CN108144049A

CN108144049A - Application of the ubiquitination pathway correlation factor in regulating regulatory T cell function

Info

Publication number: CN108144049A
Application number: CN201611096627.7A
Authority: CN
Inventors: 李斌; 杨静; 徐鹏; 李丹; 梁瑞
Original assignee: Institut Pasteur of Shanghai of CAS
Current assignee: Institut Pasteur of Shanghai of CAS
Priority date: 2016-12-02
Filing date: 2016-12-02
Publication date: 2018-06-12

Abstract

The present invention relates to application of the deubiquitination approach correlation factor in regulating regulatory T cell function.Specifically, the present invention relates to the purposes of ubiquitination pathway correlation factor, its agonist or antagonist in the preparation or reagent that adjust FOXP3 and regulatory T cells is prepared, wherein the ubiquitination correlation factor is selected from deubiquitination protein family gene coding albumen USP44 and USP7.The novel regulatory factor of the present invention can be by adjusting the balance of transcriptional regulatory activities of the FOXP3 to its target gene, regulating regulatory T cell and immune system.

Description

Application of the ubiquitination pathway correlation factor in regulating regulatory T cell function

Technical field

The present invention relates to molecular biosciences and biomedicine field.More particularly it relates to deubiquitination approach phase Close application of the factor in regulating regulatory T cell function.

Background technology

Regulatory T cells (Regulatory T cells, Tregs) are resistant to immunity of organism and the maintenance of immune homeostasis has It plays an important role, prevents the generation of autoimmunity disease, play an important roll in anti-graft rejection and tumour immunity.People's plug The important transcription factor that albumen P3 (human forkhead box P3, Foxp3) is Treg developments and function maintains, to regulation and control The development of Treg and function play an important role.In human body, FOXP3 afunction can cause immunologic function disorder, cause a variety of Endocrine disorder, enteron aisle disease and X- sex-kink syndrome is PEX (Immunodysregulation, Polyendocrinopathy, and Enteropathy,X-linked).In mouse, FOXP3 afunction can develop into lymphoproliferative disease (Scurfy), serious dermatitis, multiple organ lymphocytic infiltration and autoimmune hemolytic anemia are shown as, reason is natural type The missing of Treg.FOXP3 genes are manually integrated into expression in T cell can obtain the phenotype of Treg.Bone is carried out in mouse Marrow is fitted into experiment, finds the bone marrow cell of FOXP3 gene knockouts and can not develop into Treg, and wild type bone marrow cell then can be with. And the notable downward of FOXP3 expressions caused by the change of 3 ' noncoding region of FOXP3 genes, the immune of Treg can be seriously undermined Inhibit function, illustrate that the expression of FOXP3 has a direct impact the function of Treg.Display FOXP3 is to adjust Treg cells hair Educate the important transcription factor with function.

Transcription factor FOXP3 (Forkhead box P3) not only serves as the marker molecule of CD4+CD25+Treg, still Determine the key gene of CD4+CD25+Treg functions.Under normal circumstances, different according to the position of development and differentiation, Treg can be with It is divided into two classes:It is a kind of be subgroup in thymus gland by positive and negative screening development for natural type Treg (natural Treg, ), nTreg another kind induces differentiation to obtain FOXP3 expression and exempt from by CD4+T cells in peripheral blood under certain specific physiological conditions What epidemic disease inhibited function and came, it is called induction type Treg (induced Treg, iTreg).Both in vivo in immunological regulation Whether mechanism of action is variant, is currently not very clear.It is current that researches show that at natural type regulatory T cells (nTreg) In, FOXP3 is adjusted by poly ubiquitination and deubiquitination, and the present inventor has found in former achievements are combed, FOXP3 Fine-tune and play an important roll to functions of the iTreg in tumor microenvironment.But to induction type regulatory T cells (iTreg) research of regulation mechanism is also unclear under inflammatory conditions.In vivo under inflammatory conditions, FOXP3+Treg functions Close adjusting is most important to the development of disease, and the molecular mechanism for studying this adjusting is of great significance.It is controlled for tumour from now on The clinical treatment for the treatment of, graft rejection, virus infection, immunological diseases and inflammatory reaction etc. provides innovative clue and the new target of drug Point.

Those skilled in the art have been devoted to the technology that exploitation adjusts immune system activity, are lost for treating immune response Adjust caused disease.

Invention content

The purpose of the present invention is to provide a kind of ubiquitination pathway correlation factor answering in regulating regulatory T cell function With.

The first aspect of the present invention provides the purposes of deubiquitinating enzymes or deubiquitinating enzymes agonist, is used to prepare Preparation or kit, the preparation or kit are used for：

(1) increase the stability of FOXP3；And/or

(2) differentiation of up-regulation regulatory T cells activity or promotion regulatory T cells；And/or

(3) expression of up-regulation regulatory T cells cell factor or activity；And/or

(4) it treats and/or prevents and the too low relevant disease of regulatory T cells activity；And/or

(5) promote the differentiation of iTreg.

In another preferred example, regulatory T cells activity is too low refers to that regulatory T cells inhibit inflammation and are immunized Response function is too low.

In another preferred example, it is described to be selected from the too low relevant disease of regulatory T cells activity：Autoimmunity disease, Inflammatory reaction, tumour and communicable disease.

In another preferred example, the autoimmune disease is selected from：Multiple sclerosis, rheumatic arthritis, class wind Rejection, systemic loupus erythematosus, Crohn's disease in wet arthritis, rheumatic heart disease, organ transplant, exedens knot The autoimmunity diseases such as enteritis, ankylosing spondylitis, Autoimmune Encephalomyelitis, preferably ulcerative colitis and autoimmune Encephalomyelitis.

In another preferred example, the inflammatory reaction is selected from：Allergic inflammation, epifolliculitis, tonsillitis, pneumonia, hepatitis, Ephritis, acne, asthma, chronic inflammation, chronic prostatitis, glomerulonephritis, hypersensitivity, inflammatory bowel disease, pelvic infecton, Reperfusion injury, vasculitis or interstitial cystitis.

In another preferred example, the tumour is selected from：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.

In another preferred example, the communicable disease is selected from：The plague, cholera, severe acute respiratory syndrome, AIDS, Virus hepatitis, polio, human hepatic stellate cell, measles, Hemorrhagic fever, rabies, popular second Type encephalitis, hand-foot-and-mouth disease, dengue fever, anthrax, bacillary and amebic dysentery, pulmonary tuberculosis, Typhoid and paratyphoid, popular brain Perimyelitis, pertussis, diphtheria, neo-nataltetenus(NNT), scarlet fever, brucellosis, gonorrhoea, syphilis, leptospirosis, blood Fluke disease, malaria, influenza, mumps, rubella, acute hemorrhagic conjunctivitis, leprosy, popularity and place Property typhus, kala-azar, echinococcosis, filariasis, in addition to cholera, bacillary and amebic dysentery, Typhoid and paratyphoid Infectious diarrhea disease or fungal infection.

In another preferred example, it is one or more during the deubiquitinating enzymes are selected from the group：USPs families (Ubiquitin-specific proteases, USPs) and OUT families.

In another preferred example, it is one or more during the deubiquitinating enzymes are selected from the group：USP44、USP2、USP3、 USP4、USP5、USP7、USP10、USP12、USP14、USP17、USP18、USP21、USP22、USP30、USP39、YOD1、 CYLD and A20；Preferably, the deubiquitinating enzymes are USP44, USP7 or combination.

In another preferred example, it is one or more during the regulatory T cells are selected from the group：NTreg, pTreg and iTreg。

In another preferred example, it is one or more during the regulatory T cells cell factor is selected from the group：FOXP3、 IL-10, TGF-β and IL-2.

In another preferred example, it is one or more during the deubiquitinating enzymes agonist is selected from the group：Deubiquitinating enzymes Transcribe accelerating agent and/or inflammatory cytokine, it is preferable that the inflammatory cytokine is TGF-β.

In another preferred example, the deubiquitinating enzymes transcription accelerating agent refers to, deubiquitinating enzymes is promoted to enter nucleus Substance (such as TGF-β, SMADs families).

In another preferred example, the stability for increasing FOXP3 is to be accomplished by the following way：FOXP3 is gone Ubiquitination.

In another preferred example, the deubiquitination is the ubiquitination of catalytic elimination ubiquitin K48 couplings.

In another preferred example, the deubiquitination action site is selected from：K179, K250, K263, K268 of FOXP3.

In another preferred example, the deubiquitinating enzymes (are selected from from mammal：People).

In another preferred example, the amino acid sequence of the USP44 such as SEQ ID NO.:Shown in 1；And/or the USP7 Amino acid sequence such as SEQ ID NO.:Shown in 2.

In another preferred example, the deubiquitinating enzymes are selected from：The deubiquitinating enzymes of wild type or saltant type, are also selected From：Derivative with active fragment or deubiquitinating enzymes with deubiquitinating enzymes of the wild type deubiquitinating enzymes with identical function Object.

In another preferred example, the derivative of the deubiquitinating enzymes is selected from：Modified deubiquitination enzyme molecule, amino Acid sequence is homologous with natural deubiquitinating enzymes and has the dimerization of the protein molecular of natural deubiquitination enzymatic activity, deubiquitinating enzymes Body or polymer, the fusion protein containing deubiquitination enzyme amino acid sequence.

In another preferred example, the modified deubiquitinating enzymes are PEGylated deubiquitinating enzymes.

It is in another preferred example, described that " amino acid sequence is homologous with natural deubiquitinating enzymes and with natural deubiquitination The protein molecular of enzymatic activity " refers to its amino acid sequence compared with wild-type amino acid sequence (such as USP44, with SEQ ID NO.:1 compares), have >=85% homology, homology preferably >=90%, homology more preferably >=95%, most Homology goodly >=98%；And the protein molecular with natural deubiquitination enzymatic activity.

In another preferred example, it is described to refer to and regulatory T cells activity mistake with the relevant disease of regulatory T cells activity High or too low relevant disease or symptom.

In another preferred example, it is described to be selected from the relevant disease of regulatory T cells activity：Tumour and communicable disease.

In another preferred example, the preparation is selected from：Pharmaceutical composition, Halth-care composition, food compositions, vaccine Composition or experiment reagent.

The second aspect of the present invention provides the purposes of deubiquitinating enzymes antagonist, is used to prepare preparation or kit, institute It states preparation or kit is used for：

(1) stability of FOXP3 is reduced；And/or

(2) it lowers regulatory T cells activity or inhibits the differentiation of regulatory T cells；And/or

(3) expression or the activity of regulatory T cells cell factor are lowered；And/or

(4) it treats and/or prevents and the relevant disease of regulatory T cells hyperactivity；And/or

(5) expression of FOXP3 is raised, enhances the immune suppression function of Treg；And/or

(6) it treats and/or prevents and the relevant disease of regulatory T cells activity.

In another preferred example, it is described to be selected from the group with the relevant disease of regulatory T cells hyperactivity：Tumour and biography Infectious diseases.

In another preferred example, the deubiquitinating enzymes antagonist is selected from：Antibody, sh-RNA, miRNA, antisense oligonucleotides Acid, chemical inhibitor or combination.

In another preferred example, the antagonist is selected from：(bob presss from both sides by anti-USP44 antibody, anti-USP7 antibody, shUSP44 RNA), the chemical inhibitor of shUSP7 (short hairpin RNA), the antisense oligonucleotides for USP44 or USP7, USP44 or USP7.

In another preferred example, the chemical inhibitor is：10-hydroxycamptothecine (molecular formula：C20H16N2O5,10- hydroxycamptothecin,10-HCPT)。

In another preferred example, the regulatory T cells are selected from：What Natural regulation T cell (nTreg) and induction generated Adaptability regulatory T cells (iTreg).

In another preferred example, it is described to be selected from the group with the relevant disease of regulatory T cells activity：Cancer, virus infection, Autoimmunity disease, preferably cancer.

The third aspect of the present invention provides a kind of composition for the stability for being used to increase FOXP3, the composition choosing From：Deubiquitinating enzymes or derivatives thereof and/or deubiquitinating enzymes agonist.

In another preferred example, the deubiquitinating enzymes agonist is as described above.

In another preferred example, the composition also contains pharmaceutically acceptable carrier.

In another preferred example, the dosage form of the composition is selected from：Solid formulation, liquid formulation, preferably dry powder or Solution form.

In another preferred example, the composition is pharmaceutical composition, Halth-care composition or vaccine composition.

The fourth aspect of the present invention, provides a kind of compound of separation, and the compound is deubiquitinating enzymes and FOXP3 It is combined formed compound.

In another preferred example, the compound is binary complex.

In another preferred example, molecular weight >=80KD of the compound；The molecular weight of preferably described compound >= 100KD。

In another preferred example, the molecular weight of the compound is 100KD-500KD；Preferably 100KD-200KD.

The fifth aspect of the present invention provides the application of albumen composition described in fourth aspect present invention, for screening medicine Object or compound, the drug or compound promote or inhibit deubiquitinating enzymes and FOXP3 to form the compound.

In another preferred example, when application compound screening drug, the application includes step：

(a) in test group, in the presence of test substance, cultivate regulatory T cells, and pair without test substance is set According to group；

(b) it detects the content H1 of compound described in test group and is compared with the complex content H0 in control group, Wherein when H1 is significantly higher than H0, then it represents that the tester is the accelerating agent of deubiquitinating enzymes；When H1 is substantially less than H0, then it represents that The tester is the antagonist of deubiquitinating enzymes.

(a) in test group, test substance and (a) described compound and/or (b) deubiquitinating enzymes and FOXP3 are carried out It is incubated altogether；And the control group without test substance is set；

The sixth aspect of the present invention provides a kind of purposes of deubiquitinating enzymes antagonist, be used to prepare treatment tumour or The drug of communicable disease.

In another preferred example, the chemical inhibitor is：10-hydroxycamptothecine (C20H16N2O5,10- Hydroxycamptothecin, 10-HCPT).

The seventh aspect of the present invention provides purposes of the TGF-β in the reagent for promoting deubiquitinating enzymes transcription is prepared.

It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and have in below (eg embodiment) It can be combined with each other between each technical characteristic of body description, so as to form new or preferred technical solution.As space is limited, exist This no longer tires out one by one states.

Description of the drawings

Fig. 1 shows USP44 high expression in iTreg.

Wherein, Figure 1A sub-elects CD4 with Flow Cytometry (FACS) from human peripheral blood cell (PBMC)⁺CD25^-Teff Cell, CD4⁺CD25^hiCD127^loTreg cells, CD45RA+CD4+naive T cells, under regulatory T cells inductive condition Induction type regulatory T cells (iTreg) are obtained, extracting RNA is carried out with special primer real-time fluorescence quantitative PCR (q-RTPCR) Or with special antibody protein immunoblot experiment (WB), USP44 high expression in iTreg is as a result shown；

Figure 1B sub-elects CD45RA+CD4+naive with Flow Cytometry (FACS) from human peripheral blood cell (PBMC) T cell, obtains different helper T lymphocyte subgroups under different helper T lymphocyte inductive conditions and induction type regulatory T is thin Born of the same parents (iTreg), extracting RNA are carried out with special primer real-time fluorescence quantitative PCR (q-RTPCR) or with special antibody protein Immunoblot experiment (WB) as a result shows USP44 high expression in iTreg；

MS:Mass Spectrometry, mass spectral analysis.(*:p<0.05,**:p<0.005).

Fig. 2 shows USP44 and FOXP3 interactions.

Wherein, Fig. 2A is collected after turning expression FLAG label USP44 and MYC label Fs OXP3,48h in HEK293T cells China and foreign countries Cell carries out two-way co-immunoprecipitation (IP) with corresponding antibodies and protein immunization imprinting (IB) tests (FLAG-USP44 expression generals FLAG labels and USP44 genes connect together the USP44 albumen with FLAG labels of expression, and MYC-FOXP3a is represented MYC Label and FOXP3a (longest one in FOXP3 gene difference spliced bodies) gene connect together expression with MYC labels FOXP3 albumen, similarly hereinafter).

Fig. 2 B collect stablize expression HA-FOXP3 Jurkat T cells, with corresponding antibodies carries out two-way co-immunoprecipitation with Protein immunization imprinting experiment (IP-USP44 represent with the specific recognition antibody of USP44 in cell pyrolysis liquid to USP44 albumen into Row enrichment, INPUT represent the supernatant of untreated cell pyrolysis liquid, similarly hereinafter).

Fig. 2 C are isolated from human peripheral PBMCThe iTreg of T cell Differentiation Induction in vitro, uses corresponding antibodies Carry out co-immunoprecipitation and protein immunization imprinting experiment.

Fig. 2 D are isolated from human peripheral PBMCT cell Differentiation Induction in vitro uses corresponding antibodies into iTreg Carry out immunofluorescence experiment.sAbs:Secondary antibodies, fluorescence secondary antibody.

Fig. 3 shows that USP44 stablizes FOXP3 albumen.

Wherein, Fig. 3 A turn expression FLAG labels USP44 (wild type WT and enzyme inactive mutant in HEK293T cells China and foreign countries C282S, 0.5,1,1.5 μ g of plasmid dose) and MYC label Fs OXP3,48h after collect cell carry out protein immunization with corresponding antibodies Blotting experiments.

Fig. 3 B turn expression FLAG labels USP44 (WT and C282S) and MYC label F OXP3 in HEK293T cells China and foreign countries, point Not Yong protein synthesis inhibitor CHX processing (0,4,8,12h), collect cell carry out protein immunization imprinting experiment with corresponding antibodies (WT represents wild type, and CA represents that the cysteine of the 282nd on enzyme activity related locus, that is, amino acid sequence of USP44 is dashed forward Become serine, albumen structure is reduced to the thus enzyme activity that changes with hydrophobicity, similarly hereinafter).

Fig. 3 C, D are isolated from human peripheral PBMCThe iTreg of T cell Differentiation Induction in vitro, infection carry The slow virus (shCK, shUSP44-1, shUSP44-2) of resistance screening label, is screened after 72h, is collected cell after a week and is split Solution, protein immunization imprinting experiment and facs analysis are carried out with corresponding antibodies.In figure, shCK is to have been infected in Treg cells without appointing The packaging virus of the sequence of what targeting, thus be will not on the influential control of the expression of any gene, shUSP44-1 and ShUSP44-2 is respectively the packaging virus that the sequence for having special targeting to USP44 genes has been infected in Treg cells, 1 and 2 generations The different sequence of table, but all there is targeting, according to principles such as homologous silences, USP44 genes in this cell to USP44 genes By it is special strike it is low.

(E) expression that the cell in Fig. 3 C carries out qRT-PCR detection related genes is collected, shown here is representational Independent experiment result.

Fig. 4 shows that USP44 influences Transcription inhibitions of the FOXP3 to downstream Il2 genes.

Wherein Fig. 4 A subcontract the luciferase reporter plasmid of the gene promoters of Il2 containing people in HEK293T cells China and foreign countries, Transfection efficiency internal reference Renilla, expression FLAG labels USP44 (WT and C282S) or USP7 or STUB1 and MYC label F OXP3, Cell is collected after 48h to carry out uciferase activity measure and carry out protein immunization imprinting experiment with corresponding antibodies.

Fig. 4 B subcontracted in HEK293T cells China and foreign countries the luciferase reporter genes of the gene promoters of Il2 containing people, Renilla, Cell progress fluorescein is collected after expressing FLAG labels USP44 (0.5,1,1.5 μ g of plasmid dose) and MYC label Fs OXP3,48h Enzyme assay and carry out protein immunization imprinting experiment with corresponding antibodies.

HEK293T cell infections are carried slow virus (shCK, shUSP44-1, shUSP44- of resistance screening label by Fig. 4 C 2) it, is screened after 72h, transfection after a week includes luciferase reporter gene, Renilla and the table of people's Il2 gene promoters Up to FOXP3 or the plasmid of zero load, cell cracking is collected after 48h, carries out uciferase activity determination experiment, it is shown here to there is generation Independent experiment result of table.

Fig. 5 shows USP44 removal FOXP3 and the poly ubiquitination for relevant K48 couplings of degrading.

Wherein, Fig. 5 A cotransfection expression USP44 (WT) and mutant (C282S), MYC labels in HEK293T cells The plasmid of the ubiquitin protein (ubiquitin) of FOXP3 and His labels, MG13220 μM is added in after 44h, is received cell cracking after 4h and is used Ni-NTA magnetic bead combination His labels carry out ubiquitin precipitation, then detect protein expression and the ubiquitination water of FOXP3 with corresponding antibodies It is flat that (His-ubi expressions connect together 6 His labels and ubiquitin gene the ubiquitin protein with His labels of expression, and ubi is The abbreviation of ubiquitin protein).

HEK293T cell infections are carried slow virus (shCK, shUSP44-1, shUSP44- of resistance screening label by Fig. 5 B 2) it, is screened after 72h, after a week the matter of the ubiquitin protein of transfection expression MYC label Fs OXP3 and His labels (ubiquitin) , 20 μM of MG132 is added in after 44h, cell cracking is received after 4h and carries out ubiquitin precipitation with Ni-NTA magnetic bead combination His labels, then Protein expression and the ubiquitination level of FOXP3 are detected with corresponding antibodies.

Fig. 5 C cotransfections in HEK293T cells express USP44, the ubiquitin protein of MYC label F OXP3 and His labels (ubiquitin) and its plasmid of various mutant 20 μM of MG132, is added in after 44h, cell cracking Ni-NTA magnetic is received after 4h Pearl combines His labels and carries out ubiquitin precipitation, then detects protein expression and the ubiquitination level of FOXP3 with corresponding antibodies.

Fig. 6 shows that FOXP3 albumen K179/250/263/268 is the deubiquitination action site of USP44.

Wherein, Fig. 6 A will express FOXP3 wild types (WT) and its different mutants and the plasmid co-transfection of USP44 albumen enters Cell cracking, which is received, in HEK29T cells, after 48h carries out Protein Detection with corresponding antibodies.

Fig. 6 B cotransfections expression FOXP3 (WT) and mutant (K179/250/263/268R, i.e. 4R), USP44 and His marks The plasmid of the ubiquitin protein (ubiquitin) of label adds in 20 μM of MG132 after 44h, and cell cracking Ni-NTA magnetic beads are received after 4h Ubiquitin precipitation is carried out with reference to His labels, then detects protein expression and the ubiquitination level of FOXP3 with corresponding antibodies.

Fig. 6 C cotransfections expression FOXP3 (WT) and mutant (K179/250/263/268R, i.e. 4R and all K mutant 20R) with the plasmid of USP44 albumen, then with the ubiquitination level of corresponding antibodies detection protein expression and FOXP3, (K is lysine Abbreviation).

Fig. 7 shows that TGF-β signal promotes iTreg differentiation by raising USP44 transcriptional levels.

Wherein.Fig. 7 A are by the luciferase reporter gene carrier of the promoter sequence+17- (- 1712) comprising USP44 genes It is transfected into Jurkat T cells, phorbol exters (PMA) and the TGF- of ionomycin (Ionomycin) and various concentration is used after 36h β stimulates different time, receives cell cracking and carries out luciferase assays and corresponding antibodies detection p-SMAD2 and SMAD2 albumen It is horizontal that (P+I is jointly processed by for PMA and Inomycin, and for simulating the stimulation of TCR signal paths, PMA is phorbol exters, ionomycin It is ionomycin).

The Treg amplification in vitros that Fig. 7 B, C are detached from PBMC, separationT cell Differentiation Induction in vitro 0,1,2,3,4,5d The iTreg of generation, extracting RNA carries out qRT-PCR detection corresponding gene mRNA expressions, while is tested and used by immunoblotting analysis Specific antibodies detect the protein level of USP44, USP7, STUB1, Hsp70 and FOXP3, and shown here is representational primary Experimental result.

Fig. 8 shows STUB1 by enhancing the more ubiquitinations of USP44 come the USP44 that degrades.

Wherein, the Jurkat T cells (1 × 10 that Fig. 8 A will stablize expression USP44 albumen⁶) first with PMA (50ng/ml) and (1 μM) of Ionomycin stimulation 4h, then respectively with different temperature stimulation different time or when stimulating different with LPS (1 μ g/ml) Between, it then collects cell cracking and carries out protein immunization imprinting experiment with corresponding antibodies.

Fig. 8 B are isolated from human peripheral PBMCThe iTreg of T cell Differentiation Induction in vitro adds in 100U/ml IL-2 respectively with (LPS (1 μ g/ml), IL-6 (20ng/ml), IL-1 β (10ng/ml)) for 24 hours, collects cell cracking, with corresponding Antibody carries out protein immunization imprinting experiment.

Fig. 8 C turn the plasmid of table USP44 and MYC label STUB1 (0.5,1,1.5 μ g) in HEK293T cells China and foreign countries, after 48h It collects cell and carries out protein immunization imprinting experiment with corresponding antibodies.

Fig. 8 D turn the plasmid of expression USP44 and MYC labels STUB1 (WT, H260Q, K30A) in HEK293T cells China and foreign countries, Cell is collected after 48h and carries out protein immunization imprinting experiment with corresponding antibodies.

Fig. 8 E turn the plasmid of expression FLAG label USP44 and MYC labels STUB1 or FOXP3 in HEK293T cells China and foreign countries, Cell is collected after 48h and carries out co-immunoprecipitation and protein immunization imprinting experiment with corresponding antibodies.

Fig. 8 F turn the plasmid of expression USP44 and MYC labels STUB1 (WT, H260Q, K30A) in HEK293T cells China and foreign countries, Cell is collected after 48h and carries out co-immunoprecipitation and protein immunization imprinting experiment with corresponding antibodies.

Fig. 8 G cotransfections in HEK293T cells express USP44, the ubiquitin protein (ubiquitin) of STUB1 and HA labels Plasmid, 20 μM of MG132 is added in after 44h, cell cracking is received after 4h and carries out co-immunoprecipitation and protein immunization print with corresponding antibodies Document tests detection protein expression and the ubiquitination level of USP44.

Fig. 9 shows that USP44 and USP7 synergistic effects stablize FOXP3.

Wherein, Fig. 9 A turn the plasmid of expression MYC label USP44 and FLAG labels USP7,48h in HEK293T cells China and foreign countries Cell is collected afterwards carries out two-way co-immunoprecipitation and protein immunization imprinting experiment with corresponding antibodies.

Fig. 9 B turn expression USP44, MYC label F OXP3 and FLAG label USP7 (0.5,1,1.5 in HEK293T cells China and foreign countries μ g) plasmid, cell is collected after 48h and carries out co-immunoprecipitation and protein immunization imprinting experiment with corresponding antibodies.

Fig. 9 C cotransfections in HEK293T cells express FLAG labels USP44, USP7, MYC label F OXP3 and His label Ubiquitin protein (ubiquitin) plasmid, 20 μM of MG132 is added in after 44h, cell cracking is received after 4h and is carried out with corresponding antibodies Co-immunoprecipitation and the ubiquitination level of protein immunization imprinting experiment detection protein expression and FOXP3.

Fig. 9 D turn the slow virus packaging plasmid with shCK or shSTUB1 in HEK293T cells China and foreign countries, after 1 day, then are transferred to The plasmid of USP44 and MYC labels USP7 is expressed, collect cell after 48h carries out protein immunization imprinting experiment with corresponding antibodies.

Fig. 9 E HEK293T cells China and foreign countries turn expression FLAG labels USP44 (WT, C282S) and MYC labels STUB1 (WT, H260Q) or the plasmid of USP7 (WT, C223S), cell is collected after 48h and carries out protein immunization imprinting experiment with corresponding antibodies.

Figure 10 shows the Treg detached in USP44 knock out mice, and the expression of FOXP3 and the inhibition function of Treg are bright It is aobvious to be suppressed, separationT cell carries out differentiation iTreg in immunodeficient mouse body and is also significantly inhibited, The expression of IFN-γ and IL-17 are then significantly raised.

Wherein, Figure 10 A are by 1 × 10⁶ CD4⁺T cell (CD62L^highCD25^-) individually or with wild type Treg or The Treg cells 4 × 10 detached in USP44 knock out mice⁵Squeeze into RAG2^-/-Manufacture enteritis mould in immunodeficient mouse body Type records its weight (as schemed) in certain time and does curve graph (N=6), and control group design is as schemed.

Figure 10 B, C take the distal end intestinal tissue that enteritis mouse model of the cell after 8 weeks is squeezed into Figure 10 A carry out hematoxylin-she Red (H/E) is dyed, and is carried out white light (100 ×) under histotomy microscope and analyzed, and is carried out disease assessment marking and made column Figure.

Figure 10 D will be withThe Treg cells one detached in T cell and wild type Treg and USP44 knock out mice With squeezing into immunodeficient mouse body, the mucosal lymphatic knot (mLN) of mouse, peripheral lymph nodes (pLN), spleen are taken after a certain period of time (SPL) separation Treg cells (CD4 is carried out⁺/Thy1.1⁺), the expression of facs analysis FOXP3 is carried out with specific antibody, control group is set Meter is as schemed.

Figure 10 E will together be squeezed into from different Treg in immunodeficient mouse bodyT cell takes in certain time Go out and use specific antibody (CD4⁺/Thy1.2⁺) facs analysis IFN-γ and the expression of IL-17 are carried out, control group design is as schemed.

Figure 11 shows USP44 and FOXP3 common high expression in the cancerous tissue of the hepatocarcinoma patient sample of the HCV positives.

Wherein, Figure 11 A carry out immunohistochemistry reality in the cancerous tissue of the hepatocarcinoma patient sample of the HCV positives with USP44 antibody It tests, finds USP44 specifically expressings in mesenchyma stroma of tumors.

Figure 11 B statistical informations are shown in by cancerous tissue and the cancer of 17 patients that liver cancer (HCC) is eventually led to by HCV infection And in normal structure, USP44 and the FOXP3 common high expression in the cancerous tissue of wherein 11 samples.

Figure 11 C immunohistochemical experiment results are shown in the cancer group of 17 patients that liver cancer (HCC) is eventually led to by HCV infection It knits and takes up in normal structure with cancer, USP44 and the FOXP3 common high expression in the cancerous tissue of wherein 11 samples.

Figure 12 shows that 10-hydroxycamptothecine (10-HCPT) can be specifically inhibits stabilizations of the USP44 to FOXP3 albumen.

Cotransfection FLAG labels USP44 and MYC the label F OXP3 in HEK293T cells, and with the 10- hydroxyls of various concentration Base camplotheca acuminata alkali process, DMSO is as control, the results show that the inhibition USP44 that 10-hydroxycamptothecine (10-HCPT) can be special To the stabilization of FOXP3 albumen, and in dose-dependant.

Specific embodiment

The present inventor and in-depth study, is found surprisingly that by extensive, and deubiquitinating enzymes are for raising the stabilization of FOXP3 Property have and significantly affect, and then regulatory T cells activity can be raised and its break up and can raise regulatory T cells cell The expression of the factor, completes the present invention on this basis.

Term

Deubiquitinating enzymes and preparation method thereof

So far it has been found that about 100 kinds of deubiquitinating enzymes, major function are by hydrolyzing carboxyl terminal on ubiquitin Residue hydrolyzes ubiquitin molecule from the albumen for be connected with ubiquitin.Five families can be divided into according to its structure and function feature Race：Ubiquitin carboxy terminal hydrolase family (Ubiquitin C-terminal hydrolases, UCHs), ubiquitin-specific water Solve enzyme family (Ubiquitin-specific proteases, USPs), ovarian neoplasm GAP-associated protein GAP enzyme (Ovarian tumor, OTU), Ataxin-3 (structural domain containing Josephin), MPN (+)/JAMM protease (JAB1/MPN/Mov34metlloenzyme domain zinc-dependent metalloprotease family)。

Have now been found that deubiquitinating enzymes function includes：1. deubiquitinating enzymes activate egg before ubiquitin by way of cotranslational In vain.Ubiquitin is as a preceding protein expression and is fused on ribosomal protein or linear polymeric ubiquitin, while PolyUb gene Product, which must also remove the extra residue of carbon teminal, could activate ubiquitin.2. deubiquitination is recyclable to be included thiol esters intermediate The ubiquitin of cellule nucleopilic reagent capture, so as to participate in the ubiquitination of albumen.3. deubiquitinating enzymes reversely remove the general of target protein Elementization or the modification of ubiquitin sample.The ubiquitination of deubiquitinating enzymes antagonist protein, similar to phosphatase in kinases/phosphoric acid enzyme adjustment access Effect.4. ubiquitin enzyme is gone to be responsible for the regeneration of single ubiquitin in non-anchor more poly-ubiquitin chains.

Deubiquitinating enzymes USP44 specially remove substrate protein list gather or poly ubiquitination and prevent its enter protease Body degrades or influences it and functions, so as to influence the activities such as tumour, Apoptosis, immune response.Research is found USP44 can be acted on cyclin CDC20 by target so as to inhibit cell division, can also be by removing histone list Ubiquitination is broken up to influence stem cell.But influences of the USP44 to immune system is still unclear.

And deubiquitinating enzymes USP7 influences growth of tumour cell by stablizing p53.The study find that USP7 can stablize USP44, and functionally there is synergistic effect with USP44.

Therefore, the biochemistry furtherd investigate in regulatory T cells and Transcription factor FOXP3, IL-10, IL-2 and TGF-β is living Property, physiological function and its regulation and control molecule mechanism, for the immunocompetence and base of regulatory T cells in therapeutic control human body It is all most important in the immunization therapy of regulatory T cells.Fundamental immunity research is is converted into clinical research and understands people by this Class autoimmune disease, tumour and communicable disease provide new research proposition and challenge, are expected to become new therapeutic target Point.

Deubiquitinating enzymes of the present invention are selected from：The deubiquitinating enzymes of wild type or saltant type, are also selected from：With with open country Raw type deubiquitinating enzymes have the active fragment of deubiquitinating enzymes of identical function or the derivative of deubiquitinating enzymes.

As used in the present invention, deubiquitinating enzymes " USP44 " or " USP7 " refer to a kind of protein, which has With (Frank Stegmeier, et al.Nature.2007), (Roberta Visconti, et al.Nat Commun.2012) " USP44 " or " USP7 " base described in (Jorg van Loosdregt, et al.Immunity.2013) This identical amino acid sequence and (b) have the biological activity identical with natural " USP44 " or " USP7 ", and with basic phase Same amino acid sequence." USP44 " or " USP7 " of the present invention includes, but are not limited to：People " USP44 " or " USP7 ", recombined human " USP44 " or " USP7 ", mouse " USP44 " or " USP7 " and/or recombinant murine " USP44 " or " USP7 ".

In certain embodiments of the present invention, the nucleotides sequence of USP44 is classified as：

ATGCTAGCAATGGATACGTGCAAACATGTTGGGCAGCTGCAGCTTGCTCAAGACCATTCCAGCCTCAACCCTCAGAA ATGGCACTGTGTGGACTGCAACACGACCGAGTCCATTTGGGCTTGCCTTAGCTGCTCCCATGT TGCCTGTGGAAGATATATTGAAGAGCATGCACTCAAGCACTTTCAAGAAAGCAGTCATCCTGTTGCATTGGAGGTGA ATGAGATGTACGTTTTTTGTTACCTTTGTGATGATTATGTTCTGAATGATAACACAACTGGAGACCTGAAGTTACTA CGACGTACATTAAGTGCCATCAAAAGTCAAAATTATCACTGCACAACTCGTAGTGGGAGGTTTTTACGGTCCATGGG TACAGGTGATGATTCTTATTTCTTACATGACGGTGCCCAATCTCTGCTTCAAAGTGAAGATCAACTGTATACTGCTC TTTGGCACAGGAGAAGGATACTAATGGGTAAAATCTTTCGAACATGGTTTGAACAATCACCCATTGGAAGAAAAAAG CAAGAAGAACCATTTCAGGAAAAAATAGTAGTAAAAAGAGAAGTAAAGAAAAGACGGCAGGAATTGGAGTATCAAGT TAAAGCAGAATTGGAAAGTATGCCTCCAAGAAAGAGTTTACGTTTACAAGGGCTCGCTCAGTCGACCATAATAGAAA TAGTTTCTGTTCAGGTGCCAGCACAAACGCCAGCATCACCAGCAAAAGATAAAGTACTCTCTACCTCAGAAAATGAA ATATCTCAAAAAGTCAGTGACTCCTCAGTTAAACGAAGGCCAATAGTAACTCCTGGTGTAACAGGATTGAGAAATTT GGGAAATACTTGCTATATGAATTCTGTTCTTCAGGTGTTGAGTCATTTACTTATTTTTCGACAATGTTTTTTAAAGC TTGATCTGAACCAATGGCTGGCTATGACTGCTAGCGAGAAGACAAGATCTTGTAAGCATCCACCAGTCACAGATACA GTAGTATATCAAATGAATGAATGTCAGGAAAAAGATACAGGTTTTGTTTGCTCCAGACAATCAAGTCTGTCATCAGG ACTAAGTGGTGGAGCATCAAAAGGTAGAAAGATGGAACTTATTCAGCCAAAGGAGCCAACTTCACAGTACATTTCTC TTTGTCATGAATTGCATACTTTGTTCCAAGTCATGTGGTCTGGAAAGTGGGCGTTGGTCTCACCATTTGCTATGCTA CACTCAGTGTGGAGACTCATTCCTGCCTTTCGTGGTTACGCCCAACAAGACGCTCAGGAATTTCTTTGTGAACTTTT AGATAAAATACAACGTGAATTAGAGACAACTGGTACCAGTTTACCAGCTCTTATCCCCACTTCTCAAAGGAAACTCA TCAAACAAGTTCTGAATGTTGTAAATAACATTTTTCATGGACAACTTCTTAGTCAGGTTACATGTCTTGCATGTGAC AACAAATCAAATACCATAGAACCTTTCTGGGACTTGTCATTGGAGTTTCCAGAAAGGTATCAATGCAGTGGAAAAGA TATTGCTTCCCAGCCATGTCTGGTTACTGAAATGTTGGCCAAATTTACAGAAACTGAAGCTTTAGAAGGAAAAATCT ACGTATGTGACCAGTGTAACTCAAAGCGTAGAAGGTTTTCCTCCAAACCAGTTGTACTCACAGAAGCCCAGAAACAA CTTATGATATGCCACCTACCTCAGGTTCTCAGACTGCACCTCAAACGATTCAGGTGGTCAGGACGTAATAACCGAGA GAAGATTGGTGTTCATGTTGGCTTTGAGGAAATCTTAAACATGGAGCCCTATTGCTGCAGGGAGACCCTGAAATCCC TCAGACCAGAATGCTTTATCTATGACTTGTCCGCGGTGGTGATGCACCATGGGAAAGGATTTGGCTCAGGGCACTAC ACTGCCTACTGCTATAATTCTGAAGGAGGGTTCTGGGTACACTGCAATGATTCCAAACTAAGCATGTGCACTATGGA TGAAGTATGCAAGGCTCAAGCTTATATCTTGTTTTATACCCAACGAGTTACTGAGAATGGACATTCTAAACTTTTGC CTCCAGAGCTCCTGTTGGGGAGCCAACATCCCAATGAAGACGCTGATACCTCGTCTAATGAAATCCTTAGCTGA (SEQ ID NO.1)

The amino acid sequence of USP44 is：

MLAMDTCKHVGQLQLAQDHSSLNPQKWHCVDCNTTESIWACLSCSHVACGRYIEEHALKHFQESSHPVALEVNEMYV FCYLCDDYVLNDNTTGDLKLLRRTLSAIKSQNYHCTTRSGRFLRSMGTGDDSYFLHDGAQSLLQSEDQLYTALWHRR RILMGKIFRTWFEQSPIGRKKQEEPFQEKIVVKREVKKRRQELEYQVKAELESMPPRKSLRLQGLAQSTIIEIVSVQ VPAQTPASPAKDKVLSTSENEISQKVSDSSVKRRPIVTPGVTGLRNLGNTCYMNSVLQVLSHLLIFRQCFLKLDLNQ WLAMTASEKTRSCKHPPVTDTVVYQMNECQEKDTGFVCSRQSSLSSGLSGGASKGRKMELIQPKEPTSQYISLCHEL HTLFQVMWSGKWALVSPFAMLHSVWRLIPAFRGYAQQDAQEFLCELLDKIQRELETTGTSLPALIPTSQRKLIKQVL NVVNNIFHGQLLSQVTCLACDNKSNTIEPFWDLSLEFPERYQCSGKDIASQPCLVTEMLAKFTETEALEGKIYVCDQ CNSKRRRFSSKPVVLTEAQKQLMICHLPQVLRLHLKRFRWSGRNNREKIGVHVGFEEILNMEPYCCRETLKSLRPEC FIYDLSAVVMHHGKGFGSGHYTAYCYNSEGGFWVHCNDSKLSMCTMDEVCKAQAYILFYTQRVTENGHSKLLPPELL LGSQHPNEDADTSSNEILS(SEQ ID NO.2)

In certain embodiments of the present invention, the nucleotides sequence of USP7 is classified as：

ATGAACCACCAGCAGCAGCAGCAGCAGCAGAAAGCGGGCGAGCAGCAGTTGAGCGAGCCCGAGGACATG GAGATGGAAGCGGGAGATACAGATGACCCACCAAGAATTACTCAGAACCCTGTGATCAATGGGAATGTGGCCCTGAG TGATGGACACAACACCGCGGAGGAGGACATGGAGGATGACACCAGTTGGCGCTCCGAGGCAACCTTTCAGTTCACTG TGGAGCGCTTCAGCAGACTGAGTGAGTCGGTCCTTAGCCCTCCGTGTTTTGTGCGAAATCTGCCATGGAAGATTATG GTGATGCCACGCTTTTATCCAGACAGACCACACCAAAAAAGCGTAGGATTCTTTCTCCAGTGCAATGCTGAATCTGA TTCCACGTCATGGTCTTGCCATGCACAAGCAGTGCTGAAGATAATAAATTACAGAGATGATGAAAAGTCGTTCAGTC GTCGTATTAGTCATTTGTTCTTCCATAAAGAAAATGATTGGGGATTTTCCAATTTTATGGCCTGGAGTGAAGTGACC GATCCTGAGAAAGGATTTATAGATGATGACAAAGTTACCTTTGAAGTCTTTGTACAGGCGGATGCTCCCCATGGAGT TGCGTGGGATTCAAAGAAGCACACAGGCTACGTCGGCTTAAAGAATCAGGGAGCGACTTGTTACATGAACAGCCTGC TACAGACGTTATTTTTCACGAATCAGCTACGAAAGGCTGTGTACATGATGCCAACCGAGGGGGATGATTCGTCTAAA AGCGTCCCTTTAGCATTACAAAGAGTGTTCTATGAATTACAGCATAGTGATAAACCTGTAGGAACAAAAAAGTTAAC AAAGTCATTTGGGTGGGAAACTTTAGATAGCTTCATGCAACATGATGTTCAGGAGCTTTGTCGAGTGTTGCTCGATA ATGTGGAAAATAAGATGAAAGGCACCTGTGTAGAGGGCACCATACCCAAATTATTCCGCGGCAAAATGGTGTCCTAT ATCCAGTGTAAAGAAGTAGACTATCGGTCTGATAGAAGAGAAGATTATTATGATATCCAGCTAAGTATCAAAGGAAA GAAAAATATATTTGAATCATTTGTGGATTATGTGGCAGTAGAACAGCTCGATGGGGACAATAAATACGACGCTGGGG AACATGGCTTACAGGAAGCAGAGAAAGGTGTGAAATTCCTAACATTGCCACCAGTGTTACATCTACAACTGATGAGA TTTATGTATGACCCTCAGACGGACCAAAATATCAAGATCAATGATAGGTTTGAATTCCCAGAGCAGTTACCACTTGA TGAATTTTTGCAAAAAACAGATCCTAAGGACCCTGCAAATTATATTCTTCATGCAGTCCTGGTTCATAGTGGAGATA ATCATGGTGGACATTATGTGGT TTATCTAAACCCCAAAGGGGATGGCAAATGGTGTAAATTTGATGACGACGTGGT GTCAAGGTGTACTAAAGAGGAAGCAATTGAGCACAATTATGGGGGTCACGATGACGACCTGTCTGTTCGACACTGCA CTAATGCTTACATGTTAGTCTACATCAGGGAATCAAAACTGAGTGAAGTTTTACAGGCGGTCACCGACCATGATATT CCTCAGCAGTTGGTGGAGCGATTACAAGAAGAGAAAAGGATCGAGGCTCAGAAGCGGAAGGAGCGGCAGGAAGCCCA TCTCTATATGCAAGTGCAGATAGTCGCAGAGGACCAGTTTTGTGGCCACCAAGGGAATGACATGTACGATGAAGAAA AAGTGAAATACACTGTGTTCAAAGTATTGAAGAACTCCTCGCTTGCTGAGTTTGTTCAGAGCCTCTCTCAGACCATG GGATTTCCACAAGATCAAATTCGATTGTGGCCCATGCAAGCAAGGAGTAATGGAACAAAACGACCAGCAATGTTAGA TAATGAAGCCGACGGCAATAAAACAATGATTGAGCTCAGTGATAATGAAAACCCTTGGACAATATTCCTGGAAACAG TTGATCCCGAGCTGGCTGCTAGTGGAGCGACCTTACCCAAGTTTGATAAAGATCATGATGTAATGTTATTTTTGAAG ATGTATGATCCCAAAACGCGGAGCTTGAATTACTGTGGGCATATCTACACACCAATATCCTGTAAAATACGTGACTT GCTCCCAGTTATGTGTGACAGAGCAGGATTTATTCAAGATACTAGCCTTATCCTCTATGAGGAAGTTAAACCGAATT TAACAGAGAGAATTCAGGACTATGACGTGTCTCTTGATAAAGCCCTTGATGAACTAATGGATGGTGACATCATAGTA TTTCAGAAGGATGACCCTGAAAATGATAACAGTGAATTACCCACCGCAAAGGAGTATTTCCGAGATCTCTACCACCG CGTTGATGTCATTTTCTGTGATAAAACAATCCCTAATGATCCTGGATTTGTGGTTACGTTATCAAATAGAATGAATT ATTTTCAGGTTGCAAAGACAGTTGCACAGAGGCTCAACACAGATCCAATGTTGCTGCAGTTTTTCAAGTCTCAAGGT TATAGGGATGGCCCAGGTAATCCTCTTAGACATAATTATGAAGGTACTTTAAGAGATCTTCTACAGTTCTTCAAGCC TAGACAACCTAAGAAACTTTACTATCAGCAGCTTAAGATGAAAATCACAGACTTTGAGAACAGGCGAAGTTTTAAAT GTATATGGTTAAACAGCCAATTTAGGGAAGAGGAAATAACACTATATCCAGACAAGCATGGGTGTGTCCGGGACCTG TTAGAAGAATGTAAAAAGGCCGTGGAGCTTGGGGAGAAAGCATCAGGGAAACTTAGGCTGCTAGAAATTGTAAGCTA CAAAATCATTGGTGTTCATCAAGAAGATGAACTATTAGAATGTTTATCTCCTGCAACGAGCCGGACGTTTCGAATAG AGGAAATCCCTTTGGACCAGGTGGACATAGACAAAGAGAATGAGATGCTTGTCACAGTGGCGCATTTCCACAAAGAG GTCTTCGGAACGTTCGGAATCCCGTTTTTGCTGAGGATACACCAGGGCGAGCATTTTCGAGAAGTGATGAAGCGAAT CCAGAGCCTGCTGGACATCCAGGAGAAGGAGTTTGAGAAGTTTAAATTTGCAATTGTAATGATGGGCCGACACCAGT ACATAAATGAAGACGAGTATGAAGTAAATTTGAAAGACTTTGAGCCACAGCCCGGTAATATGTCTCATCCTCGGCCT TGGCTAGGGCTCGACCACTTCAACAAAGCCCCAAAGAGGAGTCGCTACACTTACCTTGAAAAGGCCATTAAAATCCA TAACTGA(SEQ ID NO.3)

USP7 amino acid sequences are：

MNHQQQQQQQKAGEQQLSEPEDMEMEAGDTDDPPRITQNPVINGNVALSDGHNTAEEDMEDDTSWRSEA TFQFTVERFSRLSESVLSPPCFVRNLPWKIMVMPRFYPDRPHQKSVGFFLQCNAESDSTSWSCHAQAVLKIINYRDD EKSFSRRISHLFFHKENDWGFSNFMAWSEVTDPEKGFIDDDKVTFEVFVQADAPHGVAWDSKKHTGYVGLKNQGATC YMNSLLQTLFFTNQLRKAVYMMPTEGDDSSKSVPLALQRVFYELQHSDKPVGTKKLTKSFGWETLDSFMQHDVQELC RVLLDNVENKMKGTCVEGTIPKLFRGKMVSYIQCKEVDYRSDRREDYYDIQLSIKGKKNIFESFVDYVAVEQLDGDN KYDAGEHGLQEAEKGVKFLTLPPVLHLQLMRFMYDPQTDQNIKINDRFEFPEQLPLDEFLQKTDPKDPANYILHAVL VHSGDNHGGHYVVYLNPKGDGKWCKFDDDVVSRCTKEEAIEHNYGGHDDDLSVRHCTNAYMLVYIRESKLSEVLQAV TDHDIPQQLVERLQEEKRIEAQKRKERQEAHLYMQVQIVAEDQFCGHQGNDMYDEEKVKYTVFKVLKNSSLAEFVQS LSQTMGFPQDQIRLWPMQARSNGTKRPAMLDNEADGNKTMIELSDNENPWTIFLETVDPELAASGATLPKFDKDHDV MLFLKMYDPKTRSLNYCGHIYTPISCKIRDLLPVMCDRAGFIQDTSLILYEEVKPNLTERIQDYDVSLDKALDELMD GDIIVFQKDDPENDNSELPTAKEYFRDLYHRVDVIFCDKTIPNDPGFVVTLSNRMNYFQVAKTVAQRLNTDPMLLQF FKSQGYRDGPGNPLRHNYEGTLRDLLQFFKPRQPKKLYYQQLKMKITDFENRRSFKCIWLNSQFREEEITLYPDKHG CVRDLLEECKKAVELGEKASGKLRLLEIVSYKIIGVHQEDELLECLSPATSRTFRIEEIPLDQVDIDKENEMLVTVA HFHKEVFGTFGIPFLLRIHQGEHFREVMKRIQSLLDIQEKEFEKFKFAIVMMGRHQYINEDEYEVNLKDFEPQPGNM SHPRPWLGLDHFNKAPKRSRYTYLEKAIKIHN(SEQIDNO.4)

Term " essentially identical amino acid sequence " refer to sequence it is identical or by one or more amino acid changes (missing, Increase, substitution) caused by it is different, but this change does not reduce its biological activity substantially.It is any to meet " essentially identical " want The deubiquitinating enzymes asked are included in the present invention, and no matter it is glycosylated (derives from naturally or from eukaryon life Object expression system) or it is nonglycosylated (i.e. from prokaryotic expression system or chemical synthesis).

" deubiquitinating enzymes " further include the deubiquitination zymoprotein of PEGylated deubiquitinating enzymes and covalent modification.Example Such as, it can be modified with the polyethylene glycol (PEG) that the molecular weight of various activation is 5,000~100,000 so that IL-17 producing high-moleculars, Extend its half-life period.Concrete operations can be found in Greenwald et al., Bioorg.Med.Chem.Lett.1994,4,2465； Caliceti et al.,IL Farmaco,1993,48,919；Zalipsky and Lee,《Polyethylene glycol chemistry：Biological skill Art and biomedical applications》, J.M.Harris volumes, Plenum Press, N.Y., 1992.

The deubiquitinating enzymes of the present invention can use gene recombination technology clonal expression.It is thin that the host cell of expression includes protokaryon Born of the same parents, yeast cells or higher eukaryotic cell.In addition to prokaryotic cell, eukaryocyte such as filamentous fungi (filamentous Fungi) or saccharomycete (yeast) is equally applicable to express or clones the interleukin-17 of the present invention.It is glycosylated for expressing The present invention deubiquitinating enzymes host cell resources in multi-cell organism.The example of invertebral zooblast includes insect Cell such as Drosophila S2 and Spodoptera Sf9, plant cell.The example of applicable mammalian host cell includes Chinese hamster ovary cell (CHO), COS cells.Those of ordinary skill in the art should know how to select suitable host cell.

Above-mentioned host cell can be in traditional battalion after deubiquitinating enzymes expression vector or cloning vector transfection or conversion It supports in base (nutrient media) and cultivates, the Nutrient medium is suitable for evoked promoter (promoter), selectivity after modification Transformant (selecting transformant) or amplification deubiquitinating enzymes coding gene sequence.Condition of culture is as cultivated The selection of base, temperature, pH etc. should then be known to those skilled in the art.How culturing and propagating power is made most Rule, scheme and the operating technology changed greatly can be found in Mammalian Cell Biotechnology:a Practical Approach,M.Butler,ed.(IRL Press,1991)and Sambrook et al.,supra。

The present invention deubiquitinating enzymes not only can directly be expressed by genetic recombination, can also by with polypeptide shape Mode into warm polypeptide produces, the latter can be one section of signal sequence for being located at mature protein or polypeptide N-terminal or Positioned at mature protein or other polypeptide fragments with specific cleavage site of polypeptide N-terminal.

Deubiquitinating enzymes dimer and preparation method thereof

The DNA sequence dna of coding deubiquitinating enzymes dimer of the present invention or fusion protein, can be all artificial synthesized.Also it can be used PCR amplification or synthetic method obtain the coding DNA sequence of the first monomer of deubiquitinating enzymes and/or deubiquitinating enzymes second comonomer Row, are then stitched together, and form the DNA sequence dna for encoding fusion protein of the present invention.

In order to improve the expression quantity of host cell, deubiquitinating enzymes dimer coded sequence can be transformed, such as Using the codon of host cell preference, the sequence for being unfavorable for genetic transcription and translation is eliminated.In the present invention, ferment may be used The codon of mother cell or mammalian cell preference, and using computer DNA softwares to deubiquitinating enzymes dimer gene into Row detection, excludes to be unfavorable for the sequence of genetic transcription and translation in gene, including introne shearing site, transcription terminator Deng.

After the DNA sequence dna for obtaining the new fusion protein of the coding present invention, suitable expression vector is connected into, then turn Enter suitable host cell.Finally, the host cell after culture conversion obtains the new fusion egg of the present invention by isolating and purifying In vain.

As used herein, term " carrier " includes plasmid, clay, expression vector, cloning vector, viral vectors etc..

In the present invention, for example commercially available carrier of various carriers known in the art can be selected.For example, select commercially available load Then the nucleotide sequence for encoding the new fusion protein of the present invention is operably coupled to expression regulation sequence, can form egg by body White expression vector.

As used herein, it " is operably coupled to " refer to such a situation, i.e. certain parts of linear DNA molecule being capable of shadow Ring the activity of same linear DNA molecule other parts.For example, if signal peptide DNA is as precursor expression and participates in dividing for polypeptide It secretes, then signal peptide (secretion targeting sequencing) DNA is exactly to be operably coupled to polypeptid DNA；If promoter control sequence turns Record, then it is to be operably coupled to coded sequence；If ribosome bind site is placed in the position that can translate it, that It is to be operably coupled to coded sequence.Generally, " be operably coupled to " mean it is adjoining, and for secreting leading sequence Row then mean adjacent in reading frame.

In the present invention, term " host cell " is including prokaryotic cell and eukaryocyte.Common prokaryotic host cell Example includes Escherichia coli, hay bacillus etc..Common eukaryotic host cell includes yeast cells, and insect cell and lactation are moved Object cell etc..Preferably, the host cell is eukaryocyte, more preferably it is mammalian cell.

After the host cell for obtaining conversion, the cell can be cultivated under conditions of expression fusion protein of the present invention is suitble to, So as to give expression to fusion protein.Then the fusion protein of expression is isolated again.

FOXP3

People's plug albumen P3 (human forkhead box P3, Foxp3) is important turn of Treg developments and function maintenance The factor is recorded, is played an important role to the development and function that regulate and control Treg.In human body, FOXP3 afunction can cause immune function Disorder, causes a variety of endocrine disorders, enteron aisle disease and X- sex-kink syndrome is PEX (Immunodysregulation, Polyendocrinopathy,and Enteropathy,X-linked).In mouse, FOXP3 afunction can develop into lymph Hyperblastosis disease (Scurfy) shows as serious dermatitis, multiple organ lymphocytic infiltration and autoimmune hemolytic anemia, Reason is the missing of natural type Treg.FOXP3 genes are manually integrated into expression in T cell can obtain the phenotype of Treg. Bone marrow chimerism experiment is carried out in mouse, Treg can not be developed by finding the bone marrow cell of FOXP3 gene knockouts, and wild type marrow Cell then can be with.And the notable downward of FOXP3 expressions caused by the change of 3 ' noncoding region of FOXP3 genes, it can seriously cut The immune suppression function of weak Treg illustrates that the expression of FOXP3 has a direct impact the function of Treg.In conclusion FOXP3 It is the important transcription factor for adjusting Treg cell developments and function.

Term " treatment " refers to based on healing, alleviation, improvement, mitigation, influences treatment object disease, symptom, disease constitution (predisposition) purpose and the interleukin-17 for giving the object present invention in need for the treatment of.

Term " treatment object " refers to mouse, people and other mammals.

Term " therapeutically effective amount " is the deubiquitinating enzymes that refer to reach therapeutic purposes in treatment object body or it spreads out The amount of biology, deubiquitinating enzymes agonist or antagonist.It should be appreciated by those skilled in the art that " therapeutically effective amount " Can with deubiquitinating enzymes or derivatives thereof, the administration route of deubiquitinating enzymes agonist or antagonist, excipient substance used and It is different and different from other drugs drug combination situation.

Pharmaceutical composition and method of administration

The pharmaceutical composition of the present invention include deubiquitinating enzymes of the present invention or derivatives thereof in the range of safely, effectively amount, Deubiquitinating enzymes agonist or antagonist (active constituent) and pharmacologically acceptable excipient or carrier.Wherein " safely, have Effect amount " refers to：The amount of active constituent is enough to be obviously improved the state of an illness, and is unlikely to generate serious side effect.In general, medicine group Active constituent/agent of active constituent/agent, preferably 0.05-300mg that object contains 0.001-1000mg is closed, more preferably, is contained Active constituent/agent of 0.5-200mg.

The active constituent of the present invention and its pharmacologically acceptable salt can be made into various preparations, wherein comprising safely, effectively The active constituent or its pharmacologically acceptable salt and pharmacologically acceptable excipient or carrier of the present invention in the range of amount. It wherein " safely, effectively measures " and refers to：The amount of active constituent is enough to be obviously improved the state of an illness, and is unlikely to generate serious secondary work With.Safely, effectively measuring for active constituent is determined according to concrete conditions such as the age for the treatment of object, the state of an illness, the courses for the treatment of.

" pharmacologically acceptable excipient or carrier " refers to：One or more biocompatible solids or liquid filler or Gelatinous mass, they are suitable for people's use and it is necessary to have enough purity and sufficiently low toxicity." compatibility " herein means Be in composition each component energy and the compound of the present invention and they between mutually admix, and significantly reduce the medicine of compound Effect.Pharmacologically acceptable excipient or carrier part example have cellulose and its derivates (such as sodium carboxymethylcellulose, second Base sodium cellulosate, cellulose ethanoate etc.), gelatin, talcum, kollag (such as stearic acid, magnesium stearate), calcium sulfate, plant Object oil (such as soya-bean oil, sesame oil, peanut oil, olive oil), polyalcohol (such as propylene glycol, glycerine, mannitol, sorbierite), breast Agent is (such as), wetting agent (such as lauryl sodium sulfate), colorant, flavoring agent, stabilizer, antioxidant, preservative, Apirogen water etc..

During using the present composition, can take orally, rectum, parenteral (intravenous, intramuscular or subcutaneous), part to Medicine.

The present composition can be administered alone or with other pharmaceutically acceptable compound administering drug combinations.

The sustained-release administration of the microcapsules active constituent for use in the present invention of composition containing the present invention.Recombinant protein Microcapsule controlled-release medicine-feeding technology has been successfully applied to human growth hormone recombinant (rhGH), recombinant human interferon alpha 2 (rhIFN), proleulzin With MNrgp120 (Johnson et al., Nat.Med., 2:795-799(1996)；Yasuda,Biomed.Ther 27: 1221-1223(1993)；WO 97/03692,WO 96/40072,WO 96/07399；U.S.Pat.No.5654010.

The sustained release preparation of the active constituent of the present invention can be used with good biological compatibility and wide in range biodegradability It is prepared by lactic-glycolic acid high polymer (PLGA).The catabolite of PLGA, lactic acid and hydroxyacetic acid can quickly be removed by human body.And And the degradation capability of the high polymer can with its molecular weight and composition difference, from some months extend to several years (Lewis, “Controlled release of bioactive agents form lactide/glycolide polymer,”in: M.Chasin and R.Langer(Eds.),Biodegradable Polymers as Drug Delivery Systems (Marcel Dekker:New York,1990),pp.1-41))。

It is the lactation that the active constituent of the present invention of safe and effective amount is applicable to treatment during using pharmaceutical composition Animal (such as people), wherein dosage is the effective dosage pharmaceutically thought during application, for the people of 60kg weight, every time Dosage is usually 0.01~300mg, preferably 0.5~100mg.Certainly, specific dosage is also contemplated that administration route, Bing Renjian The factors such as health situation, within the scope of these are all skilled practitioners technical ability.

One of main object of the present invention is to provide ubiquitination pathway correlation factor and its agonist or antagonist and is adjusting Purposes in FOXP3, IL-10, IL-2 and TGF-β activity.Another object of the present invention is to provide these regulatory factors adjusting Transcriptional regulatory activities of the FOXP3 to its target gene is saved, and then regulates and controls FOXP3⁺Purposes in the immunosuppressive activity of Treg.

Ubiquitination pathway correlation factor, its agonist or antagonist, which are provided, in one aspect of the present invention is preparing adjusting Purposes in FOXP3, IL-10, IL-2 and the composition of TGF-β activity, wherein the ubiquitination pathway correlation factor is selected from： USP44/USP7, induction USP44/USP7 expression or the signal factor, and/or their coded sequence that change the positioning of its caryoplasm； And/or the agonist or antagonist of the ubiquitination pathway correlation factor.

In yet another embodiment of the present invention, can be to the adjusting of FOXP3, IL-10, IL-2 and TGF-β activity Positive regulator or negative regulator, wherein, the ubiquitination pathway correlation factor or its agonist for positive regulator FOXP3, IL-10 and TGF-β activity and negative regulator IL-2 activity, the antagonist of the ubiquitination pathway correlation factor are used for negative regulator FOXP3, IL- 10 and TGF-β activity and positive regulator IL-2 activity.

In a preference, the antagonist is shUSP44, shUSP7.

In another preference, the antagonist is USP44 inhibitor (10-hydroxycamptothecin, 10- hydroxyl Base camptothecine).

In yet another embodiment of the present invention, the pro-inflammatory cytokine is selected from：IL-17 and IFN-γ.

In a preferred embodiment of the present invention, the purposes is that ubiquitination pathway correlation factor is preparing positive regulator Purposes in the composition of FOXP3 activity, wherein the ubiquitination pathway correlation factor is selected from：USP44/USP7, induction USP44/USP7 expresses or changes the signal factor of its caryoplasm positioning, and/or the agonist of the ubiquitination pathway correlation factor Or antagonist.

In the preference of the present invention, the activity imbalance is that hyperactivity or activity are too low.

In yet another embodiment of the present invention, it is described related to FOXP3, IL-10, IL-2 and TGF-β activity. The shRNA of USP44/USP7 protein-specifics can inhibit FOXP3, IL-10 and TGF-β mRNA to express, and promote IL-2mRNA tables It reaches.

In the preference of the present invention, the composition also comprising pharmaceutically, in health care conduct and learning or can in immunology The carrier of receiving.

In another aspect of this invention, a kind of combination for adjusting FOXP3, IL-10, IL-2 and TGF-β activity is provided Object, it includes：

(a) one or more ubiquitination pathway correlation factors selected from the group below：USP44/USP7, original induction USP44/ USP17 expresses or changes the signal factor, and/or their coded sequence of its caryoplasm positioning；And/or the ubiquitination pathway phase Close the agonist or antagonist of the factor.

(b) pharmaceutically, acceptable carrier in immunology or in health care conduct and learning.

In a preference, the composition is active with FOXP3, IL-10, IL-2 and TGF-β for treating or preventing The relevant disease of imbalance (i.e. hyperactivity or active too low) or symptom or as vaccine adjuvant.

In a preference, the composition also comprising one or more adjustable FOXP3, IL-10, IL-2 and Other active materials of TGF-β.

In another preference, the gene protein USP44/USP7 can be reduced and the relevant ubiquitination of FOXP3 degradations.

In a preference, the content of regulatory factor is 0.05-99.5 weight %, preferably 0.1-95 in the composition Weight %, more preferable 1-90 weight %, more preferable 5-80 weight %.

In another preferred example, the composition is injection, tablet, granule, pulvis or capsule.

In other aspects of the invention, a kind of side for adjusting FOXP3, IL-10, IL-2 and TGF-β activity is provided Method, the method includes giving one or more ubiquitination pathway correlation factors selected from the group below：USP44/USP7, induction USP44/USP7 expresses or changes the signal factor, and/or their coded sequence of its caryoplasm positioning；And/or the ubiquitination The agonist or antagonist of approach correlation factor.

In another preferred example, the present invention still further provide treatment or prevention and FOXP3, IL-10, IL-2 and TGF-β activity is lacked of proper care the method for relevant disease or symptom, and the method includes giving to need the objects of the treatment or prevention A effective amount of one or more ubiquitination pathway correlation factors selected from the group below：USP44/USP7, induction USP44/USP7 expression Or change the signal factor, and/or their coded sequence of the positioning of its caryoplasm；And/or the ubiquitination pathway correlation factor Agonist or antagonist.

In another preferred example, the method is further used for improving the immunogenicity of vaccine.

The other aspects of the present invention are apparent to those skilled in the art due to this disclosure 's.

Main advantages of the present invention are：

(1) effects of the deubiquitinating enzymes USP44 in the stability for adjusting FOXP3 is disclosed for the first time；

(2) transcription that TGF-β can be used in promoting deubiquitinating enzymes USP44 is disclosed for the first time.

(3) deubiquitinating enzymes can enhance the stability of FOXP3 and then improve regulatory T cells activity and its differentiation.

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.Those skilled in the art can make the present invention appropriate modification, change, these modifications It is within the scope of the present invention with variation.

Test method without specific conditions in the following example can be used the conventional method in this field, such as join It examines《Molecular Cloning:A Laboratory guide》(third edition, New York, CSH Press, New York：Cold Spring Harbor Laboratory Press, 1989) or according to the condition proposed by supplier.The sequencing approach of DNA is normal for this field The method of rule also can provide test by commercial company.

Unless otherwise stated, otherwise percentage and number are calculated by weight.Unless otherwise defined, it is all used in text Professional and scientific terms have the same meanings as commonly understood by one of ordinary skill in the art.It is in addition, any similar or equal to described content Deng method and material all can be applied in the method for the present invention.Preferred implement methods and materials described herein only present a demonstration it With.

Materials and methods

1. experiment material

1.1 plasmid

People's USP7, USP44 gene cloning is from human PBMC.For carrying the plasmid vector of people source FOXP3a and USP44 genes PIPHA2/MYC2/FLAG2, the pGL3-basic for carrying the promoter genes of Il-2 containing someone are purchased from Promega companies.Plasmid PIPHA2-FOXP3a and its different mutants and truncate, pIPFLAG2-USP44 and its block body, mutant pIPFLAG2- USP44-C282S, pIPMYC2-USP7-C223S, pIPMYC2-STUB1-H260Q or K30A and pIPHis6-Ub and its K0, K6, K11, K23, K29, K33, K48, K63 mutant are built by conventional method.Slow virus is packed and expression plasmid pLVX- IRES-EGFP is obtained from Institut Pasteur of Shanghai, and FUGW, del8.9 and VSV-G are obtained from Institut Pasteur of Shanghai.For building The carrier pLKO.1 of shRNA plasmids is transformed to be suitble to opening purchased from Open Biosystems companies and through this laboratory The insertion of shRNAs.Luciferase reporter gene carrier pGL4-basic is obtained from Medical College, Shanghai Communication Univ..USP44 promoters Region+17- (- 1712) is cloned from 293 T cell genomic DNAs of HEK.

1.2 antibody

Antibody used in the embodiment of the present invention includes：anti-USP44(1:1000,M2,Santa Cruz),anti- USP7(1:1000,Cell Signaling),anti-Foxy(1:2500, eBioscience), anti-STUB1 (H-231, Santa Cruz), anti-HA (1:1000, F-7, Santa Cruz), anti-FLAG (1:20000, M2, Sigma), anti- MYC(1:1000,9E10, Santa Cruz).anti-β-actin(1:1000,Sungene Biotech)and anti- GAPDH(1:5000, Sungene biotech), anti-human CD 45 RAs-PerCP/Cy5.5 (BioleGend, 304121), Anti- people CD4-FITC (BioleGend, 300506), anti-people CD25-PE (BioleGend, 302606), anti- FOXP3-APC(eBioscience,E09946-1633).The used FACS antibody of experiment is purchased from BD Pharmingen. anti-CD3/CD28Dynabeads(Gibco,1412799).The secondary antibody of horseradish peroxidase-labeled used is purchased from Jackson Lab。

1.3 enzymes and reagent

All restriction enzymes needed for molecular cloning, archaeal dna polymerase, T4DNA ligases be purchased from TAKARA or NEB companies.Point mutation KOD-plus enzymes and kit are purchased from Toyobo.CDNA Reverse Transcriptase kits and real time fluorescent quantitative PCR kit (SYBR green) is purchased from TaKaRa.It recombinates human cell factor and is purchased from R＆D system, TGF-β (R＆D, 240- B-002),IL-6(R&D,206-IL-010),IL-23(R&D,1290-1L-010)and IL1β(R&D,201-LB-005), IL-2(R&D,202-IL-010),rIL-4(R&D,204-IL-010),α-IL-4(R&D,MAB204),IL-12(R&D,219- IL-005), α-IFN-γ (R＆D, 285-IF-100).TRIzol reagents are purchased from Invitrogen.Proteasome inhibitor MG132 is purchased from Merck companies.Proteinase inhibitor C ocktail is purchased from Sigma.Protein synthesis inhibitor Cycloheximide (CHX) purchased from Sigma.Protein AG-beads are purchased from Santa Cruz.Lymphocyte separation medium (Ficoll, StemCell Technologies).LPS-EK is purchased from Invivogen.

1.4 primer

All primers order spontaneous work bioengineering Co., Ltd (Shanghai) in this subject.

It is for the primer sequence of gene cloning in this subject:

The various body primers that block of FOXP3a are designed by doctor Chen Zuojia, bibliography (Chen, Barbi et al.2013)。

It is for the primer sequence of real-time fluorescence quantitative PCR：

Remaining real-time fluorescence quantitative PCR primer, such as STUB1, IL-2, CTLA4, CD25, GITR are set by doctor Chen Zuojia Meter, and be described at (Zuojia Chen, et.al.Immunity.2013) (Chen Z 2013).

Gene order for building the primer of shRNA is：

It is for the primer sequence of point mutation：

Primer	Sequence	SEQ ID NO.
			USP44-C282S-F	GAGAAATTTGGGAAATACTAGCTATATGAATTCTGTTC	50
USP44-C282S-R	GAACAGAATTCATATAGCTAGTATTTCCCAAATTTCTC	51
			USP7-C223S-F	GAATCAGGGAGCGACTAGCTACATGAACAGCCTGC	52
USP7-C223S-R	GCAGGCTGTTCATGTAGCTAGTCGCTCCCTGATTC	53

Remaining mutant, such as the various mutation of FOXP3a and STUB1-H206Q, K30A point mutation, primer is by Chen Zuojia Doctor designs, bibliography (Chen, Barbi et al.2013).

2 experimental methods

The culture of 2.1 cell lines

Used HEK293T cells in this subject are that human embryonic kidney cells transformation cell lines are obtained through transfecting SV40 large T antigens Can be cultivated with the cell line of subculture in vitro separately containing 10% fetal calf serum, (Fetal bovine serum, FBS, are purchased from ), Excell in DMEM (Dulbecco Modified Eagle medium) culture medium of 100 μ g/ml mycillins.Cultivate item Part is 37 DEG C of temperature and 5%CO₂Saturated humidity.Jurkat T cells, i.e. people's Pancytopenia cell strain Jurkat The culture of E6.1 T cells system is containing 10%FBS (be purchased from Hyclone), 100 μ g/ml mycillins, 1 × NEAA (Gibco), and 1 In 1640 culture mediums of × Sodium Pyruvate (Gibco).Condition of culture is 37 DEG C of temperature and 5%CO₂Saturated humidity.

2.2 cell transfecting

The transfection of HEK293T cells is using polyethyleneimine Polyethylenimine (PEI) reagents and in strict accordance with saying Bright book operation.In brief, treat that HEK293T cell densities grow to 70-90% and proceed by transfection, first by 4 μ g plasmids and 100 μ l Low blood serum medium OPTI-MEM (Gibco) is sufficiently mixed, and adds the 12 abundant mixings of μ l PEI, after being stored at room temperature 10min, By cell to be transfected discard it is old training liquid change the fresh culture DMEM (complete medium) containing 10%FBS, be slowly added to more than Mixture.At 37 DEG C, 5%CO₂After cultivating 4h in incubator, discard old training liquid and change complete medium, continue at 37 DEG C, 5% CO₂48h or so is cultivated in incubator and receives sample.Jurkat T transfection using electroporation transfection instrument (Bio-Rad Laboratories, USA).It first by Jurkat T cells 50ng/ml phorbol exters (PMA) and 1 μM of ionomycin Ionomycin or is carried before transfection The magnetic pole of anti-CD3 (1 μ g/ml) and anti-CD28 (1 μ g/ml) activate 4h, in strict accordance with the program recommended on operation manual Electricity is carried out with condition to turn, later at 37 DEG C, 5%CO₂48h or so is cultivated in incubator and receives sample.

2.3 co-immunoprecipitations (Immunoprecipitation, IP) and protein immunization imprinting experiment (Western blotting,WB)

After 1 × PBS that the cell of collection is pre-chilled is washed, add in and contain protease inhibitors (1:100,P8340, ), Sigma-Aldrich PMSF (1mM)) and NaF (1mM) RIPA lysates (50mM Tris-HCl pH 7.4,150mM NaCl, 1%NP-40,0.5%Na-DOC, 1mM EDTA) on ice stand 30min cell is allowed fully to be cleaved, 12000rpm, 4 DEG C 10min is centrifuged, precipitation is abandoned and supernatant is transferred in new centrifuge tube, add in corresponding destination protein antibody and in Cool Room 4 DEG C shaking table Upper incubation 1h.The Protein A/G Agarose Beads of 10 μ l are sequentially added, continue to be incubated 1h under the same terms, later 1000rpm, 4 DEG C of centrifugation 3min sedimentation Beads add in 1 milliliter of RIPA (not having NP-40) lysate after removing supernatant, wash repeatedly It washs 4 times.After supernatant is removed in last time centrifugation, the every 2 × SDS loading buffer of pipe plus 40 μ l, after mixing by sample cell in 5-10min is boiled on drying heater for 100 DEG C, and 12000rpm room temperature centrifuges 1min after cooling, takes supernatant electrophoretic analysis.By equivalent body For long-pending Sample supernatants loading in the sample well of concentration glue, constant pressure carries out SDS-PAGE electrophoresis.Concentrate gel electrophoresis condition:80V, 30min；Separation gel deposition condition:120V,90min.Albumen on glue is gone into nitrocellulose with wet robin (100V, 90min) On film (NC).5% skim milk room temperature of film is closed 1h or 4 DEG C overnight later, then uses the corresponding primary antibody room of destination protein Temperature is incubated 1h.1 × TBST washs 3 times (10min/ time) the extra primary antibody of removing, then with horseradish peroxidase Secondary antibody is incubated at room temperature 1h.1 × TBST washs 3 times (10min/ times) and removes extra secondary antibody, adds ECL chemical substrate solution As 1 is pressed with solution B:1 mixing is reacted (kit is purchased from Millipore) with secondary antibody, and is detected and received with colour developing instrument GE LAS4000 Collection image is used to analyze.

2.4 immunofluorescence experiment

The Treg cell additions poly-L- that Jurkat-HA-FOXP3a surely turns plant cell or isolated from human PBMC In the coated planks of lysine, 300g centrifugation 5min are gently discarded supernatant, gently PFA (paraformaldehyde) room temperature of addition 4% is solid Determine 15min；It adds in the BSA containing 0.5%Triton-X 100,3% and wears film liquid room temperature closing 1h, then sequentially add and purpose The corresponding primary antibody of albumen (1h), secondary antibody (anti-mouse -488 of donkey, goat-anti rabbit -555 and core dyestuff antibody DAPI (1:5000) add together, It 0.5h) is incubated at room temperature, has been incubated washs (3 times, 5min/ times) the extra antibody of removing with 1 × PBS every time, added in anti-quencher Mounting afterwards is kept in dark place.It is finally detected fluorescence signal with Leica Confocal system SP5 instruments and is collected image and be used for Analysis.

2.5 luciferase reporter gene test experiences

People pGL3-IL-2-reporter and pGL4-USP44-reporter luciferase reporter plasmid and internal reference Renilla (pSV40) plasmids, which are transfected by PEI to HEK293T or electricity, to be gone in Jurkat T cells, and cell is collected after 48h (Jurkat T first activate 4h with 50ng/ml PMA and 1 μM of Ionomycin before being collected).It is first that the cell to get off is collected by centrifugation It is washed with 1 × PBS of precooling, discarding to be handled with reporter gene lysate after PBS makes it fully crack, 12000rpm centrifugations 10min takes supernatant to be reacted with it with double reporter gene detection kits (purchased from Promega), is then detected with Chemiluminescence Apparatus Uciferase activity, and the expression of analysis report gene is come as the internal reference of transfection efficiency using Renilla.

The extraction of 2.6 cell RNAs

Cell (1 × 10 is collected by centrifugation⁶) ice after 1ml TRIzol reagents (being purchased from Invitrogen) fully mixing is added in afterwards Upper lytic cell adds in 0.2ml chloroforms, 5min on ice is placed after acutely vibrating 15s, makes in 4 DEG C of centrifugation 15min of 12000rpm It is layered.Carefully upper strata aqueous phase is transferred in new pipe, addition 0.5ml isopropanol precipitatings RNA, it is quiet on ice after abundant mixing Put 5min.It is discarded supernatant after 4 °C of centrifugation 15min of 12000rpm.The ethanol solution for adding in 75% washs precipitation once, 4 °C of centrifugation 5min of 7500rpm.It abandons supernatant to uncap drying precipitated 5-10min after room temperature, precipitation is finally dissolved in RNase- In free water (no less than 20 μ l).

2.7 real-time fluorescence quantitative PCRs (Quantitative Real-time PCR)

Before real-time fluorescence quantitative PCR is carried out, first using SYBR PrimeScript RT-PCR (reverse Transcription PCR) RNA of extracting carries out reverse transcription and obtains cDNA by Kit (be purchased from TaKaRa).Two-step method reaction condition It is as follows:37 DEG C of 15min (reverse transcription reaction), 85 DEG C of 5s (inactivation reaction of reverse transcriptase) are obtained for real time fluorescent quantitative The cDNA templates of PCR.Then it prepares mixed liquor using SYBR green PCR mix kits (12 μ l systems) and is dispensed into PCR In each hole of plate, then real-time fluorescence is carried out on ABI Prism 7900HT (being purchased from Applied Biosystems) instrument and determined Measure PCR.Program setting:95℃2min；2 step PCR programs:95 DEG C of 15s and 60 DEG C of 60s, 40 cycles are carried out with this.Using β- Actin or GAPDH excludes the difference of general RNA in sample as internal standard gene.It can be calculated with relative quantification method by machine straight It connects to obtain multiple of the sample sets relative to the gene expression of control group.Absolute quantitation then needs first to calculate three multiple holes of every group of sample Averaging loop division of a ci poem value (cyclethreshold, CT), the difference of sample CT values and internal standard gene CT values are Δ CT, sample Δ CT and The difference of control sample Δ CT is Δ Δ CT, and the relative expression quantity for calculating target gene is 2- Δ Δ CT, and then analysis is each successively A gene.

Separation, induction and the amplification of 2.8 people's primary cells

1. the separation of PBMC in whole blood

Healthy People blood donor leukocyte samples (Shanghai City Blood Center offer) are placed in 50ml centrifuge tubes (per Guan Buchao Cross 15ml), 1 × PBS dilutes in equal volume, and volume is then dispensed in new 50ml centrifuge tubes and the lymph of dilute blood equivalent is thin Born of the same parents' separating liquid (Ficoll) is slowly added into whole blood dilution along wall, makes liquid natural layering, and interface is tried not to occur muddy It is turbid.2500rpm, which does not brake, to be centrifuged 20 minutes.Middle white PBMC cellular layers are carefully drawn with 1ml pipettors, try not to be drawn onto The liquid on cell upper strata is collected in PBMC to new centrifuge tube, adds in 1 × PBS washing cells, and 300g is centrifuged 5 minutes, gone Clearly, it is repeated 2 times.It washed once again with 1640 culture mediums, sorting is either dyed according to experiment demand Direct Pyrolysis or use X- VIVO culture mediums are resuspended, and put 37 DEG C, 5%CO₂It is cultivated in incubator, so that subsequent experimental uses.

2. peopleThe sorting of T and Treg cells and the Differentiation Induction in vitro of iTreg

By the PBMC cells detached by Healthy People blood donor leukocyte samples (Shanghai City Blood Center offer), resisted with streaming Body is incubated dyeing, and it is thin to obtain CD4+CD25hiCD127lo Treg using FACS ARIA II cell sorters (being purchased from BD) sorting Born of the same parents' (purity degree is more than 95%), CD4+CD25-CD45RA+T cell.The X- of 10% people's AB serum of people Treg cells Vivo culture mediums (add in 500U/ml IL-2 and anti-CD3/CD28beads (1:1)) amplification cultivation.It will first be expanded using preceding Treg cells removal beads tranquillization culture 2 days in containing 100U/ml IL-2 culture mediums afterwards, add anti-CD3/ CD28beads(1:4) it is stimulated with corresponding cell factor.The X-Vivo culture mediums of 10% people's AB serum of T cell (100U/ml IL-2 and 5ng/ml TGF-β is added in, and (cell and magnetic bead ratio are 1 to anti-CD3/CD28beads:1))7d It is induced to be divided into iTreg, is reused after detection FOXP3 expression.

3. the Differentiation Induction in vitro of people's Th cell subsets

People Th cell subsets breaks up under corresponding inductive condition, and in brief, initial cell is people's cell, adds in (cell and magnetic bead ratio are 1 to anti-CD3/CD28beads:1), then for Th0 cells, α-IL-4 (10 μ g/ml), α- IFN-γ(10μg/ml),IL-2(50u/ml)；It is anti-IL-4 (10 μ g/ml) for Th1 cells induction differentiation condition, rIL-12(10ng/ml),IL-2(50u/ml)；It is rIL-4 (20ng/ml), α-IFN- for Th2 cells induction differentiation condition γ(10μg/ml),IL-2(50u/ml)；It is TGF-β (1ng/ml) for Th17 cells induction differentiation condition, IL-6 (1ng/ ml),IL-23(100ng/ml)and IL1β(10ng/ml).Induction 7d makes it be divided into corresponding Th cells, detects corresponding transcription It is reused after the factor and cytokine-expressing.

2.9 ubiquitin precipitation experiments (His-pull down)

This laboratory reference (Jorg van Loosdregt, et.al.Immunity.2013) (van Loosdregt, Fleskens et al.2013).In short, by ubiquitin plasmid and the plasmid of express express target protein with 6 histidine marks It is transfected into HEK293T cells with PEI, receives 4h (20 μM) processing of MG132 before sample.Collect cell (8.0 urea of PH of solution 1 Solution, 8M Urea, 100mM Na2HPO4,10mM TRIS PH 8.0,0.2%TX-100,10mM immidazole, 1mM N-ethylmaleimide it) cracks, Ultrasonic Cell Disruptor ultrasound 1min (30s stops 10s, 30s), then 12000rpm, 20 DEG C, from Heart 30min.Supernatant is taken to sequentially add Ni-NTA beads (5 μ l), 3h, 3000rpm, 1.5min precipitation are incubated at room temperature on shaking table Then beads is washed twice with solution 1 respectively, solution 2 (6.3 urea liquids of PH, 8M Urea, 100mM Na2HPO4,10mM TRIS PH 6.3,0.2%TX-100,10mM immidazole)) it washes twice, (the 20mM TRIS PH 8.0,100mM of solution 3 NaCl, 20%glycerol, 1mM DTT, 10mM imidazole) it washes once, to remove the albumen of non-specific binding and impurity. It is then centrifuged for discarding residual supernatant, adds in 2x SDS loading and boil sample 5-10min, and carry out protein immunization imprinting experiment point Analysis.

The packaging of 2.10 slow virus and infection

By the pLKO.1-shRNA plasmids and pLKO.1-shCK of target gene and slow virus packaging plasmid △ 8.9, VSV-G Cotransfection HEK293T cell 10cm culture dishes.0.45 μM of membrane filtration of supernatant is collected after 48h, be distributed into mono- pipes of 1ml freeze- 80 DEG C spare.People Treg cells (5 × 10⁵) with anti-CD3/28beads (1:1) activation postoperative infection virus.After infection overnight (for 24 hours) normal training liquid is changed into.Efficiency of infection is observed after 48h.Cell is collected after 72h to be detected or screened.

2.11 Flow Cytometries (FACS) detect

The cell for detection is collected, is washed twice with 1 × PBS centrifugations of precooling, first contaminates viability dye working solutions dye Color excludes dead cell, extra dyestuff is washed away with 1 × PBS, then cell is resuspended into FACS buffer solution (1%BSA PBS).Add Enter cell surface antibodies and be protected from light incubation 30min on ice, washing twice with FACS buffer solution removes extra antibody, then adds in solid Surely film working solution (eBioscience) mixing is worn, 45min is stood in being protected from light on ice.Add in 1 × wear film buffer by centrifugation washing Cell is twice.Then with the diluted intracellular antibody working solution addition cell of film buffer solution is worn, it is protected from light stands 1h progress intracellulars on ice Dyeing.Add in 1 × wear film buffer by centrifugation washing cell remove extra antibody twice, finally cell is resuspended to 1 × 10⁶/ Ml, debugs FACS Fortessa instruments (being purchased from BD companies) program and upper machine is detected, and it is soft with FlowJo to collect experimental data Part is analyzed.

2.12 statistical analysis

All quantitative datas are represented as mean+/-standard error (SEM).The significance analysis of data difference passes through T- Test is completed in GraphPad Prism softwares, is represented as at least independent repetition experiment three times.(*p<0.05,**p< 0.005)

Embodiment 1, USP44 special high expression in iTreg

The present inventor first will be isolated from human peripheral (if referring to Healthy People without specified otherwise) peripheral blood list Nucleus (PBMC) carries out streaming antibody dyeing, then isolated from human peripheral PBMC with Flow Cytometry (FACS) Naive T cells (), T effector T cell (Teff) and nTreg, it is external evokedT makes it be divided into iTreg, cracking Cell Aspiration RNA carries out real-time fluorescence quantitative PCR (Quantitative Real-time PCR, qRT-PCR) and detects dependency basis As a result the expression of cause shows USP44 special high expression (Figure 1A) in iTreg.The different t helper cell of Differentiation Induction in vitro (Th) subgroup (Th1, Th2, Th17) and iTreg, it is similary to carry out qRT-PCR detections, also turn out that USP44 is special high in iTreg It expresses (Figure 1B).Illustrating the expression of the USP44 and FOXP3 in iTreg has correlation.

Embodiment 2, USP44 and FOXP3 interact

Since USP44 and FOXP3 cooperate with up-regulation in iTreg, the present inventor assumes that the two may have certain phase interaction With, in order to verify USP44 whether act on FOXP3 and and its function have correlation, the present inventor's contrived experiment verification the two Interaction.First, turn expression FLAG-USP44 and MYC-FOXP3 in HEK293T cells China and foreign countries, carry out two-way immune coprecipitated It forms sediment and protein immunization imprinting is tested, as a result show that the two can interact (Fig. 2A) in external rotary member system.Then it collects and stablizes The Jurkat T cells of HA-FOXP3 are expressed, two-way co-immunoprecipitation is carried out with corresponding antibodies and protein immunization imprinting is tested, knot Both fruit displays surely turn that (Fig. 2 B) can also be interacted in cell line.It is isolated from human peripheral PBMC again T cell, Differentiation Induction in vitro carry out co-immunoprecipitation with corresponding antibodies and protein immunization imprinting are tested, as a result show into iTreg The two albumen interacts (Fig. 2 C) in iTreg.And immunofluorescence experiment also shows the two common location (figure in iTreg 2D).There is interaction in above experiment, this promotes the present inventor further to think between fully demonstrating USP44 and FOXP3 albumen USP44 is examined to the albumen of FOXP3 and the influence of function.

Embodiment 3, USP44 stablize FOXP3 albumen

In order to understand influences of the USP44 to the albumen of FOXP3, the present inventor co-expresses in overexpression system first MYC-FOXP3 and FLAG-USP44 (dosage is incremented by 0.5,1,1.5 μ g into gradient) carries out protein immunization with corresponding anti-tag antibody As a result blotting experiments show that USP44 can stablize FOXP3 albumen, and with dose-dependant (Fig. 3 A).Since USP44 has Deubiquitination enzymatic activity, in order to verify whether its stabilization to FOXP3 albumen is related to its deubiquitination enzymatic activity, the present inventor Construct USP44 enzymes inactive mutant (C282S) and devise protein synthesis inhibitor cycloheximide CHX processing (0,4,8, It 12h) tests, as a result shows that coexpression wild type (WT) USP44 can significantly extend the half-life period of FOXP3 albumen, and enzyme inactivates Mutation C282S significantly reduces this effect, this illustrates influences of the USP44 to FOXP3 protein stabilities and its deubiquitinating enzymes It is active related, at the same also illustrate expression of the USP44 to FOXP3 influence be in its posttranslational modification level, by inhibit its into Enter proteasome pathway degradation to realize (Fig. 3 B).Further to prove shadows of the endogenic USP44 to FOXP3 protein stabilities It ringing, the present inventor knocks out USP44 endogenous in iTreg with slow-virus infection (carrying shCK, shUSP44-1, shUSP44-2), Protein immunization imprinting experiment is carried out with corresponding antibodies and with Flow Cytometry facs analysis, and as a result display is struck low USP44 and reduced The stability of endogenous FOXP3 albumen (Fig. 3 C, D).The expression of detection FOXP3 downstreams related gene is tested with qRT-PCR, as a result Low USP44 is struck in display causes FOXP3 downstream gene expression amounts to change：Reduce the downstream gene CTLA-4 of FOXP3 inductions Transcriptional level, increase FOXP3 inhibition downstream gene IL2 transcriptional level.

The sequence of shCK is as follows：

CAACAAGATGAAGAGCACCAA(SEQ ID NO.54)

The sequence of shUSP44-1 is as follows：

ACTGAGAATGGACATTCTAAA(SEQ ID NO.55)

The sequence of shUSP44-2 is as follows：

GAGTATCAAGTTAAAGCAGAA(SEQ ID NO.56)

Embodiment 4, USP44 influence Transcription inhibitions of the FOXP3 to downstream Il2 genes

In order to further verify influences of the USP44 to FOXP3 protein functions, the present inventor turns in HEK293T cells China and foreign countries The luciferase reporter plasmid of people's Il2 gene promoters, FLAG-USP44 (WT and C282S) and MYC-FOXP3 are included, with USP7 is positive control, and STUB1 is negative control, and Renilla is the internal reference of transfection efficiency, collects cell and carries out fluorescein enzyme activity Property measure and carry out protein immunization imprinting experiment with corresponding antibodies, as a result show that USP44 can significantly increase FOXP3 to downstream Il2 The transcripting suppressioning action (Fig. 4 A) of gene.Dosage experiments show the enhancing that USP44 inhibits the Il2 genetic transcriptions that FOXP3 is mediated (Fig. 4 B) is relied on into dose gradient.And after striking low endogenous USP44 with slow virus (containing shCK, shUSP44-1, shUSP44-2), The humidification of this Transcription inhibition significantly reduces (Fig. 4 C), further demonstrates influence tools of the USP44 to FOXP3 protein functions It plays an important role.Above the experiment proves that USP44 has influence, and be happened at albumen to the stability and function of FOXP3 albumen Posttranslational modification is horizontal, and proteins ubiquitin modification-proteasome degradation pathway be it is common be happened at protein translation after modify One of horizontal and mode for influencing protein stability and function, USP44 has deubiquitination enzymatic activity, this promotes the present inventor Further inquire into mechanism of the USP44 to FOXP3 protein stabilities and function effect.

Embodiment 5, USP44 removal FOXP3 and the poly ubiquitination for relevant K48 couplings of degrading

Deubiquitinating enzymes the USP44 special iTreg that is found in this subject is to the stability of its important Transcription factor FOXP3 Play an important roll with function, following the present inventor wants to know whether USP44 can remove as a deubiquitinating enzymes The ubiquitination of FOXP3.The present inventor cotransfection expression FLAG-USP44 (WT) and enzyme inactivation first in HEK293T cells Mutant (C282S), the ubiquitin protein (ubiquitin) of MYC-FOXP3 and His labels are added in before receiving sample at MG132 (20 μM) It manages 4h and prevents proteasome degradation pathway, ubiquitin precipitation is then carried out with coordinate bond covalent bond His labels with Ni-NTA magnetic beads Experiment, protein immunization imprinting result show that USP44 (WT) can significantly reduce the ubiquitination level of FOXP3 albumen and enzyme inactivation is prominent Variant (C282S) cannot (Fig. 5 A).It also still can be with even co-immunoprecipitation experiment also shows enzyme inactive mutant C282S FOXP3 interacts (data are not given and being listed), and display C282S is unable to deubiquitination FOXP3 and is not as to lead combined with it It causes, and is to rely on its deubiquitination enzymatic activity.The process reduced to prove this ubiquitination level depends on USP44, this Inventor strikes low endogenous USP44 with slow virus (carrying shCK, shUSP44-1, shUSP44-2) infection, and screening turns after a week The plasmid of dye expression MYC-FOXP3 and His-ubiquitin, it is similary to carry out ubiquitin precipitation, then albumen is detected with corresponding antibodies The ubiquitination level (Fig. 5 B) that low USP44 dramatically increases FOXP3 albumen is struck in expression and the ubiquitination level of FOXP3, as a result display. Illustrate deubiquitination enzymatic activity of the reduction of the ubiquitination level of FOXP3 dependent on USP44.With the 48th lysine of ubiquitin (K48) there is a report in the ubiquitination morning directly related with the degradation of albumen of coupling, and the 63rd lysine (K63) coupling is general Elementization modification plays an important roll the Function of albumen.In order to which understand USP44 removals is any ubiquitination, this K all on ubiquitin is first mutated into arginine (R) by inventor, i.e. then K0 carries out back mutation to K0 successively, build ubiquitin The single reservation mutant (K6, K11, K23, K29, K33, K48, K63) of different K.The present inventor's cotransfection table in overexpression Up to USP44, the ubiquitin protein (WT) of MYC-FOXP3 and His labels and its plasmid of various mutant carry out ubiquitin precipitation, so Protein expression and the ubiquitination level of FOXP3 are detected with corresponding antibodies afterwards, as a result shows USP44 major catalytics removal ubiquitin K48 The ubiquitination (Fig. 5 C) of coupling, this result disclose the mechanism that USP44 stablizes FOXP3.

Embodiment 6, FOXP3 albumen K179/250/263/268 are the deubiquitination action sites of USP44

Due to research before have shown that multiple sites on FOXP3 albumen can by ubiquitination, so, in addition to K179, the ubiquitination for being likely present other sites can also be by USP44 catalytic eliminations.In order to find out these sites, this The single lysines of all FOXP3 for being previously mentioned structure are retained mutant and USP44 cotransfections and carry out albumen by inventor Immunoblotting analysis is tested, and three times after independent experiment, obtains the single reservation site (K179/250/263/ of relatively stable four 268) it still can be stablized (Fig. 6 A) by USP44.In order to verify this four sites whether be USP44 whole action sites, this Inventor constructs the joint mutant 4R (K179/250/263/268R) of this four site lysines, and carries out outer turn of table altogether Up to ubiquitin precipitation experiments, as a result show and compared with wild type, add in USP44 after FOXP3-4R ubiquitination levels significantly increase Add, and cannot substantially be stablized (Fig. 6 B, C) by USP44.K250 is disclosed, K263, K268 are that possible other of USP44 remove ubiquitin Change action site.Also include K250, K263, K268 in the action site of STUB1, therefore this result implies USP44 and STUB1 There may be functional antagonism to the adjusting of FOXP3.

Embodiment 7, TGF-β signal promote iTreg to break up by raising USP44 transcriptional levels

The present inventor analyzes the promoter region of USP44 genes, finds the bound site of multiple TGF-β downstream kinase SMADs Point (Fig. 7 A) implies that the deubiquitinating enzymes USP44 of the special high expression of iTreg may be by TGF-β signals-modulating.In order to verify this It is assumed that the present inventor constructs the luciferase reporting base of promoter sequence+17- (- 1712) bp comprising USP44 genes first It because of plasmid, and is transfected into Jurkat T cells, phorbol exters (PMA, 50ng/ml) and ionomycin is used after 36h The TGF-β of (Ionomycin, 1 μM) and various concentration (0,1,5,10 μ g/ml) stimulation different time (0,1,2,4,8h), then Luciferase assays are carried out, as a result show that TGF-β stimulation can significantly raise the transcriptional level (Fig. 1-12) of USP44, phosphorus The SMAD2 (p-SMAD2) of acidification is horizontal to show that TGF-β access is activated when stimulating 1h.Show USP44 genes expression by TGF-β regulates and controls, this also explains why the USP44 specifically high expression in iTreg.Since TGF-β is in external evoked point of iTreg There is very important effect, the present inventor assumes that TGF-β promotes iTreg induction differentiation by raising USP44 in change.In order to demonstrate,prove Bright this point, the present inventor detach from human PBMCCell carries out Differentiation Induction in vitro 0,1,2,3,4,5d and obtains ITreg, extracting RNA carries out qRT-PCR detection corresponding gene mRNA expressions, while is resisted by immunoblotting analysis experiment with specific The protein level of USP44, USP7, STUB1, Hsp70 and FOXP3 are surveyed in physical examination, are as a result shown in iTreg atomizations, with The extension of TGF-β action time, USP44 expressions are continuously increased, FOXP3 expression also constantly up-regulation (Fig. 7 B, C), and STUB1 It is on a declining curve with Hsp70 expressions, USP7 expressions variation unobvious.It is upper that this result proves that TGF-β signal passes through USP44 transcriptional levels is adjusted to express stablizing FOXP3 and iTreg is promoted to break up.

Embodiment 8, STUB1 is by enhancing the more ubiquitinations of USP44 come the USP44 that degrades

Ubiquitin ligase STUB1 promotes FOXP3 protein degradations by modifying the ubiquitination of FOXP3K48 couplings, so as to increase The unstability of Treg be the present inventor laboratory before result of study (Zuojia Chen, et.al.Immunity.2013) (Chen Z 2013).Experiment above also turns out that USP44 and STUB1 has the coefficient site in part, and the two exists Expression in iTreg is negatively correlated, also opposite to the adjustment effect of FOXP3.These results promote the present inventor to think deeply The mutual adjustment effect of STUB1 and USP44, it is desirable to check on USP44 whether can catalytic elimination STUB1 to the general of FOXP3 Elementization is modified and whether USP44 expression is influenced with function by the operating condition of STUB1.The present inventor's contrived experiment first Corotation STUB1, USP44, FOXP3 and ubiquitin, after co-immunoprecipitation is the results show that add in USP44, by the FOXP3 of STUB1 modifications Ubiquitination level is not substantially reduced, and USP44 is inherently degraded (data are not listed) by STUB1 instead.Therefore the present invention People speculates that STUB1 can maintain the ubiquitination of FOXP3 by degrading USP44.In order to verify this inference, the present invention People constructs the Jurkat T cells system for stablizing expression USP44, takes cell (1 × 10⁶) first with PMA (50ng/ml) and (1 μM) stimulation 4h of Ionomycin, then with (37 DEG C or 42 DEG C) stimulation different times (1h, 3h) of different temperatures or use LPS respectively (1 μ g/ml) stimulation different time (0,12, for 24 hours) then collects cell cracking and carries out protein immunization imprinting reality with corresponding antibodies It tests, as a result shows that 42 DEG C of stimulation 1h USP44 start to degrade, 3h major parts USP44 has been degraded, and LPS stimulation 12h, big portion USP44 is divided to be degraded (Fig. 8 A), is illustrated in the operating condition acted in STUB1 FOXP3, the function of USP44 is because of itself It is degraded and suppressed.In order to understand in iTreg, whether USP44 is by being influenced FOXP3 destabilizing factors, the present inventor The iTreg of Differentiation Induction in vitro is taken, adds in 100U/ml IL-2 and anti-CD3/CD28beads (1:4,4 one, cells Beads) activating cell, respectively with LPS (1 μ g/ml), IL-6 (20ng/ml), IL-1 β (10ng/ml) are stimulated for 24 hours, collect cell After cracking, protein immunization imprinting experiment is carried out with corresponding antibodies, the results show that these destabilizing factors can be with Partial digestion USP44 is horizontal, and opposite STUB1 expressions raise (Fig. 8 B).These results promote the present inventor urgently to wonder, STUB1 is It is no to act on USP44.First, the present inventor in overexpression system transfection agents amount MYC-STUB1 incremental in gradient (0.5,1, 1.5 μ g) and USP44, protein immunization imprinting experiment is carried out with corresponding antibodies, as a result shows that STUB1 can degrade USP44, and in agent Amount relies on (Fig. 8 C).Then (WT, enzyme inactive mutant H260Q cannot be combined prominent corotation STUB1 with molecular chaperones Hsp70 Variant K30A) and USP44, co-immunoprecipitation and protein immunization imprinting experiment are carried out, as a result shows that STUB1 can be mutual with USP44 Effect, STUB1 enzyme inactive mutant H260Q and the mutant K30A that cannot be combined with molecular chaperones Hsp70 cannot degrade USP44, and STUB1-K30A can not combine USP44, this illustrate STUB1 to the degradation of USP44 and its ubiquitination enzymatic activity and The assistance of molecular chaperones is related (Fig. 8 D, E, F).Ubiquitin precipitation experiments show that corotation STUB1 can dramatically increase the ubiquitin of USP44 Change horizontal, illustrate STUB1 by enhancing the more ubiquitinations of USP44 come the USP44 that degrades, FOXP3's is unstable so as to promote.

Embodiment 9, USP44 and USP7 synergistic effects stablize FOXP3

Deubiquitinating enzymes USP7 has stablized FOXP3 albumen and Treg functions by interacting and removing ubiquitination There is report (Jorg van Loosdregt, et al.Immunity.2013) (van Loosdregt, Fleskens et al.2013).The deubiquitinating enzymes USP44 found in this subject equally has FOXP3 albumen interaction and by removing ubiquitin Change modification and stablize its protein level, and then enhance the effect of its function.Find that USP44 can be degraded by STUB1 simultaneously, and USP7 Also there is the action site identical with STUB1 parts, can also be degraded by LPS, therefore, the present inventor assumes USP44 and USP7 Act synergistically on FOXP3.In order to prove this it is assumed that the present inventor looks first at whether USP44 interacts with USP7.Crossing table Turn expression MYC-USP44 and FLAG-USP7 up to system China and foreign countries, find that the two can phase interaction by two-way co-immunoprecipitation experiment With (Fig. 9 A).The interaction of the two is determined, next step the present inventor wants to understand whether the two can cooperate with stable FOXP3 eggs In vain.The plasmid of contrived experiment corotation USP44, MYC-FOXP3 and FLAG-USP7 (0.5,1,1.5 μ g), and carry out co-immunoprecipitation It is tested with protein immunization imprinting, as a result shows that corotation USP44 and USP7 can stablize more FOXP3 albumen, and USP7 is in itself USP44 can be stablized, and in dose-dependant, result above proves that FOXP3 (Fig. 9 B) is stablized in USP44 and USP7 collaborations.And corotation USP44 and USP7 carries out ubiquitin precipitation experiments, and FOXP3 ubiquitination levels can be reduced by as a result turning USP44 or USP7 outside display, and Both transfect simultaneously, FOXP3 ubiquitination levels are minimum (Fig. 9 C), show that the mechanism that FOXP3 is stablized in USP44 and USP7 collaborations is logical Cross the ubiquitination of removal FOXP3.Due to above having been proven that STUB1 can degrade USP44, and Fig. 9 B show USP7 It itself can stablize USP44, and in dose-dependant.Therefore the present inventor wants to understand the relationship between this three.In HEK293T Cell China and foreign countries turn the slow virus packaging plasmid with shCK or shSTUB1, after 1 day, then are transferred to expression USP44's and MYC-USP7 Plasmid carries out protein immunization imprinting experiment with corresponding antibodies, and after as a result low STUB1 is struck in display, USP7 is steady to USP44 albumen Fixed apparent increase (Fig. 9 D).And transfect FLAG-USP44 (WT, C282S) and MYC-STUB1 (WT, H260Q) or USP7 (WT, enzyme Inactive mutant C223S) plasmid, and protein immunization imprinting experiment is carried out with corresponding antibodies, the present inventor can see, corotation Both STUB1 (WT) and USP44 (swimming lane 6) and transfection add USP7 (C223S) (swimming lane 10), and USP44 levels are minimum；And it is total to Turn both USP7 (WT) and USP44 (swimming lane 3) and transfection plus STUB1 (H260Q) (swimming lane 9), the horizontal highests of USP44； USP7 corotation USP44 (WT) protein level showed increased, and corotation USP44 (C282S) protein levels and single-turn USP7 connect in itself Closely (Fig. 9 E).To sum up as a result, the present inventor's analysis obtains USP44 and USP7 interactions, mutually stablize, and increased by cooperateing with Strong deubiquitination acts on stablizing FOXP3 protein levels.

Embodiment 10, USP44 gene knockouts influence the expression of endogenous FOXP3 and the inhibition function of Treg

In order to verify influences of the USP44 to Treg functions in animal body, the present inventor is first by 1 × 10⁶ CD4⁺ T cell (CD62L^hig^hCD25- Treg cells 4 that are) independent or being detached with wild type Treg or USP44 knock out mice ×10⁵Squeeze into RAG2-^/Manufacture colitis model in immunodeficient mouse body records its weight (as schemed) in certain time and does song Line chart (N=6) as a result shows that the decline of USP44 gene knockout group Treg mouse weights is faster than wild type Treg groups (Figure 10 A).It takes The distal end intestinal tissue that enteritis mouse model of the cell after 8 weeks is squeezed into Figure 10 A carries out hematoxylin-eosin (H/E) dyeing, goes forward side by side White light (100 ×) is analyzed under row histotomy microscope, and is carried out disease assessment marking and made block diagram.As a result USP44 is shown Gene knockout significantly affects the effect (Figure 10 B, C) that Treg inhibits inflammation.It will be withT cell and wild type Treg and The Treg cells detached in USP44 knock out mice are squeezed into together in immunodeficient mouse body, take mouse after a certain period of time Mucosal lymphatic knot (mLN), peripheral lymph nodes (pLN), spleen (SPL) carry out separation Treg cells (CD4⁺/Thy1.1⁺), with special Antibody carries out the expression of facs analysis FOXP3, control group design such as figure (Figure 10 D).As a result the notable shadow of USP44 gene knockouts is shown Ring the FOXP3 expressions of Treg.It will together be squeezed into immunodeficient mouse body from different Treg simultaneouslyT is thin Born of the same parents take out in certain time and use specific antibody (CD4⁺/Thy1.2⁺) facs analysis IFN-γ and the expression of IL-17 are carried out, it is right According to group a design such as figure (Figure 10 E).As a result display is squeezed into together with USP44 gene knockouts Treg in immune-deficient mice bodyThe IFN-γ of T cell and the expression of IL-17 significantly rise.

Embodiment 11, USP44 and the FOXP3 common high expression in the cancerous tissue of the hepatocarcinoma patient sample of the HCV positives

Immunohistochemical experiment is carried out with USP44 antibody in the cancerous tissue of the hepatocarcinoma patient sample of the HCV positives, is found USP44 specifically expressings (Figure 11 A) in mesenchyma stroma of tumors.Statistical information is shown in 17 and eventually leads to liver cancer (HCC) by HCV infection Patient cancerous tissue and cancer take up in normal structure, USP44 and the FOXP3 common high table in the cancerous tissue of wherein 11 samples Up to (Figure 11 B).Immunohistochemical experiment result is shown in the cancerous tissue of 17 patients that liver cancer (HCC) is eventually led to by HCV infection Take up in normal structure with cancer, USP44 and the FOXP3 common high expression (Figure 11 C) in the cancerous tissue of wherein 11 samples.

Embodiment 12,10- hydroxycamptothecin (10-HCPT) can be specifically inhibit USP44 to the stabilization of FOXP3 albumen

Cotransfection FLAG labels USP44 and MYC the label F OXP3 in HEK293T cells, and with the 10- hydroxyls of various concentration Base camplotheca acuminata alkali process, DMSO is as control, the results show that the inhibition USP44 that 10-hydroxycamptothecine (10-HCPT) can be special To the stabilization of FOXP3 albumen, and in dose-dependant (Figure 12).

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.

Bibliography：

Chen,Z.,J.Barbi,S.Bu,H.Y.Yang,Z.Li,Y.Gao,D.Jinasena,J.Fu,F.Lin, C.Chen,J.Zhang,N.Yu,X.Li,Z.Shan,J.Nie,Z.Gao,H.Tian,Y.Li,Z.Yao,Y.Zheng, B.V.Park,Z.Pan,J.Zhang,E.Dang,Z.Li,H.Wang,W.Luo,L.Li,G.L.Semenza,S.G.Zheng, K.Loser,A.Tsun,M.I.Greene,D.M.Pardoll,F.Pan and B.Li(2013)."The ubiquitin ligase Stub1 negatively modulates regulatory T cell suppressive activity by promoting degradation of the transcription factor Foxp3."Immunity 39(2):272- 285.

Chen Z,B.J.,Pan F,Li B,et.al.(2013)."The ubiquitin ligase Stub1 negatively modulates regulatory T cell suppressive activity by promoting degradation of the transcription factor FOXP3."Immunity 39(2):272-285.

van Loosdregt,J.,V.Fleskens,J.Fu,A.B.Brenkman,C.P.Bekker,C.E.Pals, J.Meerding,C.R.Berkers,J.Barbi,A.Grone,A.J.Sijts,M.M.Maurice,E.Kalkhoven, B.J.Prakken,H.Ovaa,F.Pan,D.M.Zaiss and P.J.Coffer(2013)."Stabilization of the transcription factor Foxp3 by the deubiquitinase USP7 increases Treg-cell- suppressive capacity."Immunity 39(2):259-271.

Sequence table

<110>Institut Pasteur of Shanghai, Chinese Academy of Sciences

<120>Application of the ubiquitination pathway correlation factor in regulating regulatory T cell function

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cccaaattat tccgcggcaa aatggtgtcc tatatccagt gtaaagaagt agactatcgg 1020

tctgatagaa gagaagatta ttatgatatc cagctaagta tcaaaggaaa gaaaaatata 1080

tttgaatcat ttgtggatta tgtggcagta gaacagctcg atggggacaa taaatacgac 1140

gctggggaac atggcttaca ggaagcagag aaaggtgtga aattcctaac attgccacca 1200

gtgttacatc tacaactgat gagatttatg tatgaccctc agacggacca aaatatcaag 1260

atcaatgata ggtttgaatt cccagagcag ttaccacttg atgaattttt gcaaaaaaca 1320

gatcctaagg accctgcaaa ttatattctt catgcagtcc tggttcatag tggagataat 1380

catggtggac attatgtggt ttatctaaac cccaaagggg atggcaaatg gtgtaaattt 1440

gatgacgacg tggtgtcaag gtgtactaaa gaggaagcaa ttgagcacaa ttatgggggt 1500

cacgatgacg acctgtctgt tcgacactgc actaatgctt acatgttagt ctacatcagg 1560

gaatcaaaac tgagtgaagt tttacaggcg gtcaccgacc atgatattcc tcagcagttg 1620

gtggagcgat tacaagaaga gaaaaggatc gaggctcaga agcggaagga gcggcaggaa 1680

gcccatctct atatgcaagt gcagatagtc gcagaggacc agttttgtgg ccaccaaggg 1740

aatgacatgt acgatgaaga aaaagtgaaa tacactgtgt tcaaagtatt gaagaactcc 1800

tcgcttgctg agtttgttca gagcctctct cagaccatgg gatttccaca agatcaaatt 1860

cgattgtggc ccatgcaagc aaggagtaat ggaacaaaac gaccagcaat gttagataat 1920

gaagccgacg gcaataaaac aatgattgag ctcagtgata atgaaaaccc ttggacaata 1980

ttcctggaaa cagttgatcc cgagctggct gctagtggag cgaccttacc caagtttgat 2040

aaagatcatg atgtaatgtt atttttgaag atgtatgatc ccaaaacgcg gagcttgaat 2100

tactgtgggc atatctacac accaatatcc tgtaaaatac gtgacttgct cccagttatg 2160

tgtgacagag caggatttat tcaagatact agccttatcc tctatgagga agttaaaccg 2220

aatttaacag agagaattca ggactatgac gtgtctcttg ataaagccct tgatgaacta 2280

atggatggtg acatcatagt atttcagaag gatgaccctg aaaatgataa cagtgaatta 2340

cccaccgcaa aggagtattt ccgagatctc taccaccgcg ttgatgtcat tttctgtgat 2400

aaaacaatcc ctaatgatcc tggatttgtg gttacgttat caaatagaat gaattatttt 2460

caggttgcaa agacagttgc acagaggctc aacacagatc caatgttgct gcagtttttc 2520

aagtctcaag gttataggga tggcccaggt aatcctctta gacataatta tgaaggtact 2580

ttaagagatc ttctacagtt cttcaagcct agacaaccta agaaacttta ctatcagcag 2640

cttaagatga aaatcacaga ctttgagaac aggcgaagtt ttaaatgtat atggttaaac 2700

agccaattta gggaagagga aataacacta tatccagaca agcatgggtg tgtccgggac 2760

ctgttagaag aatgtaaaaa ggccgtggag cttggggaga aagcatcagg gaaacttagg 2820

ctgctagaaa ttgtaagcta caaaatcatt ggtgttcatc aagaagatga actattagaa 2880

tgtttatctc ctgcaacgag ccggacgttt cgaatagagg aaatcccttt ggaccaggtg 2940

gacatagaca aagagaatga gatgcttgtc acagtggcgc atttccacaa agaggtcttc 3000

ggaacgttcg gaatcccgtt tttgctgagg atacaccagg gcgagcattt tcgagaagtg 3060

atgaagcgaa tccagagcct gctggacatc caggagaagg agtttgagaa gtttaaattt 3120

gcaattgtaa tgatgggccg acaccagtac ataaatgaag acgagtatga agtaaatttg 3180

aaagactttg agccacagcc cggtaatatg tctcatcctc ggccttggct agggctcgac 3240

cacttcaaca aagccccaaa gaggagtcgc tacacttacc ttgaaaaggc cattaaaatc 3300

cataactga 3309

<210> 4

<211> 1102

<212> PRT

<213>Artificial sequence

<400> 4

Met Asn His Gln Gln Gln Gln Gln Gln Gln Lys Ala Gly Glu Gln Gln

1 5 10 15

Leu Ser Glu Pro Glu Asp Met Glu Met Glu Ala Gly Asp Thr Asp Asp

20 25 30

Pro Pro Arg Ile Thr Gln Asn Pro Val Ile Asn Gly Asn Val Ala Leu

35 40 45

Ser Asp Gly His Asn Thr Ala Glu Glu Asp Met Glu Asp Asp Thr Ser

50 55 60

Trp Arg Ser Glu Ala Thr Phe Gln Phe Thr Val Glu Arg Phe Ser Arg

65 70 75 80

Leu Ser Glu Ser Val Leu Ser Pro Pro Cys Phe Val Arg Asn Leu Pro

85 90 95

Trp Lys Ile Met Val Met Pro Arg Phe Tyr Pro Asp Arg Pro His Gln

100 105 110

Lys Ser Val Gly Phe Phe Leu Gln Cys Asn Ala Glu Ser Asp Ser Thr

115 120 125

Ser Trp Ser Cys His Ala Gln Ala Val Leu Lys Ile Ile Asn Tyr Arg

130 135 140

Asp Asp Glu Lys Ser Phe Ser Arg Arg Ile Ser His Leu Phe Phe His

145 150 155 160

Lys Glu Asn Asp Trp Gly Phe Ser Asn Phe Met Ala Trp Ser Glu Val

165 170 175

Thr Asp Pro Glu Lys Gly Phe Ile Asp Asp Asp Lys Val Thr Phe Glu

180 185 190

Val Phe Val Gln Ala Asp Ala Pro His Gly Val Ala Trp Asp Ser Lys

195 200 205

Lys His Thr Gly Tyr Val Gly Leu Lys Asn Gln Gly Ala Thr Cys Tyr

210 215 220

Met Asn Ser Leu Leu Gln Thr Leu Phe Phe Thr Asn Gln Leu Arg Lys

225 230 235 240

Ala Val Tyr Met Met Pro Thr Glu Gly Asp Asp Ser Ser Lys Ser Val

245 250 255

Pro Leu Ala Leu Gln Arg Val Phe Tyr Glu Leu Gln His Ser Asp Lys

260 265 270

Pro Val Gly Thr Lys Lys Leu Thr Lys Ser Phe Gly Trp Glu Thr Leu

275 280 285

Asp Ser Phe Met Gln His Asp Val Gln Glu Leu Cys Arg Val Leu Leu

290 295 300

Asp Asn Val Glu Asn Lys Met Lys Gly Thr Cys Val Glu Gly Thr Ile

305 310 315 320

Pro Lys Leu Phe Arg Gly Lys Met Val Ser Tyr Ile Gln Cys Lys Glu

325 330 335

Val Asp Tyr Arg Ser Asp Arg Arg Glu Asp Tyr Tyr Asp Ile Gln Leu

340 345 350

Ser Ile Lys Gly Lys Lys Asn Ile Phe Glu Ser Phe Val Asp Tyr Val

355 360 365

Ala Val Glu Gln Leu Asp Gly Asp Asn Lys Tyr Asp Ala Gly Glu His

370 375 380

Gly Leu Gln Glu Ala Glu Lys Gly Val Lys Phe Leu Thr Leu Pro Pro

385 390 395 400

Val Leu His Leu Gln Leu Met Arg Phe Met Tyr Asp Pro Gln Thr Asp

405 410 415

Gln Asn Ile Lys Ile Asn Asp Arg Phe Glu Phe Pro Glu Gln Leu Pro

420 425 430

Leu Asp Glu Phe Leu Gln Lys Thr Asp Pro Lys Asp Pro Ala Asn Tyr

435 440 445

Ile Leu His Ala Val Leu Val His Ser Gly Asp Asn His Gly Gly His

450 455 460

Tyr Val Val Tyr Leu Asn Pro Lys Gly Asp Gly Lys Trp Cys Lys Phe

465 470 475 480

Asp Asp Asp Val Val Ser Arg Cys Thr Lys Glu Glu Ala Ile Glu His

485 490 495

Asn Tyr Gly Gly His Asp Asp Asp Leu Ser Val Arg His Cys Thr Asn

500 505 510

Ala Tyr Met Leu Val Tyr Ile Arg Glu Ser Lys Leu Ser Glu Val Leu

515 520 525

Gln Ala Val Thr Asp His Asp Ile Pro Gln Gln Leu Val Glu Arg Leu

530 535 540

Gln Glu Glu Lys Arg Ile Glu Ala Gln Lys Arg Lys Glu Arg Gln Glu

545 550 555 560

Ala His Leu Tyr Met Gln Val Gln Ile Val Ala Glu Asp Gln Phe Cys

565 570 575

Gly His Gln Gly Asn Asp Met Tyr Asp Glu Glu Lys Val Lys Tyr Thr

580 585 590

Val Phe Lys Val Leu Lys Asn Ser Ser Leu Ala Glu Phe Val Gln Ser

595 600 605

Leu Ser Gln Thr Met Gly Phe Pro Gln Asp Gln Ile Arg Leu Trp Pro

610 615 620

Met Gln Ala Arg Ser Asn Gly Thr Lys Arg Pro Ala Met Leu Asp Asn

625 630 635 640

Glu Ala Asp Gly Asn Lys Thr Met Ile Glu Leu Ser Asp Asn Glu Asn

645 650 655

Pro Trp Thr Ile Phe Leu Glu Thr Val Asp Pro Glu Leu Ala Ala Ser

660 665 670

Gly Ala Thr Leu Pro Lys Phe Asp Lys Asp His Asp Val Met Leu Phe

675 680 685

Leu Lys Met Tyr Asp Pro Lys Thr Arg Ser Leu Asn Tyr Cys Gly His

690 695 700

Ile Tyr Thr Pro Ile Ser Cys Lys Ile Arg Asp Leu Leu Pro Val Met

705 710 715 720

Cys Asp Arg Ala Gly Phe Ile Gln Asp Thr Ser Leu Ile Leu Tyr Glu

725 730 735

Glu Val Lys Pro Asn Leu Thr Glu Arg Ile Gln Asp Tyr Asp Val Ser

740 745 750

Leu Asp Lys Ala Leu Asp Glu Leu Met Asp Gly Asp Ile Ile Val Phe

755 760 765

Gln Lys Asp Asp Pro Glu Asn Asp Asn Ser Glu Leu Pro Thr Ala Lys

770 775 780

Glu Tyr Phe Arg Asp Leu Tyr His Arg Val Asp Val Ile Phe Cys Asp

785 790 795 800

Lys Thr Ile Pro Asn Asp Pro Gly Phe Val Val Thr Leu Ser Asn Arg

805 810 815

Met Asn Tyr Phe Gln Val Ala Lys Thr Val Ala Gln Arg Leu Asn Thr

820 825 830

Asp Pro Met Leu Leu Gln Phe Phe Lys Ser Gln Gly Tyr Arg Asp Gly

835 840 845

Pro Gly Asn Pro Leu Arg His Asn Tyr Glu Gly Thr Leu Arg Asp Leu

850 855 860

Leu Gln Phe Phe Lys Pro Arg Gln Pro Lys Lys Leu Tyr Tyr Gln Gln

865 870 875 880

Leu Lys Met Lys Ile Thr Asp Phe Glu Asn Arg Arg Ser Phe Lys Cys

885 890 895

Ile Trp Leu Asn Ser Gln Phe Arg Glu Glu Glu Ile Thr Leu Tyr Pro

900 905 910

Asp Lys His Gly Cys Val Arg Asp Leu Leu Glu Glu Cys Lys Lys Ala

915 920 925

Val Glu Leu Gly Glu Lys Ala Ser Gly Lys Leu Arg Leu Leu Glu Ile

930 935 940

Val Ser Tyr Lys Ile Ile Gly Val His Gln Glu Asp Glu Leu Leu Glu

945 950 955 960

Cys Leu Ser Pro Ala Thr Ser Arg Thr Phe Arg Ile Glu Glu Ile Pro

965 970 975

Leu Asp Gln Val Asp Ile Asp Lys Glu Asn Glu Met Leu Val Thr Val

980 985 990

Ala His Phe His Lys Glu Val Phe Gly Thr Phe Gly Ile Pro Phe Leu

995 1000 1005

Leu Arg Ile His Gln Gly Glu His Phe Arg Glu Val Met Lys Arg

1010 1015 1020

Ile Gln Ser Leu Leu Asp Ile Gln Glu Lys Glu Phe Glu Lys Phe

1025 1030 1035

Lys Phe Ala Ile Val Met Met Gly Arg His Gln Tyr Ile Asn Glu

1040 1045 1050

Asp Glu Tyr Glu Val Asn Leu Lys Asp Phe Glu Pro Gln Pro Gly

1055 1060 1065

Asn Met Ser His Pro Arg Pro Trp Leu Gly Leu Asp His Phe Asn

1070 1075 1080

Lys Ala Pro Lys Arg Ser Arg Tyr Thr Tyr Leu Glu Lys Ala Ile

1085 1090 1095

Lys Ile His Asn

1100

<210> 5

<211> 27

<212> DNA

<213>Artificial sequence

<400> 5

cgggatccat gctagcaatg gatacgt 27

<210> 6

<211> 27

<212> DNA

<213>Artificial sequence

<400> 6

gatatctcag ctaaggattt cattaga 27

<210> 7

<211> 28

<212> DNA

<213>Artificial sequence

<400> 7

ccgctcgaga tgctagcaat ggatacgt 28

<210> 8

<211> 29

<212> DNA

<213>Artificial sequence

<400> 8

gctctagatc agctaaggat ttcattaga 29

<210> 9

<211> 24

<212> DNA

<213>Artificial sequence

<400> 9

gatatcaggt tggtgtacct gtcc 24

<210> 10

<211> 28

<212> DNA

<213>Artificial sequence

<400> 10

cccaagctta ctgctttcct gaagtcac 28

<210> 11

<211> 28

<212> DNA

<213>Artificial sequence

<400> 11

cgggatccac aggattgaga aatttggg 28

<210> 12

<211> 26

<212> DNA

<213>Artificial sequence

<400> 12

gatatctcat acaccaggag ttacta 26

<210> 13

<211> 26

<212> DNA

<213>Artificial sequence

<400> 13

gatatctcat aacttcaggt ctccag 26

<210> 14

<211> 27

<212> DNA

<213>Artificial sequence

<400> 14

cgggatcctg tgtggactgc aacacga 27

<210> 15

<211> 28

<212> DNA

<213>Artificial sequence

<400> 15

cgggatccct acgacgtaca ttaagtgc 28

<210> 16

<211> 28

<212> DNA

<213>Artificial sequence

<400> 16

cgtcgtagac agtgccattt ctgagggt 28

<210> 17

<211> 28

<212> DNA

<213>Artificial sequence

<400> 17

ggcactgtct acgacgtaca ttaagtgc 28

<210> 18

<211> 20

<212> DNA

<213>Artificial sequence

<400> 18

tgccacctac ctcaggttct 20

<210> 19

<211> 20

<212> DNA

<213>Artificial sequence

<400> 19

ctggtctgag ggatttcagg 20

<210> 20

<211> 20

<212> DNA

<213>Artificial sequence

<400> 20

tcaaacgatt caggtggtca 20

<210> 21

<211> 20

<212> DNA

<213>Artificial sequence

<400> 21

accgcggaca agtcatagat 20

<210> 22

<211> 20

<212> DNA

<213>Artificial sequence

<400> 22

gaggaggaca tggaggatga 20

<210> 23

<211> 20

<212> DNA

<213>Artificial sequence

<400> 23

aagcgtggca tcaccataat 20

<210> 24

<211> 20

<212> DNA

<213>Artificial sequence

<400> 24

tgtccgggac ctgttagaag 20

<210> 25

<211> 20

<212> DNA

<213>Artificial sequence

<400> 25

ggctcgttgc aggagataaa 20

<210> 26

<211> 20

<212> DNA

<213>Artificial sequence

<400> 26

tgcaaaaggc ttcagagaca 20

<210> 27

<211> 20

<212> DNA

<213>Artificial sequence

<400> 27

ctctgttggg gtgaaaggag 20

<210> 28

<211> 20

<212> DNA

<213>Artificial sequence

<400> 28

tcccagagtt cctccacaac 20

<210> 29

<211> 20

<212> DNA

<213>Artificial sequence

<400> 29

attgagtgtc cgctgcttct 20

<210> 30

<211> 22

<212> DNA

<213>Artificial sequence

<400> 30

aggccaagca cgacaagtac at 22

<210> 31

<211> 22

<212> DNA

<213>Artificial sequence

<400> 31

ctgatcttgc cacacaggta gt 22

<210> 32

<211> 20

<212> DNA

<213>Artificial sequence

<400> 32

aagcagacgc agatcttcac 20

<210> 33

<211> 20

<212> DNA

<213>Artificial sequence

<400> 33

ctcgatctcc tccttgctca 20

<210> 34

<211> 20

<212> DNA

<213>Artificial sequence

<400> 34

gcaactcctg tcttgcattg 20

<210> 35

<211> 20

<212> DNA

<213>Artificial sequence

<400> 35

cagttctgtg gccttcttgg 20

<210> 36

<211> 20

<212> DNA

<213>Artificial sequence

<400> 36

agtgggactg catgtgtgtc 20

<210> 37

<211> 20

<212> DNA

<213>Artificial sequence

<400> 37

gcagtctgtc caaggtttgc 20

<210> 38

<211> 20

<212> DNA

<213>Artificial sequence

<400> 38

tggggaatga gttgaccttc 20

<210> 39

<211> 20

<212> DNA

<213>Artificial sequence

<400> 39

gcacggttct ggatcaatta 20

<210> 40

<211> 20

<212> DNA

<213>Artificial sequence

<400> 40

gagtcaacgg atttggtcgt 20

<210> 41

<211> 20

<212> DNA

<213>Artificial sequence

<400> 41

gacaagcttc ccgttctcag 20

<210> 42

<211> 21

<212> DNA

<213>Artificial sequence

<400> 42

caacaagatg aagagcacca a 21

<210> 43

<211> 21

<212> DNA

<213>Artificial sequence

<400> 43

actgagaatg gacattctaa a 21

<210> 44

<211> 21

<212> DNA

<213>Artificial sequence

<400> 44

gagtatcaag ttaaagcaga a 21

<210> 45

<211> 21

<212> DNA

<213>Artificial sequence

<400> 45

cggatgatga acttgtgcaa t 21

<210> 46

<211> 21

<212> DNA

<213>Artificial sequence

<400> 46

ttgtggttac gttatcaaat a 21

<210> 47

<211> 21

<212> DNA

<213>Artificial sequence

<400> 47

tcctaaggac cctgcaaatt a 21

<210> 48

<211> 21

<212> DNA

<213>Artificial sequence

<400> 48

gcacgacaag tacatggcgg a 21

<210> 49

<211> 21

<212> DNA

<213>Artificial sequence

<400> 49

tgccgccact atctgtgtaa t 21

<210> 50

<211> 38

<212> DNA

<213>Artificial sequence

<400> 50

gagaaatttg ggaaatacta gctatatgaa ttctgttc 38

<210> 51

<211> 38

<212> DNA

<213>Artificial sequence

<400> 51

gaacagaatt catatagcta gtatttccca aatttctc 38

<210> 52

<211> 35

<212> DNA

<213>Artificial sequence

<400> 52

gaatcaggga gcgactagct acatgaacag cctgc 35

<210> 53

<211> 35

<212> DNA

<213>Artificial sequence

<400> 53

gcaggctgtt catgtagcta gtcgctccct gattc 35

<210> 54

<211> 21

<212> DNA

<213>Artificial sequence

<400> 54

caacaagatg aagagcacca a 21

<210> 55

<211> 21

<212> DNA

<213>Artificial sequence

<400> 55

actgagaatg gacattctaa a 21

<210> 56

<211> 21

<212> DNA

<213>Artificial sequence

<400> 56

gagtatcaag ttaaagcaga a 21

Claims

1. the purposes of deubiquitinating enzymes or deubiquitinating enzymes agonist, which is characterized in that be used to prepare preparation or kit, institute It states preparation or kit is used for：

(1) increase the stability of FOXP3；And/or

(5) promote the differentiation of iTreg.

2. purposes as described in claim 1, which is characterized in that the regulatory T cells activity is too low to refer to that regulatory T is thin Born of the same parents inhibit inflammation and immune response function too low；

Described is selected from the too low relevant disease of regulatory T cells activity：Autoimmunity disease, inflammatory reaction, tumour and infection Property disease.

3. purposes as described in claim 1, which is characterized in that the deubiquitinating enzymes be selected from the group in it is one or more： USPs families (Ubiquitin-specific proteases, USPs) and OUT families；

Preferably, it is one or more during the deubiquitinating enzymes are selected from the group：USP44、USP2、USP3、USP4、USP5、 USP7, USP10, USP12, USP14, USP17, USP18, USP21, USP22, USP30, USP39, YOD1, CYLD and A20；It is excellent Selection of land, the deubiquitinating enzymes are USP44, USP7 or combination.

4. purposes as described in claim 1, which is characterized in that the stability for increasing FOXP3 is real in the following manner It is existing：Deubiquitination is carried out to FOXP3；

Preferably, the deubiquitination is the ubiquitination of catalytic elimination ubiquitin K48 couplings；

It is highly preferred that the deubiquitination action site is selected from：K179, K250, K263, K268 of FOXP3.

5. the purposes of deubiquitinating enzymes antagonist, which is characterized in that be used to prepare preparation or kit, the preparation or kit For：

(1) stability of FOXP3 is reduced；And/or

6. a kind of composition for the stability for being used to increase FOXP3, which is characterized in that the composition is selected from：Deubiquitinating enzymes Or derivatives thereof and/or deubiquitinating enzymes agonist.

7. a kind of albumen composition of separation, which is characterized in that the compound is combined institute's shape for deubiquitinating enzymes and FOXP3 Into compound.

8. the application of albumen composition described in claim 7, which is characterized in that for screening drug or compound, the drug Or compound promotes or inhibits deubiquitinating enzymes and FOXP3 to form the compound.

9. a kind of purposes of deubiquitinating enzymes antagonist, which is characterized in that be used to prepare the medicine for the treatment of tumour or communicable disease Object.

Purposes of the 10.TGF- β in the reagent for promoting deubiquitinating enzymes transcription is prepared；Preferably, the deubiquitinating enzymes are selected from It is one or more in the following group：USPs families (Ubiquitin-specific proteases, USPs) and OUT families.