CN107176976A - Application of the precursor coded polypeptide of Microrna 31 in immunoregulation medicament is prepared - Google Patents
Application of the precursor coded polypeptide of Microrna 31 in immunoregulation medicament is prepared Download PDFInfo
- Publication number
- CN107176976A CN107176976A CN201610139849.6A CN201610139849A CN107176976A CN 107176976 A CN107176976 A CN 107176976A CN 201610139849 A CN201610139849 A CN 201610139849A CN 107176976 A CN107176976 A CN 107176976A
- Authority
- CN
- China
- Prior art keywords
- polypeptide
- mipep31
- polynucleotides
- cell
- amino acid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The present invention relates to application of the precursor coded polypeptide of Microrna 31 in immunoregulation medicament is prepared.The precursor coded polypeptide of Microrna 31 of the present invention can increase the quantity of regulatory T cells, with immunoloregulation function, therefore it can be applied to immunological regulation, prevention or treatment autoimmune disease, such as multiple sclerosis.
Description
Technical field
The invention belongs to biomedicine field, more particularly it relates to prepared by the precursor coded polypeptide of Microrna 31
Application in immunoregulation medicament.
Background technology
Regulatory T cells (Regulatory Cells, abbreviation Treg) are the T of autoimmune response in a class control volume
Cell subsets, it is related closely with autoimmune disease.Regulatory T cells can be divided into naturally-produced Natural regulation
Property T cell (nTreg) and induction produce adaptability regulatory T cells (aTreg or iTreg), such as Th3, Tr1, still have in addition
CD8 Treg, NKT cells etc..
There are some researches show Treg and type i diabetes, systemic loupus erythematosus, alpastic anemia, idiopathic blood are small
The diseases such as plate reduction property purpura, autoimmune hemolytic anemia, multiple sclerosis, IBD, myasthenia gravis are close
It is related.In addition, its also with rheumatoid arthritis, autoimmune thyroiditis, autoimmune liver disease, a variety of kidney troubles
Morbidity etc. many autoimmune diseases is relevant.Therefore, Treg has for the occurrence and development of autoimmune disease
Significance, carries out furtheing investigate the pathogenesis that will be helpful to understand autoimmune disease to it, disease prognosis is judged,
Further treatment has profound significance.
Multiple sclerosis (multiple sclerosis) belongs to one kind of autoimmune disease, and it is in betiding
Chronic, the inflammatory, autoimmune disease of pivot nervous system (brain and spinal cord), it is characterized in that different in central nervous system
Normal neurological lesion (neurodegeneration) triggers the inflammatory cell based on T cell, B cell, antigen presenting cell
Infiltration, so that cause the nerve of many places demyelinate (demyelination) phenomenon occur, and in the process of myelin tissue repair
In, hardened along axonal loss.The definite pathogenic factor of multiple sclerosis is not clear, patient be likely to occur it is handicapped,
The nervous functions such as visual impairment, pain incompleteness symptom, seriously can be lethal.Multiple sclerosis there is no the medicine of radical cure at present.
Polypeptide (peptide) medicine traditionally often refers to peptide hormone.Typically by 50 or less than 50 amino acid residue groups
Into compound be included in polypeptide.Know and contained and secrete a variety of hormones and active peptides in organism, only there is in brain
Nearly 40 kinds, and people also constantly finding, separate, purify new activity polypeptid substance.The concentration of polypeptide in vivo is very
It is low, but physiologically active is very strong, and very important effect is played in regulation of physiological functions.Polypeptide drug is low due to its toxicity,
It is specific high, the unique advantage of the small grade of molecular weight itself, the optimal selection as patient.
Therefore, the polypeptide drug for autoimmune disease has important clinical value.
The content of the invention
A kind of new polypeptide is provided it is an object of the invention to provide the present invention, it can selectively increase in inflammatory environment
Plus Treg quantity, it can be applied to prepare immunoregulation medicament.
In the first aspect of the present invention there is provided a kind of polypeptide of separation, the polypeptide is selected from：
(a) there is SEQ ID NO:The polypeptide of amino acid sequence shown in 2；Or
(b) by the amino acid sequence of (a) polypeptide limited by one or more (such as 1-10；Preferably 1-5；
More preferably 1-3, such as 2) amino acid residue substitution, missing or addition formed by, and there is polypeptide that (a) limited
The polypeptide of function；Or
(c) with SEQ ID NO:Amino acid sequence shown in 2 has at least 85% (preferably at least 90%；More preferably at least
95%) the phase same sex, and the polypeptide of the function with (a) polypeptide limited.
In the preference of the present invention, described polypeptide is encoded by Microrna -31 (miRNA-31) precursor.
In another aspect of this invention there is provided a kind of polynucleotides of separation, it encodes described polypeptide.
In another aspect of this invention there is provided a kind of expression vector (including viral vector or non-virus carrier), it contains
Described polynucleotides.
In another aspect of this invention there is provided a kind of recombinant cell, wherein containing described expression vector or its genome
In include described polynucleotides.
There is provided described polypeptide or the polynucleotides for encoding it, or described expression vector in another aspect of this invention
Or application of the described recombinant cell in immunoregulation medicament is prepared.
In the preference of the present invention, described immunoregulation medicament is：Prevention or treatment autoimmune disease
Medicine.
In another preference of the present invention, described autoimmune disease includes but is not limited to：Multiple sclerosis
Disease, rheumatoid arthritis, systemic loupus erythematosus and psoriasis；Or
In another preference of the present invention, described immunoregulation medicament is to increase the number of regulatory T cells (Treg)
The medicine (by increasing Treg quantity, carrying out immunological regulation) of amount.
In another aspect of this invention there is provided a kind of method for preparing described polypeptide, methods described includes：Culture is described
Recombinant cell, so as to recombinantly express described polypeptide；Or
Methods described includes：Described polypeptide is prepared by external artificial synthesized method.
It is used for immunoregulatory pharmaceutical composition, described pharmaceutical composition there is provided one kind in another aspect of this invention
Including：Described polypeptide encodes its polynucleotides, or described expression vector or described recombinant cell；And
Pharmaceutically or physiologically acceptable carrier.
It is used for immunoregulatory medicine box there is provided one kind in another aspect of this invention, the medicine box includes：
The polypeptide stated encodes its polynucleotides；Or
Described expression vector；Or
Described recombinant cell；Or
Described pharmaceutical composition.
The other side of the present invention, due to this disclosure, is apparent to those skilled in the art
's.
Brief description of the drawings
Fig. 1, electrophoresis-immune-blotting method and GFP protein expressions and molecular weight.
Wherein, blank group only transfects the GFP plasmids of blank, and miPEP31 groups transfect miPEP-GFP and GFP empty plasmids simultaneously.
Fusion EGFP represent the band of miPEP-GFP fusion proteins.
The initiating sequence of Fig. 2, miPEP31 reading frame (ORF) can start translation GFP albumen.
A, miPEP31 initiation codon are ATG；
B, miPEP31 initiation codon are ATT.
Fig. 3, the miPEP31 of external source synthesis can be entered among cell.
The miPEP31 of A, 1nM FITC marks is co-cultured with 3T3 cells, the FITC fluorescence of Flow cytometry cell,
It can observe that FITC is entered in cell；
The miPEP31 and CD4 of B, 1nM FITC marks+T cell is co-cultured, and the FITC of Flow cytometry cell is glimmering
Light, can observe that FITC is entered in cell；
C, 3T3 cell use confocal laser scanning microscope fluorescence after contaminating nucleus with DAPI after fixing；
D, 3T3 cell are co-cultured with the miPEP31 that FITC is marked, and laser co-focusing is used after contaminating nucleus with DAPI after fixing
Micro- sem observation fluorescence；
Amplifying observation FITC fluorescence in E, upper figure.
Fig. 4, the miPEP31 of external source synthesis can promote Treg differentiation.
A, the miPEP31 of various concentrations is added in Treg differentiated system, the differentiation of Flow cytometry cell；
In B, the spleen cell that the miPEP31 of various concentrations is separated after being added to the modeling of EAE mouse 15 days, MOG is added again
Treg differentiation is detected after stimulation.
Fig. 5, the miPEP31 of external source synthesis can promote EAE maincenters Treg differentiation and treat EAE.
A, miPEP treated EAE mouse after 15 days, killed the mononuclearcell that mouse separates cental system, detection Treg's
Ratio；
The EAE scoring situations of B, miPEP treatment EAE mouse, miPEP can be obviously improved the incidence of EAE mouse；
Black point-like lines：MiPEP31 starts on the 10th day, every 3 days 1 time treatment EAE mouse scorings of 30 days；
Hollow point-like lines：Only inject the PBS control EAE mouse scorings of 30 days.
Embodiment
The present inventor's research finds that the partial nucleotide sequence of the precursor of Microrna 31 can encode one section of polypeptide of generation,
And it is referred to as the precursor coded polypeptide (miPEP31) of Microrna 31.Described miPEP31 polypeptides can increase Treg quantity, tool
There is immunoloregulation function, therefore it can be applied to immunological regulation, prevention or treatment autoimmune disease, such as multiple sclerosis
Disease.
As used herein, described " conservative variation's polypeptide " refers to the fragments of miPEP31 polypeptides, derivative and similar
Thing.Usually, being somebody's turn to do " conservative variation's polypeptide " is and SEQ ID NO:2 amino acid sequence is compared, and has at most 10, preferably
At most 5, more preferably at most 3 amino acid are replaced by the similar or close amino acid of property and form polypeptide.
As used herein, described " the precursor coded polypeptide of Microrna 31 ", " miRNA-31 precursors coded polypeptide ",
" miPEP31 polypeptides " can be with used interchangeably.
As used herein, the composition of " pharmaceutically acceptable " apply to people and/or mammal and without excessively bad
Side reaction (such as toxicity), i.e., with rational benefit/risk than material.Term " pharmaceutically acceptable carrier ", which refers to, to be used for
The carrier of Therapeutic Administration, including various excipient and diluent.The term refers to some such medicament carriers：Themselves is not
It is necessary active component, and does not have undue toxicity after administration.
As used herein, " effective dose " refers to therapeutic agent treatment, alleviates or prevent the amount of target disease or situation, or performance
Go out the amount of detectable treatment or prevention effect.
MiPEP31 polypeptides
The inventors discovered that the partial nucleotide sequence of the precursor of Microrna 31 can encode one section of polypeptide of generation, name
For miPEP31.
The miPEP31 polypeptides of the present invention can be recombinant polypeptide, synthesis polypeptide.It can be the product of chemical synthesis, or
Produced using recombinant technique from protokaryon or eucaryon host (for example, bacterium, yeast, higher plant, insect and mammalian cell)
It is raw.The method of chemical synthesis is known, such as solid-phase peptide synthesis to those skilled in the art.
The sequence of miPEP31 polypeptides of the present invention can be：
MRDWASVSSLGSGLWKERLWKSITTKRDGIAPVTRNWRGGKMLA(SEQ ID NO:2)。
Present invention additionally comprises the fragment of miPEP31 polypeptides, derivative and analog.As used herein, term " fragment ",
" derivative " and " analog " refer to be kept substantially the present invention miPEP31 polypeptide identical biological functions or activity it is many
Peptide.Polypeptide fragment, the derivative or the like of the present invention can be：
(i) there is one or more (such as 1-10,1-5,1-3 or 1-2) conservative or non-conservative amino acid residue
(preferably conservative amino acid) substituted polypeptide, and such substituted amino acid residue can be may not be by
Genetic code encoding, or
(ii) there is the polypeptide of substituted radical in one or more amino acid residues, or
(iii) mature polypeptide and another compound (such as extending the compound of polypeptide half-life period, such as polyethylene glycol)
The formed polypeptide of fusion, or
(iv) additional amino acid sequence is fused to polypeptide (such as targeting sequencing or secretion sequence formed by this peptide sequence
Or for purifying the sequence or proprotein sequence of this polypeptide, or fusion protein).According to this paper definition these fragments, derivative
Belong to scope known to those skilled in the art with analog.
In the present invention, miPEP31 polypeptides can refer to SEQ ID NO:The polypeptide of sequence shown in 2.The term is also wrapped
Including has and miPEP31 polypeptide identical functions, SEQ ID NO:The variant form (conservative variation's polypeptide) of 2 sequences.These
Variant form includes (but being not limited to)：The missing of several (such as 1-10,1-5,1-3 or 1-2) amino acid, insertion
And/or substitution, and C-terminal and/or N-terminal add one or several (be, for example, within 300, preferably 200 with
It is interior, within more preferably 100, within more preferably 50, such as 40,30,20,10,5,3,2,1) amino acid.For example, in ability
In domain, when being replaced with similar nature or similar amino acid, it will not generally change the function of protein.Again such as, at C ends
End and/or N-terminal, which add one or several amino acid, will not generally also change the function of protein.The term also includes
The active fragment and reactive derivative of miPEP31 polypeptides.
In the present invention, also including in order to increase the stability of polypeptide, half-life period, promote effect and to one or several amino
The polypeptide (not changing primary structure generally) for the modified forms that acid is constituted after being modified, including：Inner or in vitro polypeptide
Chemically derived form such as acetylation or carboxylated.Modification also includes glycosylation.Modified forms also include having phosphorylated amino acid
The sequence of residue (such as phosphotyrosine, phosphoserine, phosphothreonine).Also include being modified improving hydrolytic resistance
The polypeptide of solubility property or can be optimized.
MiPEP31 disclosed by the invention synthesis is simple, cost is low, do not have immunological rejection, can be special in inflammatory environment
Disease is uneasy to recur after different cause Treg response, good effect, treatment and Small side effects.
The complex formed after merging, be coupled or adhere to other materials present invention also offers miPEP31 polypeptides.Example
Such as, described miPEP31 polypeptides can be coupled with some fluorescence labelings (such as FITC, GFP or EGFP), consequently facilitating it was observed that
The presence situation of miPEP31 polypeptides in the cell.
The peptide that described miPEP31 polypeptides can have transmembrane ability with some is merged, to improve its penetration cell, is entered
To intracellular ability.There is the peptide of transmembrane ability to include for some：1. protein derived peptide (protein derived CPPs), such as
Penetratin, TAT and pVEC etc.；2. model peptide (model peptides) such as MAP and (Arg) 7；3. designed peptide
(designed CPPs) such as MPG and Transportan.3 classes can be also classified as from its amphipathic property：1. it is amphipathic
CPPs (PaCPPs), such as MPG, transportan, TP10, Pep-1；2. medium amphipathic CPPs (SaCPPs), such as
Penetratin, RL16；3. Non-amphiphilic CPPs (NaCPPs), such as R9.
Present invention also offers the polynucleotide sequence for encoding miPEP31 polypeptides of the present invention or its conservative variation's polypeptide.
The polynucleotides of the present invention can be DNA form or rna form.DNA can be coding strand or noncoding strand.That is, " coding is more
The polynucleotides of peptide " can be included encoding the polynucleotides of this polypeptide or also include additional code and/or non-coding
The polynucleotides of sequence.
It is many present invention also relates to the carrier of the polynucleotides comprising the present invention, and with the carrier or miPEP31 of the present invention
The host cell (recombinant cell) that the coded sequence of peptide is produced through genetic engineering, and it is of the present invention many through recombinant technique generation
The method of peptide.
Term " expression vector " refers to bacterial plasmid well known in the art, bacteriophage, yeast plasmid, plant cell virus, the food in one's mouth
Newborn zooblast virus or other carriers.In a word, as long as can be replicated in host and stably, any plasmid and carrier can
With.One key character of expression vector is to usually contain replication orgin, promoter, marker gene and translation control element.
The carrier of above-mentioned appropriate polynucleotide sequence and appropriate promoter or control sequence is included, can be used for turning
Change appropriate host cell, allow it to express polypeptide.Host cell can be prokaryotic, such as bacterial cell；Or it is low
Deng eukaryotic, such as yeast cells；Or higher eucaryotic cells, such as plant cell.Representative example has：Escherichia coli, strepto-
Pseudomonas, Agrobacterium；Fungal cell's such as yeast；Plant cell etc..
The application of miPEP31 polypeptides
Present invention also offers the application of described miPEP31 polypeptides, for preparing immunoregulation medicament, or for preparing
Increase the medicine of the quantity of regulatory T cells (Treg).It is preferred that described immunoregulation medicament is：Prevent or treat itself
The medicine of immunity disease.
In a particular embodiment of the present invention, it is determined that miPEP31 can be expressed in cell, external source is synthesized
MiPEP31 can be entered in cell, and miPEP can promote Treg to break up.Also, miPEP31 can effectively induce multiple
Property sclerosis animal model EAE mouse in tool function Treg generation, so as to mitigate EAE mouse invasions.Above-mentioned research
As a result show, miPEP31 polypeptides can be applied to increase Treg quantity, prepare immunoregulation medicament.
For example, described autoimmune disease includes：Multiple sclerosis, rheumatoid arthritis, systemic erythema
Lupus and psoriasis etc..In addition, also having to some other disease related to Treg immunoloregulation function imbalance or symptom
Potential prevention or therapeutic action.At present it is known that the disease or symptom related to Treg immunoloregulation function imbalance is selected from：It is swollen
Knurl or virus infection, inflammatory reaction, rheumatoid arthritis, organ transplant, systemic loupus erythematosus, psoriasis, Crohn's disease
Or ulcerative colitis, communicable disease etc..
For example, the tumour includes：Prostate cancer, breast cancer, liver cancer, glioma, intestinal cancer, cervix cancer, non-small cell
Lung cancer, lung cancer, cancer of pancreas, stomach cancer, carcinoma of urinary bladder, cutaneum carcinoma, striated muscle cancer, Dendritic cell, nasopharyngeal carcinoma, oophoroma, placental villi cancer,
Glioma, lymthoma, leukaemia, rectal adenocarcinoma or melanoma, etc..
For example, the inflammatory reaction includes：Allergic inflammation, epifolliculitis, tonsillitis, pneumonia, hepatitis, ephritis, acne,
Asthma, autoimmune disease, chronic inflammation, chronic prostatitis, glomerulonephritis, hypersensitivity, inflammatory bowel disease, basin
Chamber inflammation, reperfusion injury, rheumatoid arthritis, graft-rejection, vasculitis or interstitial cystitis, etc..
For example, the communicable disease includes：The plague, cholera, severe acute respiratory syndrome, AIDS, virus hepatitis,
Polio, human hepatic stellate cell, measles, Hemorrhagic fever, rabies, Japanese Type-B encephalitis, brothers
Stomatosis, dengue fever, anthrax, bacillary and amebic dysentery, pulmonary tuberculosis, Typhoid and paratyphoid, meningococal meningitis, hundred
Day cough, diphtheria, neo-nataltetenus(NNT), scarlet fever, brucellosis, gonorrhoea, syphilis, leptospirosis, snail fever, malaria
Disease, influenza, mumps, rubella, acute hemorrhagic conjunctivitis, leprosy, popularity and region macula wound
Cold, kala-azar, echinococcosis, filariasis, the infectivity in addition to cholera, bacillary and amebic dysentery, Typhoid and paratyphoid
Diarrhoeal diseases, fungal infection, etc..
Pharmaceutical composition and medicine box
The present invention also provides a kind of for immunoregulatory pharmaceutical composition, and described pharmaceutical composition includes：The present invention
Described polypeptide encodes its polynucleotides, or the expression vector containing the polynucleotides or express the polypeptide restructuring it is thin
Born of the same parents；And pharmaceutically or physiologically acceptable carrier.
Suitable pharmaceutically acceptable carrier is well known to those of ordinary skill in the art.In Remington ' s
Absolutely proving on pharmaceutically acceptable carrier can be found in Pharmaceutical Sciences.Medicine in the composition
Acceptable carrier can contain liquid on, such as water, phosphate buffer, ringer solution, physiological saline, balanced salt solution,
Glycerine or sorbierite etc..In addition, there is likely to be complementary material in these carriers, such as lubricant, glidant, wetting agent or
Emulsifying agent, pH buffer substance and stabilizer, such as albumin.
When in use, it is by the polypeptide of the present invention of safe and effective amount or encodes its many nucleosides
Acid, or the expression vector containing the polynucleotides or express the recombinant cell of the polypeptide and be applied to mammal (such as people), wherein
The safe and effective amount typically at least about 0.01 microgram/kg body weight, and in most cases it is no more than about 10 mg/kgs
Body weight.Certainly, specific dosage is also contemplated that the factors such as method of administration, patient health situation, and these are all skilled practitioners technical ability models
Within enclosing.
Accurate effective dose for a certain object depends on the build and health status, the property of illness and journey of the object
The combination of therapeutic agent and/or therapeutic agent that degree and selection are given.For certain given situation, normal experiment can be used
To determine the effective dose, clinician can interpolate that out.
Present invention also offers a kind of medicine box or kit, including：Polypeptide of the present invention encodes many of its
Nucleotides, or the expression vector containing the polynucleotides or the recombinant cell for expressing the polypeptide；Or described pharmaceutical composition.
For the ease of clinical practice, pharmaceutical composition of the invention may be embodied in injection delivery device (such as pumping needle)
In, in described injection delivery device, the described pharmaceutical composition of single administration amount can be included.Described injection administration
Device can be contained in medicine box, to facilitate storage, use.
In medicine box or kit of the present invention, operation instructions are may also include, so that those skilled in the art press
Used according to correct mode.
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention
Rather than limitation the scope of the present invention.The experimental method of unreceipted actual conditions in the following example, generally according to conventional strip
Part such as J. Pehanorm Brookers etc. are write, Molecular Cloning:A Laboratory guide, the third edition, Science Press, the condition described in 2002, or
According to the condition proposed by manufacturer.
Embodiment 1, miPEP31 sequence analyses and external synthesis
1st, miPEP31 sequence analyses
The sequence of miR-31 precursors is as follows：
ACGTAACCTAAAGCTAACAGACGGGGAAGCCATCACCAGGGTTTGGGTTGGATTCCCTACCAGTAAAATGAGGTAATGATGTG
AAATTGGTCACGTTGTTGAAGAGTTGAA
CTGTTGAACTGAGAACCTGCTATGCCAACATATTGCCATCTTTCCTGTCTGACAGCAGCTTGGCTACCTCCGTCCTG
TTCCTCCTTGTCTTGCTACAAGCCATCCATGATATGTAGGGCCCTGTGACTTGGTCTGTCTCGCCCTGACTTCTCTC
CAGTCCTATACCGAATCACTCGCTCTGTTCTAGCCACACTGGCCTTTTGGGGATGTTCTTGGCTGCACCAGGAATAT
TCCCGCCTCTACTGCCCTGTCTTATCTTTTGGGCATCAGTGGAGAACTCTTTCACCATGGCACTGTCTATAAAACCT
TACATGTGCCCAGCCACCGTTCACCTCATGACCCTGCTCTGACTTGTCAGAATCATTGGGCACTACCTGTCCATGTT
CATTTGCTTAGTTGCTGCTTGATTTACTGTACCAGGTTGTAAGTCCTTTAAGGGACACCCGTCTTCATTTCTGTTCA
CCATACCCCTAAACCCTGACGTTTGCAAGTCCTCAAGTCATGTCTTTGCGACTCTACCCTGGACTTATTGTGCAACA
GAAGTGTCAAATAATGAGATTTTAATCATGCCATGAATGGCTGTGATGAAACACTGGTTTATAAGTAACAAAGAATA
AACAAATGCTACTGATTTCTAAGCCTGCAAACCCAACATCTTAAAGGAGCCACAATAAAGTTACCATCAGGTCTACA
ACTCAGAGAAGACAAAATATTGTATGGAAAAGAGATTATATTCAAAATAAAAGTTACTTTTGCGGTTTCA(SEQ ID
NO:1)
In above-mentioned sequence, italic underscore part is miPEP31 forecasting sequences (SEQ ID NO:112-246 of 1).
The sequence for being translated as amino acid is：MRDWASVSSLGSGLWKERLWKSITTKRDGIAPVTR NWRGGKMLA(SEQ ID NO:
2)。
2nd, miPEP external synthesis
Using conventional solid-phase peptide synthesis, according to SEQ ID NO:2 amino acid sequence synthesis polypeptides, mass spectral analysis
Determine that amino acid is correct.Purity reaches that to be dissolved in PBS before more than 96%, use stand-by.
The expression of embodiment 2, miPEP31 in cell
MiPEP31 coded sequence is building up to plasmid pEGFP-N1 (Addgene) Sal1/Xhol1 restriction sites
In.So as to which the heavy grain of acquisition can form miPEP31 and GFP fusion protein after expression.
By in the Transfected Recombinant Plasmid of foregoing acquisition to adipocyte precursor cells (3T3), to be transferred to empty plasmid (pEGFP-
N1blank fat cell body cell) is control.Blank group only transfects the GFP plasmids of blank, and miPEP31 groups are transfected simultaneously
MiPEP-GFP and GFP empty plasmids.
Cell is cultivated, the expression of the method detection GFP after albumen with electrophoresis-Western blotting is extracted, as a result such as Fig. 1.
Empty plasmid group can detect GFP expression, and miPEP31 groups detect two GFP band, and wherein one point
Son amount is bigger than normal, to have merged GFP miPEP31.
In addition, the initiation codon ATG in miPEP31 coded sequences is mutated into ATT by the present inventor, the sequence is using such as
Preceding described method is building up in plasmid pEGFP-N1, is transferred to the expression of 3T3 cells.
The GFP fluorescing matters that cell before and after observation mutation is produced, as a result such as Fig. 2.By the sequence before miPEP31 reading frames
It is cloned on the GFP plasmids for removing ATG, is transferred in adipocyte precursor cells (3T3) together with initiation codon ATG, detection is thin
The GFP fluorescence that born of the same parents produce, it can be found that there is GFP fluorescence；By the sequence before miPEP31 reading frames together with initiation codon ATG
Detection GFP fluorescence, the appearance of GFP fluorescence is not found after being mutated into ATT and being cloned on the GFP plasmids for removing ATG.
The above results illustrate that miPEP31 is strictly a kind of polypeptide expressed under physiological status.
Embodiment 3, the miPEP31 of external source synthesis can be entered in cell
The miPEP31 miPEP that solid-phase peptide synthesis are obtained in embodiment 1, in its C-terminal flag F ITC.
1st, 3T3 cells
The miPEP31 that 1nM FITC are marked is co-cultured with 3T3 cells, the FITC fluorescence of Flow cytometry cell.
As a result such as Fig. 3 A, it can observe in cell there is FITC signals, illustrate that miPEP can be entered in 3T3 cells.
After 3T3 cells are fixed, nucleus is contaminated with DAPI, confocal laser scanning microscope fluorescence is used afterwards.As a result as schemed
3C.Nucleus is contaminated with DAPI, the fluorescence of blueness can be observed.
3T3 cells and 1nM the FITC miPEP31 marked are co-cultured；Afterwards, cell is fixed, nucleus is contaminated with DAPI
Confocal laser scanning microscope fluorescence is used afterwards.As a result such as Fig. 3 D~E, it is observed that existing outside in nucleus (blue-fluorescence)
Green fluorescence, is the fluorescence that FITC is produced.
Thus, it could be seen that miPEP31 can significantly enter it is intracellular.
2、CD4+T cell
By the 1nM FITC miPEP31 marked and CD4+T cell is co-cultured, and the FITC of Flow cytometry cell is glimmering
Light.As a result such as Fig. 3 B, it can observe in cell there is FITC signals, illustrating the miPEP31 of external source synthesis can significantly enter
Enter to CD4+In T cell.
Embodiment 4, miPEP promote Treg differentiation
The miPEP31 miPEP that solid-phase peptide synthesis are obtained in embodiment 1, determine its influence broken up for Treg
Effect.
1st, to the influence of the external evoked systems of Treg cells
The spleen cell of mouse is obtained, immuno magnetic cell separation mouse CD4 is utilized+CD25-T cell, is cultivated under the conditions of 37 DEG C
In 1640 culture mediums, anti-CD3 (2 μ g/ml), anti-CD28 (2 μ g/ml), TFG- β (2ng/ml) are added in the medium
Cultivate 4 days to obtain Treg with IL-2 (2ng/ml).
Treg is cultivated under the conditions of 37 DEG C, in 1640 culture mediums, is divided into several culture groups, is separately added into 0nM, 0.1nM,
The miPEP31 of 1nM and 10nM concentration, the influence that flow cytometry observation miPEP31 breaks up to Treg.
As a result such as Fig. 4 A, illustrate that the miPEP31 that various concentrations are added in the external evoked system of Treg cells can be with bright
Aobvious promotion Treg differentiation.
2nd, the influence of the lymphocyte isolated to inflammatory environment
Experimental autoimmune encephalomyelitis (EAE) mouse modeling：Heat inactivation knot is added in incomplete Freund's adjuvant
Core mycobacteria, as complete Freund's adjuvant, MOG35-55 are made into final concentration 10mg/ml.Mouse is respectively at dorsal part center line both sides 2
Point is subcutaneously injected presses 1 by MOG35-55 and CFA:1 mixing and emulsifying antigen.The immune same day and second day give mouse tail vein injection hundred
Cough toxin 200ng/ days only, inducing mouse produces EAE.
After modeling 15 days, mouse, separating spleen cell are put to death.
By 0nM, 0.1nM, the spleen that the miPEP31 of 1nM and 10nM concentration is separated after being added separately to the modeling of EAE mouse 15 days
In dirty cell, the differentiation that Treg is detected after MOG is stimulated again is added.
As a result such as Fig. 4 B, illustrate to add miPEP31 discoveries when stimulating again from the isolated lymphocyte of inflammatory environment can
To improve Treg differentiation in vitro.
Therefore, miPEP31 can selectively induce regulatory T cells to occur and reach and control curative effect in inflammatory environment
Really.
The therapeutic effect of embodiment 5, test miPEP31 to multiple sclerosis (EAE)
MiPEP31 is infused on histology, immunology, polygenes characteristic and therapeutic response and human multiple sclerosis
The very much like experimental autoimmune encephalomyelitis of disease (Experimental Autoimmune
Encephalomyelitis, EAE) in Mice Body.
It is to set up EAE models first, heat inactivation mycobacterium tuberculosis is added in incomplete Freund's adjuvant, is completely not
Family name's adjuvant, MOG35-55 is made into final concentration 10mg/ml.Mouse is subcutaneously injected by MOG35-55 for 2 points respectively at dorsal part center line both sides
1 is pressed with CFA:1 mixing and emulsifying antigen.The immune same day and second day only, lure to mouse tail vein injection pertussis toxin 200ng/
Lead mouse and produce EAE.
The miPEP31 that every disease mice starts the μ g of tail vein injection 50 on the 10th day (is dissolved in PBS, concentration is 0.5mg/
Ml), inject once within every 3 days afterwards.Observed 30 days from illness and scoring daily.Using 5 points of point systems, EAE standards of grading tool
Body is as follows：
0 point：Do not fall ill；
1 point：Flaccid tail；
2 points：Slight hind limb weakness；
3 points：Serious hind limb paralysis；
4 points：Quadriplegia；
5 points：It is at death's door or dead.
MiPEP treated EAE mouse after 15 days, killed mouse, separated the mononuclearcell of cental system, detected Treg ratio
Example.As a result such as Fig. 5 A, it is seen that compared with control group, Treg ratio is greatly improved in cental system after miPEP treatments.
Daily observation, appraisal result such as Fig. 5 B, the incidence of the EAE mouse of miPEP treatment groups are lighter, can be basic
Cure.
All documents referred in the present invention are all incorporated as reference in this application, independent just as each document
It is incorporated as with reference to such.In addition, it is to be understood that after the above-mentioned instruction content of the present invention has been read, those skilled in the art can
To be made various changes or modifications to the present invention, these equivalent form of values equally fall within the model that the application appended claims are limited
Enclose.
Claims (10)
1. a kind of polypeptide of separation, it is characterised in that the polypeptide is selected from：
(a) there is SEQ ID NO:The polypeptide of amino acid sequence shown in 2；Or
(b) by the amino acid sequence of (a) polypeptide limited is by the substitution of one or more amino acid residues, missing or adds
Formed by, and the polypeptide of the function with (a) polypeptide limited；Or
(c) with SEQ ID NO:Amino acid sequence shown in 2 has at least 85% phase same sex, and with (a) polypeptide limited
The polypeptide of function.
2. polypeptide as claimed in claim 1, it is characterised in that described polypeptide is encoded by the precursor of Microrna -31.
3. a kind of polynucleotides of separation, it encodes the polypeptide described in claim 1.
4. a kind of expression vector, it contains the polynucleotides described in claim 3.
5. a kind of recombinant cell, wherein containing including claim 3 institute in the expression vector described in claim 4 or its genome
The polynucleotides stated.
6. any described polypeptides of claim 1-2 encode its polynucleotides, or expression vector described in claim 4 or
Application of the recombinant cell in immunoregulation medicament is prepared described in claim 5.
7. application as claimed in claim 6, it is characterised in that described immunoregulation medicament is：Prevent or treat itself to exempt from
The medicine of epidemic disease disease；Or
Described autoimmune disease includes：Multiple sclerosis, rheumatoid arthritis, systemic loupus erythematosus and silver are considered to be worth doing
Disease；Or
Described immunoregulation medicament is the medicine for the quantity for increasing regulatory T cells.
8. a kind of method of the polypeptide prepared described in claim 1, it is characterised in that methods described includes：Cultivate claim 5
Described recombinant cell, so as to recombinantly express the polypeptide described in claim 1；Or
Methods described includes：The polypeptide described in claim 1 is prepared by external artificial synthesized method.
9. one kind is used for immunoregulatory pharmaceutical composition, it is characterised in that described pharmaceutical composition includes：Claim 1-
2 any described polypeptides encode its polynucleotides, or described in the expression vector or claim 5 described in claim 4
Recombinant cell；And
Pharmaceutically or physiologically acceptable carrier.
10. one kind is used for immunoregulatory medicine box, it is characterised in that the medicine box includes：
Any described polypeptides of claim 1-2 encode its polynucleotides；Or
Expression vector described in claim 4；Or
Recombinant cell described in claim 5；Or
Pharmaceutical composition described in claim 11.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610139849.6A CN107176976B (en) | 2016-03-11 | 2016-03-11 | Application of micro RNA31 precursor encoding polypeptide in preparation of immunomodulatory drugs |
| PCT/CN2017/076264 WO2017152873A1 (en) | 2016-03-11 | 2017-03-10 | Application of micro rna31 precursor encoded polypeptide for preparing immunomodulatory drugs |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610139849.6A CN107176976B (en) | 2016-03-11 | 2016-03-11 | Application of micro RNA31 precursor encoding polypeptide in preparation of immunomodulatory drugs |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107176976A true CN107176976A (en) | 2017-09-19 |
| CN107176976B CN107176976B (en) | 2020-02-14 |
Family
ID=59790061
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610139849.6A Active CN107176976B (en) | 2016-03-11 | 2016-03-11 | Application of micro RNA31 precursor encoding polypeptide in preparation of immunomodulatory drugs |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN107176976B (en) |
| WO (1) | WO2017152873A1 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108690123A (en) * | 2018-06-21 | 2018-10-23 | 上海交通大学医学院 | Application of the small peptide in preparing immunoregulation medicament |
| CN113018418A (en) * | 2021-03-09 | 2021-06-25 | 上海交通大学医学院 | Application of micro RNA31 precursor encoding polypeptide miPEP31 in preparation of hypertension drugs |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1321645A (en) * | 2000-04-29 | 2001-11-14 | 上海博德基因开发有限公司 | Novel polypeptide-KIAA0883-44 polynucleotide for coding this polypeptide |
| CN101755049A (en) * | 2007-05-29 | 2010-06-23 | 约翰内斯堡威特沃特斯兰德大学 | A primary micro RNA expression cassette |
| CN104278030A (en) * | 2013-07-05 | 2015-01-14 | 上海交通大学医学院 | Preparation method and application of small molecule compound Antagomir-31 |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP5894581B2 (en) * | 2010-05-21 | 2016-03-30 | ウニヴェルズィテート・フューア・ボーデンクルトゥーア・ウィーン | Composition for use in the treatment or diagnosis of bone disorders and / or cardiovascular disorders |
| CN102839174B (en) * | 2012-08-24 | 2013-05-29 | 中国医科大学附属第一医院 | Molecular marker miR-31 for progress of human immunodeficiency virus (HIV) infectious disease |
| CN103316342A (en) * | 2013-02-28 | 2013-09-25 | 中国人民解放军沈阳军区总医院 | Applications of miR-31 inhibitor in inhibition of angiostenosis after damage |
| CN104740648B (en) * | 2013-12-27 | 2020-02-14 | 江苏命码生物科技有限公司 | Application of miRNA-214 inhibitor in inhibiting regulatory T cells |
-
2016
- 2016-03-11 CN CN201610139849.6A patent/CN107176976B/en active Active
-
2017
- 2017-03-10 WO PCT/CN2017/076264 patent/WO2017152873A1/en active Application Filing
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1321645A (en) * | 2000-04-29 | 2001-11-14 | 上海博德基因开发有限公司 | Novel polypeptide-KIAA0883-44 polynucleotide for coding this polypeptide |
| CN101755049A (en) * | 2007-05-29 | 2010-06-23 | 约翰内斯堡威特沃特斯兰德大学 | A primary micro RNA expression cassette |
| CN104278030A (en) * | 2013-07-05 | 2015-01-14 | 上海交通大学医学院 | Preparation method and application of small molecule compound Antagomir-31 |
Non-Patent Citations (2)
| Title |
|---|
| GENBANK: "登录号：WP_035646082", 《GENBANK数据库》 * |
| 葛伏林等: "miRNA-31在肿瘤研究中的新进展", 《中华保健医学杂志》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108690123A (en) * | 2018-06-21 | 2018-10-23 | 上海交通大学医学院 | Application of the small peptide in preparing immunoregulation medicament |
| CN108690123B (en) * | 2018-06-21 | 2021-11-19 | 上海交通大学医学院 | Application of short peptide in preparation of immunoregulation medicine |
| CN113018418A (en) * | 2021-03-09 | 2021-06-25 | 上海交通大学医学院 | Application of micro RNA31 precursor encoding polypeptide miPEP31 in preparation of hypertension drugs |
| CN113018418B (en) * | 2021-03-09 | 2022-11-15 | 上海交通大学医学院 | Application of micro RNA31 precursor encoding polypeptide miPEP31 in preparation of hypertension drugs |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107176976B (en) | 2020-02-14 |
| WO2017152873A1 (en) | 2017-09-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| TW464655B (en) | Recombinant obese (OB) proteins | |
| AU784345B2 (en) | Hepatitis B core antigen fusion proteins | |
| JPS60115528A (en) | Human interleukin-2 protein, its production and pharmacological composition containing the same | |
| CN102101888B (en) | Novel polypeptide for resisting tumors caused by EB (Epstein-Barr) viruses, and application and preparation method thereof | |
| WO2024002149A1 (en) | Recombinant type-iii collagen and method for preparing same | |
| CN100564517C (en) | A kind of anti-glioma peptide of scorpion and its production and application | |
| CN106519041A (en) | Establishing method of pig immunoglobulin Fc fragment-swine classical fever E2 fusion protein in CHO cell strain, as well as preparation method and application of fusion protein | |
| CN105985427A (en) | High-affinity NY-ESO T cell receptor | |
| CN103805621B (en) | The novel preparation process of targeting antineoplastic amalgamation protein matter LPO | |
| CN111777667B (en) | Small peptide and application thereof in preparation of immunoregulation medicine | |
| CN107176976A (en) | Application of the precursor coded polypeptide of Microrna 31 in immunoregulation medicament is prepared | |
| CN112442129A (en) | Tumor enzyme response type recombinant pyropheumatin drug delivery system and anti-tumor application thereof | |
| CN108690123B (en) | Application of short peptide in preparation of immunoregulation medicine | |
| CN101117635B (en) | Fusion expression of PTD,HIF ODD and tumour inhibitory gene and uses thereof | |
| CN110256553B (en) | GLP-1 mutant and preparation method and application thereof | |
| CN112341523B (en) | Small peptide encoded by DLEU2 and application thereof in preparation of immunomodulatory drugs | |
| CN106552259A (en) | A kind of purposes of fusion protein and its treatment disease | |
| CN108250277A (en) | Application of the truncate of 31 precursor of Microrna coding polypeptide in immunoregulation medicament is prepared | |
| CN113292656A (en) | Fusion protein of mesencephalon astrocyte-derived neurotrophic factor for preventing and treating obesity | |
| CN1978466B (en) | Transduction peptide-human brain-derived neurotrophic factor fusion protein and its use | |
| CN106749561B (en) | A kind of stenotrophomonas maltophilia outer membrane protein and its application | |
| CN113501862B (en) | Polypeptide and application thereof in preparation of immunoregulation medicament | |
| ES2396231T3 (en) | Peptide complex | |
| TW201204830A (en) | Modified sodium iodide symporter proteins and uses thereof | |
| WO2021204287A1 (en) | High-affinity tcr for recognizing hpv16 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |