CN106279390A - Protein B ach1 purposes in preparing cell Wnt signal path inhibitor - Google Patents
Protein B ach1 purposes in preparing cell Wnt signal path inhibitor Download PDFInfo
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- CN106279390A CN106279390A CN201510239681.1A CN201510239681A CN106279390A CN 106279390 A CN106279390 A CN 106279390A CN 201510239681 A CN201510239681 A CN 201510239681A CN 106279390 A CN106279390 A CN 106279390A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4702—Regulators; Modulating activity
- C07K14/4703—Inhibitors; Suppressors
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
Abstract
The invention belongs to biotechnology and field of medicaments.Relate to a kind of protein B ach1 and be applied to suppress the purposes of cell canonical Wnt signal pathway, it is to intervene the relevant disease of abnormal Wnt signal path (such as, blood vessel occurs, cancer that the present invention proposes Bach1, abnormal cell proliferation, Alzheimer's disease and osteoarthritis) drug target.Bach1 albumen suppresses Wnt signal path expression of target gene by directly blocking the formation of the protein binding interference transcription complex of β-catenin/TCF, and suppression cell proliferation is the most apoptosis-induced.The present invention is that screening treatment has the medicine of related disorders to provide new target spot with abnormal Wnt signal path, protein B ach1 can be used for preparing cell Wnt signal path inhibitor further, with the medicine for preparing prevention and/or the treatment disease relevant with abnormal Wnt signal path.
Description
Technical field
The invention belongs to biotechnology and field of medicaments, relate to the new application of protein B ach1, be specifically related to one
Protein B ach1 is for preparing the purposes in cell Wnt signal path inhibitor.
Background technology
Prior art discloses Wnt signal path is intracellular highly important signal path, it and tumor, blood
Pipe regeneration, cell proliferation, tissue regeneration, fetal development and nerve, the generation of skeleton and develop close phase
Close.Research display, Wnt signal transduction pathway is broadly divided into the warp depending on β-catenin/TCF transcription complex
Allusion quotation Wnt signal transduction pathway (Wnt/ β-catenin approach) and do not rely on β-catenin/TCF transcription complex
Non-classical Wnt signal transduction pathway (Wnt/Ca2+Approach and Wnt/JNK approach).At classical Wnt signal
In transduction pathway, transcription activator β-Catenin is nucleus；In the case of there is no Wnt signal, cell
β-Catenin in matter and much albumen form multiprotein complex, and β-Catenin is degraded by ubiquitination；Work as Wnt
During signal path activation, some albumen that Wnt and receptor Fzd is combined in active cell prevent β-Catenin's
Degraded.Studies have reported that β-Catenin in intracellular accumulation and enters nucleus and T cell factor TCF lymph
The transcription factor of cell enhancer family forms complex and activates transcribing of downstream gene.Those skilled in the art recognize
For, owing to Wnt/ β-catenin signal pathway has relation, so regulating out of control with numerous cancers and disease
Wnt/ β-catenin signal pathway can be as a weight for the treatment of with Wnt/ β-catenin signal pathway relevant disease
Wanting means, therefore, the research of Wnt/ β-catenin signal pathway inhibitor is led to for abnormal Wnt signal with application
The treatment of road relevant disease is significant.
BTB-CNC autoploid 1 (BTB and CNC homology 1, Bach1) is a transcription inhibition factor
Son, belongs to basic leucine zipper protein family, and its structure height is guarded, and is widely present in mammal various
In tissue；The Bach1 albumen of people contains 736 aminoacid, and molecular weight is 92kDa.Under known Bach1 regulation and control
Trip gene has Heme oxygeanse-1 (heme oxygenase-1, HO-1) etc., but, yet there are no
Close Bach1 as Wnt signal path inhibitor and the report of related application.
Summary of the invention
It is an object of the invention to provide the new application of protein B ach1, be specifically related to protein B ach1 for preparing
Purposes in cell Wnt signal path inhibitor.
Bach1 albumen of the present invention, its aminoacid sequence polypeptide as shown in SEQ ID NO.1.
In the present invention, carry out Bach1 suppression cell Wnt/ β-catenin control path experiment, including:
By activity and the expression of target gene, the Bach1 of Bach1 suppression Wnt/ β-catenin reporter gene TOPFlash
The protein binding of suppression β-catenin and TCF4, the albumen knot of Bach1 suppression β-catenin and CBP/P300
Close, and by recruiting the tests such as the promoter region of HDAC1 with Wnt target gene is combined；And carried out Bach1
Inducing cell cycle arrest and apoptosis, suppress cell proliferation experiment, including: cell cycle analysis is tested,
Cell apoptosis assay, and cell proliferation experiment；Experimental result shows, described Bach1 is by directly blocking
The formation of the protein binding interference transcription complex of β-catenin/TCF is to realize the suppression of Wnt signal path；
Bach1 realizes Wnt signal path reporter gene by suppression Wnt signal path and expression of target gene suppresses；
Bach1 is pressed down to realize cell proliferation by Selective depression Wnt signal path inducing cell cycle arrest and apoptosis
System.
The invention discloses protein B ach1 and can be used for suppressing the new application of cell canonical Wnt signal pathway, through experiment
Confirming, Bach1 can suppress the reporter gene of classical Wnt signal path or the expression activity of genes of interest, Bach1
Albumen suppresses by directly blocking the formation of the protein binding interference transcription complex of β-catenin/TCF
Wnt signal path expression of target gene, suppression cell proliferation is the most apoptosis-induced, test result indicate that, protein B ach1
(such as, relevant disease can be activated to Wnt signal path as the disease intervening abnormal Wnt signal path relevant
Or the genetic diseases caused by Wnt signal path component mutation) drug target；It is further used for preparation regulation
The medicine of canonical Wnt signal pathway, and be used for preparing cell Wnt signal path inhibitor, in particular for preparation
Prevent and/or treat the medicine of the disease relevant with abnormal Wnt signal path.
The disease relevant to the activation of Wnt signal path of the present invention, is selected from: cancer, and blood vessel occurs, different
Often cell proliferation, diabetic retinopathy, pulmonary fibrosis, rheumatoid arthritis, scleroderma, mycete or
Virus infects, bone or cartilage disease, Alzheimer or osteoarthritis etc.；
Genetic diseases caused by Wnt signal path component mutation of the present invention, is selected from: polyp of colon,
Opsteoporosis pseudoglioma syndrome, familial exudative vitreoretinopathy, retinal blood
Pipe generation, early coronary disease, congenital extremity cut off syndrome, type 2 diabetes mellitus, Fu Erman syndrome, dimension
Er Musi tumor, skeleton development exception, focal dermal hypoplasia, autosomal recessive anonychia, neurocele lack
Fall into or alpha Thalassemia (ATRX) syndrome.
Accompanying drawing explanation
Fig. 1 is activity and the target gene of Bach1 suppression cell canonical Wnt signal pathway reporter gene TOPFlash
Expression, wherein,
Figure 1A shows, the TOPFlash reporter gene activity that Bach1 gene overexpression suppression Wnt3a activates；Figure
1B shows, the TOPFlash reporter gene activity that Bach1 gene overexpression suppression β-catenin activates；Fig. 1 C shows
Showing, Bach1 gene inhibition increases basis or the TOPFlash reporter gene activity of Wnt3a activation；Fig. 1 D shows,
Bach1 gene overexpression suppresses, and Bach1 gene reduces the table promoting Wnt/ β-catenin signal pathway target gene
Reach.
Fig. 2 is that Bach1 is combined with TCF4, the protein binding of suppression β-catenin and TCF4,
Wherein, Fig. 2 A shows the interaction utilizing co-immunoprecipitation method to analyze Bach1 Yu TCF4, Bach1
Total length or Bach1N-section have combination with TCF4；The shadow that β-catenin and TCF4 is combined by Fig. 2 B display Bach1
Ring, the protein binding of Bach1 suppression β-catenin and TCF4.
Fig. 3 is to utilize co-immunoprecipitation method to analyze the impact that β-catenin and CBP/P300 is combined by Bach1,
Wherein, Fig. 3 A and 3B shows the protein binding of Bach1 suppression β-catenin and CBP/P300；Fig. 3 C
Show the Acetylation Level of Bach1 suppression β-catenin.
Fig. 4 is that Bach1 is combined by recruiting the promoter region of HDAC1 with Wnt target gene, suppresses target gene
Transcribe,
Wherein, Fig. 4 A shows the interaction utilizing co-immunoprecipitation method to analyze Bach1 Yu HDCA1, knot
Fruit shows, Bach1 and histone deacetylase 1 (HDCA1) have combination；Fig. 4 B shows Bach1 pair
The impact of histone deacetylase activity, result shows, Bach1 process LAN increases histon deacetylase (HDAC)
Activity, the increase of enzymatic activity can be by specific Antibiotic FR 901228 Trichostatin A
(TSA) suppressed；Fig. 4 C shows and utilizes chromatin immune coprecipitation to analyze whether Bach1 opens with IL-8
Mover district combines, and result shows, Bach1 Yu IL-8 promoter region (-1-193) have combination；Fig. 4 D shows
Chromatin immune coprecipitation is utilized to analyze Bach1 process LAN to β-catenin, HDAC1 and IL-8 promoter
The impact that district combines, result shows, Bach1 process LAN is recruited the promoter region of HDAC1 with IL-8 and is combined,
The promoter region of suppression β-catenin and IL-8 is combined, thus suppresses IL-8 genetic transcription.
Fig. 5 is Bach1 inducing cell cycle arrest and apoptosis, suppresses cell proliferation,
Wherein, Fig. 5 A shows that Bach1 process LAN causes the cell S phase to block；Fig. 5 B shows Bach1 process LAN
The apoptosis of inducing cell；Fig. 5 C shows that Bach1 process LAN suppresses cell proliferation after 48 and 72 hours.
Detailed description of the invention
Experimental technique used in following embodiment if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, the most commercially obtain.
Embodiment 1 Bach1 suppression cell Wnt/ β-catenin control path test
The activity of test example 1-1:Bach1 suppression Wnt/ β-catenin reporter gene TOPFlash and the table of target gene
Reach
(1) cell is cultivated
HEK293T cell is in DMEM (the American I nvitrogen company) culture fluid containing 10% hyclone
Cultivate, 37 DEG C, CO2Concentration 5%, human microvascular endothelial cell (mvec) (HMVEC) is by Massachusetts, USA university
Professor Shao Rong grants, and cultivates in ECM culture fluid；Wnt3a conditioned medium is from Wnt3a cell strain
Prepared by (CRL-2647, purchased from U.S.'s ATCC cell bank): use the DMEM cell adding 10% hyclone
Culture medium culturing Wnt3a cell 4 days, is then centrifuged for collecting supernatant culture medium, and collating condition culture medium is from L cell
Prepared by strain (CRL-2648, purchased from U.S.'s ATCC cell bank): use the DMEM adding 10% calf serum thin
Born of the same parents' culture medium culturing L cell 4 days, is then centrifuged for collecting supernatant culture medium ,-80 DEG C long-term guarantors after liquid nitrogen flash freezer
Deposit；
(2) cell transfecting and luciferase (Luciferase, Luc) are analyzed
Cell liposome lipofectamin2000 (American I nvitrogen company) transfects to specifications
TOPFlash or FOPFlash reporter plasmid (Millipore company of the U.S.) or Bach1siRNA or right
According to siRNA, cotransfection pCMV-β-galactosidase (β-gal) plasmid is as internal reference simultaneously；Transfect is thin
After born of the same parents cultivate 18-20 hour, add the Wnt3a conditioned medium added with the small-molecule drug less than or equal to 20 μMs
Stimulate 16-20 hour, then with reporter lysis buffer (Promega company) cell lysis, use fluorescence
Element enzyme detecting system (Promega company) detection；β-gal activity analysis buffer hatches 1 hour at 37 DEG C
After, spectrophotometric determination 420nm absorbance, the Luc activity that Luc/ β-gal ratio is relative (is defined as
TOPFlash reporter gene activity), and with FOPFlash reporter gene for comparison correction；
(3) RNA extracts and reverse transcription-real-time adjustment (RT-PCR)
The extraction of cell total rna is prepared with Tripure reagent to specifications；Use M-MLV reverse transcriptase, with
Oligo (dT) 18 is primer, and the total serum IgE reverse transcription of 2 μ g is synthesized cDNA；Use specific primer amplification
The gene of VEGF, IL-8, c-Myc, cyclin D1, MMP-3 and GAPDH, primer sequence is as follows:
VEGF forward primer (5 '-ATCTTCAAGCCATCCTGTGTGC-3 '),
VEGF downstream primer (5 '-CAAGGCCCACAGGGATTTTC-3 ')；
IL-8 forward primer (5 '-CTCTCTTGGCAGCCTTCCTGA-3 '),
IL-8 downstream primer (5 '-CCCTCTGCACCCAGTTTTCCTT-3 ')；
C-Myc forward primer (5 '-ACAGCGTCTGCTCCACCT-3 '),
C-Myc downstream primer (5 '-CCTCATCTTCTTGTTCCTCCT-3 ')；
Cyclin D1 forward primer (5 '-TGGAGGTCTGCGAGGAACA-3 '),
Cyclin D1 downstream primer (5 '-CCTTCATCTTAGAGGCCACGAA-3 ')；
MMP-3 forward primer (5 '-TGCTGTTTTTGAAGAATTTGGGTT-3 '),
MMP-3 downstream primer (5 '-AGTTCCCTTGAGTGTGACTCG-3 ')；
GAPDH forward primer (5 '-CCATCTTCCAGGAGCGAGATC-3 '),
GAPDH downstream primer (5 '-GCCTTCTCCATGGTGGTGAA-3 ')；
(method is said according to SYBR green PCR master mix test kit to carry out real-time fluorescence quantitative PCR
Bright book), with 2-△△CtMethod carries out quantitative analysis；Result shows, Bach1 gene overexpression suppression Wnt3a
The TOPFlash reporter gene activity activated；The TOPFlash that Bach1 gene overexpression suppression β-catenin activates
Reporter gene activity；Bach1 gene inhibition increases basis or the TOPFlash reporter gene activity of Wnt3a activation；
Bach1 gene overexpression suppresses, and Bach1 gene reduces the table promoting Wnt/ β-catenin signal pathway target gene
Reach.
The protein binding of test example 1-2:Bach1 suppression β-catenin and TCF4
(1) protein immunization co-precipitation experiment
HEK293T cell cotransfection TCF4-HA and Bach1-flag total length or N-section or C-section Bach1 plasmid,
Cell pyrolysis liquid is collected for immunoprecipitation and immunoblotting analysis experiment after 48 hours；The step that immunoprecipitation is concrete
For: being mixed with the antibody (10 μ g) of anti-Flag by cell pyrolysis liquid albumen (1-1.5mg), 4 DEG C to hatch 3 little
Time, it is subsequently adding protein A/4 DEG C of overnight incubation of G sepharose 4B 20 μ l, will stick and have Flag antigen-antibody to be combined
The sepharose 4B RIPA buffer of thing washes 1 time, and PBS washes 4 times.It is eventually adding 2 × SDS sample-loading buffer, will
The 0 DEG C of degeneration of protein 10 5 minutes caught, then carries out SDS-polyacrylamide gel electrophoresis and carries out protein and exempt from
The epidemic disease marking；
(2) protein immunization imprinting experiment
Pass through step: cell RIPA buffer (150mM NaCl, 100mM Tris, pH8.0,1%Triton
X-100,1% NaTDC, 1%SDS, 5mM EDTA, 10mM NaF, containing protease inhibitor)
Cell lysis, hatches 30 minutes on ice, and 12000rpm is centrifuged 10 minutes and collects the supernatant containing total protein, uses
Bio-Rad protein quantification test kit detection protein concentration, equal protein carries out SDS-polyacrylamide gel electrophoresis,
And by wet robin by protein delivery to nylon membrane, with containing 0.05%Tween 20, the PBS of 5% defatted milk powder
Buffer blind 1 hour, 4 DEG C of overnight incubation of specific antibody, resist with two after washing film next day and hatch 1 hour, wash
Film 3-4 time, operates to specifications with chemiluminescence examination (PE company), detects specific on Chemiluminescence Apparatus
Protein band；
(3) TCF4 fusion protein and the Binding experiment of Bach1
The TCF4 prokaryotic expression carrier PGEX-TCF4 built is converted e. coli bl21, and picking list bacterium colony connects
Plant in the 2 × YT culture medium containing 100 nanograms/milliliter ammonia Bians, 37 DEG C of shaking overnight incubation activated spawn, 37 DEG C
Amplification culture to OD600 be about 0.9 time, add IPTG to final concentration of 1mM, 18 DEG C of inducing culture 24 hours
Rear centrifugal collection thalline；This thalline ultrasonic degradation supernatant is TCF4 fusion protein, with GST-beads 4 DEG C
Overnight incubation, then with transfection Bach1 or β-catenin 4 DEG C of overnight incubation of HEK293T cell pyrolysis liquid, in conjunction with
Albumen on beads carries out Western blot, with the corresponding albumen of antibody test；Result shows, Bach1 total length
Or Bach1N-section has combination with TCF4.
The protein binding of test example 1-3:Bach1 suppression β-catenin and CBP/P300, the acetyl of suppression β-catenin
Change
Known CBP/P300 is two kinds of acetylation of histone enzymes, and they can be combined with β-catenin, promotes
The acetylation of β-catenin, activates transcribing of downstream gene, in the present embodiment, utilizes co-immunoprecipitation method analysis
The impact that β-catenin and CBP/P300 is combined by Bach1, result shows, Bach1 suppression β-catenin with
The protein binding of CBP/P300；The Acetylation Level of Bach1 suppression β-catenin；
Test example 1-4:Bach1 is combined by recruiting the promoter region of HDAC1 with Wnt target gene, suppresses target gene
Transcribe
(1) histon deacetylase (HDAC) analysis experiment
With the nucleus of Nuclear extract extraction agent box extracting HEK293T cell, detection is thin to specifications
Histone deacetylase activity (Millipore company of the U.S.) in karyon, in excitation wavelength 360nm, sends out
The long 450nm of ejected wave reads fluorescent value；
(2) chromatin immune co-precipitation experiment
HEK293T cell adds formaldehyde fixative, and room temperature fixes 10 minutes, collects and cell precipitates ultrasonic breaking
Broken chromatin, by DNA Yu Protein G magnetic bead, 4 DEG C of overnight incubation of Bach1 antibody, is combined on beads
DNA carries out Real_time quantitative detection, and primer sequence is as follows: IL-8ChIP (-1-193) forward primer (5 '-
TATTTTAAAGATCAAAGAAAACTTTCG-3 '), IL-8ChIP (-1-193) downstream is drawn
Thing (5 '-TGCTCCGGTGGCTTTTTATATC-3 ')；IL-8ChIP (-309-418) forward primer
(5 '-TGGCTGGCTTATCTTCAC-3 '), IL-8ChIP (-309-418) downstream primer (5 '-
TAGAGTGGCAGGTGTTAG-3’)；IL-8ChIP (-833-939) forward primer (5 '-
CTGGAGCATAAACATACC-3 '), IL-8ChIP (-833-939) downstream primer (5 '-
AATAGGAGGGCTTCAATA-3’)；
Analyze the interaction of Bach1 Yu HDCA1 by co-immunoprecipitation method, result shows, Bach1 goes with histone
Acetylase 1 (HDCA1) has combination；；Bach1 process LAN increases histone deacetylase activity, enzyme
The increase of activity can be pressed down by specific Antibiotic FR 901228 Trichostatin A (TSA)
System；There is combination Bach1 Yu IL-8 promoter region (-1-193)；Bach1 process LAN recruits HDAC1 and IL-8
Promoter region combine, the promoter region of suppression β-catenin and IL-8 is combined, thus suppresses IL-8 gene
Transcribe.
Embodiment 2:Bach1 inducing cell cycle arrest and apoptosis, suppress cell proliferation test
(1) cell cycle analysis experiment
Utilize Bach1 adenovirus or comparison adenovirus infection human microvascular endothelial cell (mvec), Bach1 adenovirus
(Ad-Bach1) or comparison virus (Ad-GFP) by Shanghai Ji Kai chemical gene technology company provide；72 is little
Shi Hou, collects cell, fixes with ice ethanol, abandons supernatant after being centrifuged, and adds 1ml DNA dyeing liquor (containing PI
And RNaseA), incubated at room 30 minutes, deliver to flow cytomery；
(2) cell apoptosis assay
Utilizing the apoptosis of Annexin V/PI test kit (American I nvitrogen company) detection cell, collector is micro-
Vascular endothelial cell, every 100 μ l cell suspension add 5 μ l Alexa488annexin V(Component
A) and 1 μ l 100 μ g/ml PI working solution；Incubated at room 15 minutes；Add 400 μ l 1 × annexin-binding
Buffer (Component C), mixing, deliver to flow cytomery analysis；
(3) cell proliferation experiment
Propagation bromodeoxyuridine (BrdU) of cell participates in method detection, by Bach1 adenovirus infection
Human microvascular endothelial cell (mvec) (5000) plant in 96 orifice plates, every 6 Kong Weiyi groups, with untreated work
For comparison, in culture fluid, add BrdU in different time points and hatch 6 hours, the detection of the BrdU amount of participating according to
The method detection of test kit description, compares the proliferative conditions of cell, knot with the mean absorbance values of 450nm
Fruit shows, Bach1 process LAN causes the cell S phase to block；The apoptosis of Bach1 process LAN inducing cell；Bach1
Process LAN suppresses cell proliferation after 48 and 72 hours.
SEQUENCE LISTING
<110>Fudan University
<120>protein B ach1 purposes in preparing cell Wnt signal path inhibitor
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 736
<212> PRT
<213> Homo sapiens
<400> 1
Met Ser Leu Ser Glu Asn Ser Val Phe Ala Tyr Glu Ser Ser Val His
1 5 10 15
Ser Thr Asn Val Leu Leu Ser Leu Asn Asp Gln Arg Lys Lys
Asp Val
20 25 30
Leu Cys Asp Val Thr Ile Phe Val Glu Gly Gln Arg Phe Arg
Ala His
35 40 45
Arg Ser Val Leu
Ala Ala Cys Ser Ser Tyr Phe His Ser Arg Ile Val
50 55 60
Gly Gln Ala Asp Gly Glu Leu Asn Ile Thr Leu Pro Glu Glu
Val Thr
65
70 75 80
Val Lys Gly Phe Glu Pro Leu Ile Gln Phe Ala Tyr Thr Ala
Lys Leu
85 90 95
Ile Leu Ser Lys Glu Asn Val Asp Glu Val Cys Lys Cys Val
Glu Phe
100 105 110
Leu Ser Val His Asn Ile Glu Glu Ser Cys Phe Gln Phe Leu
Lys Phe
115 120 125
Lys Phe Leu Asp Ser Thr Ala Asp Gln Gln Glu Cys Pro Arg
Lys Lys
130 135 140
Cys Phe Ser Ser His Cys Gln Lys Thr Asp Leu Lys Leu Ser
Leu Leu
145
150 155 160
Asp Gln Arg Asp Leu Glu Thr Asp Glu Val Glu Glu Phe Leu
Glu Asn
165 170 175
Lys Asn Val Gln
Thr Pro Gln Cys Lys Leu Arg Arg Tyr Gln Gly Asn
180 185 190
Ala Lys Ala Ser Pro Pro Leu Gln Asp Ser Ala Ser Gln Thr
Tyr Glu
195 200 205
Ser Met Cys Leu Glu Lys Asp Ala Ala Leu Ala Leu Pro Ser
Leu Cys
210 215 220
Pro Lys Tyr Arg Lys Phe Gln Lys Ala Phe Gly Thr Asp Arg
Val Arg
225
230 235 240
Thr Gly Glu Ser Ser Val Lys Asp Ile His Ala Ser Val Gln
Pro Asn
245 250 255
Glu Arg Ser Glu Asn Glu Cys Leu Gly Gly Val Pro Glu Cys
Arg Asp
260 265 270
Leu Gln Val Met Leu Lys Cys Asp Glu Ser Lys Leu Ala Met
Glu Pro
275 280 285
Glu Glu Thr Lys Lys Asp Pro Ala Ser Gln Cys Pro Thr Glu
Lys Ser
290 295 300
Glu Val Thr Pro Phe Pro His Asn Ser Ser Ile Asp Pro His
Gly Leu
305
310 315 320
Tyr Ser Leu Ser Leu Leu His Thr Tyr Asp Gln Tyr Gly Asp
Leu Asn
325 330 335
Phe Ala Gly Met Gln Asn Thr Thr Val Leu Thr Glu Lys Pro
Leu Ser
340 345 350
Gly Thr Asp Val Gln Glu Lys Thr Phe Gly Glu Ser Gln Asp
Leu Pro
355 360 365
Leu Lys Ser Asp Leu Gly Thr Arg Glu Asp Ser Ser Val Ala
Ser Ser
370 375 380
Asp Arg Ser Ser Val Glu Arg Glu Val Ala Glu His Leu Ala
Lys Gly
385
390 395 400
Phe Trp Ser Asp Ile Cys Ser Thr Asp Thr Pro Cys Gln Met
Gln Leu
405 410 415
Ser Pro Ala Val
Ala Lys Asp Gly Ser Glu Gln Ile Ser Gln Lys Arg
420 425 430
Ser Glu Cys Pro Trp
Leu Gly Ile Arg Ile Ser Glu Ser Pro Glu Pro
435 440 445
Gly Gln Arg Thr Phe
Thr Thr Leu Ser Ser Val Asn Cys Pro Phe Ile
450 455 460
Ser Thr Leu Ser Thr
Glu Gly Cys Ser Ser Asn Leu Glu Ile Gly Asn
465
470 475 480
Asp Asp Tyr Val Ser
Glu Pro Gln Gln Glu Pro Cys Pro Tyr Ala Cys
485 490 495
Val Ile Ser Leu Gly Asp Asp Ser Glu Thr Asp Thr Glu Gly
Asp Ser
500 505 510
Glu Ser Cys Ser Ala Arg Glu Gln Glu Cys Glu Val Lys Leu Pro Phe
515 520 525
Asn Ala Gln Arg Ile Ile Ser Leu Ser Arg Asn Asp Phe Gln Ser Leu
530 535 540
Leu Lys Met His Lys Leu Thr Pro Glu Gln Leu Asp Cys Ile His Asp
545
550 555 560
Ile Arg Arg Arg Ser Lys Asn Arg Ile Ala Ala Gln Arg Cys Arg Lys
565 570 575
Arg Lys Leu Asp Cys Ile Gln Asn Leu Glu Ser Glu Ile Glu Lys Leu
580 585 590
Gln Ser Glu Lys Glu Ser Leu Leu Lys Glu Arg Asp His Ile Leu Ser
595 600 605
Thr Leu Gly Glu Thr Lys Gln Asn Leu Thr Gly Leu Cys Gln Lys Val
610 615 620
Cys Lys Glu Ala Ala Leu Ser Gln Glu Gln Ile Gln Ile Leu Ala Lys
625 630 635 640
Tyr Ser Ala Ala Asp Cys Pro Leu Ser Phe Leu Ile Ser Glu Lys Asp
645 650 655
Lys Ser Thr Pro Asp Gly Glu Leu Ala Leu Pro Ser Ile Phe Ser Leu
660 665 670
Ser Asp Arg Pro Pro Ala Val Leu Pro Pro Cys Ala Arg Gly Asn Ser
675 680 685
Glu Pro Gly Tyr Ala Arg Gly Gln Glu Ser Gln Gln Met Ser Thr Ala
690 695 700
Thr Ser Glu Gln Ala Gly Pro Ala Glu Gln Cys Arg Gln Ser Gly Gly
705
710 715 720
Ile Ser Asp Phe Cys Gln Gln Met Thr Asp Lys Cys Thr Thr Asp Glu
725 730 735
Claims (7)
1. protein B ach1 is for preparing the purposes in cell Wnt signal path inhibitor；Described Bach1
Its aminoacid sequence of albumen polypeptide as shown in SEQ ID NO.1.
2. the purposes as described in claim 1, it is characterised in that described protein B ach1 as target spot with
Purposes in the medicine of the disease that preparation prevents and/or treatment is relevant with abnormal Wnt signal path.
3. the purposes as described in claim 1 or 2, it is characterised in that described protein B ach1 suppression classical Wnt
The reporter gene of signal path or the expression activity of genes of interest.
4. the purposes as described in claim 1 or 2, it is characterised in that described protein B ach1 directly blocks
The formation suppression cell proliferation of the protein binding interference transcription complex of β-catenin/TCF in Wnt signal path.
5. the purposes as described in claim 2, it is characterised in that described is relevant with abnormal Wnt signal path
Disease be activate relevant disease or Wnt signal path component mutation to Wnt signal path caused by hereditary disease
Sick.
6. the purposes as described in claim 5, it is characterised in that described is relevant with abnormal Wnt signal path
Disease be: cancer, blood vessel occur, abnormal cell proliferation, diabetic retinopathy, pulmonary fibrosis, class
Rheumatic arthritis, scleroderma, mycete or virus infect, bone or cartilage disease, and Alzheimer or bone close
Joint inflammation.
7. the purposes as described in claim 5, it is characterised in that described Wnt signal path component mutation institute
The genetic diseases caused is: polyp of colon, opsteoporosis pseudoglioma syndrome, familial ooze out
Property vitreoretinopathy, retinal vessel generation, early coronary disease, congenital extremity cut off syndrome,
Type 2 diabetes mellitus, Fu Erman syndrome, wilms' tumor, skeleton development exception, focal dermal hypoplasia,
Autosomal recessive anonychia, neural tube defect or alpha Thalassemia (ATRX) syndrome.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510239681.1A CN106279390B (en) | 2015-05-12 | 2015-05-12 | Protein B ach1 is preparing the purposes in cell Wnt signal path inhibitor |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201510239681.1A CN106279390B (en) | 2015-05-12 | 2015-05-12 | Protein B ach1 is preparing the purposes in cell Wnt signal path inhibitor |
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| Publication Number | Publication Date |
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| CN106279390A true CN106279390A (en) | 2017-01-04 |
| CN106279390B CN106279390B (en) | 2019-06-07 |
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| CN109517826A (en) * | 2018-11-28 | 2019-03-26 | 复旦大学 | A kind of Bach1 gene of modification and its application |
| CN110172462A (en) * | 2017-08-20 | 2019-08-27 | 浙江大学 | Gene and its expression product and application of the occurrence and development of a kind of pair of tumour with facilitation |
| CN110194787A (en) * | 2018-02-05 | 2019-09-03 | 中国医学科学院药物研究所 | Active polypeptide of targeted inhibition Wnt/ β-catenin signal and application thereof |
| CN110343752A (en) * | 2019-07-03 | 2019-10-18 | 扬州大学 | A method of Wnt signal and histone cooperate with target gene mechanism in research PGCs atomization |
| CN111317734A (en) * | 2020-03-27 | 2020-06-23 | 广州医科大学附属第二医院 | Wnt signal pathway inhibitor and application |
| CN114452373A (en) * | 2022-03-04 | 2022-05-10 | 广州赛佰澳生物医药科技有限公司 | Application of EP0 protein in immune regulation |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110172462A (en) * | 2017-08-20 | 2019-08-27 | 浙江大学 | Gene and its expression product and application of the occurrence and development of a kind of pair of tumour with facilitation |
| CN110194787A (en) * | 2018-02-05 | 2019-09-03 | 中国医学科学院药物研究所 | Active polypeptide of targeted inhibition Wnt/ β-catenin signal and application thereof |
| CN110194787B (en) * | 2018-02-05 | 2022-05-17 | 中国医学科学院药物研究所 | Polypeptide for targeted inhibition of Wnt/beta-catenin signal activity and application thereof |
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| CN109517826B (en) * | 2018-11-28 | 2019-12-17 | 复旦大学 | Modified Bach1 gene and application thereof |
| CN110343752A (en) * | 2019-07-03 | 2019-10-18 | 扬州大学 | A method of Wnt signal and histone cooperate with target gene mechanism in research PGCs atomization |
| CN111317734A (en) * | 2020-03-27 | 2020-06-23 | 广州医科大学附属第二医院 | Wnt signal pathway inhibitor and application |
| CN114452373A (en) * | 2022-03-04 | 2022-05-10 | 广州赛佰澳生物医药科技有限公司 | Application of EP0 protein in immune regulation |
| CN114452373B (en) * | 2022-03-04 | 2023-10-03 | 广州赛佰澳生物医药科技有限公司 | Application of EP0 protein in immune regulation |
| CN116789843A (en) * | 2022-03-16 | 2023-09-22 | 伯桢生物科技(杭州)有限公司 | Wnt recombinant protein and application thereof |
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