fifth aspect of the present invention provides the delivery method of the above-mentioned pharmaceutical preparation, wherein the above-mentioned pharmaceutical preparation is injected intraocularly. More preferably, the intraocular injection is intravitreal injection.

More preferably, the disease is an eye disease.

More preferably, the eye disease is a disease related to optic nerve damage, such as acute optic nerve damage or chronic optic nerve damage.

More preferably, the optic nerve damage-related disease is autosomal dominant optic atrophy (DOA), Leber's hereditary optic neuropathy (LHON), ischemic optic neuropathy or glaucoma.

The carrier or pharmaceutical preparation can cause stable and high expression of Nrf2 gene in retinal ganglion cells.

The carrier or pharmaceutical preparation can treat diseases related to optic nerve damage in a long-term manner.

The carrier or pharmaceutical preparation can activate cell anti-inflammation or anti-oxidation response.

The carrier or pharmaceutical preparation can increase the survival rate of ganglion cells.

The carrier or pharmaceutical preparation can prevent or delay the apoptosis of ganglion cells.

The carrier or pharmaceutical preparation can eliminate free radicals of diseased cells.

The present invention provides an expression vector of human nuclear factor E2-related factor 2 protein and its application. Specifically, the present invention discloses a nucleotide sequence encoding human nuclear factor E2-related factor 2 (Nrf2) protein, which comprises The nucleic acid encoding human nuclear factor E2-related factor 2 (Nrf2) protein. Further disclosed is a recombinant expression vector comprising the above nucleic acid. The nucleic acid encoding human nuclear factor E2-related factor 2 (Nrf2) protein described in the present invention has faster expression time and higher expression level, so it can better treat optic nerve-related diseases.

In the nucleic acid molecule encoding human nuclear factor E2-related factor 2, the expression vector and its application provided by the present invention, the raw materials and reagents used can be purchased from the market.

Below in conjunction with embodiment, further set forth the present invention:

Example 1. Vector Construction

Using the existing vector pAAV-nrf2 as a template, use primeSTAR GXL premix high-fidelity amplification enzyme to perform the first round of PCR to amplify the intermediate target fragment. After the PCR, agarose gel electrophoresis was performed on the PCR product, and the target fragment was recovered according to the size of the target product. Using the PCR product of the first round as a template, a second round of nested PCR was performed to amplify the target fragment. PCR products were subjected to agarose gel electrophoresis to recover the target fragment. Carry out an enzyme digestion reaction between the target product and the target carrier, and recover the target band after electrophoresis. Use T4 ligase to ligate the target fragment and the vector, and ligate overnight at 37°C. The ligation product was transformed into stbl3 competent cells, plated, cultured overnight, single colony was picked and shaken, and the plasmid was extracted. The extracted plasmids were identified by enzyme digestion, and the correct plasmids were selected and sent to the sequencing company for sequencing identification.

Table 1: PCR Primers

PCR amplification miniNrf2-1 reaction system

PCR on-board program:

PCR amplification miniNrf2-2 reaction system

PCR on-board program:

PCR amplification miniNrf2-3 reaction system

PCR on-board program:

Primers are listed in Table 1. The electrophoresis of the constructed vector is shown in Figure 1, the enzyme digestion identification of the constructed vector is shown in Figure 2, and the map of the vector is shown in Figure 3.

Figure 1 shows that pscAAV-mini Nrf2-1 is about 5kd, pscAAV-mini Nrf2-2 is about 5kd, and pscAAV-mini Nrf2-3 is about 4.7kd, which is consistent with the theoretical size. After digestion in Figure 2, the size is in line with expectations. Combined with the sequencing results, it was confirmed that the vector was constructed correctly.

Example 2. Expression and functional detection of vectors in in vitro experiments

2.1 Vector expression

The expression of the vector was detected by Western blot. The 6-well plates were divided into 8 groups, and 5×10 ⁵ cells were inoculated in each well. After 24 hours of inoculation, the cell count was around 1×10 ⁶ , and 3ug of plasmids were transfected. After culturing for 48 hours, HEK293 cells in different experimental groups were collected, removed the culture medium and washed twice with PBS. Add 150 microliters of lysate to each well, and place on ice to fully lyse. Collect the cell lysate, centrifuge at 10,000-14,000 g at 4°C for 3-5 minutes, and take the supernatant. After the protein concentration was determined by the BCA method, the loading amount of the experimental group and the control group was calculated based on 50 μg of total protein, and SDS-PAGE gel electrophoresis and Western blot were performed. After incubation with primary antibody (anti-flag 1:2000) and secondary antibody (anti-rabbit HRP 1:10000), chemiluminescence imaging was taken.

The results are shown in Figure 4. The protein sizes of pscAAV-mini Nrf2-1, pscAAV-mini Nrf2-2, and pscAAV-mini Nrf2-3 were in line with the theoretical values, and all of them were significantly expressed. It shows that the three vectors were constructed successfully and can express the target protein in large quantities.

2.2 Cell localization

The nuclear localization of the vector was detected by immunofluorescence staining. Digest HEK293 cells, plate 6-well plates according to the requirement of 4*10^5 cells/well. After culturing overnight, PEI was transfected with 3ug plasmid/well, which were blank group, pAAV-GFP group, pAAV-dN2 group, pAAV-dN2N6 group, and pAAV-dN2N6N7 group. Cells were harvested 30 hours after transfection. The medium was discarded, washed twice with PBS, 1ml of 4% PFA fixative was added to each well, and fixed at room temperature for 10min. Discard the fixative, leave a small portion of PBS in the six-well plate, and store it temporarily at 4°C. The cell slides were moved to a 24-well plate, washed with PBS, punched with 1% Triton for 15 minutes, and blocked for 2 hours at room temperature. Primary antibody (anti-flag 1:200), overnight at 4°C. The secondary antibody was incubated at room temperature for 1 hour. After adding DAPI staining solution and mounting the slides, observe under a fluorescence microscope. The result is shown in Figure 5,

The blank group and the pAAV-CAG-GFP group did not express the target protein, but the cells in the pscAAV-mini Nrf2-1, pscAAV-mini Nrf2-2, and pscAAV-mini Nrf2-3 groups could specifically express the target protein, and the target protein was obviously aggregated In the nucleus, it indicates that the vector normally expresses the protein, and the target protein is localized in the nucleus.

2.3 qPCR detection of the function of each Nrf2 mutant

HEK293 cells (1.5ug, 3ug) were transfected with pAAV-Nrf2 mutant plasmid, and RNA was collected from the cells 31 hours after transfection, and the mRNA levels of NQO-1 and HO-1, the downstream target genes of Nrf2, were detected by qPCR. The results are shown in Figure 6a and Figure 6b.

In Figure 6a, HO-1 is the downstream target gene of the Nrf2 gene. After transfection of cells with pscAAV-mini Nrf2-1, pscAAV-miniNrf2-2, and pscAAV-mini Nrf2-3, HO-1 was expressed to varying degrees. Among them, the downstream activation effect of miniNrf2-2 carrier is the strongest. The NQO1 gene downstream of nrf2 in Figure 6b is also expressed to varying degrees.

Based on the above experimental results, mini Nrf2-1, 2, and 3 can well express the target protein and enter the nucleus to regulate the expression of downstream target genes, and the function is stronger than that of the full-length Nrf2 vector.

Example 3. Virus preparation

The three-plasmid system (pAAV2-, pHelper, pRC) was transfected at 85% confluency of HEK293 cells (PEI transfection reagent was used for transfection). A total of 30 micrograms of plasmids were transfected into a 10 cm culture dish (the ratio of the three plasmids was 10:10:10). The medium was not changed on the day of transfection, and the medium was changed to DMEM with 5% FBS the next day, and the cells were harvested within 72 hours. Put it in a freezing tube, freeze and thaw at -70°C/37°C three times, each time for at least 10 minutes, and vortex vigorously for 2 minutes after each thaw. Then centrifuge at 14,000 rpm for 5 minutes, and take the supernatant. The virus was purified by cesium chloride solution gradient centrifugation.

Fluorescent quantitative PCR method was used to detect the physical titer of the virus. Using SYBRⅡ (takara) and target fragment primers (20uM), the packaged virus was accurately quantified with the target plasmid, and the virus to be tested was quantified by fluorescence. The PCR reaction conditions in the PCR eight-tube (Bio-rad) were pre-denaturation: 95°C 10 minutes; cycle: 95°C for 15 seconds, 60°C for 1 minute. It was finally determined that the genome titer of the viral vector described in this application was 1×10 ¹³ vg/mL.

Example 4 In vivo retinal expression and functional detection of scAAV-mini Nrf1-1, -2, -3

1. Expression detection in mouse retina

The mice were administered 1E10 vg/eye through the vitreous cavity of the eyeball. After 4 weeks of administration, the mice were sacrificed by breaking the spine, and the eyeballs were fixed with 4% PFA. After OCT embedding, frozen sections were taken. 1% TritonX-100 was punched for 15 minutes, blocked for 30 minutes, antigen retrieval, primary antibody (anti-flag 1:200) overnight at 4°C, secondary antibody (anti-rabbit Alexa594 1:1000) at room temperature for 2 hours. Observe the expression distribution of mini Nrf protein in the retina. Figure 7a-c results show that mini Nrf21-3 protein is mainly expressed in retinal RGC cells.

2. Activation of downstream target genes in mice

The mice were administered 1E10vg/eye through the vitreous cavity of the eyeball. After 4 weeks of administration, the mice were sacrificed by breaking the spine, and the eyeballs were taken, and the retina was peeled off. Total RNA was extracted by TRIZOL method, cDNA was reverse transcribed, and the expression of NQO1 and HO-1 genes downstream of NRF2 transcription factors were detected by qPCR.

The results are shown in Figure 8, mini Nrf2-1, -2, -3 can activate the expression of Nrf2 downstream target genes NQO1 (as shown in 8a) and HO-1 (as shown in 8b) in mice, proving that these vectors It is also functional in the body.

The above is only a preferred embodiment of the present invention, it should be pointed out that, for those of ordinary skill in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications can also be made. It should be regarded as the protection scope of the present invention.

sequence listing

<110> Wuhan Neophth Biotechnology Co., Ltd.

<120> Nucleic acid molecule encoding human nuclear factor E2-related factor 2, expression vector and application thereof

<130> MP21016163

<160> 13

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1503

<212>DNA

<213> Artificial Sequence

<400> 1

ggatccgcca ccatggatgc tttgtacttt gatgactgca tgcagctttt ggcgcagaca 60

ttcccgtttg tagatgacaa tgaggtttct tcggctacgt ttcagtcact tgttcctgat 120

attcccggtc acatcgagag cccagtcttc attgctacta atcaggctca gtcacctgaa 180

acttctgttg ctcaggtagc ccctgttgat ttagacggta tgcaacagga cattgagcaa 240

gtttgggagg agctattatc cattcctgag ttacagtgtc ttaatattga aaatgacaag 300

ctggttgaga ctaccatggt tccaagtcca gaagccaaac tgacagaagt tgacaattat 360

catttttact catctatacc ctcaatggaa aaagaagtag gtaactgtag tccacatttt 420

cttaatgctt ttgaggattc cttcagcagc atcctctcca cagaagaccc caaccagttg 480

acagtgaact cattaaattc agatgccaca gtcaacacag attttggtga tgaattttat 540

tctgctttca tagctgagcc cagtatcagc aacagcatgc cctcacctgc tactttaagc 600

cattcactct ctgaacttct aaatgggccc attgatgttt ctgatctatc actttgcaaa 660

gctttcaacc aaaaccaccc tgaaagcaca gcagaattca atgattctga ctccggcatt 720

tcactaaaca caagtcccag tgtggcatca ccagaacact cagtggaatc ttccagctat 780

ggagacacac tacttggcct cagtgattct gaagtggaag agctagatag tgcccctgga 840

agtgtcaaac agaatggtcc taaaacacca gtacattctt ctggggatat ggtacaaccc 900

ttgtcaccat ctcaggggca gagcactcac gtgcatgatg cccaatgtga gaacacacca 960

gagaaagaat tgcctgtaag tcctggtcat cggaaaaccc cattcacaaa agacaaacat 1020

tcaagccgct tggaggctca tctcacaaga gatgaactta gggcaaaagc tctccatatc 1080

ccattccctg tagaaaaaat cattaacctc cctgttgttg acttcaacga aatgatgtcc 1140

aaagagcagt tcaatgaagc tcaacttgca ttaattcggg atatacgtag gaggggtaag 1200

aataaagtgg ctgctcagaa ttgcagaaaa agaaaactgg aaaatatagt agaactagag 1260

caagatttag atcatttgaa agatgaaaaa gaaaaattgc tcaaagaaaa aggagaaaat 1320

gacaaaagcc ttcacctact gaaaaaacaa ctcagcacct tatatctcga agttttcagc 1380

atgctacgtg atgaagatgg aaaaccttat tctcctagtg aatactccct gcagcaaaca 1440

agagatggca atgttttcct tgttcccaaa agtaagaagc cagatgttaa gaaaaacctc 1500

gag 1503

<210> 2

<211> 1356

<212>DNA

<213> Artificial Sequence

<400> 2

ggatccgcca ccatggccca gcacatccag tcagaaacca gtggatctgc caactactcc 60

caggttgccc aattcccaa atcagatgct ttgtactttg atgactgcat gcagcttttg 120

gcgcagacat tcccgtttgt agatgacaat gaggtttctt cggctacgtt tcagtcactt 180

gttcctgata ttcccggtca catcgagagc ccagtcttca ttgctactaa tcaggctcag 240

tcacctgaaa cttctgttgc tcaggtagcc cctgttgatt tagacggtat gcaacaggac 300

attgagcaag tttgggagga gctattatcc attcctgagt tacagtgtct taatattgaa 360

aatgacaagc tggttgagac taccatggtt ccaagtccag aagccaaact gacagaagtt 420

gacaattatc atttttactc atctataccc tcaatggaaa aagaagtagg taactgtagt 480

ccacattttc ttaatgcttt tgaggattcc ttcagcagca tcctctccac agaagacccc 540

aaccagttga cagtgaactc attaaattca gatgccacag tcaacacaga ttttggtgat 600

gaattttatt ctgctttcat agctgagccc agtatcagca acagcatgcc ctcacctgct 660

actttaagcc attcactctc tgaacttcta aatggggccca aacagaatgg tcctaaaaca 720

ccagtacatt cttctgggga tatggtacaa cccttgtcac catctcaggg gcagagcact 780

cacgtgcatg atgcccaatg tgagaacaca ccagagaaag aattgcctgt aagtcctggt 840

catcggaaaa ccccattcac aaaagacaaa cattcaagcc gcttggaggc tcatctcaca 900

agagatgaac ttagggcaaa agctctccat atcccattcc ctgtagaaaa aatcattaac 960

ctccctgttg ttgacttcaa cgaaatgatg tccaaagagc agttcaatga agctcaactt 1020

gcattaattc gggatatacg tagggaggggt aagaataaag tggctgctca gaattgcaga 1080

aaaagaaaac tggaaaatat agtagaacta gagcaagatt tagatcattt gaaagatgaa 1140

aaagaaaaat tgctcaaaga aaaaggagaa aatgacaaaa gccttcacct actgaaaaaa 1200

caactcagca ccttatct cgaagttttc agcatgctac gtgatgaaga tggaaaacct 1260

tattctccta gtgaatactc cctgcagcaa acaagagatg gcaatgtttt ccttgttccc 1320

aaaagtaaga agccagatgt taagaaaaac ctcgag 1356

<210> 3

<211> 1032

<212>DNA

<213> Artificial Sequence

<400> 3

ggatccgcca ccatggccca gcacatccag tcagaaacca gtggatctgc caactactcc 60

caggttgccc aattcccaa atcagatgct ttgtactttg atgactgcat gcagcttttg 120

gcgcagacat tcccgtttgt agatgacaat gaggtttctt cggctacgtt tcagtcactt 180

gttcctgata ttcccggtca catcgagagc ccagtcttca ttgctactaa tcaggctcag 240

tcacctgaaa cttctgttgc tcaggtagcc cctgttgatt tagacggtat gcaacaggac 300

attgagcaag tttgggagga gctattatcc attcctgagt tacagtgtct taatattgaa 360

aatgacaagc tggttaaaca gaatggtcct aaaacaccag tacattcttc tggggatatg 420

gtacaaccct tgtcaccatc tcaggggcag agcactcacg tgcatgatgc ccaatgtgag 480

aacacaccag agaaagaatt gcctgtaagt cctggtcatc ggaaaaccccc attcacaaaa 540

gacaaacatt caagccgctt ggaggctcat ctcacaagag atgaacttag ggcaaaagct 600

ctccatatcc cattccctgt agaaaaaatc attaacctcc ctgttgttga cttcaacgaa 660

atgatgtcca aagagcagtt caatgaagct caacttgcat taattcggga tatacgtagg 720

aggggtaaga ataaagtggc tgctcagaat tgcagaaaaa gaaaactgga aaatatagta 780

gaactagagc aagattaga tcatttgaaa gatgaaaaag aaaaattgct caaagaaaaa 840

ggagaaaatg acaaaagcct tcacctactg aaaaaacaac tcagcacctt atatctcgaa 900

gttttcagca tgctacgtga tgaagatgga aaaccttatt ctcctagtga atactccctg 960

cagcaaacaa gagatggcaa tgttttcctt gttcccaaaa gtaagaagcc agatgttaag 1020

aaaaacctcg ag 1032

<210> 4

<211> 46

<212>DNA

<213> Artificial Sequence

<400> 4

agctggatcc gccaccatgg atgctttgta ctttgatgac tgcatg 46

<210> 5

<211> 42

<212>DNA

<213> Artificial Sequence

<400> 5

atcgctcgag gtttttctta acatctggct tcttactttt gg 42

<210> 6

<211> 38

<212>DNA

<213> Artificial Sequence

<400> 6

agctggatcc gccaccatgg cccagcacat ccagtcag 38

<210> 7

<211> 49

<212>DNA

<213> Artificial Sequence

<400> 7

ctggtgtttt aggacattc tgtttgggcc catttagaag ttcagagag 49

<210> 8

<211> 49

<212>DNA

<213> Artificial Sequence

<400> 8

ctctctgaac ttctaaatgg gcccaaacag aatggtccta aaacaccag 49

<210> 9

<211> 54

<212>DNA

<213> Artificial Sequence

<400> 9

ctggtgtttt aggacattc tgtttaacca gcttgtcatt ttcaatatta agac 54

<210> 10

<211> 54

<212>DNA

<213> Artificial Sequence

<400> 10

gtcttaatat tgaaaatgac aagctggtta aacagaatgg tcctaaaaca ccag 54

<210> 11

<211> 501

<212> PRT

<213> Artificial Sequence

<400> 11

Gly Ser Ala Thr Met Asp Ala Leu Tyr Phe Asp Asp Cys Met Gln Leu

1 5 10 15

Leu Ala Gln Thr Phe Pro Phe Val Asp Asp Asn Glu Val Ser Ser Ala

20 25 30

Thr Phe Gln Ser Leu Val Pro Asp Ile Pro Gly His Ile Glu Ser Pro

35 40 45

Val Phe Ile Ala Thr Asn Gln Ala Gln Ser Pro Glu Thr Ser Val Ala

50 55 60

Gln Val Ala Pro Val Asp Leu Asp Gly Met Gln Gln Asp Ile Glu Gln

65 70 75 80

Val Trp Glu Glu Leu Leu Ser Ile Pro Glu Leu Gln Cys Leu Asn Ile

85 90 95

Glu Asn Asp Lys Leu Val Glu Thr Thr Met Val Pro Ser Pro Glu Ala

100 105 110

Lys Leu Thr Glu Val Asp Asn Tyr His Phe Tyr Ser Ser Ile Pro Ser

115 120 125

Met Glu Lys Glu Val Gly Asn Cys Ser Pro His Phe Leu Asn Ala Phe

130 135 140

Glu Asp Ser Phe Ser Ser Ile Leu Ser Thr Glu Asp Pro Asn Gln Leu

145 150 155 160

Thr Val Asn Ser Leu Asn Ser Asp Ala Thr Val Asn Thr Asp Phe Gly

165 170 175

Asp Glu Phe Tyr Ser Ala Phe Ile Ala Glu Pro Ser Ile Ser Asn Ser

180 185 190

Met Pro Ser Pro Ala Thr Leu Ser His Ser Leu Ser Glu Leu Leu Asn

195 200 205

Gly Pro Ile Asp Val Ser Asp Leu Ser Leu Cys Lys Ala Phe Asn Gln

210 215 220

Asn His Pro Glu Ser Thr Ala Glu Phe Asn Asp Ser Asp Ser Gly Ile

225 230 235 240

Ser Leu Asn Thr Ser Pro Ser Val Ala Ser Pro Glu His Ser Val Glu

245 250 255

Ser Ser Ser Tyr Gly Asp Thr Leu Leu Gly Leu Ser Asp Ser Glu Val

260 265 270

Glu Glu Leu Asp Ser Ala Pro Gly Ser Val Lys Gln Asn Gly Pro Lys

275 280 285

Thr Pro Val His Ser Ser Gly Asp Met Val Gln Pro Leu Ser Pro Ser

290 295 300

Gln Gly Gln Ser Thr His Val His Asp Ala Gln Cys Glu Asn Thr Pro

305 310 315 320

Glu Lys Glu Leu Pro Val Ser Pro Gly His Arg Lys Thr Pro Phe Thr

325 330 335

Lys Asp Lys His Ser Ser Arg Leu Glu Ala His Leu Thr Arg Asp Glu

340 345 350

Leu Arg Ala Lys Ala Leu His Ile Pro Phe Pro Val Glu Lys Ile Ile

355 360 365

Asn Leu Pro Val Val Asp Phe Asn Glu Met Met Ser Lys Glu Gln Phe

370 375 380

Asn Glu Ala Gln Leu Ala Leu Ile Arg Asp Ile Arg Arg Arg Gly Lys

385 390 395 400

Asn Lys Val Ala Ala Gln Asn Cys Arg Lys Arg Lys Leu Glu Asn Ile

405 410 415

Val Glu Leu Glu Gln Asp Leu Asp His Leu Lys Asp Glu Lys Glu Lys

420 425 430

Leu Leu Lys Glu Lys Gly Glu Asn Asp Lys Ser Leu His Leu Leu Lys

435 440 445

Lys Gln Leu Ser Thr Leu Tyr Leu Glu Val Phe Ser Met Leu Arg Asp

450 455 460

Glu Asp Gly Lys Pro Tyr Ser Pro Ser Glu Tyr Ser Leu Gln Gln Thr

465 470 475 480

Arg Asp Gly Asn Val Phe Leu Val Pro Lys Ser Lys Lys Pro Asp Val

485 490 495

Lys Lys Asn Leu Glu

500

<210> 12

<211> 452

<212> PRT

<213> Artificial Sequence

<400> 12

Gly Ser Ala Thr Met Ala Gln His Ile Gln Ser Glu Thr Ser Gly Ser

1 5 10 15

Ala Asn Tyr Ser Gln Val Ala His Ile Pro Lys Ser Asp Ala Leu Tyr

20 25 30

Phe Asp Asp Cys Met Gln Leu Leu Ala Gln Thr Phe Pro Phe Val Asp

35 40 45

Asp Asn Glu Val Ser Ser Ala Thr Phe Gln Ser Leu Val Pro Asp Ile

50 55 60

Pro Gly His Ile Glu Ser Pro Val Phe Ile Ala Thr Asn Gln Ala Gln

65 70 75 80

Ser Pro Glu Thr Ser Val Ala Gln Val Ala Pro Val Asp Leu Asp Gly

85 90 95

Met Gln Gln Asp Ile Glu Gln Val Trp Glu Glu Leu Leu Ser Ile Pro

100 105 110

Glu Leu Gln Cys Leu Asn Ile Glu Asn Asp Lys Leu Val Glu Thr Thr

115 120 125

Met Val Pro Ser Pro Glu Ala Lys Leu Thr Glu Val Asp Asn Tyr His

130 135 140

Phe Tyr Ser Ser Ile Pro Ser Met Glu Lys Glu Val Gly Asn Cys Ser

145 150 155 160

Pro His Phe Leu Asn Ala Phe Glu Asp Ser Phe Ser Ser Ile Leu Ser

165 170 175

Thr Glu Asp Pro Asn Gln Leu Thr Val Asn Ser Leu Asn Ser Asp Ala

180 185 190

Thr Val Asn Thr Asp Phe Gly Asp Glu Phe Tyr Ser Ala Phe Ile Ala

195 200 205

Glu Pro Ser Ile Ser Asn Ser Met Pro Ser Pro Ala Thr Leu Ser His

210 215 220

Ser Leu Ser Glu Leu Leu Asn Gly Pro Lys Gln Asn Gly Pro Lys Thr

225 230 235 240

Pro Val His Ser Ser Gly Asp Met Val Gln Pro Leu Ser Pro Ser Gln

245 250 255

Gly Gln Ser Thr His Val His Asp Ala Gln Cys Glu Asn Thr Pro Glu

260 265 270

Lys Glu Leu Pro Val Ser Pro Gly His Arg Lys Thr Pro Phe Thr Lys

275 280 285

Asp Lys His Ser Ser Arg Leu Glu Ala His Leu Thr Arg Asp Glu Leu

290 295 300

Arg Ala Lys Ala Leu His Ile Pro Phe Pro Val Glu Lys Ile Ile Asn

305 310 315 320

Leu Pro Val Val Asp Phe Asn Glu Met Met Ser Lys Glu Gln Phe Asn

325 330 335

Glu Ala Gln Leu Ala Leu Ile Arg Asp Ile Arg Arg Arg Gly Lys Asn

340 345 350

Lys Val Ala Ala Gln Asn Cys Arg Lys Arg Lys Leu Glu Asn Ile Val

355 360 365

Glu Leu Glu Gln Asp Leu Asp His Leu Lys Asp Glu Lys Glu Lys Leu

370 375 380

Leu Lys Glu Lys Gly Glu Asn Asp Lys Ser Leu His Leu Leu Lys Lys

385 390 395 400

Gln Leu Ser Thr Leu Tyr Leu Glu Val Phe Ser Met Leu Arg Asp Glu

405 410 415

Asp Gly Lys Pro Tyr Ser Pro Ser Glu Tyr Ser Leu Gln Gln Thr Arg

420 425 430

Asp Gly Asn Val Phe Leu Val Pro Lys Ser Lys Lys Pro Asp Val Lys

435 440 445

Lys Asn Leu Glu

450

<210> 13

<211> 344

<212> PRT

<213> Artificial Sequence

<400> 13

Gly Ser Ala Thr Met Ala Gln His Ile Gln Ser Glu Thr Ser Gly Ser

1 5 10 15

Ala Asn Tyr Ser Gln Val Ala His Ile Pro Lys Ser Asp Ala Leu Tyr

20 25 30

Phe Asp Asp Cys Met Gln Leu Leu Ala Gln Thr Phe Pro Phe Val Asp

35 40 45

Asp Asn Glu Val Ser Ser Ala Thr Phe Gln Ser Leu Val Pro Asp Ile

50 55 60

Pro Gly His Ile Glu Ser Pro Val Phe Ile Ala Thr Asn Gln Ala Gln

65 70 75 80

Ser Pro Glu Thr Ser Val Ala Gln Val Ala Pro Val Asp Leu Asp Gly

85 90 95

Met Gln Gln Asp Ile Glu Gln Val Trp Glu Glu Leu Leu Ser Ile Pro

100 105 110

Glu Leu Gln Cys Leu Asn Ile Glu Asn Asp Lys Leu Val Lys Gln Asn

115 120 125

Gly Pro Lys Thr Pro Val His Ser Ser Gly Asp Met Val Gln Pro Leu

130 135 140

Ser Pro Ser Gln Gly Gln Ser Thr His Val His Asp Ala Gln Cys Glu

145 150 155 160

Asn Thr Pro Glu Lys Glu Leu Pro Val Ser Pro Gly His Arg Lys Thr

165 170 175

Pro Phe Thr Lys Asp Lys His Ser Ser Arg Leu Glu Ala His Leu Thr

180 185 190

Arg Asp Glu Leu Arg Ala Lys Ala Leu His Ile Pro Phe Pro Val Glu

195 200 205

Lys Ile Ile Asn Leu Pro Val Val Asp Phe Asn Glu Met Met Ser Lys

210 215 220

Glu Gln Phe Asn Glu Ala Gln Leu Ala Leu Ile Arg Asp Ile Arg Arg

225 230 235 240

Arg Gly Lys Asn Lys Val Ala Ala Gln Asn Cys Arg Lys Arg Lys Leu

245 250 255

Glu Asn Ile Val Glu Leu Glu Gln Asp Leu Asp His Leu Lys Asp Glu

260 265 270

Lys Glu Lys Leu Leu Lys Glu Lys Gly Glu Asn Asp Lys Ser Leu His

275 280 285

Leu Leu Lys Lys Gln Leu Ser Thr Leu Tyr Leu Glu Val Phe Ser Met

290 295 300

Leu Arg Asp Glu Asp Gly Lys Pro Tyr Ser Pro Ser Glu Tyr Ser Leu

305 310 315 320

Gln Gln Thr Arg Asp Gly Asn Val Phe Leu Val Pro Lys Ser Lys Lys

325 330 335

Pro Asp Val Lys Lys Asn Leu Glu

340

Claims (17)

1. A nucleic acid molecule characterized in that it has:

a nucleotide sequence shown as SEQ ID No.1, 2 or 3; or (b)

(II) a complementary nucleotide sequence to the nucleotide sequence shown in any of SEQ ID No.1, 2 or 3; or (b)

(III) a nucleotide sequence having at least 95% sequence identity to the nucleotide sequence of (I) or (II).

2. The nucleic acid molecule of claim 1, having a nucleotide sequence with at least 98% sequence identity to the nucleotide sequence of (i) or (ii).

3. The polypeptide or protein encoded by the nucleic acid molecule of claim 1 or 2, having an amino acid sequence as set forth in SEQ ID No. 11, SEQ ID No. 12 or SEQ ID No. 13.

4. An expression vector comprising the nucleic acid molecule of claim 1 or 2.

5. The expression vector of claim 4, wherein the expression vector is a viral vector comprising a double-stranded adeno-associated viral vector.

6. The expression vector of claim 5, wherein the serotype of the viral vector is selected from AAV2, AAV5, AAV7, or AAV8, or a combination thereof.

7. The expression vector of claim 5 or 6, wherein the double stranded adeno-associated viral vector further comprises a promoter.

8. The expression vector of claim 7, wherein the promoter comprises one or more of SYN, CMV, or CAG.

9. Use of a nucleic acid molecule according to claim 1 or 2, a polypeptide or protein according to claim 3, an expression vector according to any one of claims 4 to 8 for the preparation of a medicament for the prevention and/or treatment of diseases associated with optic nerve damage.

10. The use according to claim 9, wherein the disorder associated with optic nerve damage includes, but is not limited to, autosomal dominant optic atrophy, leber's hereditary optic neuropathy, ischemic optic neuropathy or glaucoma.

11. The use according to claim 9 or 10, wherein the disorder associated with optic nerve injury is acute optic nerve injury or chronic optic nerve injury.

12. The use according to any one of claims 9 to 11, wherein the activity of the nucleic acid molecule according to claim 1 or 2, the polypeptide or protein according to claim 3, the expression vector according to any one of claims 4 to 8 comprises, but is not limited to, any of the following:

(1) Causing stable high expression of Nrf2 gene in retinal ganglion cells; and/or

(2) Long-acting treatment of diseases related to optic nerve injury; and/or

(3) Activating cellular anti-inflammatory or anti-oxidative responses; and/or

(4) Increasing the survival rate of ganglion cells; and/or

(5) Preventing or delaying apoptosis of ganglion cells; and/or

(6) And eliminating free radicals of pathological cells.

13. A medicament comprising a nucleic acid molecule according to claim 1 or 2, a polypeptide or protein according to claim 3, an expression vector according to any one of claims 4 to 8, and a pharmaceutically acceptable adjuvant.

14. A pharmaceutical combination comprising a nucleic acid molecule according to claim 1 or 2, a polypeptide or protein according to claim 3, an expression vector according to any one of claims 4 to 8, and any other active ingredient.

15. Pharmaceutical formulation, characterized by comprising a nucleic acid molecule according to claim 1 or 2, a polypeptide or protein according to claim 3, an expression vector according to any one of claims 4 to 8, and a pharmaceutically acceptable carrier or excipient.

16. The pharmaceutical formulation of any one of claims 15, wherein the pharmaceutical formulation comprises a liquid formulation.

17. A method of delivering a drug, characterized in that the drug according to claim 13, the pharmaceutical combination according to claim 14, the pharmaceutical formulation according to claim 15 or 16 is administered to the eye by intravitreal administration.

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