CN110090295A - Purposes of the Ephrin-B1 in the drug of preparation treatment inflammatory bowel disease - Google Patents

Purposes of the Ephrin-B1 in the drug of preparation treatment inflammatory bowel disease Download PDF

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Publication number
CN110090295A
CN110090295A CN201810085585.XA CN201810085585A CN110090295A CN 110090295 A CN110090295 A CN 110090295A CN 201810085585 A CN201810085585 A CN 201810085585A CN 110090295 A CN110090295 A CN 110090295A
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ephrin
mouse
inflammatory bowel
bowel disease
drug
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CN110090295B (en
Inventor
王晨辉
张华芝
郭晓利
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Wuhan Kanglitong Technology Co Ltd
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Wuhan Kanglitong Technology Co Ltd
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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
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian

Abstract

The invention discloses purposes of the Ephrin-B1 in the drug of preparation treatment inflammatory bowel disease, belong to biomedicine field.Ephrin-B1 extracellular region coded sequence is building up on expression vector pINFUSE-hIgG2-Fc2 by the present invention, pass through mammalian cell expression recombinant protein Ephrin-B1-Fc, with the mouse for suffering from colitis of Ephrin-B1-Fc intraperitoneal injection DSS induction, it was found that the mouse enteritis that Ephrin-B1-Fc can be induced with substantially reduced DSS, mitigate mouse weight decline, reduce mouse death rate, show that Ephrin-B1 can be used to prepare the drug for the treatment of inflammatory bowel disease, Ephrin-B1 is people Ephrin-B1 (SEQ ID NO.1) or mouse Ephrin-B1 (SEQ ID NO.3).The present invention provides new direction for the treatment of inflammatory bowel disease.

Description

Purposes of the Ephrin-B1 in the drug of preparation treatment inflammatory bowel disease
Technical field
The present invention relates to biomedicine fields, and in particular to Ephrin-B1 is in the drug of preparation treatment inflammatory bowel disease Purposes.
Background technique
Inflammatory bowel disease (Inflammatory bowel disease, IBD) be involve total colectomy one kind it is chronic itself Immunity disease mainly includes ulcerative colitis (Ulcerative colitis, UC) and Crohn disease (Crohn's Disease, CD).In recent years, inflammatory bowel disease increases year by year in China's disease incidence, it has also become digestive system common disease and slow Property diarrhea major reason, and patient is mostly person between twenty and fifty, therefore is paid more and more attention.Inflammatory bowel disease lacks special effective at present Ground treatment method, the frequent protracted course of disease of the state of an illness, and also chronic inflamatory substantially increases patients' colorectal cancer Risk1.It therefore is the hot spot in autoimmune disease research to the research of inflammatory bowel disease molecule, cell mechanism.Sulfuric acid Portugal is poly- Sugared sodium (DSS) is widely used for building the model of inflammatory bowel disease in mouse, with higher with the inflammatory bowel disease of the mankind Similitude is the ideal model for studying human inflammatory bowel disease2。
Erythropoietin human liver cell receptor (Erythropoietin-producing hepatoma cell line, Eph) by Body is one of maximum member of tyrosine kinase receptor family, and the signal transduction between EPHB receptor and Ephrin-B ligand mediates Numerous physiology, pathologic function, in embryonic development, neuronal migration, Intestinal epitheliual cell proliferation, enterocyte positioning, blood Pipe generation etc. played an important role3.EPHB receptor signaling pathways also play in the occurrence and development for inhibiting tumour Important role is presently believed to be important tumor suppressor gene4。
Bibliography:
1.de Souza HS1,Fiocchi.Immunopathogenesis of IBD:current state of the art.Nat Rev Gastroenterol Hepatol.2016Jan；13(1):13-27.
2.Whittem CG,Williams AD,Williams CS.Murine Colitis modeling using Dextran Sulfate Sodium(DSS).J Vis Exp.2010Jan 19；(35).
3.Boyd AW,Bartlett PF,Lackmann M.Therapeutic targeting of EPH receptors and their ligands.Nat Rev Drug Discov.2014Jan；13(1):39-62.
4.Pasquale EB.Eph receptors and ephrins in cancer:bidirectional signalling and beyond.Nat Rev Cancer.2010Mar；10(3):165-80.
Summary of the invention
It is an object of the invention to solve the technical problems existing in the prior art, the ligand of EPHB receptor is provided New application of the Ephrin-B1 in the drug of preparation treatment inflammatory bowel disease.
The coded sequence of Ephrin-B1 extracellular region is building up on expression vector pINFUSE-hIgG2-Fc2 by the present invention, is led to Mammalian cell expression recombinant protein Ephrin-B1-Fc is crossed, suffers from colon with Ephrin-B1-Fc intraperitoneal injection DSS induction Scorching mouse, the mouse enteritis that discovery Ephrin-B1-Fc can be induced with substantially reduced DSS mitigate mouse weight decline, reduce Mouse death rate, this shows that Ephrin-B1 can be used in treating inflammatory bowel disease.
The invention provides the following technical scheme:
The purposes of Ephrin-B1 or composition comprising Ephrin-B1 in the drug of preparation treatment inflammatory bowel disease.Institute The Ephrin-B1 stated is people Ephrin-B1 or mouse Ephrin-B1.Wherein, the amino acid sequence of people Ephrin-B1 such as SEQ Shown in ID NO.1, coding nucleotide sequence is as shown in SEQ ID NO.2；The amino acid sequence of mouse Ephrin-B1 such as SEQ ID Shown in NO.3, coding nucleotide sequence is as shown in SEQ ID NO.4.
Ephrin-B1 extracellular region polypeptide or composition comprising Ephrin-B1 extracellular region polypeptide are inflammatory in preparation treatment Purposes in the drug of enteropathy.The Ephrin-B1 extracellular region polypeptide is the 28th leucine of people Ephrin-B1 to respectively 236 serines, the 30th lysine of mouse Ephrin-B1 to the 229th serine, coding nucleotide is respectively SEQ ID The position the 88-687 nucleotide of sequence shown in the position the 82-708 nucleotide of sequence shown in NO.2, SEQ ID NO.4.
The fused polypeptide that is made of the heavy chain Fc structural domain of Ephrin-B1 extracellular region and Immunoglobulin IgG2 or comprising institute State purposes of the composition of fused polypeptide in the drug of preparation treatment inflammatory bowel disease.
A kind of carrier for expressing above-mentioned fused polypeptide, skeleton carrier are preferably pINFUSE-hIgG2-Fc2, include coding The nucleotide of Ephrin-B1 extracellular region.
It is a kind of for producing the host carrier system of above-mentioned fused polypeptide, for the host cell comprising above-mentioned carrier.It is described Host cell is bacterial cell, yeast cells, insect cell or mammalian cell, preferably 293F, COS7, Chinese hamster ovary celI.
A method of above-mentioned fused polypeptide is produced, is above-mentioned host carrier system in the condition for allowing to generate fused polypeptide Fused polypeptide caused by lower recycling thus, after cultivation, centrifuging and taking supernatant carries out pure the host cell such as comprising above-mentioned carrier Change.
Above-mentioned inflammatory bowel disease includes ulcerative colitis (Ulcerative colitis, UC), Crohn disease (Crohn' S disease, CD)；The administration route for treating the drug of inflammatory bowel disease is oral administration or intravenously administrable.
Present invention discover that Ephrin-B1 can be used in treating inflammatory bowel disease, provided newly for the treatment of inflammatory bowel disease Direction.
Detailed description of the invention
Fig. 1 is race glue, coomassie brilliant blue staining figure after FC albumen and Ephrin-B1 protein purification.
Fig. 2 is the changes of weight figure after DSS inducing mouse injection FC albumen, Ephrin-B1 albumen.
Fig. 3 is the colonic tissue H&E colored graph after DSS inducing mouse injection FC albumen, Ephrin-B1 albumen.
Specific embodiment
With reference to the accompanying drawings and examples, specific embodiments of the present invention will be described in further detail.Implement below Example is not intended to limit the scope of the invention for illustrating the present invention.Unless otherwise specified, technological means used in embodiment The conventional means being well known to those skilled in the art.
Embodiment 1
(1) experiment mice
C57BL/6 mouse is bought from Hubei Province drug safety evaluation center, and at Hubei Province drug safety evaluation center SPF grades of Animal Lab. raisings.It chooses 8 week old male mices and does zoopery.Mouse is randomly divided into 2 groups, every group 10, one group Inject reference protein Fc, another group of injection recombinant protein Ephrin-B1-Fc.
(2) pINFUSE-hIgG2-Fc2-Ephrin-B1-Fc vector construction
The full cDNA sequence of mouse Ephrin-B1 (shown in SEQ ID NO.4) is synthesized by company, as template, is passed through PCR amplification, digestion, (extracellular region amino acid is mouse Ephrin-B1 to connection building mouse Ephrin-B1 extracellular region expression vector The 30th lysine of albumen is to the 229th serine).Expression vector is pINFUSE-hIgG2-Fc2, public purchased from Invivogen Department.
The upstream primer of carrier construction is: 5 '-CATGCCATGGGCCACCCTGGCCAAGAACCTGGAGCC-3 ',
Downstream primer is: 5 '-GGAAGATCTAGAGTCCCCGCTGCCACC-3 '.
PCR reaction system are as follows: 10 × PCR buffer, 51 μ L of μ L, dNTPs, up/down swim each 1 μ L of primer (10 μM), mould 1 μ L of plate DNA 1ng, PFU archaeal dna polymerase, adds ddH2O to 50 μ L.
PCR response procedures are as follows: 95 DEG C 5 minutes, (95 DEG C 1 minute, 58 DEG C 30 seconds, 72 DEG C 2 minutes) circulation 30 times, 72 DEG C 10 minutes, 4 DEG C.
PCR reaction system after reaction is run into nucleic acid electrophoresis glue, gel extraction DNA fragmentation.By the DNA piece after gel extraction DNA fragmentation is recycled in section and 37 DEG C of pINFUSE-hIgG2-Fc2 empty carrier digestion 2 hours (NCoI and BglII digestion).It will recycling DNA fragmentation and carrier afterwards carries out 16 DEG C of connections overnight.5 μ L connection products are taken to convert DH5 α competent cell.Conversion plate is put Enter 37 DEG C of bacterium incubators to stay overnight, transfer within second day monoclonal colonies, carries out bacterium colony PCR identification, will identify correct bacterium colony upgrading Grain simultaneously send company to be sequenced.Correct plasmid, which is sequenced, will carry out mentioning in plasmid, prepare transfection cell.NCoI and BglII enzyme is purchased from NEB Company.DNA gel extraction kit and DNA solution QIAquick Gel Extraction Kit are purchased from Omega, and article No. is D2500-01 and D2500- 02, recovery method is referring to the description of product.DNA T4 ligase is purchased from THERMO FISHER, and article No. is EL0014, connection method ginseng According to the description of product.DH5 α competent cell is purchased from Tiangeng biochemical technology company, and article No. is CB101.
(3) preparation of recombinant protein Ephrin-B1-Fc
The expression vector pINFUSE-hIgG2-Fc2-Ephrin-B1 and pINFUSE-hIgG2-Fc2 that build is unloaded Body be transfected into 293F cell (1L cultivate cell, 1 × 106/ mL), in 37 DEG C, 5%CO2, 130rpm shakes 6 days.Transfection reagent is The PolyPlus transfection reagent of PolyPlus transfection company, article No. are FECTO PRO, and the specific dyeing method that fills is shown in production Product explanation.Collect within the 6th day after transfection cells and supernatant, centrifugation removal cell (4 DEG C, 1500rpm, 5 minutes), 0.45 μm of filter membrane Filtering.With protein G prepacked column purification of recombinant proteins, (prepacked column grinds company purchased from Wu Hanhui to supernatant, article No. after filtering HZ1012-1), purification process is shown in the description of product.By the ultrafiltration strain ultrafiltration of Milipore, (article No. is elution albumen after purification UFC501096), albumen melts in 1 × PBS after ultrafiltration, crosses 0.22 μm of filter membrane, surveys concentration, -80 DEG C of preservations.
(4) foundation of DSS inducing mouse colitis model
Acute colitis mouse model is generated, DSS (36-50kD, DSS are purchased from MP Biomedicals company) is added to In mouse drinking water (concentration 3%, mass percent), mouse is exposed to DSS water 5 days.
(5) enteritis of DSS induction is treated by injection recombinant protein Ephrin-B1-Fc
8 week old C57BL/6 mouse change light water into after being exposed to 3%DSS water 5 days, start two groups of mouse within the 0th day and distinguish abdomen Chamber injects recombinant protein Ephrin-B1-Fc, reference protein Fc (1 × PBS solution), and 50 μ g//times, injection is primary every two days, One co-injection 5 times.Mouse weight is weighed daily, is depicted as changes of weight figure.10th day execution mouse, takes colonic tissue to be H&E Dyeing.
The result is shown in Figure 1,2,3.Fig. 1 is race glue, coomassie brilliant blue staining figure after FC albumen and Ephrin-B1 protein purification. Fig. 2 is mouse weight variation diagram, it can be seen that the mouse weight of the substantially reduced DSS induction of injection recombinant protein Ephrin-B1-Fc Decline, and two groups of results have extremely significant sex differernce.Injection recombinant protein Ephrin-B1-Fc group mouse has arrived post-weight and has opened Begin to restore, mouse state is also obviously good compared with FC protein injection group mouse, shows what recombinant protein Ephrin-B1-Fc induced DSS Mouse enteritis has apparent therapeutic effect.Fig. 3 is colonic tissue H&E colored graph, it can be seen that recombinant protein Ephrin-B1-Fc Significantly improve the colitis situation of DSS induction.As seen in Figure 3, FC protein injection group mouse intestinal loses hidden Nest structure, and have a large amount of inflammatory cell infiltration；Inject recombinant protein Ephrin-B1-Fc group mouse intestinal crypts structure preservation compared with Good, inflammatory cell infiltration is less.These results suggest that injection recombinant protein Ephrin-B1-Fc has obviously the DSS enteritis induced Therapeutic effect.
Sequence table
<110>Wuhan Kang Litong Science and Technology Ltd.
<120>purposes of the Ephrin-B1 in the drug of preparation treatment inflammatory bowel disease
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<210> 1
<211> 346
<212> PRT
<213> Homo sapiens
<400> 1
Met Ala Arg Pro Gly Gln Arg Trp Leu Gly Lys Trp Leu Val Ala Met
1 5 10 15
Val Val Trp Ala Leu Cys Arg Leu Ala Thr Pro Leu Ala Lys Asn Leu
20 25 30
Glu Pro Val Ser Trp Ser Ser Leu Asn Pro Lys Phe Leu Ser Gly Lys
35 40 45
Gly Leu Val Ile Tyr Pro Lys Ile Gly Asp Lys Leu Asp Ile Ile Cys
50 55 60
Pro Arg Ala Glu Ala Gly Arg Pro Tyr Glu Tyr Tyr Lys Leu Tyr Leu
65 70 75 80
Val Arg Pro Glu Gln Ala Ala Ala Cys Ser Thr Val Leu Asp Pro Asn
85 90 95
Val Leu Val Thr Cys Asn Arg Pro Glu Gln Glu Ile Arg Phe Thr Ile
100 105 110
Lys Phe Gln Glu Phe Ser Pro Asn Tyr Met Gly Leu Glu Phe Lys Lys
115 120 125
His His Asp Tyr Tyr Ile Thr Ser Thr Ser Asn Gly Ser Leu Glu Gly
130 135 140
Leu Glu Asn Arg Glu Gly Gly Val Cys Arg Thr Arg Thr Met Lys Ile
145 150 155 160
Ile Met Lys Val Gly Gln Asp Pro Asn Ala Val Thr Pro Glu Gln Leu
165 170 175
Thr Thr Ser Arg Pro Ser Lys Glu Ala Asp Asn Thr Val Lys Met Ala
180 185 190
Thr Gln Ala Pro Gly Ser Arg Gly Ser Leu Gly Asp Ser Asp Gly Lys
195 200 205
His Glu Thr Val Asn Gln Glu Glu Lys Ser Gly Pro Gly Ala Ser Gly
210 215 220
Gly Ser Ser Gly Asp Pro Asp Gly Phe Phe Asn Ser Lys Val Ala Leu
225 230 235 240
Phe Ala Ala Val Gly Ala Gly Cys Val Ile Phe Leu Leu Ile Ile Ile
245 250 255
Phe Leu Thr Val Leu Leu Leu Lys Leu Arg Lys Arg His Arg Lys His
260 265 270
Thr Gln Gln Arg Ala Ala Ala Leu Ser Leu Ser Thr Leu Ala Ser Pro
275 280 285
Lys Gly Gly Ser Gly Thr Ala Gly Thr Glu Pro Ser Asp Ile Ile Ile
290 295 300
Pro Leu Arg Thr Thr Glu Asn Asn Tyr Cys Pro His Tyr Glu Lys Val
305 310 315 320
Ser Gly Asp Tyr Gly His Pro Val Tyr Ile Val Gln Glu Met Pro Pro
325 330 335
Gln Ser Pro Ala Asn Ile Tyr Tyr Lys Val
340 345
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<213> Homo sapiens
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atggctcggc ctgggcagcg ttggctcggc aagtggcttg tggcgatggt cgtgtgggcg 60
ctgtgccggc tcgccacacc gctggccaag aacctggagc ccgtatcctg gagctccctc 120
aaccccaagt tcctgagtgg gaagggcttg gtgatctatc cgaaaattgg agacaagctg 180
gacatcatct gcccccgagc agaagcaggg cggccctatg agtactacaa gctgtacctg 240
gtgcggcctg agcaggcagc tgcctgtagc acagttctcg accccaacgt gttggtcacc 300
tgcaataggc cagagcagga aatacgcttt accatcaagt tccaggagtt cagccccaac 360
tacatgggcc tggagttcaa gaagcaccat gattactaca ttacctcaac atccaatgga 420
agcctggagg ggctggaaaa ccgggagggc ggtgtgtgcc gcacacgcac catgaagatc 480
atcatgaagg ttgggcaaga tcccaatgct gtgacgcctg agcagctgac taccagcagg 540
cccagcaagg aggcagacaa cactgtcaag atggccacac aggcccctgg tagtcggggc 600
tccctgggtg actctgatgg caagcatgag actgtgaacc aggaagagaa gagtggccca 660
ggtgcaagtg ggggcagcag cggggaccct gatggcttct tcaactccaa ggtggcattg 720
ttcgcggctg tcggtgccgg ttgcgtcatc ttcctgctca tcatcatctt cctgacggtc 780
ctactactga agctacgcaa gcggcaccgc aagcacacac agcagcgggc ggctgccctc 840
tcgctcagta ccctggccag tcccaagggg ggcagtggca cagcgggcac cgagcccagc 900
gacatcatca ttcccttacg gactacagag aacaactact gcccccacta tgagaaggtg 960
agtggggact acgggcaccc tgtctacatc gtccaagaga tgccgcccca gagcccggcg 1020
aacatctact acaaggtctg a 1041
<210> 3
<211> 345
<212> PRT
<213> Mus musculus
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Met Ala Arg Pro Gly Gln Arg Trp Leu Ser Lys Trp Leu Val Ala Met
1 5 10 15
Val Val Leu Thr Leu Cys Arg Leu Ala Thr Pro Leu Ala Lys Asn Leu
20 25 30
Glu Pro Val Ser Trp Ser Ser Leu Asn Pro Lys Phe Leu Ser Gly Lys
35 40 45
Gly Leu Val Ile Tyr Pro Lys Ile Gly Asp Lys Leu Asp Ile Ile Cys
50 55 60
Pro Arg Ala Glu Ala Gly Arg Pro Tyr Glu Tyr Tyr Lys Leu Tyr Leu
65 70 75 80
Val Arg Pro Glu Gln Ala Ala Ala Cys Ser Thr Val Leu Asp Pro Asn
85 90 95
Val Leu Val Thr Cys Asn Lys Pro His Gln Glu Ile Arg Phe Thr Ile
100 105 110
Lys Phe Gln Glu Phe Ser Pro Asn Tyr Met Gly Leu Glu Phe Lys Lys
115 120 125
Tyr His Asp Tyr Tyr Ile Thr Ser Thr Ser Asn Gly Ser Leu Glu Gly
130 135 140
Leu Glu Asn Arg Glu Gly Gly Val Cys Arg Thr Arg Thr Met Lys Ile
145 150 155 160
Val Met Lys Val Gly Gln Asp Pro Asn Ala Val Thr Pro Glu Gln Leu
165 170 175
Thr Thr Ser Arg Pro Ser Lys Glu Ser Asp Asn Thr Val Lys Thr Ala
180 185 190
Thr Gln Ala Pro Gly Arg Gly Ser Gln Gly Asp Ser Asp Gly Lys His
195 200 205
Glu Thr Val Asn Gln Glu Glu Lys Ser Gly Pro Gly Ala Gly Gly Gly
210 215 220
Gly Ser Gly Asp Ser Asp Ser Phe Phe Asn Ser Lys Val Ala Leu Phe
225 230 235 240
Ala Ala Val Gly Ala Gly Cys Val Ile Phe Leu Leu Ile Ile Ile Phe
245 250 255
Leu Thr Val Leu Leu Leu Lys Leu Arg Lys Arg His Arg Lys His Thr
260 265 270
Gln Gln Arg Ala Ala Ala Leu Ser Leu Ser Thr Leu Ala Ser Pro Lys
275 280 285
Gly Gly Ser Gly Thr Ala Gly Thr Glu Pro Ser Asp Ile Ile Ile Pro
290 295 300
Leu Arg Thr Thr Glu Asn Asn Tyr Cys Pro His Tyr Glu Lys Val Ser
305 310 315 320
Gly Asp Tyr Gly His Pro Val Tyr Ile Val Gln Glu Met Pro Pro Gln
325 330 335
Ser Pro Ala Asn Ile Tyr Tyr Lys Val
340 345
<210> 4
<211> 1038
<212> DNA
<213> Mus musculus
<400> 4
atggcccggc ctgggcagcg ttggctcagc aagtggcttg tggctatggt cgtgctgacg 60
ctgtgccggc ttgccacgcc gttggccaag aacctggagc ccgtgtcctg gagctctctt 120
aaccctaagt tcctaagtgg gaagggcttg gtgatctacc cgaagattgg agacaagctg 180
gacatcatct gcccccgagc agaagcaggg cggccctacg agtactacaa gctgtacctg 240
gtgcggccag agcaggcggc tgcttgcagc actgtgcttg atcccaatgt actggtcact 300
tgcaacaagc cacaccagga aatccgcttc accatcaagt tccaagagtt cagccccaac 360
tacatgggcc tggaattcaa aaagtaccac gattactaca ttacatcaac gtccaatggg 420
agcttggagg gactggagaa ccgggaggga ggtgtgtgtc gcacccgcac tatgaagatc 480
gttatgaagg ttgggcaaga tccaaatgct gtgacacccg agcagttgac taccagccgg 540
ccaagcaagg agtcagacaa cactgtcaag acagccacac aggctcctgg tcggggatcc 600
cagggtgact ctgacggcaa gcatgagact gtgaaccagg aagagaagag tggcccaggt 660
gcaggtggcg gtggcagcgg ggactctgac agcttcttca actccaaggt agcattgttc 720
gcagccgtcg gcgccggctg tgtcatcttc ctgctcatca tcatcttctt gacagtccta 780
ctactcaagc tccgcaagcg ccatcgcaag catacacagc agcgggcggc tgccctctcg 840
ctcagtaccc tagccagccc caaagggggt agtggtacag cgggcaccga gcccagcgac 900
atcatcatcc ccttacggac tacagagaac aactactgcc cccactatga gaaggtgagt 960
ggggactacg ggcatcctgt ctacatcgtc caggagatgc cccctcagag cccggcgaac 1020
atctactaca aggtttga 1038

Claims (10)

  1. The purposes of 1.Ephrin-B1 or composition comprising Ephrin-B1 in the drug of preparation treatment inflammatory bowel disease.
  2. 2. purposes according to claim 1, it is characterised in that: the Ephrin-B1 is people Ephrin-B1 or mouse The amino acid sequence of Ephrin-B1, people Ephrin-B1 are as shown in SEQ ID NO.1, the amino acid sequence of mouse Ephrin-B1 As shown in SEQ ID NO.3.
  3. 3. purposes according to claim 2, it is characterised in that: the coding nucleotide sequence of people Ephrin-B1 such as SEQ ID Shown in NO.2；The coding nucleotide sequence of mouse Ephrin-B1 is as shown in SEQ ID NO.4.
  4. 4.Ephrin-B1 extracellular region polypeptide or composition comprising Ephrin-B1 extracellular region polypeptide treat inflammatory bowel in preparation Purposes in the drug of disease.
  5. 5. the fused polypeptide that is made of the heavy chain Fc structural domain of Ephrin-B1 extracellular region and Immunoglobulin IgG2 or comprising described Purposes of the composition of fused polypeptide in the drug of preparation treatment inflammatory bowel disease.
  6. 6. purposes according to claim 5, it is characterised in that: the Ephrin-B1 extracellular region behaviour Ephrin-B1 the 28th Position leucine is to the 30th lysine of the 236th serine or mouse Ephrin-B1 to the 229th serine.
  7. 7. a kind of carrier of fused polypeptide described in expression claim 5, it is characterised in that: skeleton carrier pINFUSE- HIgG2-Fc2, the nucleotide comprising encoding Ephrin-B1 extracellular region.
  8. 8. a kind of for producing the host carrier system of fused polypeptide described in claim 5, it is characterised in that: to include power Benefit require 7 described in carrier host cell.
  9. 9. a kind of method for producing fused polypeptide described in claim 5, it is characterised in that: host according to any one of claims 8 Carrier system recycles generated fused polypeptide under conditions of allowing to generate fused polypeptide.
  10. 10. purposes according to claim 1-6, it is characterised in that: the inflammatory bowel disease includes exedens Colitis, Crohn disease.
CN201810085585.XA 2018-01-29 2018-01-29 Application of Ephrin-B1 in preparation of medicine for treating inflammatory bowel disease Active CN110090295B (en)

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