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

Abstract

The present invention relates to the novel oxidation-resistant polypeptide based on PAQR3 functions, it is prepared and purposes.Specifically, the present invention provides a kind of polypeptide, and the polypeptide contains SEQ ID NO:1 fragment, or by SEQ ID NO:1 fragment composition；Wherein, the SEQ ID NO:1 fragment is located at SEQ ID NO:1 the 1st between the 35th amino acids residue, being preferably placed at SEQ ID NO:1 the 1st between the 30th amino acids residue；With the SEQ ID NO:1 piece segment length at least 15 amino acid residues, preferably long at least 18 amino acid residues, more preferably long at least 20 amino acid residues.The invention further relates to the coded sequence of the polypeptide, nucleic acid constructs, and pharmaceutical composition, and associated uses.

Description

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

Technical field

The present invention relates to the novel oxidation-resistant polypeptide based on PAQR3 functions, it is prepared and purposes.

Background technology

All organisms are all constantly by from either internally or externally (as from poisonous substance, exogenous material, heavy metal And ionising radiation) oxidative pressure challenge.Above-mentioned substance causes macromolecular by producing active oxygen (ROS) and electrophilic reagent Damage, DNA mutation, Apoptosis and cell transformation, so as to produce injury to cell and body.Therefore, cell has a set of Protection system mitigates these pressure.In mammalian cell, transcription factor Nrf2 (nuclear factor red blood cell 2- correlation factors 2) Leucine zipper (bZIP) albumen basic as one, play in the expression of removing toxic substances and antioxidant genes is coordinated and most attached most importance to Effect (Moi P, Chan K, Asunis I, Cao A, Kan YW, (1994) Isolation of NF-E2-related wanted Factor 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-Nrf2system, Free radical biology＆medicine).Nrf2 by Anti-oxidant and detoxification genes transcriptional activations are played key effect (Suzuki＆ by active oxygen and/or electrophilic reagent activation Yamamoto, 2015, ibid), as exogenous metabolism enzyme (NAD (P) H quinone oxidoreductases -1 (NqoI) (Venugopal R, Jaiswal AK(1996)Nrf1 and Nrf2 positively and c-Fos and Fra1 negatively regulate the human antioxidant response element-mediated expression of NAD(P) H:Quinone oxidoreductase1 gene, Proc Natl Acad Sci USA 93:14960-14965), ferroheme Oxygenase -1 (HO-1) cracking ferroheme formation 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 chemistry 274:26071-26078), Glutamate-cysteine ligase (GCLC) and its modifying agent 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 glutathione S-transferase (GST) family catalysis GSH is total to endogenous and exogenous electrophilic reagent Yoke (Hayes JD, Chanas SA, Henderson CJ, McMahon M, Sun C, Moffat GJ, Wolf CR, Yamamoto M(2000)The Nrf2transcription factor contributes both to the basal 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 the one small Maf albumen of the complicated co-activation factor of formation (SMAF) when, Nrf2 activates these genes by being combined with the Antioxidant responsive element (ARE) positioned at downstream target gene promoter Expression (Itoh K, Chiba T, Takahashi S, Ishii T, Igarashi K, Katoh Y, Oyake T, Hayashi N, Satoh K, Hatayama I, Yamamoto M, Nabeshima Y, (1997) An Nrf2/small Maf heterodimer mediates the induction of phase II detoxifying enzyme genes through antioxidant response elements.Biochemical and biophysical research communications 236:313-322)。

Nrf2 protein level activity mainly controls (Itoh K, Mimura J, Yamamoto M (2010) by Keap1 Discovery of the negative regulator of Nrf2, Keap1:a historical overview.Antioxid Redox Signal 13:1665-1678).Under quiescent condition, Keap1 is as Cul3- bases E3 ubiquitin ligases substrate adapter, constantly promote target protein Nrf2 ubiquitination so the Nrf2 that degrades (Itoh K, Wakabayashi N, Katoh Y, Ishii T, Igarashi K, Engel JD, Yamamoto M (1999) Keap1represses 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 Keap1functions as an adaptor for Cul3-based E3 ligase to regulate proteasomal degradation of Nrf2.Mol Cell Biol 24:7130- 7139).Aoxidize and electric stress under, have stress semiotic function Keap1, lose with Nrf2 interaction activity, from And reduce Nrf2 degraded.As a result, Nrf2 accumulates and is transferred on nucleus, raises the expression of antioxidant genes while mitigates Oxidative pressure.Nrf2-Keap1 systems are as a kind of medicine toxicity and pressure induced conditions, such as canceration, inflammation and nerve The strategy of degenerative disease has been widely studied (Suzuki T, Motohashi H, Yamamoto M (2013) Toward clinical application of the Keap1-Nrf2pathway.Trends in pharmacological sciences 34:340-346).Recently, Nrf2 activator dimethyl fumarate, it has obtained U.S.'s food and medicine Management board (FDA) ratifies, treatment (Fox RJ, Miller DH, Phillips JT, Hutchinson for multiple sclerosis M, Havrdova E, Kita M, Yang M, Raghupathi K, Novas M, Sweetser MT, Viglietta V, Dawson KT, Investigators CS (2012) Placebo-controlled phase 3study of oral BG- 12or glatiramer in multiple sclerosis.The New England journal of medicine 367:1087-1097；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 3study of oral BG-12for relapsing multiple sclerosis.The New England journal of medicine 367:1098-1107).Thus, it is found that new tune The molecule of section Nrf2-Keap2 paths will provide new target spot for the intervention of oxidative stress relevant disease and treatment.

PAQR3 is a member of progesterone and adiponectin receptor (PAQR) family.PAQR3 is distributed in height in mammalian cell (Feng L, Xie X, Ding Q, Luo X, He J, Fan F, Liu W, Wang Z, Chen Y (2007) on dictyosome Spatial regulation of Raf kinase signaling by RKTG.Proc Natl Acad Sci USA 104:14348-14353；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).PAQR3 main Physiological Function is by Raf/MEK/ERK and AKT Signal path activity suppression come play the activity of its tumor suppression (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 melanoma cells.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.Proc Natl 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 via sequestration of the G betagamma subunit to the Golgi apparatus.Mol Cell Biol 30:78-90；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 our current research, we have found that there is PAQR3 regulation Nrf2-Keap1 signals to lead to by internal and external experiment The function on road, and find that P1-20 polypeptides have clear and definite anti-oxidation function by the basic research, for following oxidative stress phase The intervention and treatment for a variety of diseases closed have potential value.

The content of the invention

The present invention provides a kind of polypeptide, and the polypeptide contains SEQ ID NO:1 fragment, wherein, the SEQ ID NO:1 piece Section is located at SEQ ID NO:1 the 1st between the 35th amino acids residue, and piece segment length at least 15 amino acid residues.

In one or more embodiments, piece segment length at least 18 amino acid residues, preferably at least 20 amino acid Residue.

In one or more embodiments, the fragment is located at SEQ ID NO:1 the 1st to the 30th amino acids residue it Between.

In one or more embodiments, the fragment is located at SEQ ID NO:1 the 1st to the 25th amino acids residue it Between.

In one or more embodiments, the fragment is located at SEQ ID NO:1 the 1st to the 23rd amino acids residue it Between.

In one or more embodiments, the fragment is by SEQ ID NO:1 the 1st to the 20th amino acids residue (SEQ ID NO:3) form.

In one or more embodiments, the polypeptide is by SEQ ID NO:1 fragment composition.

In one or more embodiments, the polypeptide also wears the peptide of film containing promotion.

In one or more embodiments, the peptide for promoting to wear film is selected from：RQIKIWFQNRRMKWKK(SEQ ID NO:22)、YGRKKRRQRRR(SEQ ID NO:23), KQAIPVAK- acid amides (SEQ ID NO:24)、RRRRNRTRRNRRRVR- Acid amides (SEQ ID NO:25), few arginine (R₉- R₁₂)、KLTRAQRRAAARKNKRNTRGC(SEQ ID NO:26)、 ALWKTLLKKVLKAPKKKRKVC(SEQ ID NO:27)、RKKRRQRRR(SEQ ID NO:28)、 DAATATRGRSAASRPTERPRAPARSASRPRRPVE(SEQ ID NO:29)、GWTLNSAGYLLGKINLKALAALAKKIL- Acid amides (SEQ ID NO:30), AGYLLGKINLKALAALAKKIL- acid amides (SEQ ID NO:31)、YTAIAWVKAFIRKLRK- Acid amides (SEQ ID NO:32) etc..

In one or more embodiments, the polypeptide is by promoting to wear the peptide and SEQ ID NO of film:1 fragment Composition.

In one or more embodiments, the polypeptide such as SEQ ID NO:Shown in 33.

The present invention also provides and encodes a kind of polynucleotide sequence, is selected from：

(1) polynucleotide sequence of polypeptide of the present invention is encoded；With

(2) complementary series of (1) described polynucleotide sequence.

The present invention also provides nucleic acid constructs, and the nucleic acid constructs contains the nucleotides sequence of coding said polypeptide of the present invention Row, available for expressing the polypeptide.

The present invention also provides a kind of pharmaceutical composition, and described pharmaceutical composition contains polypeptide of the present invention and pharmaceutically may be used The carrier of receiving.

The present invention also provides polypeptide of the present invention or pharmaceutical composition and benefits from Nrf2 transcription factors in preparation treatment or prevention Activation disease medicine in purposes.

In one or more embodiments, the disease of the activation for benefiting from Nrf2 transcription factors is selected from cancer, god Through degenerative disease and inflammatory disease.

In one or more embodiments, the disease of the activation for benefiting from Nrf2 transcription factors is selected from：Breast cancer, Cutaneum carcinoma, the cancer of intestines and stomach and respiratory tract, colon cancer, stomach cancer, the cancer of the esophagus, lung cancer, carcinoma of mouth, pharynx cancer, carcinoma of endometrium and Cancer of pancreas.

In one or more embodiments, the disease of the activation for benefiting from Nrf2 transcription factors be selected from cancer of pancreas, Colon cancer and/or the stomach cancer as caused by the effect of helicobacter pylori.

In one or more embodiments, the disease of the activation for benefiting from Nrf2 transcription factors is idiocrasy disease Disease, it is preferably selected from atopic rhinitis, conjunctivitis, dermatitis and asthma.

In one or more embodiments, the disease of the activation for benefiting from Nrf2 transcription factors is neurodegeneration disease Disease, it is selected from：Multiple sclerosis (MS), such as relapsing remitting MS, Secondary cases progressive MS, primary progressive MS, progress Property recurrent MS；ALS (ALS)；Alzheimer disease；Parkinson's；Huntington's disease；Acute hemorrhagic White matter encephalomyelitis；Hurst diseases；Encephalomyelitis, such as acute diseminated encephalomyelitis；Optic neuritis；Spinal cord injury；It is acute Necrotic myelitis；Transverse myelitis；Chronic progressive myelopathy；Progressive multifocal leukoencephalopathy (PML)；Radioactivity ridge Marrow disease；HTLV-1 relevant cords disease；Single-phase isolated demyelinate；Central pontine myelinolysis；Leukodystrophy (leucodystrophy), for example, addison-Schilder disease, Metachromatic leukodystrophy malnutrition, Krabbe diseases, Canavan diseases, Alexander diseases, Pelizaeus-Merbacher diseases, deorienting white matter disease and eye tooth refer to syndrome； With inflammatory demyelinate polyneuritis, such as chronic inflammatory demyelinate polyneuritis (CIDP) and acute inflammation demyelinate DPN (AIDP).

The present invention also provides polypeptide of the present invention, its coded sequence, pharmaceutical composition and benefits from oxygen preparing treatment or prevention Change stress disease medicine in purposes.

In one or more embodiments, the disease for benefiting from oxidative stress is the oxygen with Nrf2-Keap1 paths Change disease that stress be related.

In one or more embodiments, the disease related to the oxidative stress of Nrf2-Keap1 paths is selected from slow Property ephritis, tuberculosis, diabetes, angiocardiopathy, nerve degenerative diseases, noxious material or tissue damage caused by procarcinogen.

In one or more embodiments, the nerve degenerative diseases are selected from：Motor neuron disease, amyotrophic lateral sclerosis Lateral sclerosis (ALS) macula area related to the age to primary lateral sclerosis (PLS), Huntington's chorea is degenerated.

The present invention also provides a kind of method of Activated in Vitro Nrf2 approach, and methods described includes making the thin of expression Nrf2 approach Born of the same parents contact enough polypeptides of the present invention or the composition containing the polypeptide.

The present invention also provides polypeptide as described herein and activates purposes in Nrf2 approach in vitro.

Brief description of the drawings

Fig. 1：The synthetic peptide of PAQR3 functions is blocked to influence Nrf2 activity.A、B：P1-20 peptides reduce PAQR3 and Nrf2 and Interaction between Keap1.HEK293T cells are transiently transfected with shown plasmid.After transfection 24 hours, with control peptide (20 μ g/ Ml) or P1-20 peptides (20 μ g/ml) handle cell 12 hours, and then the antibody shown in carries out Western blotting (IB) analysis and is immunized Precipitate (IP) analysis.C：P1-20 peptides reduce Nrf2 ubiquitination.Transiently transfected with the ubiquitin of Nrf2 and the HA mark of Flag marks HEK293T cells.24 hours after transfection, cell is handled 12 hours with control peptide or P1-20 peptides, and handled 6 hours with MG132. Western blotting (IB) analysis is carried out with shown antibody on cell lysate and immunoprecipitation (IP) is analyzed.D：P1-20 peptides reduce thin Intracellular activity oxygen content.HepG2 cells control peptide and P1-20 peptides are handled 12 hours, then with (300 μM) processing 12 of hydrogen peroxide Hour, flow cytometry analysis intracellular reactive oxygen content is used after being dyed with dichlorofluorescein.E：P1-20 peptides improve Nrf2 target bases The expression of cause.With control peptide (20 μ g/ml), P1-20 peptides (20 μ g/ml) and (100 μM) of tBHQ processing HepG2 cells 12 hours. From the isolated total serum IgE of cell, real-time RT-PCR is carried out with it.Institute's value is standardized based on beta-actin.Number P is represented according to average value ± SD, * is expressed as<0.05, * * represents p<0.01, ns represents not notable.

Embodiment

Polypeptide and its coded sequence

The present invention relates to a kind of peptide sequence, the polypeptide includes SEQ ID NO:1 fragment, the fragment are located at SEQ ID NO:Between 1 the 1st to the 35th, and long at least 15 amino acid residues.

As used herein, " separation " it is (former if crude to refer to that material is separated from its primal environment Beginning environment is natural surroundings).As the polynucleotides under the native state in active somatic cell and polypeptide do not isolate and purify, But same polynucleotides or polypeptide with being separated in other existing materials, then isolate and purify such as from native state." point From amino acid sequence " refer to the amino acid sequence substantially free of natural relative other albumen, lipid, carbohydrate or its Its material.

Term " fragment " refers to the polypeptide being only made up of a part for complete full-length polypeptide sequence and structure.The present invention relates to PAQR3 fragment.PAQR3 amino acid sequence such as SEQ ID NO:Shown in 1.Especially, the present invention relates to PAQR3 N-terminal piece Section, specifically positioned at SEQ ID NO:1 the 1st amino acids residue preferably grows at least 15 to the fragment between the 35th amino acids Individual amino acid residue, for example, at least 18 amino acid residues, at least ten amino acid residue etc..

Preferably, the fragment at least ID containing SEQ NO:1 the 1st to the 15th amino acids residue.Therefore, the fragment SEQ ID NO can be originated in:1 the 1st amino acid residue, and terminate at SEQ ID NO:1 the 15th, 16,17,18,19,20, 21st, 22,23,24,25,26,27,28,29,30,31,32,33,34 or 35 amino acids residue.Preferably, the fragment by SEQ ID NO:1 1-20 amino acids residue forms.

It should be understood that in gene cloning operation, it is often necessary to design suitable restriction enzyme site, this certainly will be in expressed ammonia Base acid sequence end introduces one or more incoherent residues, and this has no effect on the activity of aim sequence.And for example in order to Construction of fusion protein, the expression for promoting recombinant protein, obtain the automatic recombinant protein being secreted into outside host cell or beneficial to restructuring The purifying of albumen, it is often necessary to be added to some amino acid other in the N- ends, C- ends or the albumen of recombinant protein In appropriate area, it may for example comprise but be not limited to, suitable joint peptide, signal peptide, leader peptide, end extension etc..Amino of the present invention The aminoterminal or c-terminus of acid sequence can also contain one or more polypeptide fragments, as protein tag.Any suitable label It may be used to the present invention.For example, described label can be FLAG, HA, HA1, c-Myc, Poly-His, Poly-Arg, Strep-TagII, AU1, EE, T7,4A6, ε, B, gE and Ty1.These labels can be used for purifying albumen.

The polypeptide of the present invention can also contain in the N-terminal of the fragment to be promoted to wear the peptide fragment of film.This kind of peptide fragment well known in the art, For example, reference can be made to Siegmund Reissmann, Cell penetration:scope and limitations by the Application of cell-penetrating peptides, J.Pept.Sci.2014；20:760–784.Herein by it Full content is included herein by reference.Especially, the peptide fragment for film being worn suitable for promotion of the invention includes above-mentioned document The sequence that numbering listed by table 1 is 1-92.Do not enumerate herein.The peptide of film is worn in the promotion that can be additionally used in the present invention Section includes RKKRRQRRR.

Therefore, polypeptide of the invention can be by SEQ ID NO:1 fragment composition, or can be by SEQ ID NO:1 institute The fragment stated and suitable protein tag composition, or can be by SEQ ID NO:1 fragment wears the sequence of film with promotion Composition, or can be by SEQ ID NO:The sequence composition of film is worn in 1 fragment, protein tag and promotion.

The amino acid sequence of the present invention can be the product of chemical synthesis, or use recombinant technique from protokaryon or eucaryon place Caused recombinant polypeptide in main (for example, bacterium, yeast, filamentous fungi, higher plant, insect and mammalian cell).According to Host used in recombinant production scheme, polypeptide of the invention can be glycosylated, or can be nonglycosylated.

The polynucleotides of the present invention can be DNA form or rna form.DNA form includes cDNA, genomic DNA or people The DNA of work synthesis.DNA can be single-stranded or double-strand.DNA can be coding strand or noncoding strand.Encoding mature polypeptide Coding region sequence can be with SEQ ID NO:DNA sequence dna shown in 33 is identical or the variant of degeneracy.As used herein, " variant of degeneracy " refers to encode identical amino acid sequence but the differentiated nucleotide sequence of nucleotide sequence in the present invention.

Polypeptide and polynucleotides in the present invention preferably provide in a separate form, are more preferably purified to homogeneous.

The nucleotide sequence of the present invention can generally use PCR TRAPs, recombination method or artificial synthesized method to obtain.For PCR TRAPs, primer can be designed according to relevant nucleotide sequence, especially open reading frame sequence disclosed in this invention, And by the use of commercially available cDNA storehouses or the cDNA storehouses as prepared by conventional method well known by persons skilled in the art are as template, amplification and Obtain relevant sequence.When sequence is longer, it is often necessary to carry out twice or multiple PCR is expanded, the piece for then again amplifying each time Section is stitched together by proper order.

Once obtain relevant sequence, it is possible to obtain relevant sequence in large quantity with recombination method.This is typically will It is cloned into carrier, then is transferred to cell, then by conventional method from the host cell after propagation isolated relevant sequence.

In addition, relevant sequence can be also synthesized with artificial synthesized method, when especially fragment length is shorter.Generally, lead to After first synthesizing multiple small fragments, the very long fragment of sequence can be obtained by being then attached again.

At present, it is already possible to obtain encoding the DNA sequence dna of amino acid sequence of the present invention by chemical synthesis completely.Then The DNA sequence dna can be introduced into various existing DNA moleculars (or such as carrier) as known in the art and cell.

Produced the present invention also relates to the carrier of the polynucleotides comprising the present invention, and with the carrier of the present invention through genetic engineering Raw host cell, and the method through recombinant technique generation polypeptide of the present invention.Preferably, carrier of the invention is expression Carrier.

By the recombinant DNA technology of routine, can be used to express or produce this hair using the polynucleotide sequence of the present invention Bright amino acid sequence.In general there are following steps：

(1) polynucleotides of the present invention or the variant of its degeneracy are used, or is carried with the recombination expression containing the polynucleotides Body converts or suitable host cell of transduceing；

(2) host cell cultivated in suitable culture medium；

(3) separation, protein purification from culture medium or cell.

The polynucleotide sequence of the present invention can be inserted into recombinant expression carrier.Term " recombinant expression carrier " refers to ability Bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, reverse transcription known to domain Viral or other carriers.As long as it can be replicated in host and stably, any plasmid and carrier can be used.The one of expression vector Individual key character is to usually contain replication orgin, promoter, marker gene and translation control element.Expression vector also includes translation The ribosome bind site and transcription terminator of starting.

Method well-known to those having ordinary skill in the art can be used for structure and contain nucleotide sequence of the present invention and suitable transcription/translation The expression vector of control signal.These methods include recombinant DNA technology in vi, DNA synthetic technologys, In vivo recombination technology etc..Institute The nucleotide sequence stated can be effectively connected in the appropriate promoter in expression vector, to instruct mRNA to synthesize.These promoters Representative example has：Lac the or trp promoters of Escherichia coli；Bacteriophage lambda PL promoters；It is early immediately that eukaryotic promoter includes CMV Phase promoter, HSV thymidine kinase promoters, early and late SV40 promoters, retroviruse LTRs and some other The promoter that the controllable gene known is expressed in protokaryon or eukaryotic or its virus.

In addition, expression vector preferably includes one or more selected markers, it is used to select conversion to provide The phenotypic character of host cell, such as the dihyrofolate reductase of eukaryotic culture, neomycin resistance and green fluorescence egg (GFP) in vain, or tetracycline or amicillin resistance for Escherichia coli.

Comprising above-mentioned appropriate DNA sequence dna and the carrier of appropriate promoter or control sequence, it is suitable to can be used for conversion When host cell, allow it to marking protein.

Host cell can be prokaryotic, such as bacterial cell；Or low eukaryotic, such as yeast cells；It is thread true Bacterium cell or higher eucaryotic cells, such as mammalian cell.Representative example has：Escherichia coli, streptomyces；Mouse typhus The bacterial cell of salmonella；Fungal cell such as yeast, filamentous fungi, plant cell；Drosophila S2 or Sf9 insect cell； CHO, COS, 293 cells or Bowes melanoma cells zooblast etc..

When the polynucleotides of the present invention are expressed in higher eucaryotic cells, if will when inserting enhancer sequence in the carrier Transcription can be strengthened.Enhancer is DNA cis-acting factors, generally about has 10 to 300 base-pairs, acts on and open Mover is to strengthen the transcription of gene.

Persons skilled in the art are aware that how to select appropriate carrier, promoter, enhancer and host cell.

It can be carried out with recombinant DNA conversion host cell with routine techniques well known to those skilled in the art.When host is original When core biology is such as Escherichia coli, can absorb DNA competent cell can harvest after exponential phase of growth, use CaCl₂Method processing, institute With the step of it is generally well-known in the art.Another method is to use MgCl₂.If desired, conversion can also use the side of electroporation Method is carried out.When host is eucaryote, following DNA transfection methods can be selected：Calcium phosphate precipitation, conventional mechanical methods are such as Microinjection, electroporation, liposome packaging etc..

The transformant of acquisition can use conventional method culture, express the polypeptide of the coded by said gene of the present invention.According to used Host cell, culture medium used may be selected from various conventional mediums in culture.Under conditions of suitable for host cell growth Cultivated.After host cell growth is to appropriate cell density, with suitable method (such as temperature transition or chemical induction) The promoter of selection is induced, cell is further cultured for a period of time.

Recombinant polypeptide in the above methods can be expressed or be secreted into extracellular in the cell or on cell membrane.Such as Fruit needs, can utilize its physics, chemical and other characteristic be separated by various separation methods and the albumen of purification of Recombinant.This A little methods are well-known to those skilled in the art.The example of these methods includes but is not limited to：The renaturation process of routine, use Protein precipitant handles (salting-out method), centrifugation, the broken bacterium of infiltration, super processing, ultracentrifugation, sieve chromatography (gel filtration), suction The combination of attached chromatography, ion-exchange chromatography, high performance liquid chroma- tography (HPLC) and other various liquid chromatography technologies and these methods.

The disease related to the activation of Nrf2 transcription factors or the oxidative stress relevant disease with Nrf2-Keap1 paths

The disease related to the activation of Nrf2 transcription factors (or benefit from activation or the Nrf2 approach of Nrf2 transcription factors The disease of activation) or the disease related to the oxidative stress of Nrf2-Keap1 paths be well known in the art, for example Disclosed in CN200980149899.0, CN201280023826.9, CN201410077763.6 and CN201380073599.5 etc., These document entire disclosures are included herein by reference herein.

Specifically, Nrf2 (nuclear factor red blood cell 2- correlation factors 2, AAB32188) is a kind of transcription factor, is being aoxidized Heterodimer is formed with little albumen matter Maf when stress activate, with reference to Antioxidant responsive element (ARE), the base of activation Nrf2 regulations The transcription of cause.A kind of Nrf2/ARE approach, main determining factor of II phases gene induction, has fully characterized it and has passed through The activation of II phase gene expressions carries out liver removing toxic substances and chemoprophylactic effect.The gene of ARE regulations also can be by serving as endogenous Antioxidant system and the maintenance for contributing to redox homeostasis.At present, the list of the gene of Nrf2 regulations comprises more than 200 coding be related to removing toxic substances and Antioxidation reaction protein and enzyme gene (Kwak etc., J.Biol.Chem., 2003,278: 8135):Quinone oxidoreductase, NQO2, g- glutamyl cysteine synthase (g-GCS), glucuronyl transferase, ferritin and DELTA rHO-1 (HO-1) etc..

Under basal conditions, Nrf2 chelates actin combination Kelch sample ECH- conjugated proteins 1 in cytoplasm (Keap1；Accession No.NP_987096), Cullin3 ubiquitin ligase adaptor protein matter.More particularly, Nrf2 N- Terminal domains, referred to as Neh2 domains, interacted with Keap1 C- ends Kelch spline structures domain.Respond allogene or oxygen Change stress, Nrf2 discharges from Keap1/Nrf2 compounds, so as to promote the gene that Nrf2 nuclear translocation and adjoint ARE- are mediated The activation of transcription.

Nrf2 pathway activations are proved have protection benefit in some neurodegenerative disease models.See, e.g. Calkins MJ etc., Toxicol Sci 2010；115:557–568.Neurodegenerative disease includes multiple sclerosis (MS) (example Such as relapsing remitting MS, Secondary cases progressive MS, primary progressive MS, progressive recurrent MS), ALS (ALS), Alzheimer disease, Parkinson's and Huntington's disease.Other examples of neurodegenerative disease include acute hemorrhagic White matter encephalomyelitis, Hurst diseases, encephalomyelitis (such as acute diseminated encephalomyelitis), optic neuritis, spinal cord injury, urgency Property necrotic myelitis, transverse myelitis, chronic progressive myelopathy, progressive multifocal leukoencephalopathy (PML), radioactivity Myelopathy, HTLV-1 relevant cords be sick, single-phase isolated demyelinate, central pontine myelinolysis, leukodystrophy (example Such as, addison-Schilder disease, Metachromatic leukodystrophy malnutrition, Krabbe diseases, Canavan diseases, Alexander Disease, Pelizaeus-Merbacher diseases, deorienting white matter disease, eye tooth refer to syndrome), inflammatory demyelinate multifocal neurological Scorching (for example, chronic inflammatory demyelinate polyneuritis (CIDP) and acute inflammation Demyelinating Polyneuropathy (AIDP)), Guillain-Barre syndrome (GBS), polyneuritis, myasthenia gravis (MG), EatonLambert syndromes (ELS) and brain Myelitis.

Treatment and prevention of the Nrf2 pathway activations also to following disease are beneficial, including idiopathic pulmonary fibrosis (IPF), sclerderm Sick tuberculosis, ALI (ALI)/acute respiratory distress (ARDS), asthma (such as chronic asthma), radiation-induced fibrosis meat Sample knurl disease, pulmonary hypertension, bronchopulmonary dysplasia (BPD), lung transplant rejection reaction, lung's GVHD complication, transplanting Interstitial pneumonia syndrome (IPS), COPD, silicosis, asbestosis, sarcoidosis (lung), primary hardening in recipient Property cholangitis (PSC), the hepatic fibrosis of alcohol induction, oneself immunity hepatitis, chronic viral hepatitis (HepB, C), primary Property biliary cirrhosis (PBC), nonalcoholic fatty liver disease (NASH), Liver rejection, GVHD liver complication, move Plant venous occlusive disease, FSGS (FSGS), nephrosis, IgA nephrosis, the sclerderm of recipient Acute renal failure (AKI after CABG), lupus kidney after disease, GVHD kidney complication (AKI Delayed grafting things function), CABG Scorching, hypertension induction kidney fibrosis, the nephrosis of HIV- correlations, the peritoneal fibrosiss of peritoneal dialysis induction, retroperitoneal fibrosis, spy Hair property glomerulosclerosis, kidney transplantation exclusion reaction, Alport syndromes, ISR, subarachnoid hemorrhage (SAH), heart move Plant rejection, apoplexy, vanity surgery, chronic trauma, burn, surgical adhesions, cheloid, donor graft epithelium shape again Into, myelofibrosis, corneal transplantation, Systemic sclerosis, the fibrosis of radiation-actuate, kneecap week fibrosis, Dupuytren contractions Contracting, diabetes, atopic rhinitis, conjunctivitis, dermatitis etc..

The disease related to the activation of Nrf2 transcription factors also includes cancer, is preferably selected from following cancer：Breast cancer；Skin Skin cancer；The cancer of intestines and stomach and respiratory tract, such as colon cancer, stomach cancer, the cancer of the esophagus；Lung cancer；Carcinoma of mouth；Pharynx cancer；Carcinoma of endometrium and Cancer of pancreas.It is highly preferred that it is used to prevent and treat cancer of pancreas, colon cancer and/or the stomach as caused by the effect of helicobacter pylori Cancer.

On the other hand, the disease related to the oxidative stress of Nrf2-Keap1 paths includes but is not limited to：Motor neuron Disease, amyotrophic lateral sclerosis, primary lateral sclerosis, Huntington's chorea, Alzheimer's, Parkinson's disease and The macula area of age correlation is degenerated.

Pharmaceutical composition

The present invention provides pharmaceutical composition, and it includes polypeptide of the present invention and pharmaceutically acceptable excipient and (such as carried Body).

Contain the polypeptide of the present invention for treating or preventing effective dose in pharmaceutical composition." effective dose " refers to the dosage of certain composition It is enough to produce desired reaction.Specific effective dose depends on many factors, the particular condition such as to be treated, the body of patient Concrete conditions in the establishment of a specific crime (such as weight in patients, age or sex), treatment duration, the therapy (if any) granted jointly and used Specific formula." effective dose " also refers under the dosage, and the toxicity or counter productive of polypeptide of the present invention are not as good as in caused by it Positive curative effect.

Pharmaceutically acceptable excipient is typically safe and nontoxic, and broadly may include to be used to make in pharmaceutical industries Any known substance of standby pharmaceutical composition, such as filler, diluent, coagulating agent, binder, lubricant, glidant, stably Agent, colouring agent, wetting agent, disintegrant etc..When selection is applied to deliver the excipient of synthetic peptide, this medicine group need to be mainly considered The administering mode of compound, the known technique of those skilled in the art.Suitable Examples of carriers includes but is not limited to magnesium carbonate, hard Fatty acid magnesium, cross-linked carboxymethyl cellulose sodium, microcrystalline cellulose, talcum, silica, sugar, lactose, pectin, dextrin, starch, gelatin, Tragacanth, methylcellulose, sodium carboxymethylcellulose, low melt wax, cocoa butter etc..

The content of polypeptide described in pharmaceutical composition of the present invention is about 1-1000 μM；Preferably about 10-500 μM；It is more excellent It is about 25-250 μM.For example, the concentration of polypeptide can be about 1,2,3,4,5,6,7,8,9,10,15,20,25,30,35,40, 45、50、55、60、65、70、75、80、85、90、95、100、150、200、250、300、350、400、450、500、550、600、 650th, 700,750,800,850,900,950 or 1,000 μM.

Aforementioned pharmaceutical compositions can be prepared according to known pharmaceutical procedures, for example《Remington's Pharmaceutical Science》(the 17th edition, Alfonoso R.Gennaro are compiled, Mike publishing company, Easton, Pennsylvania (1985)) it is documented in detail in a book.

The pharmaceutical composition of the present invention can be various suitable formulations, including but not limited to capsule, injection etc..It can lead to Cross polypeptide or its pharmaceutical composition that conventional method of administration gives the present invention, these approach are including but not limited to oral, it is intranasal, In transdermal, subcutaneous, intracutaneous, vagina, ear, intraocular, intramuscular, buccal, rectal, transmucosal or via suction or intravenous administration etc..

Method and purposes

The present invention is previously described each treating, preventing or improving including the use of the polypeptide of the present invention or its pharmaceutical composition The kind disease related to the activation of Nrf2 transcription factors or the oxidative stress relevant disease related with Nrf2-Keap1 paths.

Therefore, the present invention includes treatment, prevents or improve the previously described various activation for benefiting from Nrf2 transcription factors Or the method for the disease or the disease related to the oxidative stress of Nrf2-Keap1 paths of the activation of Nrf2 approach, methods described bag Include the polypeptide of the invention or its pharmaceutical composition that effective dose is given to the object for having this needs.Effective dose effectively obtains it The amount of expected purpose (curing, prevent or improve the disease).Should especially it depend on for the effective actual amount of application-specific In illness to be treated.

The dosage and frequency (single or multiple dosage) of administration can change according to many factors, and the factor includes giving Medicine approach；The age of recipient, sex, health status, body weight, the property and the order of severity of disease to be treated；Exist other Disease or other health related problems；The species for the treatment of simultaneously；And the complication caused by any disease or therapeutic scheme.

" object " refers to can benefit from the mammal for applying composition as described herein or method.Object is often referred to people.

The polypeptide of the present invention or its pharmaceutical composition can be with well known in the art for treating or preventing above-mentioned various diseases Medicine be together administered.For example, can be together administered with treating MS medicine, these medicines include but is not limited to imuran, sprinkle Buddhist nun Song Long, mycophenolate, mitoxantrone, teriflunomide, piroxicam and phenidone；Can together it be administered with treating ALS medicine, this A little medicines include Riluzole and dexpramipexole；Can together it be administered with treating the medicine of Alzheimer disease, such as Roger's row Ketone, vitamin E, donepezil, Tacrine, Rivastigmine, galanthamine and Memantine；Etc..Together administration includes giving simultaneously Medicine and consecutive administration.

Therefore the present invention also includes polypeptide of the present invention, its coded sequence, expression vector and the medicine containing polypeptide of the present invention Composition is being prepared for the previously described various activation for benefiting from Nrf2 transcription factors for the treatment of, ameliorating or preventing or Nrf2 ways The disease of the activation in footpath or with the application in the medicine of the oxidative stress relevant disease of Nrf2-Keap1 paths.

The present invention also includes a kind of method of activation Nrf2 approach, and methods described includes making the enough present invention of cells contacting Polypeptide or the composition containing the polypeptide.This method can be in-vitro method.Cell can be the cell of any expression Nrf2 approach.

In order to be more fully understood by invention described herein, following embodiments are illustrated.It should be understood that these embodiments are only used In the purpose of example, and it is not to be seen as limiting the invention in any way.

Materials and methods：

Cell culture

DMEM (Dulbecco ' s Modified Eagle ' s Medium) (Gibco), hyclone (Fetal Bovine Serum (Hyclone), Trypin-EDTA, penicillin-streptomycin (Invitrogen), DMSO (Merck), PEI (Polyethylenimine, branched, MW-25,000), Polybrene (Sigma), Hochest 33342 (Molecular Probes, Eugene, OR), transfection reagent PolyJet^TM DNA In Vitro Transfection Reagent (SignaGen Laboratories), each specification Tissue Culture Dish, cryopreservation tube, centrifuge tube (Corning).

Molecular biology reagents

Small kit, PCR glue reclaim kit of taking out of plasmid is Tiangeng Products；ProteinA/GPLUS Agarose are Santa Cruz Products；Protease inhibitor cocktail, phosphotase inhibitor cocktail For Pierce Products；Pvdf membrane, 0.22 μm and 0.45 μm of filter are Millipore Products；Import EP is managed and rifle Head is Axygen Products；Collagenase I are Worthington (Lakewood, NJ) Products；Trizol Regeant, AdEasy^TMAdenoviral Vector System are Stragagen Products；BLOCK-iT^TM Adenoviral RNAi Expression System are Invitrogene Products；AMV- reverse transcriptases, various limitations Property restriction endonuclease, T4DNA ligases, Taq archaeal dna polymerases and dNTP are purchased from TaKaRa companies；Nucleus and cytoplasmic protein extraction examination Agent box is Sangon Biotech (Shanghai) Co., Ltd.) limited company；General medicine is purchased from western Bath.

Instrument

Micropipettor (Gilson), refrigerator (Siemens), ultra low temperature freezer (Nuair), Vertial electrophorestic tank, wet film of walking around Instrument and the supporting power supplys of Power/PAC200 (Bio-Rad), 5415D types table model high speed centrifuge (Eppendorf), 5417R types are cold Freeze desk centrifuge (Eppendorf), Avanti J-20XP types high-speed refrigerated centrifuges (Beckman), PCR instrument (Eppendorf and Bio-Rad), the type fluorogenic chemiluminescence ELIASAs (Berthold) of L Max II 384, low speed refrigerated centrifuge (Heraeus), Ultrospec2100pro ultraviolet specrophotometers (Amersham), sterile super-clean bench (Thermo), Dolphin Doc image analysis systems (Wealtec), Chemi Doc image analysis systems (Bio-Rad), electric homogenizer (IKA), AL104 Type precision balance (Mettler Toledo), Horizontal electrophoresis tank and supporting power supply (multiple day science and technology), constant incubator (Medcenter), cell culture incubator (Thermo), liquid nitrogen container (Thermolyne), inverted microscope (Olympus) are desk-top true Empty pump, sealing machine, decolorization swinging table, JT502N assay balances, blender, Hit-block, Ultrasonic Pulverization instrument, thermostat water bath, PH Meter, sterile super-clean bench (domestic).

Primer sequence

The structure of recombinant plasmid

The total length Nrf2, Keap1, MafG, MafK, MafF of people by the cDNA reverse transcriptions of HEK293T cells, owns Clone is by sequencing.

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

RNA extracting：The cell of appropriate amount is taken fully to be cracked with 1ml Trizol reagents, room temperature is placed 5~10 minutes, is added Enter 0.2ml chloroforms, acutely concussion 15 seconds, are stored at room temperature 3 minutes 12000g, and 4 DEG C centrifuge 15 minutes, take supernatant be placed in it is another newly from Heart pipe, add 0.5ml isopropanols room temperature and place centrifugation in 10 minutes, 12000g, 4 DEG C 10 minutes, supernatant is removed, with 75% ethanol wash Precipitation two airing residual ethanol, precipitation is dissolved in appropriate DEPC water to three times, and purity and concentration are detected with Nano-Drop.

RT-PCR：1 μ g total serum IgEs are taken to be used for reverse transcription, using the AMV reverse transcriptases of Takara companies, according to its specification Reverse transcription is carried out, cDNA is obtained and is detected for ensuing PCR.PCR primer sequence is shown in materials and methods.

The operating method of quantitative fluorescent PCR is referring to TOYOBO Realtime PCR Master Mix specifications.

Cell culture

HEK293 and HepG2 cultures are in Dulbecco ' s modified Eagle ' s medium (DMEM) pH7.2+10% In hyclone (FBS), and penicillin and 100 μ g/ml streptomysin plus 100 μ g/ml.Condition of culture be 37 DEG C, 5% CO₂。

Cell transient transfection

HEK293T is transfected with polyethyleneimine (polyethylenime, PEI) method.Cell is in the day before transfection with appropriate Density divides disk, and changing serum-free antibiotic-free training liquid when cell growth is to about 80% into, PEI methods transfect again.Every 1 μ during transfection GDNA, add 2 μ l PEI to mix, culture dish is added after being stored at room temperature 20 minutes, liquid is changed after 6 hours.To HEK293T transfect after 24 to 48 hours harvestings.

HepG2 is transfected with liposome PolyJet methods (SignaGen Laboratories, Rockville, MD, USA), is turned Dyeing method is specifically shown in specification, harvests within 36 hours to 48 hours after transfection.

Protein extraction and Western blotting

Protein extraction：The cell of culture is washed twice with the PBS of precooling, exhausts PBS, and add suitable volumes is mixed with protease suppression The denaturation RIPA lysates of preparation and inhibitor of phospholipase enzymes (150mM NaCl, 10mM Tris pH7.2,0.1%SDS, 1% Triton X-100,1% deoxycholic acid, 5mMEDTA), scrape and be transferred to EP pipes, place on ice, 4 DEG C, 12,000g 15 points of centrifugations Clock, supernatant is taken, survey protein concentration with Bradford methods if necessary.Add+50 μM of DTT of 4 × SDS sample-loading buffers, 100 DEG C are boiled 10 points Clock, immediately loading or -20 DEG C of storages.

Western blotting：The molecular weight of albumen checked as needed first matches somebody with somebody the discontinuous denaturation polypropylene acyl of suitable concn Amine glue, cell sample 30ug or so, tissue sample 50ug or so is used for western blot analysis.Before transferring film, pvdf membrane methanol Bubble 15 seconds, then the subsequent application according to anode-sponge-filter paper-pvdf membrane-glue-filter paper-sponge-negative electrode is good, puts Into wet film instrument of walking around, covered with ice, 350mA transfer 60-120 minutes, depending on molecular weight.After transferring film terminates, according to The molecular size range that marker is shown is cut on demand, and 5% skim milk or 3%BSA are closed 1 hour, and primary antibody 4 spends night.Second Its TBST is washed 3 × 5 minutes, and upper secondary antibody room temperature 1-2 hours, TBST is washed 3 × 5 minutes, ECL colour developing tablettings.

Co-immunoprecipitation (co-IP)

Typically use 6cm Tissue Culture Dish.Tissue Culture Dish is placed on ice, ice-cold PBS is carefully washed twice, is inhaled Dry PBS, adds co-IP buffer solutions (150mM NaCl, 120mM Tris pH7.5,1%NP-40,5mM EDTA) to protein concentration About 1 μ g/ μ l, cell is scraped into the centrifuge tube of ice precooling from culture dish, 4 DEG C of rotation cracking 30min, 12000 × rpm centrifugations 10min, take supernatant to be managed to new EP, take 30 μ l to add 100 DEG C of SDS sample-loading buffers to boil 5min, freeze and be used as input in -20 DEG C. Reset and add corresponding antibodies on remaining, concentration is 1 μ g/ml, and 4 DEG C of rotations are overnight.Second day every milliliter of cell pyrolysis liquid adds 40~60 μ l Albumin A/g pearls, 4 DEG C of 3h, 5,000 × g, 4 DEG C of rotations centrifuge 30 seconds, remove supernatant, add 1ml co-IP lysis buffers to wash pearl, phase Same centrifugal condition, is repeated 3 times and (can be needed to adjust number according to experiment).Finally plus 30 μ l co-IP buffer solutions and SDS loadings are delayed Fliud flushing, 100 DEG C of albuminous degeneration 5min, Western blotting is done together with mono- off glue of input.

Statistical analysis

The analysis of experimental data is using double tail Student ' s t-test, and all statistics are with average value ± standard error Represent.

As a result：

P1-20 polypeptide blocks PAQR3 and Nrf2 and Keap1 interaction, promote the expression of cellular anti-oxidant gene：

Nrf2 paths are a kind of primary treatment targets of current oxidative stress relevant disease.Based on present invention discover that PAQR3 the present inventor's supposition blocking PAQR3 and Nrf2 interaction, may result in the negative regulation function of Nrf2 paths The activation of Nrf2 paths.

Present invention discover that 20 amino acid residues of PAQR3 N-terminal take part in Nrf2 and Keap1 interaction, based on this One finds, the present invention devises a kind of synthetic peptide (P1-20), and the synthetic peptide covers PAQR3N ends 1-20 amino acid residues (MHQKLLKSAHYIELGSYQYW).The N-terminal of the peptide fragment adds Tat sequences (RKKRRQRRR), to promote penetrating for cell.

By co-immunoprecipitation experiment, present invention discover that P1-20 polypeptides can block PAQR3 and Nrf2 and Keap1 phase Interaction, and control peptide RKKRRQRRR does not have function (Fig. 1, A and B) then, shows that P1-20 polypeptides can destroy PAQR3 and both Protein interaction.Meanwhile P1-20 can also reduce Nrf2 ubiquitination (Fig. 1, C).

HepG2 cells are handled with control peptide and P1-20 peptides 12 hours, then with the processing 12 hours of (300 μM) of hydrogen peroxide, are used Flow cytometry analysis intracellular reactive oxygen content is used after dichlorofluorescein dyeing.As a result find, P1-20 peptides can reduce into the cell Active o content (Fig. 1, D)

Next influences of the analysis P1-20 to Nrf2 expression of target gene.P1-20 polypeptides can dramatically increase background and tBHQ The expression of multiple Nrf2 of activation target gene, and control peptide does not act on (Fig. 1, E).

Therefore, these as shown by data, P1-20 polypeptides can have by blocking PAQR3 and Nrf2-Keap1 interaction Effect ground activation Nrf2 activity, promote the anti-oxidation function of cell.

Claims (10)

1. a kind of polypeptide, it is characterised in that the polypeptide contains SEQ ID NO:1 fragment, or by SEQ ID NO:1 fragment Composition；

Wherein, the SEQ ID NO:1 fragment is located at SEQ ID NO:1 the 1st between the 35th amino acids residue, preferably Positioned at SEQ ID NO:1 the 1st between the 30th amino acids residue；With

The SEQ ID NO:1 piece segment length at least 15 amino acid residues, preferably long at least 18 amino acid residues, more preferably Long at least 20 amino acid residues.

2. polypeptide as claimed in claim 1, it is characterised in that the SEQ ID NO:1 fragment is located at SEQ ID NO:1 1 between the 25th amino acids residue, being more preferably located at SEQ ID NO:1 the 1st between the 23rd amino acids residue, more It is preferred that by SEQ ID NO:1 the 1st to the 20th amino acids residue (SEQ ID NO:3) form.

3. polypeptide as claimed in claim 1 or 2, it is characterised in that the polypeptide also wears the peptide of film containing promotion, it is preferable that The peptide for promoting to wear film is selected from：

RQIKIWFQNRRMKWKK；

YGRKKRRQRRR；

KQAIPVAK- acid amides；

RRRRNRTRRNRRRVR- acid amides；

Few arginine (R₉- R₁₂)；

KLTRAQRRAAARKNKRNTRGC；

ALWKTLLKKVLKAPKKKRKVC；

RKKRRQRRR；

DAATATRGRSAASRPTERPRAPARSASRPRRPVE；

GWTLNSAGYLLGKINLKALAALAKKIL- acid amides；

AGYLLGKINLKALAALAKKIL- acid amides；With

YTAIAWVKAFIRKLRK- acid amides.

4. such as the polypeptide any one of claim 1-3, it is characterised in that the polypeptide is by promoting to wear the peptide and SEQ of film ID NO:1 fragment composition, it is preferable that the amino acid sequence of the polypeptide such as SEQ ID NO:Shown in 33.

5. a kind of polynucleotide sequence, is selected from：

(1) polynucleotide sequence of the polypeptide any one of claim 1-4 is encoded；With

(2) complementary series of the polynucleotide sequence described in (1).

6. a kind of pharmaceutical composition, it is characterised in that described pharmaceutical composition contains more any one of claim 1-4 Peptide and pharmaceutically acceptable carrier.

7. the pharmaceutical composition described in polypeptide or claim 6 any one of claim 1-4 is preparing treatment or pre- Purposes in the medicine of the anti-disease for benefiting from oxidative stress.

8. purposes as claimed in claim 7, it is characterised in that it is described benefit from oxidative stress disease be and Nrf2-Keap1 The related disease of the oxidative stress of path, is preferably selected from chronic nephritis, tuberculosis, diabetes, angiocardiopathy, nervus retrogression disease Disease, noxious material or tissue damage caused by procarcinogen；Preferably, the nerve degenerative diseases are selected from：Motor neuron disease Disease, amyotrophic lateral sclerosis (ALS) macula area related to the age to primary lateral sclerosis (PLS), Huntington's chorea Degenerate.

9. the pharmaceutical composition described in polypeptide or claim 6 any one of claim 1-4 is preparing treatment or pre- Purposes in the medicine of the disease of the anti-activation for benefiting from Nrf2 transcription factors.

10. purposes as claimed in claim 9, it is characterised in that the disease choosing of the activation for benefiting from Nrf2 transcription factors From cancer, neurodegenerative disease and inflammatory disease；

Preferably, the disease of the activation for benefiting from Nrf2 transcription factors is selected from：Breast cancer, cutaneum carcinoma, intestines and stomach and breathing Cancer, colon cancer, stomach cancer, the cancer of the esophagus, lung cancer, carcinoma of mouth, pharynx cancer, carcinoma of endometrium and the cancer of pancreas in road；

The disease of the activation for benefiting from Nrf2 transcription factors is atopic diseases, be preferably selected from atopic rhinitis, conjunctivitis, Dermatitis and asthma；

The disease of the activation for benefiting from Nrf2 transcription factors is neurodegenerative disease, is selected from：Multiple sclerosis (MS), example Such as relapsing remitting MS, Secondary cases progressive MS, primary progressive MS, progressive recurrent MS；ALS (ALS)；Alzheimer disease；Parkinson's；Huntington's disease；Acute hemorrhagic white matter encephalomyelitis；Hurst diseases；Brain ridge Marrow is scorching, such as acute diseminated encephalomyelitis；Optic neuritis；Spinal cord injury；Acute necrotizing myelitis；Transverse myelitis； Chronic progressive myelopathy；Progressive multifocal leukoencephalopathy (PML)；Radiation myelopathy；HTLV-1 relevant cords disease；It is single-phase Isolated demyelinate；Central pontine myelinolysis；Leukodystrophy (leucodystrophy), such as adrenal gland white matter battalion Support it is bad, Metachromatic leukodystrophy is malnutritive, Krabbe diseases, Canavan diseases, Alexander diseases, Pelizaeus- Merbacher diseases, deorienting white matter disease and eye tooth refer to syndrome；With inflammatory demyelinate polyneuritis, such as chronic inflammation Property demyelinate polyneuritis (CIDP) and acute inflammation Demyelinating Polyneuropathy (AIDP).