CN108250277A - Application of the truncate of 31 precursor of Microrna coding polypeptide in immunoregulation medicament is prepared - Google Patents
Application of the truncate of 31 precursor of Microrna coding polypeptide in immunoregulation medicament is prepared Download PDFInfo
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Abstract
The present invention relates to 31 precursors of Microrna to encode application of the truncate of polypeptide in immunoregulation medicament is prepared.Present invention is disclosed 31 precursor of Microrna coding polypeptides (miPEP31) and its truncate of reservation function, most short function domain polypeptide to be known as miniPEP31, it may have increases the function of Treg quantity.The truncate and most short functional fragment 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 the truncation of 31 precursor of Microrna coding polypeptide
The application of body and its most short function domain polypeptide in immunoregulation medicament is prepared.
Background technology
Regulatory T cells (Regulatory Cells, abbreviation Treg) are the T of autoimmune response in a kind of control volume
Cell subsets, with being related closely for autoimmune disease.Regulatory T cells can be divided into naturally-produced Natural regulation
Property T cell (nTreg) and induction generate adaptability regulatory T cells (aTreg or iTreg), such as Th3, Tr1, in addition still have
CD8Treg, NKT cell etc..
Some researches show that Treg and type-1 diabetes mellitus, systemic loupus erythematosus, alpastic anemia, idiopathic blood are small
The diseases such as plate reduction property purpura, autoimmune hemolytic anemia, multiple sclerosis, inflammatory bowel disease, 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 related.Therefore, Treg has the occurrence and development of autoimmune disease
Significance carries out it pathogenesis that further investigation will be helpful to understand autoimmune disease, disease prognosis is judged,
Further treatment has profound significance.
Multiple sclerosis (multiple sclerosis) belongs to one kind of autoimmune disease, 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) causes the inflammatory cell based on T cell, B cell, antigen presenting cell
, there is demyelinate (demyelination) phenomenon, and in the process of myelin tissue repair so as to cause the nerve of many places in infiltration
In, it is 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 incompleteness symptom such as visual impairment, pain, seriously can be lethal.Multiple sclerosis there is no the drug of radical cure at present.
Polypeptide (peptide) drug traditionally often refers to peptide hormone.Generally 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 are also constantly having found, are detaching, purifying new activity polypeptid substance.Polypeptide is steady with the increase of length
Qualitative and internal half-life period all phase strain differential or shortening.Also not necessarily full sequence is all to play work(to the polypeptide of physiological conditions
Necessary to energy.By exploring its most short functional domain functioned, we can increase the stability of polypeptide drugs and partly decline
Phase reduces synthesis cost, reduces bio-toxicity.The concentration of polypeptide in vivo is very low, but physiological activity is very strong, is given birth to adjusting
Very important effect is played when managing function.For polypeptide drug since its toxicity is low, specificity is high, and the small grade of molecular weight itself is unique
Advantage, become the optimal selection of patient.
Therefore, there is important clinical value for the polypeptide drug of autoimmune disease.
Invention content
The purpose of the present invention is to provide the present invention to provide a kind of new polypeptide and its truncate and most short function domain polypeptide,
They can selectively increase the quantity of Treg in inflammatory environment, can be applied to prepare immunoregulation medicament.
In the first aspect of the present invention, a kind of polypeptide of separation is provided, which is selected from：
(a) come from SEQ ID NO:2, and N-terminal originates in SEQ ID NO:The amino acid of 2 2-13 any bits, C
End terminates at SEQ ID NO:The polypeptide that the amino acid of 2 42-44 any bits is formed；
(b) amino acid sequence of any polypeptide for being limited (a) is by 1-5 (more preferably 1-3, such as 2) amino acid
Substitution, missing or the addition of residue and formed, and there is the polypeptide of the polypeptide；Or
(c) amino acid sequence of any polypeptide limited with (a) has at least 85% (preferably at least 90%；More preferably
At least 95%) the phase same sex, and there is the polypeptide of the polypeptide.
In a preference, the polypeptide of (a) is selected from：
SEQ ID NO:(the SEQ ID NO of amino acid sequence shown in 3:13-42 in 2) polypeptide；
SEQ ID NO:The polypeptide of amino acid sequence shown in 2-44 in 2；
SEQ ID NO:The polypeptide of amino acid sequence shown in 3-44 in 2；
SEQ ID NO:The polypeptide of amino acid sequence shown in 4-44 in 2；
SEQ ID NO:The polypeptide of amino acid sequence shown in 5-44 in 2；
SEQ ID NO:The polypeptide of amino acid sequence shown in 6-44 in 2；
SEQ ID NO:The polypeptide of amino acid sequence shown in 7-44 in 2；
SEQ ID NO:The polypeptide of amino acid sequence shown in 8-44 in 2；
SEQ ID NO:The polypeptide of amino acid sequence shown in 9-44 in 2；
SEQ ID NO:The polypeptide of amino acid sequence shown in 10-44 in 2；
SEQ ID NO:The polypeptide of amino acid sequence shown in 11-44 in 2；
SEQ ID NO:The polypeptide of amino acid sequence shown in 12-44 in 2；
SEQ ID NO:The polypeptide of amino acid sequence shown in 13-44 in 2；
SEQ ID NO:The polypeptide of amino acid sequence shown in 1-43 in 2；Or
SEQ ID NO:The polypeptide of amino acid sequence shown in 1-42 in 2.
In another preferred example, the polypeptide is encoded by Microrna -31 (miRNA-31) precursor.
In another aspect of this invention, a kind of polynucleotides of separation, the coding polypeptide are provided.
In another aspect of this invention, a kind of expression vector (including viral vectors or non-virus carrier) is provided, is contained
The polynucleotides.
In another aspect of this invention, a kind of recombinant cell is provided, wherein containing the expression vector or its genome
In include the polynucleotides.
In another aspect of this invention, the polypeptide is provided or encodes its polynucleotides or the expression vector
Or application of the recombinant cell in immunoregulation medicament is prepared.
In a preference, the immunoregulation medicament is：Prevention or the drug for the treatment of autoimmune disease.
In another preferred example, the autoimmune disease includes：Multiple sclerosis, rheumatoid arthritis,
Systemic loupus erythematosus and psoriasis etc..
In another preferred example, the immunoregulation medicament is the drug for the quantity for increasing regulatory T cells (Treg)
(by increasing the quantity of Treg, carrying out immunological regulation).
In another aspect of this invention, a kind of method for preparing the polypeptide is provided, the method includes：Described in culture
Recombinant cell, so as to recombinantly express the polypeptide；Or the method includes：Institute is prepared by external artificial synthesized method
The polypeptide stated.
In another aspect of this invention, it provides a kind of for immunoregulatory pharmaceutical composition, the pharmaceutical composition
Including：Any polypeptide in front or the polynucleotides or the expression vector or the recombinant cell for encoding it；With
And pharmacological or physiological acceptable carrier.
In another aspect of this invention, one kind is provided for immunoregulatory medicine box, and the medicine box includes：
Any polypeptide in front encodes its polynucleotides；Or
The expression vector；Or
The recombinant cell；Or
The pharmaceutical composition.
The other aspects of the present invention are apparent to those skilled in the art due to this disclosure
's.
Description of the drawings
Figure 1A, mass spectral analysis determine miPEP31 amino acid correctness, and the molecular weight of Mass Spectrometer Method is 5003.88.
The purity analysis of Figure 1B, HPLC, purity 95.37%.
Fig. 2, 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 the reading frame (ORF) of Fig. 3, miPEP31 can start translation GFP albumen.
The initiation codon of A, miPEP31 are ATG；
The initiation codon of B, miPEP31 are ATT.
The miPEP31 that Fig. 4, external source synthesize can be entered in cell.
The miPEP31 of A, 1nM FITC labels 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 labels+T cell co-cultures, 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 and the miPEP31 of FITC labels are co-cultured, and laser co-focusing is used after contaminating nucleus with DAPI after fixed
Micro- sem observation fluorescence；
E, the miPEP31 and CD4 that FITC is marked+T cell co-cultures, the FITC fluorescence of Flow cytometry cell.
The miPEP31 that Fig. 5, external source synthesize can promote the differentiation of Treg.
A, the miPEP31 of various concentration are added in the differentiated system of Treg, the differentiation of Flow cytometry cell；
In the spleen cell that B, the miPEP31 of various concentration are detached after being added to the modeling of EAE mouse 15 days, MOG is added in again
The differentiation of Treg is detected after stimulation.
The most short function domain polypeptide miniPEP of Fig. 6, miPEP31 can promote the differentiation of Treg.
A according to the amino acid sequence from N-terminal to C-terminal, from N reduces by 1 amino acid and is obtained to the 21st amino acid one by one respectively
To 1-21 polypeptides；It reduces by 1 amino acid one by one from C-terminal and obtains 42-22 polypeptides to the 21st amino acid.In above-mentioned TREG
The 1-42 polypeptides of 10nM synthesis, the influence that flow cytometry observation polypeptide breaks up Treg are added in the differentiated system of cell；
Wherein NC is negative control (being added without polypeptide).
B-C, in vitro according to above-mentioned amino acid sequence synthesis polypeptide：miniPEP31；It is added to above-mentioned TREG differentiation later
In system, the differentiation ratio of TREG is observed.
D, miniPEP31 are for human peripheralCD4+The influence that T cell is broken up to Treg.
The miPEP31 that Fig. 7, external source synthesize can promote the differentiation of EAE maincenters Treg and treat EAE.
A, miniPEP treated EAE mouse after 15 days, killed the mononuclearcell of mouse separation cental system, detected Treg
Ratio；
The EAE scoring situations of B, miniPEP treatment EAE mouse, miPEP can be obviously improved the incidence of EAE mouse；
The dotted lines of black：MiniPEP31 starts on the 10th day, every 3 days 1 time treatment EAE mouse scorings of 30 days；
Hollow dotted lines：The only control EAE mouse scorings of 30 days of injection PBS.
Specific embodiment
The present inventor generates a segment polypeptide the study found that the partial nucleotide sequence of the precursor of Microrna 31 can encode,
And it is known as 31 precursor of Microrna coding polypeptide (miPEP31).The miPEP31 polypeptides can increase Treg quantity, tool
There is immunoloregulation function.On the basis of the discovery, the present inventor further studies its Core Feature sequence and reservation function
Truncate, most short function domain polypeptide is known as miniPEP31, it may have increases the function of Treg quantity.The truncate and
Most short functional fragment can be applied to immunological regulation, prevention or treatment autoimmune disease, such as multiple sclerosis.
As used herein, described " 31 precursor of Microrna encodes polypeptide ", " miRNA-31 precursors encode polypeptide ",
" miPEP31 polypeptides " may be used interchangeably.
As used herein, " the most short function domain polypeptides of miPEP31 ", " miPEP31 Core Features sequences polypeptide ",
" miniPEP31 polypeptides " can be used mutually.
As used herein, the ingredient 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 substance.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
Active constituent is necessary, and does not have excessive toxicity after applying.
As used herein, " effective quantity " refers to therapeutic agent treatment, the amount for alleviating or preventing target disease or situation or performance
Go out the detectable amount for treating or preventing effect.
MiPEP31 polypeptides or its truncate
The inventors discovered that the partial nucleotide sequence of the precursor of Microrna 31, which can encode, generates a segment polypeptide, name
For miPEP31.
The miPEP31 polypeptides of the present invention can be recombinant polypeptide, synthesis polypeptide.It can be chemical synthesis product or
It is produced from protokaryon or eucaryon host (for example, bacterium, yeast, higher plant, insect and mammalian cell) using recombinant technique
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：MRDWASVSSLGSGLWKERLWKSITTKRDGIAPVTRNW
RGGKMLA(SEQ ID NO:2)。
Segment, derivative and analogue the invention also includes miPEP31 polypeptides.As used herein, term " segment ",
" derivative " and " analog " refer to the more of the identical biological function of the miPEP31 polypeptides for being kept substantially the present invention or activity
Peptide.The segments of miPEP31 polypeptides, derivative or the like can be：
(i) there are one or multiple (such as 1-10,1-5,1-3 or 1-2) conservative or non-conservative amino acid residues
(preferably conservative amino acid) substituted polypeptide, and such substituted amino acid residue can be may not be by
Genetic code encoding or
(ii) in one or more amino acid residues with substituent group polypeptide or
(iii) mature polypeptide and another compound (for example extending the compound of polypeptide half-life period, such as polyethylene glycol)
The formed polypeptide of fusion or
(iv) polypeptide (such as targeting sequencing or secretion sequence that additional amino acid sequence is fused to this peptide sequence and is formed
Or for purifying the sequence of this polypeptide or proprotein sequence or fusion protein).According to the definition of this paper these segments, derivative
It is belonged 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 also wraps
Including has and miPEP31 polypeptide identical functions, SEQ ID NO:The variant form (conservative variation's polypeptides) 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 is inserted into
And/or substitution and C-terminal and/or N-terminal addition it is one or several (be, for example, 300 within, preferably 200 with
It is interior, within more preferably 100, within more preferably 50, for example, 40,30,20,10,5,3,2,1) amino acid.For example, in ability
In domain, when being substituted with similar nature or similar amino acid, the function of protein is not usually changed.For another example, 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 further includes
The active fragment and reactive derivative of miPEP31 polypeptides.
The present invention main contributions be to obtain the truncate of miPEP31 polypeptides, the polypeptide is miPEP31 polypeptides
Retentive activity truncate, be selected from：(a) come from SEQ ID NO:2, and N-terminal originates in SEQ ID NO:2-13 of 2
The amino acid of any bit, C-terminal terminate at SEQ ID NO:The polypeptide that the amino acid of 2 42-44 any bits is formed；(b)
The amino acid sequence of any polypeptide that (a) is limited by 1-5 (more preferably 1-3, such as 2) amino acid residue substitution,
It lacks or adds and formed, and there is the polypeptide of the polypeptide；Or the amino of any polypeptide that (c) is limited with (a)
Acid sequence has at least 85% (preferably at least 90%；More preferably at least 95%) the phase same sex, and there are the more of the polypeptide
Peptide.
In the present invention, also include 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 usually) for the modified forms that acid is formed after being modified, including：In vivo or in vitro polypeptide
Chemical derivative form such as acetylation or carboxylated.Modification further includes glycosylation.Modified forms are further included with phosphorylated amino acid
The sequence of residue (such as phosphotyrosine, phosphoserine, phosphothreonine).It further includes and is modified to improve hydrolytic resistance
It can or optimize the polypeptide of solubility property.For example, in the truncate, the new of modification formation is carried out for partial amino-acid
, retain original function polypeptide.
MiPEP31 disclosed by the invention synthesis is simple, at low cost, 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.MiPEP31 disclosed by the invention
Truncate be further simplified in synthesis, and therapeutic effect is extremely notable.
It is formed after merging, be coupled or adhere to other materials the present invention also provides miPEP31 polypeptides or its truncate
Complex.For example, the miPEP31 polypeptides or its truncate can be with some fluorescent markers (such as FITC, GFP or EGFP)
Coupling, consequently facilitating observe miPEP31 polypeptides or its truncate in the cell there are situations.
The miPEP31 polypeptides or its truncate can be merged with some peptides with transmembrane ability, be worn with improving it
Saturating cell enters intracellular ability.Some peptides with transmembrane ability include：1. protein derived peptide (protein
Derived CPPs), such as penetratin, TAT and pVEC；2. model peptide (model peptides) such as MAP and (Arg) 7
Deng；Designed peptide 3. (designed CPPs) such as MPG and Transportan.3 classes can be also classified as from its amphipathic property：
1. 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.
The present invention also provides encode miPEP31 polypeptides of the present invention or its truncate or the multinuclear of its conservative variation's polypeptides
Nucleotide sequence.The polynucleotides of the present invention can be DNA form or rna form.DNA can be coding strand or noncoding strand.
That is, " polynucleotides of coding polypeptide " can be included encoding the polynucleotides of this polypeptide or further include additional code
And/or the polynucleotides of non-coding sequence.
Carrier the present invention also relates to the polynucleotides comprising the present invention and the carrier or miPEP31 with the present invention are more
The genetically engineered host cell of the coded sequence of peptide or its truncate (recombinant cell) and through recombinant technique generate this
The method for inventing the polypeptide.
Term " expression vector " refers to bacterial plasmid well known in the art, bacteriophage, yeast plasmid, plant cell virus, feeds
Newborn zooblast virus or other carriers.As long as in short, can replicate and stablize in host, any plasmid and carrier can
With.One important feature of expression vector is to usually contain replication orgin, promoter, marker gene and translation control element.
Comprising above-mentioned appropriate polynucleotide sequence and the carrier of appropriate promoter or control sequence, can be used for turning
Change appropriate host cell, allow it to expression polypeptide.Host cell can be prokaryotic cell, such as bacterial cell；It is or low
Wait eukaryocytes, 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 or its truncate
The present invention also provides the applications of the miPEP31 polypeptides or its truncate, are used to prepare immunoregulation medicament,
Or it is used to prepare the drug for the quantity for increasing regulatory T cells (Treg).Preferably, the immunoregulation medicament is：Prevention
Or the drug for the treatment of autoimmune disease.
In a specific embodiment of the present invention, it is determined that miPEP31 or its truncate can express in cell, and external source is closed
Into miPEP31 or its truncate can enter in cell, miPEP31 or its truncate can promote Treg to break up.Also,
MiPEP31 or its truncate can effectively induce the Treg of tool function in the animal model EAE mouse of multiple sclerosis
It generates, so as to mitigate EAE mouse invasions.Using the method for reducing amino acid one by one, the present inventor has found in miPEP31 and plays
The polypeptide miniPEP31 of key effect.Above-mentioned result of study shows that miPEP31 polypeptides can be applied to increase the quantity of Treg,
Its core sequence is miniPEP31, available for preparing immunoregulation medicament.
For example, the autoimmune disease includes：Multiple sclerosis, rheumatoid arthritis, systemic erythema
Lupus and psoriasis etc..In addition, also have to the relevant disease of the immunoloregulation function of some other and Treg imbalance or symptom
Potential prevention or therapeutic effect.At present it is known that the relevant disease of immunoloregulation function imbalance or symptom with Treg are 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, gastric 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 one kind for immunoregulatory pharmaceutical composition, the pharmaceutical composition includes：The present invention
The polypeptide or encode its polynucleotides containing the polynucleotide expression vector or express the polypeptide recombination it is thin
Born of the same parents；And pharmacological or physiological acceptable carrier.
Suitable pharmaceutically acceptable carrier is well known to those of ordinary skill in the art.In Remington ' s
Absolutely proving about pharmaceutically acceptable carrier can be found in Pharmaceutical Sciences.Medicine in the composition
Acceptable carrier can contain liquid on, as water, phosphate buffer, ringer solution, physiological saline, balanced salt solution,
Glycerine or sorbierite etc..In addition, there is likely to be complementary substance in these carriers, as lubricant, glidant, wetting agent or
Emulsifier, 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 polynucleotides or contain this
The expression vector of polynucleotides expresses the recombinant cell of the polypeptide and is applied to mammal (such as people), the wherein 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/kg weight.Certainly, have
Body dosage is also contemplated that the factors such as administration route, patient health situation, within the scope of these are all skilled practitioners technical ability.
Build and health status, the property of the illness and journey of the object are depended on for the accurate effective quantity of certain an object
The combination of therapeutic agent and/or therapeutic agent that degree and selection are given.For the situation that Mr. Yu gives, routine experiment can be used
Determine the effective quantity, clinician can judge.
The present invention also provides a kind of medicine box or kit, including：Polypeptide of the present invention encodes the more of its
Nucleotide or containing the polynucleotide expression vector or the recombinant cell for expressing the polypeptide；Or the 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 the injection delivery device, the pharmaceutical composition of single administration amount can be included.The 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
It is used according to correct mode.
With reference to specific embodiment, the present invention is further explained.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.Test method without specific conditions in the following example, usually according to conventional strip
Part such as J. Pehanorm Brookers etc. are write, Molecular Cloning:A Laboratory guide, the third edition, Science Press, condition described in 2002 or
According to the normal 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：
In above-mentioned sequence, italic underscore part is miPEP31 forecasting sequences.The sequence for being translated as amino acid is：
2nd, the external synthesis of miPEP31 and miniPEP31
Using conventional solid-phase peptide synthesis, according to SEQ ID NO:2 amino acid sequence synthesis polypeptides, mass spectral analysis
Determine amino acid correctly such as Figure 1A (miPEP31).Purity is 95.37%, such as Figure 1B.It is for use that PBS is dissolved in before use.
The expression of embodiment 2, miPEP31 in cell
The coded sequence of miPEP31 is building up to the Sal1/Xhol1 restriction sites of plasmid pEGFP-N1 (Addgene)
In.So as to which the heavy grain of acquisition can form the fusion protein of miPEP31 and GFP after expression.
By in the Transfected Recombinant Plasmid of aforementioned acquisition to adipocyte precursor cells (3T3), to be transferred to empty plasmid (pEGFP-
N1blank adipocyte body cell) is control.Blank group only transfects the GFP plasmids of blank, and miPEP31 groups transfect simultaneously
MiPEP-GFP and GFP empty plasmids.
Cell is cultivated, the method for electrophoresis-immunoblotting is used to detect the expression of GFP after extracting albumen, as a result such as Fig. 2.
Empty plasmid group can detect the expression of GFP, and miPEP31 groups detect the band of two GFP, and wherein one point
Son amount is bigger than normal, to have merged the miPEP31 of GFP.
In addition, the initiation codon ATG in miPEP31 coded sequences is mutated into ATT by the present inventor, the sequence is using such as
The preceding method is building up in plasmid pEGFP-N1, is transferred to the expression of 3T3 cells.
The GFP fluorescing matters that the front and rear cell of observation mutation generates, as a result such as Fig. 3 A, B.Before miPEP31 reading frames
Sequence is cloned into together with initiation codon ATG on the GFP plasmids of removal ATG, is transferred in adipocyte precursor cells (3T3), is examined
The GFP fluorescence that cell generates is surveyed, it can be found that there is GFP fluorescence；By the sequence before miPEP31 reading frames together with initiation codon
The fluorescence of detection GFP, is not found going out for GFP fluorescence after ATG is mutated into ATT and is cloned on the GFP plasmids of removal ATG
It is existing.
The above results illustrate that miPEP31 is strictly a kind of polypeptide expressed under physiological status.
The miPEP31 that embodiment 3, external source synthesize can be entered in cell
The miPEP31miPEP that solid-phase peptide synthesis obtains 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. 4 A, it can observe that there are FITC signals in cell, illustrate that miPEP can be entered in 3T3 cells.
After 3T3 cells are fixed, nucleus is contaminated with DAPI, later with confocal laser scanning microscope fluorescence.As a result as schemed
4B.Nucleus is contaminated with DAPI, can observe the fluorescence of blue.
The miPEP31 of 3T3 cells and 1nM FITC labels is co-cultured；Later, the cells are fixed, contaminates nucleus with DAPI
Confocal laser scanning microscope fluorescence is used afterwards.As a result such as Fig. 4 C, D, it is observed that existing outside in nucleus (blue-fluorescence)
Green fluorescence, the fluorescence generated for FITC.
Thus, it could be seen that miPEP31 can be entered significantly into the cell.
2、CD4+T cell
By the 1nM FITC miPEP31 marked and CD4+T cell co-cultures, and the FITC of Flow cytometry cell is glimmering
Light.As a result such as Fig. 4 E, can observe in cell there are FITC signals, illustrate external source synthesis miPEP31 can significantly into
Enter to CD4+In T cell.
Embodiment 4, miPEP promote Treg cell differentiations
The miPEP31miPEP that solid-phase peptide synthesis obtains in embodiment 1 measures it for Treg cell differentiations
Influence.
1st, to the influence of the external evoked system of Treg cells
The spleen cell of mouse is obtained, utilizes immuno magnetic cell separation mouse CD4+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
4 days are cultivated with IL-2 (2ng/ml) to obtain Treg.
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 Treg.
As a result such as Fig. 5 A, illustrate that the miPEP31 that various concentration is added in the external evoked system of Treg cells can be bright
The differentiation of aobvious promotion Treg.
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 back side 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
Day cough toxin 200ng/, only inducing mouse generated EAE.
After modeling 15 days, mouse, separating spleen cell are put to death.
By 0nM, spleen that 0.1nM, the miPEP31 of 1nM and 10nM concentration are detached 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 in.
As a result such as Fig. 5 B, illustrating to add in miPEP31 discoveries when stimulating again from the isolated lymphocyte of inflammatory environment can
To improve the differentiation of Treg in vitro.
Therefore, miPEP31 can selectively induce regulatory T cells to occur in inflammatory environment and reach treatment effect
Fruit.
Embodiment 5, miniPEP31 are the most short ordered sequence that Treg differentiation is improved in miPEP31
On the basis of, according to the amino acid sequence from N-terminal to C-terminal, reducing by 1 amino acid one by one from N respectively, (C-terminal is not
Become) obtain 1-21 polypeptides to the 21st amino acid；Reduce by 1 amino acid (N-terminal is constant) one by one from C-terminal to the 21st amino acid
Obtain 42-22 polypeptides.
MiPEP31 sequences：
No. 1 peptide：SEQ ID NO:2-44 in 2；
No. 2 peptides：SEQ ID NO:3-44 in 2；
No. 3 peptides：SEQ ID NO:4-44 in 2；
No. 4 peptides：SEQ ID NO:5-44 in 2；
No. 5 peptides：SEQ ID NO:6-44 in 2；
No. 6 peptides：SEQ ID NO:7-44 in 2；
No. 7 peptides：SEQ ID NO:8-44 in 2；
No. 8 peptides：SEQ ID NO:9-44 in 2；
No. 9 peptides：SEQ ID NO:10-44 in 2；
No. 10 peptides：SEQ ID NO:11-44 in 2；
No. 11 peptides：SEQ ID NO:12-44 in 2；
No. 12 peptides：SEQ ID NO:13-44 in 2；
No. 13 peptides：SEQ ID NO:14-44 in 2；
No. 14 peptides：SEQ ID NO:15-44 in 2；
No. 15 peptides：SEQ ID NO:16-44 in 2；
No. 16 peptides：SEQ ID NO:17-44 in 2；
No. 17 peptides：SEQ ID NO:18-44 in 2；
No. 18 peptides：SEQ ID NO:19-44 in 2；
No. 19 peptides：SEQ ID NO:20-44 in 2；
No. 20 peptides：SEQ ID NO:21-44 in 2；
No. 21 peptides：SEQ ID NO:22-44 in 2；
No. 42 peptides：SEQ ID NO:1-43 in 2；
No. 41 peptides：SEQ ID NO:1-42 in 2；
No. 40 peptides：SEQ ID NO:1-41 in 2；
No. 39 peptides：SEQ ID NO:1-40 in 2；
No. 38 peptides：SEQ ID NO:1-39 in 2；
No. 37 peptides：SEQ ID NO:1-38 in 2；
No. 36 peptides：SEQ ID NO:1-37 in 2；
No. 35 peptides：SEQ ID NO:1-36 in 2；
No. 34 peptides：SEQ ID NO:1-35 in 2；
No. 33 peptides：SEQ ID NO:1-34 in 2；
No. 32 peptides：SEQ ID NO:1-33 in 2；
No. 31 peptides：SEQ ID NO:1-32 in 2；
No. 30 peptides：SEQ ID NO:1-31 in 2；
No. 29 peptides：SEQ ID NO:1-30 in 2；
No. 28 peptides：SEQ ID NO:1-29 in 2；
No. 27 peptides：SEQ ID NO:1-28 in 2；
No. 26 peptides：SEQ ID NO:1-27 in 2；
No. 25 peptides：SEQ ID NO:1-26 in 2；
No. 24 peptides：SEQ ID NO:1-25 in 2；
No. 23 peptides：SEQ ID NO:1-24 in 2；
No. 22 peptides：SEQ ID NO:1-23 in 2.
In vitro 1-42 polypeptides are synthesized according to above-mentioned listed amino acid sequence.
Treg cells are obtained as described in Example 5；Under the conditions of 37 DEG C, Treg cells are cultivated in 1640 culture mediums.At this
The 1-42 polypeptides of 10nM synthesis, the shadow that flow cytometry observation polypeptide breaks up Treg are added in the differentiated system of Treg cells
It rings.As a result as shown in Figure 6A, the effect of the promotion Treg differentiation of 1-12 polypeptides does not change, and 13-40 polypeptides promote Treg
The effect of differentiation reduces, and the effect of the promotion Treg differentiation of 41 and No. 42 polypeptides does not change.
The above results show：Sequence (the i.e. SEQ ID NO of 13rd amino acid to the 42nd amino acid:13-42 in 2) be
Shortest ordered sequence.I.e. above-mentioned SEQ ID NO:In 2 sequences, italic underscore part and the most short functional domain for miPEP31
It is named as miniPEP31 by polypeptide, the present inventor, and sequence is：GLWKERLWKSITTKRDGIAPVTRNWRGGKM(SEQ ID
NO:3)。
In vitro according to amino acid sequence SEQ ID NO:3 synthesis polypeptides：miniPEP31.It is added to above-mentioned Treg points afterwards
In change system, as shown in Fig. 6 B-C, the differentiation ratio of Treg is obviously improved, and is illustrated that miniPEP31 has and is promoted Treg differentiation
Function.Wherein, Control is that the random of miniPEP31 upsets sequence.
Meanwhile the expression of flow cytometry FOXP3.As a result as it can be seen that miniPEP31, which has, promotes human peripheralCD4+The function that T cell is broken up to Treg, such as Fig. 6 D.
Embodiment 6 tests therapeutic effects of the miniPEP31 to multiple sclerosis (EAE)
MiniPEP31 is infused in hard with human multiple on histology, immunology, polygenes characteristic and therapeutic response
Change very much like experimental autoimmune encephalomyelitis (the Experimental Autoimmune of disease
Encephalomyelitis, EAE) in Mice Body.
It is to establish EAE models first, heat inactivation mycobacterium tuberculosis is added in incomplete Freund's adjuvant, as completely not
Family name's adjuvant, MOG35-55 are made into final concentration 10mg/ml.Mouse is subcutaneously injected by MOG35-55 for 2 points respectively at back side 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/
It leads mouse and generates EAE.
The 10th day miPEP31 for starting 50 μ g of tail vein injection of every disease mice (is dissolved in PBS, a concentration of 0.5mg/
Ml), injection in every 3 days later is primary.It observes 30 days from illness and scores daily.Using 5 points of point systems, EAE standards of grading tool
Body is as follows：
0 point：It does 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.
MiniPEP treated EAE mouse after 15 days, kills mouse, detaches the mononuclearcell of cental system, detect Treg's
Ratio.As a result such as Fig. 7 A, it is seen that compared with the control group, the ratio of Treg is greatly carried in cental system after miniPEP treatments
It is high.
Daily observation, appraisal result such as Fig. 7 B, the incidence of the EAE mouse of miniPEP treatment groups is lighter, can be by base
This healing.
All references mentioned in the present invention is incorporated herein by reference, independent just as each document
It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can
To be made various changes or modifications to the present invention, such equivalent forms equally fall within the model that the application the appended claims are limited
It encloses.
Sequence table
<110>Medical College, Shanghai Communication Univ.
<120>Application of the truncate of 31 precursor of Microrna coding polypeptide in immunoregulation medicament is prepared
<130> 169590
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 1009
<212> DNA
<213>Homo sapiens (Homo Sapiens)
<400> 1
acgtaaccta aagctaacag acggggaagc catcaccagg gtttgggttg gattccctac 60
cagtaaaatg aggtaatgat gtgaaattgg tcacgttgtt gaagagttga aatgagagat 120
tgggcatcag tatccagctt aggttccggc ctgtggaagg agagattgtg gaaaagcata 180
acaacgaaga gggatggtat tgctcctgta actcggaact ggagaggagg caagatgctg 240
gcatagctgt tgaactgaga acctgctatg ccaacatatt gccatctttc ctgtctgaca 300
gcagcttggc tacctccgtc ctgttcctcc ttgtcttgct acaagccatc catgatatgt 360
agggccctgt gacttggtct gtctcgccct gacttctctc cagtcctata ccgaatcact 420
cgctctgttc tagccacact ggccttttgg ggatgttctt ggctgcacca ggaatattcc 480
cgcctctact gccctgtctt atcttttggg catcagtgga gaactctttc accatggcac 540
tgtctataaa accttacatg tgcccagcca ccgttcacct catgaccctg ctctgacttg 600
tcagaatcat tgggcactac ctgtccatgt tcatttgctt agttgctgct tgatttactg 660
taccaggttg taagtccttt aagggacacc cgtcttcatt tctgttcacc atacccctaa 720
accctgacgt ttgcaagtcc tcaagtcatg tctttgcgac tctaccctgg acttattgtg 780
caacagaagt gtcaaataat gagattttaa tcatgccatg aatggctgtg atgaaacact 840
ggtttataag taacaaagaa taaacaaatg ctactgattt ctaagcctgc aaacccaaca 900
tcttaaagga gccacaataa agttaccatc aggtctacaa ctcagagaag acaaaatatt 960
gtatggaaaa gagattatat tcaaaataaa agttactttt gcggtttca 1009
<210> 2
<211> 44
<212> PRT
<213>Homo sapiens (Homo Sapiens)
<400> 2
Met Arg Asp Trp Ala Ser Val Ser Ser Leu Gly Ser Gly Leu Trp Lys
1 5 10 15
Glu Arg Leu Trp Lys Ser Ile Thr Thr Lys Arg Asp Gly Ile Ala Pro
20 25 30
Val Thr Arg Asn Trp Arg Gly Gly Lys Met Leu Ala
35 40
<210> 3
<211> 30
<212> PRT
<213>Homo sapiens (Homo Sapiens)
<400> 3
Gly Leu Trp Lys Glu Arg Leu Trp Lys Ser Ile Thr Thr Lys Arg Asp
1 5 10 15
Gly Ile Ala Pro Val Thr Arg Asn Trp Arg Gly Gly Lys Met
20 25 30
Claims (13)
1. a kind of polypeptide of separation, which is characterized in that the polypeptide is selected from：
(a) come from SEQ ID NO:2, and N-terminal originates in SEQ ID NO:The amino acid of 2 2-13 any bits, C-terminal are whole
Terminate in SEQ ID NO:The polypeptide that the amino acid of 2 42-44 any bits is formed；
(b) amino acid sequence of any polypeptide for being limited (a) by 1-5 amino acid residues substitution, missing or addition and
It is formed, and there is the polypeptide of the polypeptide；Or
(c) amino acid sequence of any polypeptide limited with (a) has at least 85% phase same sex, and have the function of the polypeptide
Polypeptide.
2. polypeptide as described in claim 1, which is characterized in that the polypeptide of (a) is selected from：
SEQ ID NO:The polypeptide of amino acid sequence shown in 3；
SEQ ID NO:The polypeptide of amino acid sequence shown in 2-44 in 2；
SEQ ID NO:The polypeptide of amino acid sequence shown in 3-44 in 2；
SEQ ID NO:The polypeptide of amino acid sequence shown in 4-44 in 2；
SEQ ID NO:The polypeptide of amino acid sequence shown in 5-44 in 2；
SEQ ID NO:The polypeptide of amino acid sequence shown in 6-44 in 2；
SEQ ID NO:The polypeptide of amino acid sequence shown in 7-44 in 2；
SEQ ID NO:The polypeptide of amino acid sequence shown in 8-44 in 2；
SEQ ID NO:The polypeptide of amino acid sequence shown in 9-44 in 2；
SEQ ID NO:The polypeptide of amino acid sequence shown in 10-44 in 2；
SEQ ID NO:The polypeptide of amino acid sequence shown in 11-44 in 2；
SEQ ID NO:The polypeptide of amino acid sequence shown in 12-44 in 2；
SEQ ID NO:The polypeptide of amino acid sequence shown in 13-44 in 2；
SEQ ID NO:The polypeptide of amino acid sequence shown in 1-43 in 2；Or
SEQ ID NO:The polypeptide of amino acid sequence shown in 1-42 in 2.
3. polypeptide as claimed in claim 1 or 2, which is characterized in that the polypeptide is encoded by -31 precursor of Microrna.
4. a kind of polynucleotides of separation encode the polypeptide described in claims 1 or 2.
5. a kind of expression vector contains the polynucleotides described in claim 4.
6. a kind of recombinant cell, wherein containing claim 4 institute is included in the expression vector described in claim 5 or its genome
The polynucleotides stated.
7. any polypeptides of claim 1-3 or encode its polynucleotides or claim 5 described in expression vector or
Application of the recombinant cell in immunoregulation medicament is prepared described in claim 6.
8. the use as claimed in claim 7, which is characterized in that the immunoregulation medicament is：Prevent or treat itself to exempt from
The drug of epidemic disease disease.
9. application as claimed in claim 8, which is characterized in that the autoimmune disease includes：Multiple sclerosis,
Rheumatoid arthritis, systemic loupus erythematosus and psoriasis etc..
10. the use as claimed in claim 7, which is characterized in that the immunoregulation medicament is to increase regulatory T cells
The drug of quantity.
A kind of 11. method for preparing the polypeptide described in claims 1 or 2, which is characterized in that the method includes：Cultivate right
It is required that the recombinant cell described in 6, so as to recombinantly express polypeptide described in claim 1；Or
The method includes：The polypeptide described in claims 1 or 2 is prepared by external artificial synthesized method.
12. one kind is used for immunoregulatory pharmaceutical composition, which is characterized in that the pharmaceutical composition includes：Claim
Any polypeptides of 1-3 or encode its polynucleotides claim 5 described in expression vector or claim 6 described in
Recombinant cell；And
Pharmacological or physiological acceptable carrier.
13. one kind is used for immunoregulatory medicine box, which is characterized in that the medicine box includes：
Any polypeptides of claim 1-3 encode its polynucleotides；Or
Expression vector described in claim 5；Or
Recombinant cell described in claim 6；Or
Pharmaceutical composition described in claim 12.
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| 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 |
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| 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 |
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