CN107459571A - Novel oxidation-resistant polypeptide based on PAQR3 functions, it is prepared and purposes - Google Patents

Abstract

The invention relates to a novel antioxidant polypeptide based on the function of PAQR3, its preparation and use. Specifically, the present invention provides a polypeptide, the polypeptide contains a fragment of SEQ ID NO: 1, or consists of a fragment of SEQ ID NO: 1; wherein, the fragment of SEQ ID NO: 1 is located in SEQ ID NO: 1 between amino acid residues 1 to 35, preferably between amino acid residues 1 to 30 of SEQ ID NO: 1; and said fragment of SEQ ID NO: 1 is at least 15 amino acid residues long The base is preferably at least 18 amino acid residues in length, more preferably at least 20 amino acid residues in length. The present invention also relates to the coding sequence of the polypeptide, nucleic acid construct, and pharmaceutical composition, and related uses.

Description

Novel antioxidant polypeptide based on PAQR3 function, its preparation and use

technical field

The invention relates to a novel antioxidant polypeptide based on the function of PAQR3, its preparation and use.

Background technique

All living organisms are constantly challenged by oxidative stress, either internally or externally (eg, from poisons, xenobiotics, heavy metals and ionizing radiation). The above substances cause macromolecular damage, DNA mutation, cell apoptosis, and cell transformation by generating reactive oxygen species (ROS) and electrophiles, thereby causing damage to cells and the body. Cells therefore have a protective system to relieve these stresses. In mammalian cells, the transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2), as a basic leucine zipper (bZIP) protein, plays the most important role in coordinating the expression of detoxification and antioxidant genes (Moi P, Chan K, Asunis I, Cao A, Kan YW, (1994) Isolation of NF-E2-relatedfactor 2(Nrf2), a NF-E2-like basic leucine zipper transcriptional activator that binds to the tandem NF-E2/AP1repeat of the beta-globin locus control region, Proc Natl Acad Sci USA 91:9926-9930; Suzuki T, Yamamoto M, (2015) Molecular basis of the Keap1-Nrf2 system, Free radical biology & medicine). Nrf2 is activated by reactive oxygen species and/or electrophiles and plays a key role in the transcriptional activation of antioxidant and detoxification genes (Suzuki & Yamamoto, 2015, supra), such as exogenous metabolic enzymes (NAD(P)H quinone oxidoreductase-1 (NqoI)(Venugopal R, Jaiswal AK(1996) Nrf1 and Nrf2 positively and c-Fos and Fra1 negativelyregulate the human antioxidant response element-mediated expression of NAD(P)H:quinone oxidoreductase1 gene, Proc Natl Acad Sci USA 93:14960 -14965), heme oxygenase-1 (HO-1) cleaves heme to form biliverdin (Alam J, Stewart D, Touchard C, Boinapally S, Choi AM, Cook JL (1999) Nrf2, a Cap'n 'Collar transcription factor, regulates induction of the heme oxygenase-1gene.The Journal of biological chemistry274:26071-26078), glutamic acid cysteine ligase (GCLC) and its modifier GCLM are electrophilic compound antidote (Solis WA, Dalton TP, Dieter MZ, Freshwater S, Harrer JM, He L, Shertzer HG, Nebert DW (2002) Glutamate-cysteine ligase modifier subunit: mouse Gclm gene structure and regulation by agents that cause oxidative stress, Biochemical pharmacology 63:1739-1754) , and the glutathione S-transferase (GST) family catalyzes the conjugation of GSH to endogenous and exogenous electrophiles (Hayes JD, Chanas SA, Henderson CJ, McMahon M, Sun C, Moffat GJ, Wolf CR ，YamamotoM(2000)The Nrf2transcription factor contributes both to the b asal expression of glutathione S-transferases in mouse liver and to their induction by the chemopreventive synthetic antioxidants, butylated hydroxyanisole and ethoxyquin, Biochem Soc Trans 28:33-41). When forming a complex coactivator small Maf protein (SMAF), Nrf2 activates the expression of downstream target genes by binding to antioxidant response elements (AREs) located in their promoters (Itoh K, Chiba T, Takahashi S, Ishii T, Igarashi K, Katoh Y, Oyake T, HayashiN, Satoh K, Hatayama I, Yamamoto M, Nabeshima Y, (1997) An Nrf2/small Mafheterodimer mediated the induction of phase II detoxifying enzyme genesthrough antioxidant responsive elements.Biochemical and biochemical Research Communications 236:313-322).

The protein level activity of Nrf2 is mainly controlled by Keap1 (Itoh K, Mimura J, Yamamoto M (2010) Discovery of the negative regulator of Nrf2, Keap1: a historical overview. Antioxid Redox Signal 13:1665-1678). In the resting state, Keap1 acts as a substrate adapter for Cul3-based E3 ubiquitin ligase, which continuously promotes the ubiquitination of the target protein Nrf2 and then degrades Nrf2 (Itoh K, Wakabayashi N, Katoh Y, Ishii T, Igarashi K , Engel JD, Yamamoto M (1999) Keap1 represses nuclear activation of antioxidant responsive elements by Nrf2through binding to the amino-terminal Neh2domain. Genes Dev 13:76-86; Kobayashi A, Kang MI, Okawa H, Ohtsuji M, Zenke Y, Chiba T , Igarashi K, Yamamoto M (2004) Oxidative stress sensor Keap1 functions as an adapter for Cul3-based E3ligase to regulate proteasomal degradation of Nrf2. Mol Cell Biol 24:7130-7139). Under oxidative and electrical stress, Keap1, which has the function of stress signaling, loses the activity of interacting with Nrf2, thereby reducing the degradation of Nrf2. As a result, Nrf2 accumulates and translocates to the nucleus, upregulates the expression of antioxidant genes and reduces oxidative stress. The Nrf2-Keap1 system has been extensively studied as a strategy to treat drug toxicity and stress-induced conditions, such as carcinogenesis, inflammation, and neurodegenerative diseases (Suzuki T, Motohashi H, Yamamoto M (2013) Toward clinical application of the Keap1-Nrf2 pathway. Trends in pharmacological sciences 34:340-346). Recently, an Nrf2 activator, dimethyl fumarate, has been approved by the US Food and Drug Administration (FDA) for the treatment of multiple sclerosis (Fox RJ, Miller DH, Phillips JT, Hutchinson M, Havrdova E, Kita M, Yang M, Raghupathi K, Novas M, Sweetser MT, Viglietta V, Dawson KT, Investigators CS (2012) Placebo-controlled phase 3 study of oral BG-12or glatiramer in multiple sclerosis. The New England journal of medicine367:1087- 1097; Gold R, Kappos L, Arnold DL, Bar-Or A, Giovannoni G, Selmaj K, Tornatore C, Sweetser MT, Yang M, Sheikh SI, Dawson KT, Investigators DS (2012) Placebo-controlled phase 3 study of oral BG -12 for relapsing multiplesclerosis. The New England journal of medicine 367:1098-1107). Therefore, the discovery of new molecules that regulate the Nrf2-Keap2 pathway will provide new targets for the intervention and treatment of oxidative stress-related diseases.

PAQR3 is a member of the progesterone and adiponectin receptor (PAQR) family. PAQR3 is distributed on the Golgi apparatus in mammalian cells (Feng L, Xie X, Ding Q, Luo X, He J, Fan F, Liu W, Wang Z, Chen Y(2007) Spatial regulation of Raf kinase signaling by RKTG.Proc Natl Acad Sci USA104:14348-14353; Luo X, Feng L, Jiang X, Xiao F, Wang Z, Feng GS, Chen Y (2008) Characterization of the topology and functional domains of RKTG. The Biochemical journal 414:399-406) . The main physiological function of PAQR3 is to exert its tumor suppressor activity through the inhibition of Raf/MEK/ERK and AKT signaling pathway activity (Fan F, Feng L, He J, Wang X, Jiang X, Zhang Y, Wang Z, Chen Y(2008) RKTG sequesters B-Raf to the Golgi apparatus and inhibits the proliferation and tumorigenicity of human malignant melanomacells. Carcinogenesis 29:1157-1163; Feng L, Xie X, Ding Q, Luo X, He J, Fan F, Liu W , Wang Z, Chen Y (2007) Spatial regulation of Raf kinase signaling by RKTG. ProcNatl Acad Sci USA 104:14348-14353; Jiang Y, Xie X, Zhang Y, Luo X, Wang X, Fan F, Zheng D, Wang Z, Chen Y (2010) Regulation of G-protein signaling by RKTG viasequestration of the G betagamma subunit to the Golgi apparatus. Mol Cell Biol30:78-90; Wang X, Wang L, Zhu L, Pan Y, Xiao F, Liu W, Wang Z, Guo F, Liu Y, Thomas WG, Chen Y, (2013) PAQR3modulates insulin signaling by shunting phosphoinositide 3-kinase p110alpha to the Golgi apparatus. Diabetes 62:444-456).

In this study, we found that PAQR3 has the function of regulating the Nrf2-Keap1 signaling pathway through in vivo and in vitro experiments, and through this basic research, we found that the P1-20 polypeptide has a clear antioxidant function, which is useful for various oxidative stress-related diseases in the future. Disease intervention and treatment has potential value.

Contents of the invention

The present invention provides a polypeptide, which comprises a fragment of SEQ ID NO: 1, wherein the fragment of SEQ ID NO: 1 is located between amino acid residues 1 to 35 of SEQ ID NO: 1, and the fragment At least 15 amino acid residues in length.

In one or more embodiments, the fragment is at least 18 amino acid residues, preferably at least 20 amino acid residues in length.

In one or more embodiments, the fragment is located between amino acid residues 1 to 30 of SEQ ID NO:1.

In one or more embodiments, the fragment is located between amino acid residues 1 to 25 of SEQ ID NO:1.

In one or more embodiments, the fragment is located between amino acid residues 1 to 23 of SEQ ID NO:1.

In one or more embodiments, the fragment consists of amino acid residues 1 to 20 of SEQ ID NO: 1 (SEQ ID NO: 3).

In one or more embodiments, the polypeptide consists of said fragment of SEQ ID NO:1.

In one or more embodiments, the polypeptide further comprises a peptide that facilitates membrane penetration.

In one or more embodiments, the peptide that promotes membrane penetration is selected from the group consisting of: RQIKIWFQNRRMKWKK (SEQ ID NO:22), YGRKKRRQRRR (SEQ ID NO:23), KQAIPVAK-amide (SEQ ID NO:24), RRRRNRTRRNRRRVR-amide (SEQ ID NO:25), oligoarginine (R ₉ -R ₁₂ ), KLTRAQRRAAARKNKRNTRGC (SEQ ID NO:26), ALWKTLLKKVLKAPKKKRKVC (SEQ ID NO:27), RKKRRQRRR (SEQ ID NO:28), DAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO:27) ID NO:29), GWTLNSAGYLLGKINLKALAALAKKIL-amide (SEQ ID NO:30), AGYLLGKINLKALAALAKKIL-amide (SEQ ID NO:31), YTAIAWVKAFIRKLRK-amide (SEQ ID NO:32), etc.

In one or more embodiments, the polypeptide consists of a peptide that facilitates membrane penetration and the fragment of SEQ ID NO:1.

In one or more embodiments, the polypeptide is shown in SEQ ID NO:33.

The present invention also provides a coding polynucleotide sequence selected from:

(1) a polynucleotide sequence encoding a polypeptide of the present invention; and

(2) A complementary sequence to the polynucleotide sequence of (1).

The present invention also provides a nucleic acid construct, which contains the nucleotide sequence encoding the polypeptide of the present invention and can be used to express the polypeptide.

The present invention also provides a pharmaceutical composition, which contains the polypeptide of the present invention and a pharmaceutically acceptable carrier.

The present invention also provides the use of the polypeptide or the pharmaceutical composition of the present invention in the preparation of a medicament for treating or preventing a disease benefited from the activation of Nrf2 transcription factor.

In one or more embodiments, the disease benefiting from activation of the Nrf2 transcription factor is selected from cancer, neurodegenerative diseases and inflammatory diseases.

In one or more embodiments, the disease benefiting from the activation of Nrf2 transcription factor is selected from: breast cancer, skin cancer, cancer of the gastrointestinal tract and respiratory tract, colon cancer, gastric cancer, esophageal cancer, lung cancer, oral cancer, Pharyngeal, endometrial, and pancreatic cancers.

In one or more embodiments, the disease benefiting from the activation of the Nrf2 transcription factor is selected from pancreatic cancer, colon cancer and/or gastric cancer caused by the action of Helicobacter pylori.

In one or more embodiments, the disease benefiting from the activation of the Nrf2 transcription factor is an atopic disease, preferably selected from the group consisting of atopic rhinitis, conjunctivitis, dermatitis and asthma.

In one or more embodiments, the disease benefiting from the activation of Nrf2 transcription factor is a neurodegenerative disease selected from: multiple sclerosis (MS), such as relapsing remitting MS, secondary progressive MS, primary Episodic progressive MS, progressive relapsing MS; Amyotrophic lateral sclerosis (ALS); Alzheimer's disease; Parkinson's disease; Huntington's disease; acute hemorrhagic leukoencephalomyelitis; Hurst disease; encephalomyelitis , such as acute disseminated encephalomyelitis; optic neuritis; spinal cord injury; acute necrotizing myelitis; transverse myelitis; chronic progressive myelopathy; progressive multifocal leukoencephalopathy (PML); radiation myelopathy; HTLV- 1-related myelopathy; monophasic isolated demyelination; midpontine myelination; leucodystrophy, eg, adrenoleukodystrophy, heterochromatic leukodystrophy, Krabbe disease, Canavan disease, Alexander disease, Pelizaeus-Merbacher disease, vanishing white matter disease, and oculodenthal syndrome; and inflammatory demyelinating polyneuritis, such as chronic inflammatory demyelinating polyneuritis (CIDP) and acute inflammatory demyelinating polyneuritis Neuropathy (AIDP).

The present invention also provides the use of the polypeptide of the present invention, its coding sequence, and pharmaceutical composition in the preparation of medicines for treating or preventing diseases benefited from oxidative stress.

In one or more embodiments, the disease benefiting from oxidative stress is a disease associated with oxidative stress of the Nrf2-Keap1 pathway.

In one or more embodiments, the disease associated with the oxidative stress of the Nrf2-Keap1 pathway is selected from chronic nephritis, lung disease, diabetes, cardiovascular disease, neurodegenerative disease, tissue damage caused by toxic substances or carcinogens .

In one or more embodiments, the neurodegenerative disease is selected from the group consisting of motor neuron disease, amyotrophic lateral sclerosis (ALS) and primary lateral sclerosis (PLS), Huntington's disease, and age-related Macular degeneration.

The present invention also provides a method for activating Nrf2 pathway in vitro, said method comprising contacting the cells expressing Nrf2 pathway with a sufficient amount of the polypeptide of the present invention or a composition containing the polypeptide.

The present invention also provides the use of the polypeptides described herein for activating the Nrf2 pathway in vitro.

Description of drawings

Figure 1: Synthetic peptides that block PAQR3 function affect Nrf2 activity. A, B: P1-20 peptide reduces the interaction between PAQR3 and Nrf2 and Keap1. HEK293T cells were transiently transfected with the indicated plasmids. 24 hours after transfection, cells were treated with control peptide (20 μg/ml) or P1-20 peptide (20 μg/ml) for 12 hours, followed by immunoblotting (IB) analysis and immunoprecipitation (IP) analysis with the indicated antibodies. C: P1-20 peptide reduces ubiquitination of Nrf2. HEK293T cells were transiently transfected with Flag-tagged Nrf2 and HA-tagged ubiquitin. 24 hours after transfection, cells were treated with control peptide or P1-20 peptide for 12 hours and with MG132 for 6 hours. Cell lysates were subjected to immunoblot (IB) analysis and immunoprecipitation (IP) analysis with the indicated antibodies. D: P1-20 peptide reduces the content of active oxygen in cells. HepG2 cells were treated with control peptide and P1-20 peptide for 12 hours, and then treated with hydrogen peroxide (300 μM) for 12 hours, stained with dichlorofluorescein, and analyzed for intracellular reactive oxygen species by flow cytometry. E: P1-20 peptide increases the expression of Nrf2 target genes. HepG2 cells were treated with control peptide (20 μg/ml), P1-20 peptide (20 μg/ml) and tBHQ (100 μM) for 12 hours. Total RNA was isolated from cells and used for real-time RT-PCR. Values were normalized based on β-actin. Data are expressed as mean ± SD, * indicates p < 0.05, ** indicates p < 0.01, ns indicates not significant.

detailed description

Peptides and their coding sequences

The present invention relates to a polypeptide sequence, which comprises a fragment of SEQ ID NO: 1, which is located between the 1st and 35th positions of SEQ ID NO: 1, and has at least 15 amino acid residues in length.

As used herein, "isolated" means that the material is separated from its original environment (if the material is native, the original environment is the natural environment). For example, polynucleotides and polypeptides in the natural state in living cells are not isolated and purified, but the same polynucleotides or polypeptides are isolated and purified if they are separated from other substances that exist together in the natural state. "Isolated amino acid sequence" means that the amino acid sequence is substantially free of other proteins, lipids, carbohydrates or other substances with which it is naturally associated.

The term "fragment" refers to a polypeptide that consists of only a portion of the complete full-length polypeptide sequence and structure. The present invention relates to fragments of PAQR3. The amino acid sequence of PAQR3 is shown in SEQ ID NO:1. In particular, the present invention relates to an N-terminal fragment of PAQR3, specifically a fragment between amino acid residue 1 and amino acid 35 of SEQ ID NO: 1, preferably at least 15 amino acid residues in length, for example at least 18 amino acids residues, at least 10 amino acid residues, etc.

Preferably, the fragment at least contains the amino acid residues 1 to 15 of SEQ ID NO:1. Thus, the fragment may start at amino acid residue 1 of SEQ ID NO:1 and end at amino acid residue 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, Amino acid residue at position 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35. Preferably, the fragment consists of amino acid residues 1-20 of SEQ ID NO:1.

It should be understood that in gene cloning operations, it is often necessary to design appropriate restriction sites, which inevitably introduces one or more irrelevant residues at the end of the expressed amino acid sequence, which does not affect the activity of the target sequence. Another example is to construct a fusion protein, promote the expression of a recombinant protein, obtain a recombinant protein that is automatically secreted outside the host cell, or facilitate the purification of a recombinant protein, it is often necessary to add some amino acids to the N-terminal, C-terminal or the recombinant protein. Other suitable regions within the protein include, for example, but are not limited to, suitable linker peptides, signal peptides, leader peptides, terminal extensions, and the like. The amino-terminal or carboxy-terminal of the amino acid sequence of the present invention may also contain one or more polypeptide fragments as protein tags. Any suitable label can be used in the present invention. For example, the tag can be FLAG, HA, HA1, c-Myc, Poly-His, Poly-Arg, Strep-TagII, AU1, EE, T7, 4A6, ε, B, gE and Ty1. These tags can be used to purify proteins.

The polypeptide of the present invention may also contain a peptide segment promoting membrane penetration at the N-terminus of the fragment. Such peptides are well known in the art, see, for example, Siegmund Reissmann, Cell penetration: scope and limitations by the application of cell-penetrating peptides, J. Pept. Sci. 2014; 20:760-784. This article incorporates its entire content into this article by reference. In particular, the membrane-penetrating-promoting peptides suitable for the present invention include the sequences numbered 1-92 listed in Table 1 of the above literature. This article does not list them all here. Peptides that can also be used in the present invention to promote membrane penetration include RKKRRQRRR.

Therefore, the polypeptide of the present invention may consist of the fragment described in SEQ ID NO: 1, or may consist of the fragment described in SEQ ID NO: 1 and a suitable protein tag, or may consist of the fragment described in SEQ ID NO: 1 in combination with a The sequence composition of the membrane, or may consist of the fragment of SEQ ID NO: 1, a protein tag, and a sequence that facilitates membrane penetration.

The amino acid sequences of the present invention may be chemically synthesized products, or recombinant polypeptides produced from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, filamentous fungi, higher plants, insects, and mammalian cells) using recombinant techniques. Depending on the host used in the recombinant production protocol, the polypeptides of the invention may be glycosylated, or may be non-glycosylated.

A polynucleotide of the invention may be in the form of DNA or RNA. Forms of DNA include cDNA, genomic DNA or synthetic DNA. DNA can be single-stranded or double-stranded. DNA can be either the coding strand or the non-coding strand. The sequence of the coding region encoding the mature polypeptide may be the same as the DNA sequence shown in SEQ ID NO: 33 or a degenerate variant. As used herein, "degenerate variants" in the present invention refer to nucleic acid sequences that encode the same amino acid sequence but differ in nucleotide sequence.

The polypeptides and polynucleotides of the invention are preferably provided in isolated form, more preferably purified to homogeneity.

The nucleotide sequence of the present invention can usually be obtained by PCR amplification, recombination or artificial synthesis. For the PCR amplification method, primers can be designed according to the relevant nucleotide sequences disclosed in the present invention, especially the open reading frame sequence, and the cDNA prepared by a commercially available cDNA library or a conventional method known to those skilled in the art can be used. The library is used as a template to amplify related sequences. When the sequence is long, it is often necessary to carry out two or more PCR amplifications, and then splice together the amplified fragments in the correct order.

Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. Usually, it is cloned into a vector, then transformed into a cell, and then the relevant sequence is isolated from the proliferated host cell by conventional methods.

In addition, related sequences can also be synthesized by artificial synthesis, especially when the fragment length is relatively short. Often, fragments with very long sequences are obtained by synthesizing multiple small fragments and then ligating them.

At present, the DNA sequence encoding the amino acid sequence of the present invention can be obtained completely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or eg vectors) and cells known in the art.

The present invention also relates to a vector comprising the polynucleotide of the present invention, a host cell produced by genetic engineering using the vector of the present invention, and a method for producing the polypeptide of the present invention by recombinant technology. Preferably, the vector of the invention is an expression vector.

The polynucleotide sequences of the present invention can be used to express or produce the amino acid sequences of the present invention by conventional recombinant DNA techniques. Generally speaking, there are the following steps:

(1) Transform or transduce a suitable host cell with the polynucleotide of the present invention or its degenerate variant, or with a recombinant expression vector containing the polynucleotide;

(2) host cells cultured in a suitable medium;

(3) Separation and purification of protein from culture medium or cells.

The polynucleotide sequences of the present invention can be inserted into recombinant expression vectors. The term "recombinant expression vector" refers to bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus or other vectors well known in the art. Any plasmid and vector can be used as long as it can be replicated and stabilized in the host. An important feature of expression vectors is that they usually contain an origin of replication, a promoter, marker genes, and translational control elements. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

Methods well known to those skilled in the art can be used to construct expression vectors containing the nucleic acid sequences of the present invention and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology and the like. Said nucleic acid sequence can be operably linked to an appropriate promoter in the expression vector to direct mRNA synthesis. Representative examples of these promoters are: E. coli lac or trp promoter; lambda phage PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, reverse LTRs of transcription viruses and other promoters known to control the expression of genes in prokaryotic or eukaryotic cells or their viruses.

In addition, the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.

Vectors containing the above-mentioned appropriate DNA sequences and appropriate promoters or control sequences can be used to transform appropriate host cells so that they can express proteins.

The host cell may be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; a filamentous fungal cell, or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: Escherichia coli, Streptomyces spp; bacterial cells of Salmonella typhimurium; fungal cells such as yeast, filamentous fungi, plant cells; insect cells of Drosophila S2 or Sf9; CHO, COS, 293 cells, or Bowes black Tumor cells, animal cells, etc.

When the polynucleotide of the present invention is expressed in higher eukaryotic cells, if an enhancer sequence is inserted into the vector, the transcription will be enhanced. Enhancers are cis-acting elements of DNA, usually about 10 to 300 base pairs in length, that act on promoters to enhance gene transcription.

Those of ordinary skill in the art will know how to select appropriate vectors, promoters, enhancers and host cells.

Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as E. coli, competent cells capable of taking up DNA can be harvested after the exponential growth phase and treated with the _CaCl2 method using procedures well known in the art. Another method is to use _MgCl2 . Transformation can also be performed by electroporation, if desired. When the host is eukaryotic, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

The obtained transformant can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. The medium used in the culture can be selected from various conventional media according to the host cells used. The culture is carried out under conditions suitable for the growth of the host cells. After the host cells have grown to an appropriate cell density, the selected promoter is induced by an appropriate method (such as temperature shift or chemical induction), and the cells are cultured for an additional period of time.

The recombinant polypeptide in the above method can be expressed inside the cell, or on the cell membrane, or secreted outside the cell. The recombinant protein can be isolated and purified by various separation methods by taking advantage of its physical, chemical and other properties, if desired. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitating agents (salting out method), centrifugation, osmotic disruption, supertreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

Diseases associated with activation of the Nrf2 transcription factor or with oxidative stress of the Nrf2-Keap1 pathway

Diseases associated with activation of Nrf2 transcription factors (or diseases benefiting from activation of Nrf2 transcription factors or activation of Nrf2 pathways) or diseases associated with oxidative stress of Nrf2-Keap1 pathways are well known in the art, for example in CN200980149899.0 , CN201280023826.9, CN201410077763.6 and CN201380073599.5, etc., the entire contents of these documents are incorporated herein by reference.

Specifically, Nrf2 (nuclear factor erythroid 2-related factor 2, AAB32188), a transcription factor, forms a heterodimer with the small protein Maf upon activation by oxidative stress, binds the antioxidant response element (ARE), and activates Nrf2 Regulated transcription of genes. The Nrf2/ARE pathway, a major determinant of phase II gene induction, has been well characterized for its role in hepatic detoxification and chemoprevention through activation of phase II gene expression. ARE-regulated genes may also contribute to the maintenance of redox homeostasis by acting as endogenous antioxidant systems. Currently, the list of Nrf2-regulated genes contains more than 200 genes encoding proteins and enzymes involved in detoxification and antioxidant responses (Kwak et al., J. Biol. Chem., 2003, 278:8135), such as glutathione peroxidation enzymes, glutathione-S-transferases (GSTs), NAD(P)H:quinone oxidoreductase, NQO2, g-glutamylcysteine synthase (g-GCS), glucuronyl Transferase, ferritin and heme oxygenase-1 (HO-1), etc.

Under basal conditions, Nrf2 sequesters actin-bound Kelch-like ECH-binding protein 1 (Keap1; Accession No. NP_987096), the Cullin3 ubiquitin ligase adapter protein, in the cytoplasm. More specifically, the N-terminal domain of Nrf2, called the Neh2 domain, interacts with the C-terminal Kelch-like domain of Keap1. In response to xenobiotics or oxidative stress, Nrf2 is released from the Keap1/Nrf2 complex, thereby promoting the nuclear translocation of Nrf2 and the concomitant ARE-mediated activation of gene transcription.

Nrf2 pathway activation has demonstrated protective benefits in several neurodegenerative disease models. See, eg, Calkins MJ et al., Toxicol Sci 2010; 115:557-568. Neurodegenerative diseases include multiple sclerosis (MS) (eg, relapsing remitting MS, secondary progressive MS, primary progressive MS, progressive relapsing MS), amyotrophic lateral sclerosis (ALS), Al Alzheimer's disease, Parkinson's disease and Huntington's disease. Other examples of neurodegenerative diseases include acute hemorrhagic leukoencephalomyelitis, Hurst's disease, encephalomyelitis (eg, acute disseminated encephalomyelitis), optic neuritis, spinal cord injury, acute necrotizing myelitis, transverse myelitis, chronic Progressive myelopathy, progressive multifocal leukoencephalopathy (PML), radiation myelopathy, HTLV-1-related myelopathy, monophasic isolated demyelination, midpontine myelination, leukodystrophy (eg, adrenal white matter Malnutrition, heterochromatic leukodystrophy, Krabbe disease, Canavan disease, Alexander disease, Pelizaeus-Merbacher disease, vanishing white matter disease, oculodactyly syndrome), inflammatory demyelinating polyneuritis (eg, chronic Inflammatory Demyelinating Polyneuritis (CIDP) and Acute Inflammatory Demyelinating Polyneuropathy (AIDP)), Guillain-Barré Syndrome (GBS), Polyneuritis, Myasthenia Gravis (MG), Eaton Lambert syndrome (ELS) and encephalomyelitis.

Nrf2 pathway activation is also beneficial in the treatment and prevention of diseases including idiopathic pulmonary fibrosis (IPF), scleroderma lung disease, acute lung injury (ALI)/acute respiratory distress (ARDS), asthma (such as chronic asthma), Radiation-induced fibrotic sarcoidosis, pulmonary hypertension, bronchopulmonary dysplasia (BPD), lung transplant rejection, pulmonary GVHD complications, interstitial pneumonia syndrome (IPS) in transplant recipients, COPD, Silicosis, asbestosis, sarcoidosis (lung), primary sclerosing cholangitis (PSC), alcohol-induced hepatic fibrosis, autoimmune hepatitis, chronic viral hepatitis (HepB,C), primary biliary Liver cirrhosis (PBC), nonalcoholic steatohepatitis (NASH), liver transplant rejection, liver complications of GVHD, veno-occlusive disease in transplant recipients, focal segmental glomerulosclerosis (FSGS), Diabetic nephropathy, IgA nephropathy, scleroderma, renal complications of GVHD (AKI delays graft function), acute renal failure after CABG (AKI after CABG), lupus nephritis, hypertension-induced renal fibrosis, HIV-associated Renal disease, peritoneal dialysis-induced peritoneal fibrosis, retroperitoneal fibrosis, idiopathic glomerulosclerosis, renal transplant rejection, Alport syndrome, restenosis, subarachnoid hemorrhage (SAH), cardiac transplant rejection, Stroke, cosmetic surgery, chronic trauma, burns, surgical adhesions, keloids, donor graft re-epithelialization, myelofibrosis, corneal transplantation, systemic sclerosis, radiation-induced fibrosis, peripatellar fibrosis, Dupuytren's contracture , diabetes, atopic rhinitis, conjunctivitis, dermatitis, etc.

Diseases associated with the activation of Nrf2 transcription factors also include cancers, preferably selected from the group consisting of breast cancer; skin cancer; gastrointestinal and respiratory tract cancers, such as colon cancer, gastric cancer, esophageal cancer; lung cancer; oral cavity cancer; pharyngeal cancer ; endometrial cancer and pancreatic cancer. More preferably, it is used for the prevention and treatment of pancreatic cancer, colon cancer and/or gastric cancer caused by the action of Helicobacter pylori.

On the other hand, diseases associated with oxidative stress of the Nrf2-Keap1 pathway include but are not limited to: motor neuron disease, amyotrophic lateral sclerosis, primary lateral sclerosis, Huntington's disease, Alzheimer's disease , Parkinson's disease, and age-related macular degeneration.

pharmaceutical composition

The present invention provides a pharmaceutical composition comprising a polypeptide of the present invention and a pharmaceutically acceptable excipient (eg, carrier).

The therapeutically or prophylactically effective amount of the polypeptide of the present invention may be contained in the pharmaceutical composition. "Effective amount" means an amount of an ingredient sufficient to produce the desired response. The specific effective amount depends on factors such as the particular condition to be treated, the physical condition of the patient (such as the patient's weight, age, or sex), the duration of treatment, co-administered therapies (if any), and the specific drug used. formula. "Effective amount" also means that under this dosage, the toxicity or negative effect of the polypeptide of the present invention is less than the positive therapeutic effect brought by it.

Pharmaceutically acceptable excipients are generally safe, non-toxic, and can broadly include any known substances used in the pharmaceutical industry for the preparation of pharmaceutical compositions, such as fillers, diluents, coagulants, binders, lubricants agent, glidant, stabilizer, colorant, wetting agent, disintegrant, etc. The mode of administration of the pharmaceutical composition should be considered primarily in the selection of suitable excipients for the delivery of synthetic peptides, which are well known to those skilled in the art. Examples of suitable carriers include, but are not limited to, magnesium carbonate, magnesium stearate, croscarmellose sodium, microcrystalline cellulose, talc, silicon dioxide, sugar, lactose, pectin, dextrin, starch, gelatin, starch, Tragacanth gum, methylcellulose, sodium carboxymethylcellulose, low melting point wax, cocoa butter, etc.

The content of the polypeptide in the pharmaceutical composition of the present invention is about 1-1000 μM; preferably about 10-500 μM; more preferably about 25-250 μM. For example, the concentration of the polypeptide can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, or 1,000 μM.

The above-mentioned pharmaceutical composition can be prepared according to known pharmaceutical procedures, such as "Remington's Pharmaceutical Science" (17th edition, edited by Alfonoso R. Gennaro, Mike Publishing Company, Easton, Pennsylvania (1985)). Detailed record.

The pharmaceutical composition of the present invention can be in various suitable dosage forms, including but not limited to capsules, injections and the like. The polypeptide of the present invention or its pharmaceutical composition can be administered through conventional routes of administration, including but not limited to oral, intranasal, transdermal, subcutaneous, intradermal, vaginal, ear, intraocular, intramuscular, intramuscular, Buccal, rectal, transmucosal or via inhalation or intravenous administration, etc.

method and use

The present invention includes using the polypeptide of the present invention or its pharmaceutical composition to treat, prevent or improve various diseases related to the activation of Nrf2 transcription factor or oxidative stress related diseases related to Nrf2-Keap1 pathway as mentioned above.

Therefore, the present invention includes methods for treating, preventing or improving various diseases benefiting from activation of Nrf2 transcription factor or activation of Nrf2 pathway or diseases related to oxidative stress of Nrf2-Keap1 pathway as described above, said method comprising An effective amount of the polypeptide of the present invention or a pharmaceutical composition thereof is administered to a subject in need thereof. An effective amount is an amount effective to achieve its intended purpose (cure, prevent or ameliorate the disease). The actual amount effective for a particular application will depend, inter alia, on the condition to be treated.

The dosage and frequency of administration (single or multiple doses) may vary according to various factors, including the route of administration; age, sex, state of health, weight of the recipient, nature and severity of the disease to be treated the extent of other diseases or other health-related problems; the type of concomitant treatment; and complications from any disease or treatment regimen.

A "subject" refers to a mammal that would benefit from administration of a composition or method described herein. A subject usually refers to a person.

The polypeptide of the present invention or its pharmaceutical composition can be administered together with drugs known in the art for the treatment or prevention of the various diseases mentioned above. For example, it can be administered with drugs to treat MS, including but not limited to azathioprine, prednisolone, mycophenolate mofetil, mitoxantrone, teriflunomide, piroxicam, and phenidone; Given with drugs for ALS, including riluzole and dpramipexole; may be given with drugs for Alzheimer's disease, such as rosiglitazone, vitamin E, donepezil, tacrine , Rastigmine, Galantamine, and Memantine; and others. Co-administration includes simultaneous administration and sequential administration.

The present invention therefore also includes the polypeptides of the present invention, their coding sequences, expression vectors, and pharmaceutical compositions containing the polypeptides of the present invention for the treatment, improvement or prevention of various activations of Nrf2 transcription factors or Nrf2 pathways as described above. Drug application in diseases of activation or diseases associated with oxidative stress of Nrf2-Keap1 pathway.

The invention also includes a method of activating the Nrf2 pathway comprising contacting a cell with a sufficient amount of a polypeptide of the invention or a composition comprising the polypeptide. The method can be an in vitro method. The cell can be any cell expressing the Nrf2 pathway.

In order that the invention described herein may be more fully understood, the following examples are set forth. It should be understood that these examples are for illustrative purposes only and should not be construed as limiting the invention in any way.

Materials and Methods:

cell culture

DMEM (Dulbecco's Modified Eagle's Medium) (Gibco), fetal bovine serum (Fetal Bovineserum (Hyclone), Trypin-EDTA, penicillin-streptomycin (Invitrogen), DMSO (Merck), PEI (Polyethyleneimine, branched, MW-25,000), Polybrene (Sigma), Hochest 33342 (Molecular Probes, Eugene, OR), transfection reagent PolyJet ^TM DNA In Vitro Transfection Reagent (SignaGen Laboratories), cell culture dishes of various specifications, cryopreservation tubes, and centrifuge tubes (Corning).

Molecular Biology Reagents

Plasmid extraction kit and PCR gel recovery kit are products of Tiangen Company; proteinA/GPLUS Agarose are products of Santa Cruz Company; Protease inhibitor cocktail, phosphotase inhibitor cocktail are products of Pierce Company; PVDF membrane, 0.22μm and 0.45μm filters are Millipore Products of the company; imported EP tubes and gun tips are products of Axygen; Collagenase I is a product of Worthington (Lakewood, NJ); Trizolregeant, AdEasy ^TM Adenoviral Vector System is a product of Stragagen; BLOCK-iT ^TM Adenoviral RNAi Expression System is a product of Invitrogene ; AMV-reverse transcriptase, various restriction endonucleases, T4DNA ligase, Taq DNA polymerase and dNTP were purchased from TaKaRa Company; nuclear and cytoplasmic protein extraction kits were Sangon Bioengineering (Shanghai) Co., Ltd.; General medicines were purchased from Sebastian.

instrument

Micropipette (Gilson), Refrigerator (Siemens), Ultra-low Temperature Refrigerator (Nuair), Vertical Electrophoresis Tank, Wet Transfer Apparatus and Power/PAC200 Supporting Power Supply (Bio-Rad), 5415D Desktop High Speed Centrifuge (Eppendorf), 5417R refrigerated desktop centrifuge (Eppendorf), Avanti J-20XP refrigerated high-speed centrifuge (Beckman), PCR instrument (Eppendorf and Bio-Rad), L MaxⅡ 384 fluorescent chemiluminescence microplate reader (Berthold), low-speed refrigerated centrifugation machine (Heraeus), Ultrospec2100pro UV spectrophotometer (Amersham), sterile clean bench (Thermo), DolphinDoc image analysis system (Wealtec), Chemi Doc image analysis system (Bio-Rad), electric homogenizer (IKA), AL104 precision balance (Mettler Toledo), horizontal electrophoresis tank and supporting power supply (Furi Technology), constant temperature incubator (Medcenter), cell culture incubator (Thermo), liquid nitrogen tank (Thermolyne), inverted microscope (Olympus), desktop vacuum pump , sealing machine, decolorizing shaker, JT502N analytical balance, mixer, Hit-block, ultrasonic pulverizer, constant temperature water bath, PH meter, sterile ultra-clean bench (made in China).

Primer sequence

Construction of recombinant plasmids

Human full-length Nrf2, Keap1, MafG, MafK, and MafF were all reverse-transcribed from cDNA of HEK293T cells, and all clones were sequenced.

RNA extraction, reverse transcription, RT-PCR and fluorescent quantitative PCR

Extraction of RNA: Take an appropriate amount of cells and lyse them with 1ml Trizol reagent, place at room temperature for 5-10 minutes, add 0.2ml chloroform, shake vigorously for 15 seconds, let stand at room temperature for 3 minutes at 12000g, centrifuge at 4°C for 15 minutes, and take the supernatant Put in another new centrifuge tube, add 0.5ml of isopropanol and let it stand at room temperature for 10 minutes, then centrifuge at 12000g at 4°C for 10 minutes, remove the supernatant, wash the precipitate with 75% alcohol two to three times, dry the residual ethanol, and dissolve the precipitate in Appropriate amount of DEPC water, use Nano-Drop to test the purity and concentration.

RT-PCR: Take 1 μg of total RNA for reverse transcription, apply AMV reverse transcriptase from Takara Company, perform reverse transcription according to its instructions, and obtain cDNA for subsequent PCR detection. See Materials and Methods for PCR primer sequences.

For the operation method of fluorescent quantitative PCR, refer to the instruction manual of TOYOBO Realtime PCR Master Mix.

cell culture

HEK293 and HepG2 were cultured in Dulbecco's modified Eagle's medium (DMEM) pH 7.2+10% fetal bovine serum (FBS), plus 100 μg/ml penicillin and 100 μg/ml streptomycin. The culture conditions were 37°C, 5% CO ₂ .

transient transfection of cells

HEK293T was transfected with polyethyleneimine (PEI) method. The day before transfection, the cells were divided into plates at an appropriate density, and when the cells grew to about 80%, they were replaced with serum-free and antibiotic-free medium, and then transfected by PEI method. For every 1 μg of DNA during transfection, add 2 μl of PEI and mix well, let it stand at room temperature for 20 minutes, add it to the culture dish, and change the medium after 6 hours. Cells were harvested 24 to 48 hours after transfection for HEK293T.

HepG2 was transfected by the liposome PolyJet method (SignaGen Laboratories, Rockville, MD, USA). The transfection method is detailed in the instruction manual, and harvested 36 hours to 48 hours after transfection.

Protein extraction and western blotting

Protein extraction: the cultured cells were washed twice with pre-cooled PBS, the PBS was aspirated, and an appropriate volume of denatured RIPA lysate (150mM NaCl, 10mM Tris pH7.2, 0.1% SDS, 1% Triton X‐100, 1% deoxycholic acid, 5mM EDTA), scrape off and transfer to EP tube, place on ice, centrifuge at 12,000g for 15 minutes at 4°C, take the supernatant, and measure protein by Bradford method if necessary concentration. Add 4×SDS sample buffer + 50 μM DTT, cook at 100°C for 10 minutes, load immediately or store at -20°C.

Western blotting: First, according to the molecular weight of the protein to be checked, match the appropriate concentration of discontinuous denatured polyacrylamide gel, about 30ug for cell samples, and about 50ug for tissue samples for Western blot analysis. Before transferring the membrane, soak the PVDF membrane in methanol for 15 seconds, then install it in the order of anode-sponge-filter paper-PVDF membrane-glue-filter paper-sponge-cathode, put it in a wet transfer membrane apparatus, cover it with ice, and transfer it at 350mA 60-120 minutes, depending on the molecular weight. After the membrane transfer, cut according to the molecular weight shown by the marker, block with 5% skimmed milk or 3% BSA for 1 hour, and spend 4 nights with the primary antibody. On the second day, wash with TBST for 3×5 minutes, apply the secondary antibody at room temperature for 1-2 hours, wash with TBST for 3×5 minutes, develop color with ECL and press.

Co-immunoprecipitation (co-IP)

Generally, a 6cm cell culture dish is used. Place the cell culture dish on ice, carefully wash twice with ice-cold PBS, blot dry the PBS, add co-IP buffer (150mM NaCl, 120mM Tris pH7.5, 1% NP-40, 5mM EDTA) to The protein concentration is about 1 μg/μl. Scrape the cells from the culture dish into an ice-precooled centrifuge tube, rotate and lyse at 4°C for 30 minutes, centrifuge at 12000×rpm for 10 minutes, take the supernatant to a new EP tube, take 30 μl and add SDS loading buffer The solution was boiled at 100°C for 5 minutes, and frozen at -20°C as input. The remaining supernatant was added with the corresponding antibody at a concentration of 1 μg/ml, and rotated overnight at 4°C. On the next day, add 40-60 μl protein A/g beads per ml of cell lysate, rotate at 4°C for 3 hours, centrifuge at 5,000×g for 30 seconds at 4°C, remove the supernatant, add 1ml co-IP lysis buffer to wash the beads, The same centrifugation conditions were repeated 3 times (the number of times can be adjusted according to the needs of the experiment). Finally, add 30 μl of co-IP buffer and SDS loading buffer, denature the protein at 100°C for 5 minutes, and run the gel together with the input for Western blotting.

Statistical analysis

The analysis of the experimental data was performed by a two-tailed Student's t-test, and all statistical data were expressed as mean ± standard error.

result:

P1-20 polypeptide blocks the interaction of PAQR3 with Nrf2 and Keap1, and promotes the expression of cellular antioxidant genes:

The Nrf2 pathway is currently a major therapeutic target in oxidative stress-related diseases. Based on the negative regulatory function of PAQR3 on the Nrf2 pathway discovered in the present invention, the inventors speculate that blocking the interaction between PAQR3 and Nrf2 may lead to the activation of the Nrf2 pathway.

The present invention found that the N-terminal 20 amino acid residues of PAQR3 were involved in the interaction between Nrf2 and Keap1. Based on this discovery, the present invention designed a synthetic peptide (P1-20), which covered the N-terminal 1-20 of PAQR3. Amino acid residues (MHQKLLKSAHYIELGSYQYW). A Tat sequence (RKKRRQRRR) was added to the N-terminus of the peptide to facilitate cell penetration.

Through co-immunoprecipitation experiments, the present invention found that the P1-20 polypeptide can block the interaction between PAQR3 and Nrf2 and Keap1, while the control peptide RKKRRQRRR has no function (Fig. 1, A and B), indicating that the P1-20 polypeptide can destroy the interaction between PAQR3 and Nrf2 and Keap1. These two proteins interact. At the same time, P1-20 was also able to reduce the ubiquitination of Nrf2 (Fig. 1, C).

HepG2 cells were treated with control peptide and P1-20 peptide for 12 hours, then treated with hydrogen peroxide (300 μM) for 12 hours, stained with dichlorofluorescein, and analyzed by flow cytometry for intracellular reactive oxygen species. It was found that the P1-20 peptide can reduce the content of reactive oxygen species in cells (Fig. 1, D)

The effect of P1-20 on the expression of Nrf2 target genes was next analyzed. The P1-20 peptide was able to significantly increase the expression of multiple Nrf2 target genes both background and tBHQ activated, whereas the control peptide had no effect (Fig. 1, E).

Therefore, these data suggest that the P1-20 polypeptide can effectively activate the activity of Nrf2 and promote the antioxidant function of cells by blocking the interaction between PAQR3 and Nrf2-Keap1.

Claims (10)

1. A polypeptide, characterized in that, the polypeptide contains a fragment of SEQ ID NO: 1, or consists of a fragment of SEQ ID NO: 1; Wherein, the fragment of SEQ ID NO: 1 is located between the 1st to 35th amino acid residues of SEQ ID NO: 1, preferably between the 1st to 30th amino acid residues of SEQ ID NO: 1; with The fragment of SEQ ID NO: 1 is at least 15 amino acid residues in length, preferably at least 18 amino acid residues in length, more preferably at least 20 amino acid residues in length. 2. The polypeptide according to claim 1, wherein the fragment of said SEQ ID NO: 1 is located between the 1st and 25th amino acid residues of SEQ ID NO: 1, more preferably located in SEQ ID NO: 1 between amino acid residues 1 to 23, more preferably composed of amino acid residues 1 to 20 of SEQ ID NO: 1 (SEQ ID NO: 3). 3. The polypeptide according to claim 1 or 2, wherein the polypeptide also contains a peptide that promotes membrane penetration, preferably, the peptide that promotes membrane penetration is selected from: RQIKIWFQNRRMKWKK; YGRKKRRQRRR; KQAIPVAK-amide; RRRRNRRTRRNRRRVR-amide; Oligoarginine (R ₉ -R ₁₂ ); KLTRAQRRAAARKNKRNTRGC; ALWKTLLKKVLKAPKKKRKVC; RKKRRQRRR; DAATATRGRSAASRPTERPRAPARSASRPRRPVE; GWTLNSAGYLLGKINLKALAALAKKIL-amide; AGYLLGKINLKALAALAKKIL-amide; and YTAIAWVKAFIRKLRK-amide. 4. The polypeptide according to any one of claims 1-3, wherein the polypeptide consists of a peptide that promotes membrane penetration and the fragment of SEQ ID NO: 1, preferably, the amino acid sequence of the polypeptide As shown in SEQ ID NO:33. 5. A polynucleotide sequence selected from the group consisting of: (1) a polynucleotide sequence encoding the polypeptide of any one of claims 1-4; and (2) A complementary sequence to the polynucleotide sequence described in (1). 6. A pharmaceutical composition, characterized in that the pharmaceutical composition contains the polypeptide according to any one of claims 1-4 and a pharmaceutically acceptable carrier. 7. Use of the polypeptide according to any one of claims 1-4 or the pharmaceutical composition according to claim 6 in the preparation of a medicament for the treatment or prevention of diseases benefited from oxidative stress. 8. The use according to claim 7, wherein the disease benefiting from oxidative stress is a disease related to oxidative stress in the Nrf2-Keap1 pathway, preferably selected from chronic nephritis, lung disease, diabetes, cardiovascular Tissue damage caused by disease, neurodegenerative disease, toxic substances or carcinogens; preferably, said neurodegenerative disease is selected from: motor neuron disease, amyotrophic lateral sclerosis (ALS) and primary lateral sclerosis (PLS), Huntington's disease, and age-related macular degeneration. 9. Use of the polypeptide according to any one of claims 1-4 or the pharmaceutical composition according to claim 6 in the preparation of a medicament for the treatment or prevention of diseases benefited from the activation of Nrf2 transcription factor. 10. The use according to claim 9, wherein the disease benefiting from activation of the Nrf2 transcription factor is selected from cancer, neurodegenerative diseases and inflammatory diseases; Preferably, the disease benefiting from the activation of Nrf2 transcription factor is selected from the group consisting of: breast cancer, skin cancer, cancer of the gastrointestinal tract and respiratory tract, colon cancer, gastric cancer, esophageal cancer, lung cancer, oral cavity cancer, pharyngeal cancer, endometrial cancer Carcinoma and pancreatic cancer; The disease benefiting from the activation of the Nrf2 transcription factor is an atopic disease, preferably selected from the group consisting of atopic rhinitis, conjunctivitis, dermatitis and asthma; Said disease benefiting from activation of the Nrf2 transcription factor is a neurodegenerative disease selected from the group consisting of: multiple sclerosis (MS), e.g. relapsing remitting MS, secondary progressive MS, primary progressive MS, progressive relapsing Amyotrophic lateral sclerosis (ALS); Alzheimer's disease; Parkinson's disease; Huntington's disease; acute hemorrhagic leukoencephalomyelitis; Hurst's disease; encephalomyelitis, such as acute disseminated encephalomyelitis ; optic neuritis; spinal cord injury; acute necrotizing myelitis; transverse myelitis; chronic progressive myelopathy; progressive multifocal leukoencephalopathy (PML); radiation myelopathy; HTLV-1-related myelopathy; monophasic isolated Demyelination; midpontine myelination; leucodystrophy, eg, adrenoleukodystrophy, heterochromatic leukodystrophy, Krabbe disease, Canavan disease, Alexander disease, Pelizaeus-Merbacher disease, vanishing leukodystrophy and eye-dentate syndrome; and inflammatory demyelinating polyneuritis, such as chronic inflammatory demyelinating polyneuritis (CIDP) and acute inflammatory demyelinating polyneuropathy (AIDP).