Nucleic acid encoding NADH ubiquinone oxidoreductase subunit 4 and use thereof

Abstract

The present invention relates to the field of gene engineering technology, and in particular to a nucleic acid encoding NADH ubiquinone oxidoreductase subunit 4 and its use. The present invention performs codon optimization on the coding nucleic acid sequence of ND4, and experiments show that drug treatment prepared by using the optimized sequence of the present invention can significantly improve mitochondrial dysfunction lesions of patient cells caused by ND4 mutation.

Description

Technical Field

[0001] The present invention relates to the field of gene engineering technology, in particular to nucleic acid encoding NADH ubiquinone oxidoreductase subunit 4 and application thereof. Background Art

[0002] ND4 (NADH-ubiquinone oxidoreductase chain 4, NADH-ubiquinone oxidoreductase subunit 4) is the core subunit of mitochondrial complex I (complex 1), and together with the other six subunits, it constitutes the hydrophobic segment of complex I. Complex I plays an important role in the mitochondrial respiratory chain. It can oxidize NADH produced by the tricarboxylic acid cycle and β-oxidation, reduce ubiquinone, provide the power for proton transport, and thus help protons cross the mitochondrial inner membrane. The mitochondrial DNA 11778 site mutation is caused by a mutation in the 11778th nucleotide of mitochondrial DNA (mtDNA). This mutation changes the 340th amino acid encoded by the ND4 gene on the respiratory chain from arginine to histidine. Although they are all basic amino acids, the arginine at this position is highly conserved. Because the mutation may reduce the efficiency of electron flow and affect the activity of the enzyme, thereby reducing the production of ATP in optic nerve cells, the cells gradually undergo apoptosis, resulting in Leber hereditary optic neuropathy (LHON) in patients.

[0003] Leber hereditary optic neuropathy (LHON) is a maternally inherited disease caused by a point mutation in the mitochondrial genome, with a global incidence of 1 / 30,000. The characteristics of LHON patients are: the disease usually occurs at the age of 20-30 years old, and as the disease progresses, the patient's bilateral vision decreases sharply until eventually blindness. The main cause is the death of the patient's retinal ganglion cells (RGCs), and the death of RGC cells is attributed to the patient's mitochondrial gene mutations. Most LHON-related mutations occur in the coding genes of the subunits of complex I, of which more than half occur at the G11778A site of ND4. This mutation accounts for 89.2% of Chinese LHON patients and has the worst prognosis.

[0004] At present, traditional drugs have limited effects on the treatment of LHON patients, while AAV-mediated gene therapy has shown great potential in treating this type of single gene mutation genetic disease. However, the effect of AAV-mediated gene therapy depends on the gene sequence, and the efficacy of wild-type AAV gene sequences in previous treatments still needs to be improved. Summary of the invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a nucleic acid encoding NADH ubiquinone oxidoreductase subunit 4 (ND4) and its use, in order to increase the expression level of ND4 and improve the therapeutic effect.

[0006] The present invention provides a nucleic acid encoding NADH ubiquinone oxidoreductase subunit 4, comprising at least one of I) to III):

[0007] 1), a nucleic acid having a nucleotide sequence as shown in SEQ ID NO: 1;

[0008] II), a nucleic acid having a sequence having at least 70% homology with the nucleotide sequence as shown in I) and encoding a protein having an amino acid sequence as shown in SEQ ID NO: 2; preferably having at least 80% homology; more preferably having at least 85% homology; more preferably having at least 90% homology; more preferably having at least 95% homology; more preferably having at least 96% homology; more preferably having at least 97% homology; more preferably having at least 98% homology; more preferably having at least 99% homology.

[0009] III), a nucleic acid that is partially complementary or completely complementary to I) or II).

[0010] The present invention also provides a transcription unit of NADH ubiquinone oxidoreductase subunit 4, which comprises: a promoter, the nucleic acid encoding NADH ubiquinone oxidoreductase subunit 4 (ND4) of the present invention, and a terminator.

[0011] The transcription unit of the present invention comprises, from the 5' end to the 3' end, the following in sequence:

[0012] Promoter, the coding nucleic acid, regulatory segment A and terminator;

[0013] Or a promoter, the encoding nucleic acid, a regulatory segment A, a regulatory segment B and a terminator.

[0014] In the present invention, the regulatory fragment A and regulatory fragment B are selected from COX10 UTR and SV40 signal fragment.

[0015] The recombinant vector of the present invention comprises a backbone vector and the nucleic acid of the present invention.

[0016] The recombinant vector of the present invention is a viral vector; the viral vector is selected from at least one of a DNA viral vector, a retroviral vector, a lentiviral vector, an adenoviral vector, and an adeno-associated viral vector; wherein the serotype of the adeno-associated viral vector is AAV2, AAV5, AAV6, AAV7, AAV8, or AAV9. The backbone vector of the recombinant vector of the present invention is pAAV2.

[0017] In some embodiments, the recombinant vector comprises the following sequentially linked fragments: AAV2 5'ITR, CMV enhancer, CMV promoter, Chimeric intron, MTS fragment, nucleic acid fragment of the sequence shown in SEQ ID NO: 1, tag fragment, COX10 UTR, SV40 signal fragment, AAV2 3'ITR;

[0018] In some other embodiments, the recombinant vector includes the following sequentially connected components: AAV2 5'ITR, CMV enhancer, CMV promoter, Chimeric intron, MTS fragment, nucleic acid fragment of sequence shown in SEQ ID NO: 1, tag fragment, COX10 UTR, AAV2 3'ITR;

[0019] In other embodiments, the recombinant vector includes the following sequentially connected components: AAV2 5'ITR, CMV enhancer, CMV promoter, Chimeric intron, MTS fragment, nucleic acid fragment of the sequence shown in SEQ ID NO: 1, tag fragment, SV40 signal fragment, and AAV2 3'ITR.

[0020] The nucleic acid, transcription unit, and recombinant vector of the present invention are used in the preparation of a drug for preventing and treating mitochondrial dysfunction. The prevention and treatment of the present invention includes prevention and / or treatment. The mitochondrial dysfunction disease to be prevented and treated in the present invention is optic neuropathy; in some embodiments, the optic neuropathy is hereditary optic neuropathy. In some specific embodiments, the present invention provides the use of the nucleic acid, transcription unit, and recombinant vector in the preparation of a drug for preventing and treating Leber's hereditary optic neuropathy.

[0021] The present invention also provides a medicine, which comprises: the nucleic acid, transcription unit or recombinant vector described in the present invention, and may further comprise a pharmaceutically acceptable carrier and excipient.

[0022] The drug of the present invention comprises: the recombinant vector, Lipofectamine 2000 reagent and serum-free DMEM medium. In some embodiments, the concentration of the recombinant vector is 0.8 μg / 100 μL to 1.0 μg / 100 μL.

[0023] The present invention also provides a method for delivering a drug, which comprises injecting the drug preparation of the present invention into the eye, preferably into the vitreous cavity.

[0024] The present invention also provides a method for preventing and treating Leber's hereditary optic neuropathy, which comprises administering the drug of the present invention by intravitreal injection.

[0025] The present invention performs codon optimization on the coding nucleic acid sequence of ND4, and experiments show that drug treatment prepared by the optimized sequence of the present invention can significantly improve the mitochondrial dysfunction lesions of patient cells caused by ND4 mutation. 293 cells were treated with the AAV2-CMV-ND4opt1 drug, and the optimized ND4 can be efficiently expressed in the cell line, and the expression efficiency is higher than that of the unoptimized ND4 sequence. At the same time, after the patient cells were treated with the AAV2-CMV-ND4opt1 drug, the ability to grow in galactose medium was restored, and the ability to produce ATP was improved and close to that of wild-type cells, proving that the mitochondrial dysfunction was corrected. Therefore, this AAV2-CMV-ND4opt1 drug has the effect of preventing or treating Leber hereditary optic neuropathy. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1A , Figure 1B The sequence alignment of rND4 and ND4opt1 is shown, and the codon sequences that differ after optimization are bolded and underlined;

[0027] Figure 2 : shows a vector map, wherein A is a pAAV2-CMV-rND4-COX10UTR-SV40 vector map, B is a pAAV2-CMV-ND4opt1-COX10UTR-SV40 vector map, C is a pAAV2-CMV-ND4opt1-COX10UTR vector map, and D is a pAAV2-CMV-ND4opt1-SV40 vector map; wherein, the vector shown in B comprises AAV2 5'ITR, CMV enhancer, CMV promoter, chimeric intron, MTS (mitochondrial targeting sequence), codon-optimized ND4opt1 or rND4 cDNA, COX10 UTR element, SV40 polyA element sequence and AAV2 3'ITR;

[0028] Figure 3Figure 3 shows the expression efficiency of pAAV2-CMV-rND4-COX10UTR-SV40 plasmid, pAAV2-CMV-ND4opt1-COX10UTR-SV40 plasmid, pAAV2-CMV-ND4opt1-COX10UTR plasmid and pAAV2-CMV-ND4opt1-SV40 plasmid in HEK293 cells. HEK293 cells were transfected with pAAV2-CMV-rND4-COX10UTR-SV40, pAAV2-CMV-ND4opt1-COX10 UTR-SV40, pAAV2-CMV-ND4opt1-COX10UTR and pAAV2-CMV-ND4opt1-SV40 plasmids, respectively. After 48 hours, the cells were lysed to extract RNA and protein, respectively. qPCR and Western Blot were used to detect the expression of ND4 mRNA and protein expression levels. It was found that there were significant differences in mRNA and protein expression between ND4opt1 and rND4 after codon optimization. The mRNA (A) and protein levels (B) increased after optimization.

[0029] Figure 4 Determination of LHON cell viability in galactose screening medium: LHON fibroblast cells with ND4 mutation were infected with AAV2-CMV-rND4-COX10UTR-SV40, AAV2-CMV-ND4opt1-COX10UTR-SV40, AAV2-CMV-ND4opt1-COX10UTR and AAV2-CMV-ND4opt1-SV40 viruses respectively, one group was not infected with the virus, and the control group was normal fibroblast cells; 48 hours after infection, the medium was replaced with DMEM (galactose) or DMEM (glucose) medium, and the cells were collected and counted after 3 days of culture, and the ratio of the number of cells (galactose / glucose) was used as the survival rate;

[0030] Figure 5Determination of ATP synthesis in LHON cells mitochondria: LHON fibroblast cells with ND4 mutation were infected with AAV2-CMV-rND4-COX10UTR-SV40, AAV2-CMV-ND4opt1-COX10 UTR-SV40, AAV2-CMV-ND4opt1-COX10UTR and AAV2-CMV-ND4opt1-SV40 viruses, respectively. One group was not infected with the virus and the control group was normal fibroblast cells. 48 hours after infection, equal amounts of cells were collected, resuspended in ATP synthesis buffer supplemented with specific substrates (5mM 2-deoxy-D-glucose+5mM pyruvate), and incubated at 37°C for 2 hours. The cells were lysed to determine the ATP content. DETAILED DESCRIPTION

[0031] The present invention provides nucleic acids encoding NADH ubiquinone oxidoreductase subunit 4 and uses thereof. Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters to achieve the same. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0032] Unless otherwise defined, all scientific and technical terms used herein have the same meaning as understood by those of ordinary skill in the art. For definitions and terms in the art, professionals can refer specifically to Current Protocols in Molecular Biology (Ausubel). The abbreviations for amino acid residues are standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

[0033] The "codon" is a sequence composed of three nucleotides (e.g., deoxyribonucleotides and ribonucleotides), which together constitute a genetic code unit encoding an amino acid. The codon optimization of the present invention includes optimizing sequence fragments that affect gene expression and protein localization, and these sequence fragments include, but are not limited to, codon usage preference, elimination of secondary structures that are not conducive to expression (such as hairpin structures), changing GC content, CpG dinucleotide content, secondary structure of mRNA, hidden splicing sites, early polyadenylation sites, internal ribosome entry sites and binding sites, negative CpG islands, RNA unstable regions, repetitive sequences (direct repeats, inverted repeats, etc.) and restriction sites that may affect cloning.

[0034] The present invention performs codon optimization on the cDNA sequence of ND4, and the wild-type ND4 gene is the cDNA sequence of the human ND4 gene (SEQ ID NO: 3); the amino acid sequence encoded by the wild-type ND4 gene is shown in SEQ ID NO: 2, specifically: MLKLIVPTIMLLPLTWLSKKHMIWINTTTHSLIISIIPLLFFNQINNNLFSCSPTFSSDPLTTPLLMLTTWLLPLTIMASQRHLSSEPLSRKKLYLSMLISLQISLIMTFTATELIMFYIFFETTLIPTLAIITRWGNQPERLNAGTYFLFYTLVGSLPLLIALIYTHNTLGSLNILLLTLTAQELSNSWANNLMWLAYTMAFMVKMPLYGLHLWLPKAHVEAP IAGSMVLAAVLLKLGGYGMMRLTLILNPLTKHMAYPFLVLSLWGMIMTSSICLRQTDLKSLIAYSSISHMALVVTAILIQTPWSFTGAVILMIAHGLTSSLLFCLANSNYERTHSRIM ILSQGLQTLLPLMAFWWLLASLANLALPPTINLLGELSVLVTTFSWSNITLLLTGLNMLVTALYSLYMFTTTQWGSLTHHINNMKPSFTRENTLMFMHLSPILLLSLNPDIITGFSS.

[0035]

[0036] The present invention first performs codon optimization on the ND4 cDNA sequence to obtain ND4 opt1, uses a CMV promoter and modifies post-transcriptional regulatory elements.

[0037] In the present invention, the transcription unit refers to a DNA sequence starting from a promoter and ending at a terminator. A regulatory segment may also be included on both sides or between the promoter and the terminator, and the regulatory segment may include a promoter, an enhancer, a transcription termination signal, a polyadenylation sequence, a replication origin, a nucleic acid restriction site, and a homologous recombination site operably connected to the nucleic acid sequence, such as an enhancer of the promoter, a poly (A) signal, etc. In some embodiments, a COX10 UTR element (SEQ ID NO: 4) or a SV40 polyA element (SEQ ID NO: 5) is added to the transcription unit, or a combination of COX10 UTR and SV40 polyA elements is used to construct three expression vector plasmids containing different transcription units: pAAV2-CMV-ND4opt1-COX10UTR, pAAV2-CMV-ND4opt1-SV40, and pAAV2-CMV-ND4opt1-COX10UTR-SV40.

[0038] In the art, the expression of exogenous genes is usually achieved by cloning the target gene coding region sequence (CDS) into the corresponding plasmid or viral vector, and using the promoter constructed on the skeleton to drive the expression of the target gene. The skeleton vector can be viral or non-viral (e.g., plasmid). The vector preferably contains one or more regulatory sequences to guide the expression of the nucleic acid sequence in the retinal target cells. The regulatory sequence may include a promoter, an enhancer, a transcription termination signal, a polyadenylation sequence, a replication origin, a nucleic acid restriction site, and a homologous recombination site operably linked to the nucleic acid sequence. The vector may also include a selective marker, such as a resistance protein marker, an amino acid screening marker, or a green fluorescent protein. In an embodiment of the present invention, the selected promoter is a CMV promoter, and the terminator is a CMV terminator. In the present invention, the recombinant vector also includes a CMV enhancer, a Chimeric intron, an MTS fragment, and a tag fragment. In an embodiment of the present invention, the tag fragment is a 3×FLAG tag.

[0039] In some embodiments, the recombinant vector comprises the following sequentially linked components: AAV2 5'ITR, CMV enhancer, CMV promoter, Chimeric intron, MTS fragment, nucleic acid fragment of the sequence shown in SEQ ID NO: 1, 3×FLAG tag, COX10 UTR, SV40 signal fragment, AAV2 3'ITR;

[0040] In some other embodiments, the recombinant vector includes the following sequentially linked components: AAV2 5'ITR, CMV enhancer, CMV promoter, Chimeric intron, MTS fragment, nucleic acid fragment of sequence shown in SEQ ID NO: 1, 3×FLAG tag, COX10UTR, AAV2 3'ITR;

[0041] In other embodiments, the recombinant vector includes the following sequentially connected components: AAV2 5'ITR, CMV enhancer, CMV promoter, Chimeric intron, MTS fragment, nucleic acid fragment of the sequence shown in SEQ ID NO: 1, 3×FLAG tag, SV40 signal fragment, and AAV2 3'ITR.

[0042] In this application, the term "adeno-associated virus vector" or "AAV" generally refers to the adenovirus itself or its derivatives. Adeno-associated virus (AAV) generally refers to a class of single-stranded DNA viruses belonging to the family Parvoviridae and the genus Dependovirus. The AAV genome may contain inverted terminal repeats (ITRs) at both ends of the DNA chain and two open reading frames (ORFs). The open reading frames may include rep and cap. rep is composed of multiple overlapping genes encoding the Rep protein required for the AAV life cycle, and cap contains overlapping nucleotide sequences encoding capsid proteins, which may include VP1, VP2 and VP3. The capsid proteins interact to form a capsid. AAV has a variety of common serotypes and more than 100 viral variants. In the present application, AAV capsid, ITR and other selected AAV components are selected from any AAV, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV8bp, AAV7M8 and AAV Anc80, any known or mentioned AAV variants or AAVs yet to be discovered or their variants or mixtures.

[0043] In this application, the term "serotype" generally refers to the detection of epitopes on the surface of the adeno-associated virus capsid by serological methods and the typing of the adeno-associated virus. Adeno-associated virus has a variety of common serotypes and more than 100 virus variants. In this application, AAV capsids, ITRs and other selected AAV components are selected from any AAV, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV8bp, AAV7M8 and AAVAnc80, any known or mentioned AAV variants or AAVs yet to be discovered or their variants or mixtures.

[0044] The backbone vector used in the construction of the recombinant vector of the present invention is AAV2. Specifically, the backbone vector is pAAV2. The present invention also relates to a vector comprising the recombinant nucleic acid of the present invention, a host cell produced by genetic engineering using the vector or protein coding sequence of the present invention, and a method for expressing ND4 protein using the host cell by recombinant technology, as well as a method for expressing ND4 protein.

[0045] The present invention transiently transfects HEK293 cells with expression vector plasmids before and after optimization, and simultaneously detects the expression of ND4 at the RNA level and protein level, and finds that the expression efficiency of ND4 in the optimized vector is significantly improved. Subsequently, the AAV2-CMV-ND4opt1 virus drug is used to infect LHON patient cells. On the one hand, the cell culture conditions after infection are replaced with galactose selection medium, and the cell growth is monitored to prove its ability to restore oxidative phosphorylation; on the other hand, the content of ATP synthesized by mitochondria of cells after infection is measured, and it is determined that the ability of mitochondria of cells in the infected group to synthesize ATP is significantly higher than that of the control group, and the AAV2-CMV-ND4opt1 drug can restore the catalytic activity of complex I of LHON patient cells. In summary, it is proved that the AAV2-CMV-ND4opt1 drug has the effect of preventing or treating Leber's hereditary optic neuropathy.

[0046] The drug provided by the present invention includes the nucleic acid, transcription unit or recombinant vector described in the present invention. In some embodiments, the drug described in the present invention includes: the recombinant vector and a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient includes a pharmaceutically acceptable carrier, excipient or osmotic pressure regulator. "Pharmaceutically acceptable carrier or excipient" refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with the active ingredients of the present invention and with each other without significantly reducing the efficacy of the active ingredients. In some specific embodiments, the drug includes the recombinant vector described in the present invention, Lipofectamine 2000 reagent and serum-free DMEM culture medium. Wherein, the concentration of the recombinant vector is 0.8μg / 100μL~1.0μg / 100μL. Wherein, the concentration of the Lipofectamine 2000 reagent is 1μL / 100μL~3μL / 100μL.

[0047] The drugs described in the present invention also include other drugs that improve ND4 levels or activity. The other drugs that improve ND4 protein levels or activity include other ND4 variants or drugs that regulate ND4 expression. In the present invention, the pharmaceutical preparation is applied to the eye, and preferably, the drug can be applied to the eye through the vitreous cavity or intravitreal administration. In any mode of administration, the drug is provided as an injectable liquid. Preferably, the injectable liquid is provided in a capsule, an ampoule or a prefilled syringe.

[0048] The present invention uses AAV2-CMV-ND4opt1 drug to treat 293 cells. After optimization, ND4 can be efficiently expressed in the cell line, and the expression efficiency is higher than that of the unoptimized ND4 sequence. At the same time, after the patient cells are treated with AAV2-CMV-ND4opt1 drug, the ability to grow in galactose medium is restored, and the ability to produce ATP is improved and close to that of wild-type cells, proving that the mitochondrial dysfunction is corrected. Therefore, this AAV2-CMV-ND4opt1 drug has the effect of preventing or treating Leber's hereditary optic neuropathy.

[0049] The test materials used in the present invention are all common commercial products and can be purchased on the market. The present invention is further described below in conjunction with the embodiments:

[0050] Example 1 Codon-optimized and post-transcriptional regulatory element-modified ND4 has higher expression efficiency

[0051] 1. Plasmid vector construction

[0052] 1. Synthesize the codon-optimized ND4 sequence (denoted as ND4 opt1) and clone it into the pUC57 plasmid backbone, and cut the ND4 opt1 coding sequence from the backbone with BamH I and Cla I. Double-digest the pAAV2-CMV-COX10UTR-SV40, pAAV2-CMV-COX10UTR, and pAAV2-CMV-SV40 plasmid backbones with BamH I and ClaI, respectively, and then connect the digested fragments with different backbones to obtain:

[0053] pAAV2-CMV-ND4opt1-COX10UTR-SV40 plasmid,

[0054] pAAV2-CMV-ND4opt1-COX10UTR plasmid,

[0055] pAAV2-CMV-ND4opt1-SV40 plasmid.

[0056] 2. The ligation product was transformed into E. coli, and a single colony was picked for restriction enzyme digestion verification and sequencing verification.

[0057] 2. Plasmid transfection into cells

[0058] 1. One day before transfection, trypsinize HEK293 cells and count them. Plate the cells so that the confluence reaches 70%-80% on the day of transfection.

[0059] 2. For each well of cells, dilute 0.8 μg-1.0 μg of plasmid DNA with 50 μl serum-free DMEM medium; dilute 1 μl-3 μl of LIPOFECTAMINE 2000 reagent with 50 μl DMEM medium.

[0060] 3. Mix the diluted DNA and diluted LIPOFECTAMINE 2000 and incubate at room temperature for 20 minutes.

[0061] 4. Add the above complex directly into each well, shake the culture plate and mix gently.

[0062] 5. Incubate at 37°C, 5% CO2 for 48 hours.

[0063] 6. Discard the culture medium and rinse with PBS. After trypsin digestion, collect the cells by centrifugation for later use.

[0064] 3. qPCR determination of mRNA content

[0065] 1. Total RNA extraction

[0066] 1) Collect 10 5 1 ml of lysis buffer was added to each cell, and the supernatant was collected after centrifugation at 13000 g for 10 min.

[0067] 3) Add 250 μl of chloroform, invert the centrifuge tube for 15 seconds, mix thoroughly, let stand for 3 minutes; centrifuge at 13,000 g for 8 minutes at 4°C.

[0068] 4) Transfer the supernatant to a new centrifuge tube, add 0.8 times the volume of isopropanol, and mix by inversion; place at -20°C for 15 min; centrifuge at 13,000 g for 10 min at 4°C; the white precipitate at the bottom of the tube is RNA.

[0069] 5) Remove the liquid by aspiration, add 1.5 ml of 75% ethanol to wash the precipitate; centrifuge at 13000 g for 5 min at 4°C; remove the liquid completely, place the centrifuge tube on a clean bench and blow for 3 min; add 20 μl of RNase-free water to dissolve the RNA; incubate at 55°C for 5 min.

[0070] 2. Reverse transcription

[0071] 1) Take a PCR tube and add a solution containing 2 μg RNA; add 1 μl oligo(dT); and make up to 12 μl with RNase-free deionized water.

[0072] 2) Incubate at 70°C for 5 min on a PCR instrument, then quickly cool on ice; add 4 μl 5× buffer, 2 μl 10 mM dNTPs, 1 μl RNA inhibitor and 1 μl reverse transcriptase in sequence, and mix by aspiration; Incubate at 42°C for 60 min on a PCR instrument, then incubate at 80°C for 5 min to inactivate the reverse transcriptase.

[0073] 3. Quantitative PCR

[0074] 1) Take a 0.2 ml PCR tube and prepare the following reaction system, prepare 3 tubes for each reverse transcription product: 2×qPCRMix12.5μl; 7.5μM gene primer; 2.0μl reverse transcription product; 2.5μld dH2O; 8.0μl.

[0075] 2) Take a 0.2 ml PCR tube and prepare the following reaction system, prepare 3 tubes for each reverse transcription product: 2×qPCR Mix 12.5 μl; 7.5 μM internal reference primer 2.0 μl; reverse transcription product 2.5 μl; ddH2O 8.0 μl.

[0076] Target gene flag amplification primers:

[0077] Forward primer 5'-AGACCATGACGGTGAT-3'

[0078] Reverse primer 5′-CTTGTCATCGTCATCCT-3′

[0079] Primers for amplification of the internal reference gene actin:

[0080] Forward primer 5′-GGACTTCGAGCAAGAGATGG-3′

[0081] Reverse primer 5′-AGGAAGGAAGGCTGGAAGAG-3′

[0082] 3) PCR amplification

[0083] Pre-denaturation 95℃, 5min,

[0084] 40 cycles of 95℃, 15s→60℃, 60s,

[0085] Melting curve: 60℃→95℃, temperature rises by 1℃ every 20s

[0086] 4) Result processing ΔΔ CT method: A = CT (target gene, sample to be tested) - CT (internal standard gene, sample to be tested); B = CT (target gene, control sample) - CT (internal standard gene, control sample); K = AB; expression multiple = 2-K .

[0087] 4. Western Blot

[0088] 1. Protein sample preparation: add PMSF to the lysate at a ratio of 1:100.

[0089] 2. Lyse cells on ice for 30 min using a strong lysis buffer; centrifuge at 12,000 rpm for 15 min at 4°C and collect the supernatant for later use.

[0090] 3. Determine protein concentration using the BCA method.

[0091] 4. Electrophoresis: a. Prepare the corresponding separation gel (5 ml / piece) according to the size of the protein to be detected, and wait for the separation gel to solidify. b. Prepare 5% concentrated gel (2 ml / piece), fill the glass plate, and insert the comb. c. Add 5 μl of pre-stained protein molecule marker SDS-PAGE into the sample well, and use 10 μl of 1× SDS-PAGE protein loading buffer to load into the blank sample well next to the sample well.

[0092] 5. Transfer: Place a wet cushion on the white transfer clamp, and place three stacked wet filter papers on the cushion. Place the wet PVDF membrane, glue, filter paper, cushion, and black clamp on the filter paper in sequence. Place the assembled clamp into the electrophoresis tank filled with transfer buffer, and place the transfer tank in an ice bath for 2 hours for transfer.

[0093] 6. Blocking: After the transfer, rinse for 1-2 minutes, use a dropper to drain the buffer, add 5% skim milk powder, shake slowly on a side-swinging shaker, and block at room temperature for 15-60 minutes. Add TBS washing solution and wash for 5 minutes. Wash 3 times in total.

[0094] 7. Primary antibody incubation: Dilute an appropriate amount of primary antibody with 5% skim milk powder / PBS + 2% BSA according to the ratio, incubate slowly at 4°C overnight or at room temperature on a side-to-side shaker for 2 hours. Wash after incubation.

[0095] 8. Secondary antibody incubation: Add the diluted secondary antibody and incubate slowly on a side-swing shaker at room temperature for 40 minutes to 1 hour. Wash after incubation.

[0096] 9. Protein detection: Use ECL reagents to detect proteins. Take 1 ml of each and mix well. Add it on the surface of the protein membrane and incubate it in the dark for 1-2 minutes. Use tweezers to place the protein membrane neatly on plastic paper and expose it on a gel imager.

[0097] V. Experimental Results and Discussion

[0098] By optimizing codon usage preference, DNA repeat sequence, mRNA secondary structure, GC content and other parameters, we obtained an optimized sequence ND4opt1 ( Figure 1A , Figure 1B ). ND4opt1 was then constructed onto the AAV2 vector backbone to obtain a series of vectors with different UTR and polyA combinations ( Figure 2 ), and then the same amount of plasmid was transfected into HEK293 cells.

[0099] First, we detected the expression of ND4 at the mRNA level and found that the level of codon-optimized ND4 (pAAV2-CMV-ND4opt1-COX10UTR-SV40) in cells was significantly higher than that of rND4, while vectors with other combinations of post-transcriptional regulatory elements (pAAV2-CMV-ND4opt1-COX10UTR and pAAV2-CMV-ND4opt1-SV40) further increased the abundance of ND4 mRNA in HEK293 cells, which was 9-37 times that of rND4 ( Figure 3 The above results indicate that by codon optimization and adding different combinations of post-transcriptional regulatory elements, we obtained ND4 that was more stably expressed in cells.

[0100] Secondly, we extracted proteins from HEK293 cells transfected with different plasmids and detected them, and obtained results that were basically consistent with the mRNA level: the expression level of rND4 in HEK293 cells was low, while the ND4 vectors with codon optimization and expression element modification (pAAV2-CMV-ND4opt1-COX10UTR-SV40, pAAV2-CMV-ND4opt1-COX10UTR and pAAV2-CMV-ND4opt1-SV40) could detect strong signals in immunoblotting experiments ( Figure 3 In summary, the optimized ND4 has a longer half-life in cells, is more stable, and has a higher translation efficiency.

[0101] Example 2 AAV2-CMV-ND4opt1 drug can effectively restore the mitochondrial dysfunction phenotype of LHON patient cells

[0102] 1. Virus Packaging

[0103] 1. HEK293T cells with a polymerization degree of more than 90% should be plated at a ratio of 1:3.

[0104] 2. About 1 to 2 hours before plasmid transfer, change to serum-free culture medium and use transfection reagent to transfer the target gene plasmid and auxiliary plasmid into HEK293T.

[0105] 3. After 24 hours of plasmid transformation, replace with new serum-free medium

[0106] 4. Collect the virus 72 hours after transfection. Blow off the cells with the culture medium and centrifuge; then harvest the culture medium supernatant and cell precipitate separately. Use PEG8000 to precipitate the virus in the culture medium supernatant, and collect the virus precipitate after precipitation overnight.

[0107] 5. The virus mixture was purified by iodixanol density gradient centrifugation and then concentrated using an ultrafiltration tube.

[0108] 2. Glucose / Galactose Culture Experiment

[0109] 1. Digest and count fibroblast cells from patients with ND4 mutation LHON.

[0110] 2. Inoculate 6-well plates at a density of 1E5 cells / well and culture in DMEM (glucose) at 37°C and 5% CO2.

[0111] The groups are as follows (3 parallel wells per group):

[0112]

[0113]

[0114] 3. 16 hours after cell plating, the experimental groups were infected with viruses according to the experimental requirements in the table above.

[0115] 4. 48 hours after virus infection, experimental groups 1-12 were first treated with DMEM (glucose - ) medium, then replace the medium with DMEM (galactose) medium for the 7-12 experimental groups and with DMEM (glucose) medium for the 1-6 experimental groups, and continue culturing.

[0116] 5. After 3 days of culture, trypsinize the cells, centrifuge at 200g for 5 min, discard the supernatant, and resuspend in PBS. Count the cells and make statistics.

[0117] 3. ATP detection of mitochondrial synthesis

[0118] 1. Digest and count fibroblast cells from patients with ND4 mutation LHON.

[0119] 2. Inoculate 6-well plates at a density of 2E5 cells / well and culture in DMEM (glucose) at 37°C and 5% CO2.

[0120] The groups are as follows (3 parallel wells per group):

[0121] Experimental Group Cell treatment 1 Control fibroblast 2 LHON fibroblast 3 LHON fibroblast+AAV2-CMV-rND4-COX10UTR-SV40 4 LHON fibroblast+AAV2-CMV-ND4opt1-COX10UTR-SV40 5 LHON fibroblast+AAV2-CMV-ND4opt1-COX10UTR 6 LHON fibroblast+AAV2-CMV-ND4opt1-SV40

[0122] 3. 16 hours after cell plating, experimental groups 3-6 were infected with viruses according to experimental requirements.

[0123] 4. 48 hours after virus infection, trypsinize the cells, centrifuge at 200g for 5 min, discard the supernatant, wash 2E6 cells once with PBS and discard the supernatant.

[0124] 5. Configure ATP synthesis buffer (SB): 156mM NaCl, 3mM KCl, 2mM MgSO4, 1.25mM

[0125] KH2PO4, 2mM CaCl2, 20mM HEPES, pH 7.35

[0126] 6. Resuspend the cells in SB, add 5mM 2-deoxy-D-glucose + 5mM pyruvate, incubate at 37°C for 2 hours, and lyse the cells to detect ATP.

[0127] 7. ATP detection

[0128] 7.1 ATP standard curve configuration

[0129] 4μM ATP: Take 8μl of 500μM ATP standard and add 992μl Tris-acetate buffer, mix well.

[0130] 2μM ATP: Take 500μL of 4μM ATP and add 500μl Tris-acetate buffer and mix well.

[0131] 1μM ATP: Take 500μL of 2μM ATP and add 500μl Tris-acetate buffer and mix well.

[0132] 0.5μM ATP: Take 500μL of 1μM ATP and add 500μl Tris-acetate buffer and mix well.

[0133] 0.25μM ATP: Take 500μL of 0.5μM ATP and add 500μl Tris-acetate buffer and mix well.

[0134] 0.125μM ATP: Take 500μL of 0.25μM ATP and add 500μL of Tris-acetate buffer and mix well.

[0135] 0.0625μM ATP: Take 500μL of 0.125μM ATP and add 500μL of Tris-acetate buffer and mix well.

[0136] 0μM ATP:Tris-acetate buffer.

[0137] 7.2 Working fluid configuration

[0138] Prepare ATP detection reagent and ATP detection diluent in a ratio of 1:9 and mix well.

[0139] 7.3 Use a completely black ELISA plate, add 100 μl of working solution to each well, and keep it away from light for a few minutes to reduce ATP background.

[0140] 7.4 Dispense 280 μl of the sample and standard into another 96-well plate, use a dispenser to draw 100 μl of the sample or standard into the working solution, mix well, and immediately detect chemiluminescence.

[0141] 4. Experimental Results and Discussion

[0142] Previous experiments have confirmed that the in vitro expression efficiency of ND4 vectors with codon optimization and post-transcriptional regulatory element modification is better than that of rND4 vectors. To further test the function of AAV2-CMV-ND4opt1 drug-expressed protein, we performed pharmacodynamic verification on fibroblast cells of drug-treated ND4 mutation LHON patients.

[0143] First, we infected ND4 mutant LHON fibroblast cells with different viral drugs, then cultured the cells in screening medium and tested cell viability after a period of time. The results showed that:

[0144] 1) The viability of normal fibroblast cells in the control group was basically unaffected by the screening pressure, with nearly 100% of the cells surviving, while the viability of ND4 mutant LHON fibroblast cells that were not treated with drugs decreased significantly under the screening conditions, with the proportion of surviving cells being around 48% at the end of the culture;

[0145] 2) The viability of ND4 mutant fibroblast LHON cells treated with AAV2-CMV-rND4-COX10UTR-SV40 was restored to a certain extent, reaching 63%; the viability of ND4 mutant LHON fibroblast cells treated with AAV2-CMV-ND4opt1-COX10UTR-SV40, AAV2-CMV-ND4opt1-COX10UTR and AAV2-CMV-ND4opt1-SV40 was higher than that of AAV2-CMV-rND4-COX10UTR-SV40, reaching an average of 65%-80% ( Figure 4 ). Glucose was removed from the screening medium, and the added galactose forced the cells to be unable to produce enough ATP through the glycolysis pathway, and they could only rely on mitochondrial oxidative phosphorylation to produce ATP to maintain the energy required for cell survival; and the LHON fibroblast cells with ND4 mutations had impaired mitochondrial function, so their survival ability in the screening medium decreased. The above results prove that the optimized drug can not only compensate for the mitochondrial function damage caused by ND4 mutation to a certain extent, but also has a stronger effect than the unoptimized rND4.

[0146] Secondly, we infected ND4 mutant LHON fibroblast cells with different viral drugs, then added complex I-specific substrates and glycolysis inhibitors to detect the ability of cell mitochondria to synthesize ATP. The results showed that:

[0147] 1) Compared with the control group, the ability of mitochondria to synthesize ATP in ND4 mutant LHON fibroblast cells without drug treatment was significantly reduced;

[0148] 2) The ability of mitochondria to synthesize ATP in ND4 mutant LHON fibroblast cells treated with AAV2-CMV-rND4-COX10UTR-SV40 was restored to a certain extent; the ability of mitochondria to synthesize ATP in ND4 mutant LHON fibroblast cells treated with AAV2-CMV-ND4opt1-COX10UTR-SV40, AAV2-CMV-ND4opt1-COX10UTR and AAV2-CMV-ND4opt1-SV40 was restored to a higher level than that in AAV2-CMV-rND4-COX10UTR-SV40 ( Figure 5). Complex I-specific substrates and glycolysis inhibitors were added during cell incubation. The ATP produced by the cells at this time reflected the activity of cell complex I to a certain extent. The above results proved that the optimized drug was more effective than the unoptimized rND4 and could restore the complex I dysfunction caused by ND4 mutation.

[0149] Based on the above results, we demonstrated the therapeutic effects of AAV2-CMV-ND4opt1-COX10UTR-SV40, AAV2-CMV-ND4opt1-COX10UTR and AAV2-CMV-ND4opt1-SV40 gene therapy drugs on Leber hereditary optic neuropathy caused by ND4 mutation, laying the foundation for further clinical application development.

[0150] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention. Sequence Listing <110> NeuroFos (Suzhou) Biotechnology Co., Ltd. <120> Nucleic acid encoding NADH ubiquinone oxidoreductase subunit 4 and use thereof <130> MP21008131 <160> 5 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1377 <212> DNA <213> Artificial Sequence <400> 1 atgttgaaat tgatcgtgcc tacaatcatg ctgctgcccc tgacctggct ttccaaaaaa 60 cacatgattt ggattaacac aacaacccat tccctgatca tctccatcat tcctctgctg 120 ttcttcaacc agatcaataa taatcttttt tcctgtagcc ccactttttc ctcagaccct 180 ttgaccactc ccctgctcat gctgactact tggcttctgc cccttacaat tatggcttcc 240 caaagacacc tgagcagcga acctctgtcc cgcaaaaaac tgtacctgag catgcttatc 300 agccttcaaa tctcactcat tatgactttc acagcaaccg agttgatcat gttttacatc 360 ttcttcgaaa ctactctgat ccccacgctg gccattataa cccgctgggg aaaccagccg 420 gaaagactga acgccggaac ctacttcctg ttctacacac ttgttggtag cttgccgttg 480 ctgattgccc tgatatatac ccacaacacg ctgggatcat tgaacattct gctcttgacc 540 ctgacggccc aggagctgtc taattcttgg gcaaacaacc tgatgtggct tgcctacacc 600 atggccttta tggtcaagat gcccctgtat ggattgcacc tgtggctgcc taaagctcat 660 gtggaagcac ccatagcagg cagtatggta cttgcagcgg tgcttctgaa actgggtgga 720 tatggaatga tgagattgac attgattttg aatccgctga caaagcacat ggcctacccc 780 ttccttgtgc tgagtttgtg gggaatgatc atgacgtctt caatctgctt gaggcagaca 840 gatctgaaat ctctcatcgc ctacagctct atatcccata tggcgctcgt ggtcacggct 900 atcctcatcc agacaccgtg gtcctttacc ggagcggtca ttttgatgat agcacatggt 960 ttgacctcat ccctgctgtt ctgcttggct aactccaact acgagcgcac acattcacgc 1020 atcatgatcc tttcccaggg gctccaaacc ctcctcccgc tcatggcatt ttggtggctt 1080 ctcgctagcc tggctaacct ggccctccct cccaccatca acttgctggg cgagctgtcc 1140 gtgctggtta ccactttcag ctggagtaat attacccttc tccttacagg cctgaacatg 1200 ctggtgaccg cgctttactc tctgtacatg tttactacaa cacagtgggg ctctctgact 1260 caccacatca acaatatgaa gccatcattt accagagaga acacactgat gtttatgcat 1320 ctgagcccta tcctcctctt gtccctgaat ccggatatta taacggggtt tagcagc 1377 <210> 2 <211> 459 <212> PRT <213> human <400> 2 Met Leu Lys Leu Ile Val Pro Thr Ile Met Leu Leu Pro Leu Thr Trp 1 5 10 15 Leu Ser Lys Lys His Met Ile Trp Ile Asn Thr Thr Thr His Ser Leu 20 25 30 Ile Ile Ser Ile Ile Pro Leu Leu Phe Phe Asn Gln Ile Asn Asn Asn 35 40 45 Leu Phe Ser Cys Ser Pro Thr Phe Ser Ser Asp Pro Leu Thr Thr Pro 50 55 60 Leu Leu Met Leu Thr Thr Trp Leu Leu Pro Leu Thr Ile Met Ala Ser 65 70 75 80 Gln Arg His Leu Ser Ser Glu Pro Leu Ser Arg Lys Lys Leu Tyr Leu 85 90 95 Ser Met Leu Ile Ser Leu Gln Ile Ser Leu Ile Met Thr Phe Thr Ala 100 105 110 Thr Glu Leu Ile Met Phe Tyr Ile Phe Phe Glu Thr Thr Leu Ile Pro 115 120 125 Thr Leu Ala Ile Ile Thr Arg Trp Gly Asn Gln Pro Glu Arg Leu Asn 130 135 140 Ala Gly Thr Tyr Phe Leu Phe Tyr Thr Leu Val Gly Ser Leu Pro Leu 145 150 155 160 Leu Ile Ala Leu Ile Tyr Thr His Asn Thr Leu Gly Ser Leu Asn Ile 165 170 175 Leu Leu Leu Thr Leu Thr Ala Gln Glu Leu Ser Asn Ser Trp Ala Asn 180 185 190 Asn Leu Met Trp Leu Ala Tyr Thr Met Ala Phe Met Val Lys Met Pro 195 200 205 Leu Tyr Gly Leu His Leu Trp Leu Pro Lys Ala His Val Glu Ala Pro 210 215 220 Ile Ala Gly Ser Met Val Leu Ala Ala Val Leu Leu Lys Leu Gly Gly 225 230 235 240 Tyr Gly Met Met Arg Leu Thr Leu Ile Leu Asn Pro Leu Thr Lys His 245 250 255 Met Ala Tyr Pro Phe Leu Val Leu Ser Leu Trp Gly Met Ile Met Thr 260 265 270 Ser Ser Ile Cys Leu Arg Gln Thr Asp Leu Lys Ser Leu Ile Ala Tyr 275 280 285 Ser Ser Ile Ser His Met Ala Leu Val Val Thr Ala Ile Leu Ile Gln 290 295 300 Thr Pro Trp Ser Phe Thr Gly Ala Val Ile Leu Met Ile Ala His Gly 305 310 315 320 Leu Thr Ser Ser Leu Leu Phe Cys Leu Ala Asn Ser Asn Tyr Glu Arg 325 330 335 Thr His Ser Arg Ile Met Ile Leu Ser Gln Gly Leu Gln Thr Leu Leu 340 345 350 Pro Leu Met Ala Phe Trp Trp Leu Leu Ala Ser Leu Ala Asn Leu Ala 355 360 365 Leu Pro Pro Thr Ile Asn Leu Leu Gly Glu Leu Ser Val Leu Val Thr 370 375 380 Thr Phe Ser Trp Ser Asn Ile Thr Leu Leu Leu Thr Gly Leu Asn Met 385 390 395 400 Leu Val Thr Ala Leu Tyr Ser Leu Tyr Met Phe Thr Thr Thr Gln Trp 405 410 415 Gly Ser Leu Thr His His Ile Asn Asn Met Lys Pro Ser Phe Thr Arg 420 425 430 Glu Asn Thr Leu Met Phe Met His Leu Ser Pro Ile Leu Leu Leu Ser 435 440 445 Leu Asn Pro Asp Ile Ile Thr Gly Phe Ser Ser 450 455 <210> 3 <211> 1377 <212> Ms <213> human <400> 3 atgctaaaac taatcgtccc aacaattatg ttactaccac tgacatggct ttccaaaaaa 60 cacatgattt ggatcaacac aaccacccac agcctaatta ttagcatcat ccctctacta 120 ttttttaacc aaatcaacaa caacctattt agctgttccc caaccttttc ctccgacccc 180 ctaacaaccc ccctcctaat gctaactacc tggctcctac ccctcacaat catggcaagc 240 caacgccact tatccagtga accactatca cgaaaaaaac tctacctctc tatgctaatc 300 tccctacaaa tctccttaat tatgacattc acagccacag aactaatcat gttttatatc 360 ttcttcgaaa ccacacttat ccccaccttg gctatcatca cccgatgggg caaccagcca 420 gaacgcctga acgcaggcac atacttccta ttctacaccc tagtaggctc ccttccccta 480 ctcatcgcac taatttacac tcacaacacc ctaggctcac taaacattct actactcact 540 ctcactgccc aagaactatc aaactcctgg gccaacaact taatgtggct agcttacaca 600 atggctttta tggtaaagat gcctctttac ggactccact tatggctccc taaagcccat 660 gtcgaagccc ccatcgctgg gtcaatggta cttgccgcag tactcttaaa actaggcggc 720 tatggtatga tgcgcctcac actcattctc aaccccctga caaaacacat ggcctacccc 780 ttccttgtac tatccctatg gggcatgatt atgacaagct ccatctgcct acgacaaaca 840 gacctaaaat cgctcattgc atactcttca atcagccaca tggccctcgt agtaacagcc 900 attctcatcc aaaccccctg gagcttcacc ggcgcagtca ttctcatgat cgcccacggg 960 cttacatcct cattactatt ctgcctagca aactcaaact acgaacgcac tcacagtcgc 1020 atcatgatcc tctctcaagg acttcaaact ctactcccac taatggcttt ttggtggctt 1080 ctagcaagcc tcgctaacct cgccttaccc cccactatta acctactggg agaactctct 1140 gtgctagtaa ccacgttctc ctggtcaaat atcactctcc tacttacagg actcaacatg 1200 ctagtcacag ccctatactc cctctacatg tttaccacaa cacaatgggg ctcactcacc 1260 caccacatta acaacatgaa accctcattc acacgagaaa acaccctcat gttcatgcac 1320 ctatccccca ttctcctcct atccctcaac cccgacatca ttaccgggtt ttcctct 1377 <210> 4 <211> 1434 <212> DNA <213> Artificial Sequence <400> 4 gagcactggg acgcccaccg cccctttccc tccgctgcca ggcgagcatg ttgtggtaat 60 tctggacac aagagagaa attgctgggt ttagacaag attataacg aattcggtgc 120 tcagtgatca cttgacagtt tttttttt ttataatatta cccaaaatgc tcccaaata 180 agaaatgcat cagctcagtc agtgaataca aaaaaggaat tattttccc tttgagggtc 240 ttttacat ctctcctcca accccaccct ctattctgtt tctctccct cacatggggg 300 tacacataca cagctccctc ttttggttcc atccttacca ccacacca cgcacactcc 360 acatgcccag cagagtggca cttggtggcc agaagtgtg agcctcatga tctgctgtct 420 gtagttctgt gagctcaggt ccccaagg cctcggagca cccccttcct tgtgactgag 480 ccaggggctg cattttttgt ttccccacc cacacattc caccacatg tccttctc 540 ataccaata gctaggacccc ggctgctgtg cactgggact ggggattcca catgtttgcc 600 660 cattcagaa ctccaaggag tcaggcat ctttatagtt cacgttaaca tatagacact 720 gttggaagca gttccttcta aaagggtagc cctggactta attackacccg gataccctg 780 gcccccaccc cattactgta cctctggagt cactactgtg ggtcgccact cctctgctac 840 acagcacggc tttttcaagg ctgtattgag aagggaagtt aggaagaagg gtgtgctggg 900 ctaaccagcc cacagagctc acattcctgt cccttgggtg aaaaatacat gtccatcctg 960 atatctcctg aattcagaaa ttagcctcca catgtgcaat ggctttaaga gccagaagca 1020 gggttctggg aattttgcaa gttacctgtg gccaggtgtg gtctcggtta ccaaatacgg 1080 ttacctgcag ctttttagtc ctttgtgctc ccacgggtct acagagtccc atctgcccaa 1140 aggtcttgaa gcttgacagg atgttttcga ttactcagtc tcccagggca ctactggtcc 1200 gtaggattcg attggtcggg gtaggagagt taaacaacat ttaaacagag ttctctcaaa 1260 aatgtctaaa gggattgtag gtagataaca tccaatcact gtttgcactt atctgaaatc 1320 ttccctcttg gctgccccca ggtatttact gtggagaaca ttgcatagga atgtctggaa 1380 aaagcttcta caacttgtta cagccttcac atttgtagaa gctttgcggc cgct 1434 <210> 5 <211> 122 <212> DNA <213> Artificial Sequence <400> 5 taagatacat tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta 60 tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag 120 tt 122

Claims

1. A nucleic acid encoding NADH ubiquinone oxidoreductase subunit 4, the nucleotide sequence of which is shown in SEQ ID NO:

1.

2. A transcription unit of NADH ubiquinone oxidoreductase subunit 4, comprising: A promoter, the nucleic acid according to claim 1 and a terminator.

3. The transcription unit according to claim 2, characterized in that Its 5' end to 3' end includes: A promoter, the nucleic acid according to claim 1, a regulatory segment A and a terminator; or a promoter, the nucleic acid of claim 1, a regulatory segment A, a regulatory segment B and a terminator.

4. A recombinant vector comprising a backbone vector and the nucleic acid according to claim 1.

5. The recombinant vector according to claim 4, characterized in that It is a viral vector; The viral vector is selected from at least one of a retroviral vector, a lentiviral vector, an adenoviral vector, and an adeno-associated viral vector; wherein the serotype of the adeno-associated viral vector is AAV2, AAV5, AAV6, AAV7, AAV8 or AAV9.

6. The recombinant vector according to claim 4 or 5, characterized in that Its backbone vector is pAAV2.

7. The recombinant vector according to claim 6, characterized in that It includes the following sequentially connected components: AAV2 5'ITR, CMV enhancer, CMV promoter, Chimeric intron, MTS fragment, nucleic acid fragment of sequence shown in SEQ ID NO: 1, tag fragment, COX10 UTR, SV40 signal fragment, AAV2 3'ITR; Or comprising sequentially linked AAV2 5'ITR, CMV enhancer, CMV promoter, Chimeric intron, MTS fragment, nucleic acid fragment of sequence shown in SEQ ID NO: 1, tag fragment, COX10 UTR, AAV2 3'ITR; Or it may include sequentially connected AAV2 5'ITR, CMV enhancer, CMV promoter, Chimeric intron, MTS fragment, nucleic acid fragment of the sequence shown in SEQ ID NO: 1, tag fragment, SV40 signal fragment, and AAV2 3'ITR.

8. Use of the nucleic acid according to claim 1, the transcription unit according to claim 2 or 3, or the recombinant vector according to any one of claims 4 to 7 in the preparation of a drug for treating Leber hereditary optic neuropathy caused by ND4 mutation.

9. A drug, characterized in that include: The nucleic acid according to claim 1, the transcription unit according to claim 2 or 3, or the recombinant vector according to any one of claims 4 to 7.

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