CN101704893A - Human Scurfin protein span with membrane penetration sequence, fragment and preparation method - Google Patents

Abstract

The invention relates to the technical fields of gene engineering and the biomedicine of protein transduction, in particular to fusion proteins of a human Scurfin span with a membrane penetration sequence, a Forkhead removing domain human Scurfin fragment and a proline-rich removing domain human Scurfin fragment and a preparation method. The fusion proteins of the human Scurfin span with the membrane penetration sequence, the Forkhead removing domain human Scurfin fragment and the proline-rich removing domain human Scurfin fragment respectively have amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3. The invention adopts a mode of constructing PTD-hScurfin to obtain the fusion proteins for the first time, transduces a target protein into Jurkat T and peripheral CD4+CD25-T cells by the direct mediation and fusion of a protein membrane penetration structure field to obtain Treg in mass, and breaks through the bottleneck constraining Treg application in the hope of being applied to the repulsion-resisting treatment of clinical organ transplantation finally.

Description

Band is worn people Scurfin albumen total length and the fragment and the preparation method of film sequence

Technical field

The present invention relates to the field of biomedicine technology of genetically engineered and protein transduction.Specifically, the present invention relates to the people Scurfin total length of wearing the film sequence, remove jaw architecture territory (Δ Forkhead domain, Δ FKH) fusion rotein of the recombination to construct of people Scurfin fragment and removal proline rich structural domain (Δ Proline-rich domain, Δ PRD) the segmental fusion rotein of people Scurfin: PTD-hScurfin, PTD-Δ hFKH and PTD-Δ hPRD gene order and preparation thus is to Jurkat T cell (the acute T lymphocytic leukemia cell of people system) and human peripheral CD4 ⁺The T cell suppresses propagation, regulates the effect of cytokine secretion and inducing immune tolerance, and is applied to the purposes in the medicine of the preparation of regulatory T like cell, prevention and treatment treatment inflammatory immune related diseases (comprising autoimmune disease, transplant rejection relative disease) and tolerance induction or tumour.

Background technology

1.Treg general introduction

From reported first CD4 such as the nineteen ninety-five Sakaguchi of Kyoto Univ Japan ⁺CD25 ^HiRegulatory T cells (regulatory Tcells, Treg) after, Treg causes whole world related discipline scholar's extensive concern immediately.Classification to Treg is varied, and the mode classification of extensively being admitted is to grow and the difference of its specificity and mechanism of action etc. is divided into natural Treg (Natural Treg) and acquired or inducibility Treg (Adaptive or Induced Treg) with it according to Treg at present.Natural Treg mainly expresses CD4, CD25, hScurfin, CD134 (OX40), GITR (TNFRSF18), CD62L (L-selectin) and CD152 (CTLA-4).Although its quantity seldom, has very strong immune suppression function.(activated T reg can either directly contact the mediation immunosuppression with T effector cell by the MHC molecule of self, suppresses CD8 by the iuntercellular contact inhibition for it ⁺The activation of T cell; Also can express collaborative stimulation molecule by suppressing APC indirectly, thus the propagation of retarding effect T cell and the generation of IL-2.) and the effect of secrete cytokines retarding effect immunocyte, play a significant role in the formation of autoimmunization tolerance with in keeping.Its function induce dependence antigen, the enforcement of function does not then have the antigen specificity, but there are some researches show that recently also there is certain antigen-specific in its function.Acquired Treg then mainly brings into play restraining effect by the secrete cytokines mode.Find that subsequently Treg mainly is subjected to a kind of jaw wing spiral transcription factor family member p3 (forkhead box transcription factorclass p3, the Foxp3) regulation and control of the Protein S curfin of genes encoding.Generally when the gene of representing the people source and albumen, represent, and mouse source property is represented with Foxp3 and scurfin, represents and scurfin with Foxp3 when non-refering in particular to FOXP3 and Scurfin.Also useful FOXP3/Foxp3 representative is proteic, and present patent application unifies to represent respectively with hFOXP3 and hScurfin people's gene and albumen, and mFoxp3 and mScurfin represent gene and the albumen of mouse respectively.Represent gene and albumen respectively with Foxp3 and scurfin during non-refering in particular to.

2. people Scurfin gene and its coded product

People Scurfin (human Scurfin, hScurfin) gene is jaw wing spiral transcription factor family member p3 (humanforkhead box transcription factor class p3, hFOXP3), be positioned at human chromosome Xp11.23-Xp13.3, GenBank accession number: AF277993.Belong to jaw/wing spiral transcription regulaton factor family, contain 11 exons, conservative at human camber.

People FOXP3 (human FOXP3, hFOXP3) coded product of gene is the protein hScurfin of a 48-kDa, jaw spiral (the Forkhead/winged helix that its characteristic structure is the PROTEIN C end, FKH) structure, the protein that contains this structural domain belongs to one of dna binding factor family member. except the jaw district, hScurfin albumen also comprise the proline rich of a N-terminal structural domain (Proline-rich domain, PRD).In recent years the active performance of inhibition that studies show that these structural domains and hScurfin has close getting in touch.Between FKH and PRD, also comprise a C2H2 zinc fingers and a leucine zipper sequence, but do not have the PKB/AKT site of phosphorylation.HScurfin belongs to transcriptional regulatory, and main effect is to suppress to transcribe, and mainly is expressed in adenoid CD4 ⁺The T cell, very important to the effect of keeping of immune stable state.

3.Scurfin be Treg relative specificity sign

For studies show that of mouse, lack mScurfin and can cause a kind of fatal lymphoproliferative diseases rapidly, similar to the mouse that lacks CTLA-4.If change the mFoxp3 gene over to mouse that CTLA-4 knocks out, or CTLA-4 defective mouse is carried out mScurfin cross expression, can postpone the generation of lymphatic system proliferative disease, prolong 140 day life-span of this mouse, confirm the CTLA-4 function of the alternative disappearance of mScurfin.And immune disorder, polyendocrinopathy and enteropathy syndromes syndrome (the immune dysregulation of the X chromosome interlocking due to hScurfin sudden change performance and the Treg shortage, polyendocrinopathy, enteropathy, X-liked syndrome, IPEX), be also referred to as that the relevant autoimmunity of X--(X-linkedautoimmunity-allergic disregulation syndrome, XLAAD) situation is similar for allergy imbalance syndromes.

Previously for mScurfin studies show that mScurfin is high expression level in mouse Treg, and at CD4 ⁺CD25 ^-Express very lowly in the cell, cross the proteic mouse of expression mScurfin, then produce more Treg.A plurality of breadboard mScurfin that studies confirm that participate in CD4 ⁺CD25 ^HiCell development, Sakaguchi etc. can make the T cells of spleen change CD4 into by the mFoxp3 gene is passed through the retrovirus transfection ⁺CD25 ^HiThe T cell illustrates that mScurfin albumen can directly or indirectly induce the associated molecule of Treg to express.Confirmer CD4 ⁺CD25 ^HiThe T cell is partly expressed hScurfin, and hScurfin ⁺This group cell have immune suppression function.But different with mouse is to unite under the anti-CD28 monoclonal antibody stimulation CD4 at TCR ⁺CD25 ^-The T cell can transfer CD4 to ⁺CD25 ^HiThe T cell is expressed hScurfin simultaneously, and has the inhibition function of Treg sample.Calendar year 2001, the mFoxp3 gene that Wildin and Bennett propose hFOXP3 gene and sf mutant mice simultaneously has homology.In research, it is found that the Scurfin gene may be to participate in an autoimmune crucial instrumentality to autoimmune disease.With Scurfin gene Foxp3 transduction Scurfin ^-Non-modulability CD4 ⁺The T cell, can make the latter obtain the phenotype of Treg, and has anergy (promptly incompetent), active similar to natural Treg with phenotype, in vivo, all can mediate immunosuppressive action outward, the expression that Scurfin is described indicates that immunomodulatory suppresses active, and it has the growth of regulation and control Treg and the effect of function.Confirmed at present that the mRNA of Foxp3 genetic transcription and proteins encoded Scurfin are specific expressed in Treg (finding that although have to be reported on a small quantity in other cells great majority are non-lasting expression), and other CD4 ⁺The T cell subsets comprises tranquillization CD4 ⁺T cell, activated T h1/Th2 even NK T cell are all seldom expressed Scurfin, so think the specific molecular marker thing of Treg.

4.hScurfin biological action

2003, discoveries such as Hori S, mScurfin is expressed in Treg specifically, and closely related with generation, growth and the function of Treg, and this studies this special cells group in vivo, outward for molecular level foundation and specific marker is provided.There are bibliographical information hScurfin and a series of membrane molecules of mediation Treg function and the expression of cytokine (as CTLA-4, GITR, IL-10 and TGF-β) and the performance of function thereof that very close contact is arranged in addition.Experiment in vitro shows, the acceptor that hScurfin relies on IL-2 is as transcription repressor, but definite target spot is still not fully aware of in the body.

Though hScurfin is still waiting further research in aspect roles such as Treg cells whose development and function performances, but it is that control Treg grows and the key gene of functional effect that research up to now can show hScurfin, also disclose simultaneously peripheral t reg source have two kinds may: 1. periphery originally the T cell might be divided into Treg; 2. may produce CD25 in the thymus gland with similar Treg function ^-Cell, might be converted into CD25 after this cell activation ⁺Treg.

Discoveries such as Roli K, the mouse immune system of mscurfin high expression level is in holddown.Find that the T cell quantity obviously reduces, and is lower than normal level after will the mFoxp3 gene importing mouse, and the multiplication capacity of these T cells and molten cell ability are all lower, excretory IL-2 level reduces under the stimulation of TCR.Though the thymus development of this class mouse is normal, its peripheral immune organ, especially lymphoglandula show acellular state.This explanation mScurfin albumen has influenced mouse periphery immune development and functional status.

(1) keeps immunological homeostasis

Immunological homeostasis is one of basic function of body immune system, and immunity system reaches the homeostasis of environment in the immunity system by immunological tolerance and immunomodulatory.Autoimmune disorder be that some autoreactive T cells are escaped clonal deletion or immunosuppression because own or non-oneself regulatory mechanism of identification is got muddled, identification peripheral tissues antigen.Under normal circumstances, all there is autoreactive T cell in all individualities, but autoimmune disease occurs over just on 5% the person, and this illustrates that certain existence can regulate and control the cell or the factor of those potential autoreactive T cells.To remove CD25 ⁺The T Cell Deficient Mice Infected is given in the single positive T cell adoptive transfer of the CD4 of cell, can cause host's various organ specificity autoimmune diseases; And, then can prevent the generation of autoimmune disease with Treg and the common adoptive transfer of the single positive T cell of CD4.This phenomenon prompting Treg is a principal element of keeping the autoimmunization tolerance.The organ specificity autoimmune disease can take place because of the obvious minimizing of peripheral t reg in the 3 days thymectomized mouse that are born.If can utilize the powerful immunoregulation effect of Treg, go the activation of specificity inhibition autoreactive T cell, just might find the new methods of treatment of autoimmune disease.

Calendar year 2001 Brunkow etc. find the mFoxp3 gene first when the mutant mice (Sf mouse) of autoimmunity syndromes scurfy defective is studied, and the mutant gene locus that confirms the Sf mouse is to have inserted two adenylic acid (AMP) residues in mFoxp3 gene the 8th intron, cause phase shift mutation, form the albumen of FKH disappearance.Studies have shown that the Forkhead structural domain is appraised and decided the position to it and played a crucial role with combining of DNA etc.The mScurfin albumen that lacks the Forkhead structural domain can not be brought into play the function of transcription factor, causes CD4 ⁺T lymphocyte hyper-proliferative, infiltration.The hFOXP3 transgenation can cause IPEX, and it shows as comprehensive immune disorder, and with the type i diabetes and the thyroiditis of autoimmune endocrine, early onset thereof; In a part of case, can comprise eczema, food allergy and acidophilia inflammation etc. with serious dystopy specific reaction.IPEX patient's hFOXP3 gene has 13 independently catastrophe points, wherein has 6 sudden changes can cause the amino acid replacement.By the analysis discovery to a large amount of IPEX patient hFOXP3 genes, the mankind's IPEX 60% is because the sudden change of hFOXP3 genes encoding zone causes, and other 10% is that mRNA expression decline causes.

Sakaguchi etc. studies show that, when human and mouse trouble autoimmune disorder, and CD4 ⁺The expression deletion that occurs Scurfin on the T cell.Inject in the mFoxp3 genetic flaw mouse body if normal mouse is separated the Treg that obtains, can stop the generation of this mouse autoimmune disease.In addition, use retrovirus to transform the mFoxp3 gene and enter un-activation CD4 ⁺CD25 ^-The T cell makes it to become and spontaneous CD4 ⁺Treg has the cell of similar function.

Rundensky studies show that the mScurfin transcription factor can be by CD4 ⁺CD25 ^HiTreg expresses specifically, and becomes the necessary factor when T plays a role.Find that in fatal autoimmunity syndromes sf mice study mFoxp3 transgenation and mFoxp3 gene null mutation cause CD4 ⁺CD25 ^HiTreg lacks, and this shortage is different from CD4 ⁺CD25 ^-The self-defect of T cell.CD4 ⁺CD25 ^HiTreg transfers to the effect that can preferentially enlarge and play the treatment disease in the mouse of newborn mFoxp3 gene deletion.

(2) suppress tumour

Immune escape of tumor mechanism mainly comprises the weak and antigenic modulation of the antigenicity of tumour, MHC-I expresses low, secretory immune supressor such as TGF β, IL-10 and the collaborative stimulation molecule of shortage etc. the research of immunotherapy of tumors at present mainly concentrates on breaks immunological tolerance and improves host's t cell responses. and many tumour antigens are autoantigen, because Treg helps the autoantigen tolerance, by removing tissue specificity Treg antitumor action is enhanced, has also removed activatory CD8 when the CD25 monoclonal antibody is removed Treg but use in the body ⁺CD25 ⁺The T cell, thus anti-tumour effect weakened.Also find simultaneously to reject in the body CD25 ⁺Cell is not enough to treat the tumour that has taken place, and therefore must combine with other anti-tumor methods.HScurfin albumen for hFOXP3 expresses also exists the effect of other one side.Yang Liu and Pan Zheng report hScurfin can be expressed in the mammary epithelial cell recently, and down-regulated expression in breast cancer cell is crossed expression hScurfin and suppressed oncogene ErbB2; Undergo mutation or RNAi oncogene SKP2 up-regulated when disturbing hFOXP3 genetic expression when the hFOXP3 gene in addition, and find to suppress SKP2 the protein mediated tumor growth of hScurfin is suppressed very crucial, these evidences have proved that all Scurfin albumen is tumor suppressor gene.Numerous in addition bibliographical information Foxp3 expression of gene product S curfin can suppress NF-AT and NF-κ B, and the existence of NF-AT and NF-κ B and tumour and propagation have important effect.Our experiment shows that also hScurfin can suppress the propagation of T lymphoma cell line Jurkat.In sum, thus hScurfin may be able to become the important means of oncotherapy.

(3) graft-rejection is influenced

CD4 ⁺CD25 ^HiHScurfin ⁺Treg plays an important role in transplantation tolerance, is the main cell that brings out the body transplantation tolerance.Treg has the effect that suppresses transplant rejection, and along with allosome effector T cell quantity increases, its protective effect weakens.Remove Treg in the normal mouse body and cause rejection to strengthen, and reduce the survival of graft; On the contrary, if lack the post-transplantation mouse inoculation homology Treg of T cell and sensitized T cell not, graft survival time can obviously prolong.In MLR in vitro, under the recessive allele LS, Treg can suppress CD4 effectively ⁺T cell and CD8 ⁺The proliferative response of T cell.In addition, utilization recessive allele irritation cell and high dosage IL-2 can induce and expansion of antigen specific T reg external.Existing experiment in vitro clones at the antigenic CD4 of human graft ⁺The T cell subsets, the result shows, causes that with Th1 or Th2 clone tolerance is opposite, Treg induces tolerance by the transfer of adopting property.The ability that in not being subjected to the Naive mouse body of exotic antigen sensitization, has natural inhibition transplant rejection, just the ability at its resisting transplant rejection of transplantation tolerance mouse strengthens to some extent, therefore Treg can regulate and control heterogenic effector T cell, thereby helps setting up the allogeneic tolerance.When transplanting, the activation of Treg has certain restraining effect to graft-rejection.If before transplanting or at the transplanting commitment, can produce enough Treg, can reduce non-specific immunity and suppress amount of drug, even can replace the inhibitive ability of immunity medicine fully.

Graft versus host disease (GVH disease) (GVHD) is that the donor lymphocyte of living in the recipient's body interplantation after the recessive allele hematopoietic stem cell transplantation is the graft-rejection of principal character to the organogenetic immunity damage of host, is recessive allele hematopoietic stem cell transplantation biggest obstacle.Along with the understanding to Treg, people begin to Treg the effect in GVHD and also study.Discover the Treg that removes in the donor lymphocyte or before transplanting, the host mouse is removed CD25 ⁺Cell all can increase the GVHD morbidity; Can obviously alleviate GVHD and use transferring for mouse Treg of fresh separated purifying, for clinical prevention GVHD provides new method, but Treg accounts for CD4 ⁺The ratio of T cell is lower, and clinical application Treg is restricted.Can obtain a fairly large number of Treg by cultivating in recent years, adopt the Treg of amplification to transfer the generation that also can obviously reduce lethality GVHD, so this method get a good chance of being applied to clinical in future at amplification in vitro.The hyaluronic acid specific transgenic CD4 of transduction hFOXP3 gene ⁺CD25 ^-The T cell can make male sex's skin graft avoid syngeneic women repulsion.

5.HIV TAT-PTD effect characteristics and mechanism

Carrying out protein transduction by cell-penetrating peptides is a kind of effective ways that present external albumen transmits.The most frequently used protein transduction peptide is exactly the 48-56 amino acids residue of first exons coding of HIV-TAT gene, its have nexin transduction domain (protein transduction domain, PTD), its effect characteristics are as follows:

1. speed is fast---several minutes, onset in several hours.

2. thermal adaptability is wide.Can carry out at 4 ℃ and 37 ℃; It goes into the kinetic mechanism that born of the same parents' process is followed a non-acceptor, non-energy and non-endocytosis.

3. the quantity that enters of protein depends on the concentration of fusion rotein in the nutrient solution

4. lead fusion rotein to penetrate culturing cell efficient height.

5. as if the abdominal cavity that fusion rotein is injected into animal, in very short time, can in nearly all tissue and organ, see fusion rotein.

Brought into the destiny of the molecule of cell by TAT-PTD: the molecule of being brought into cell by TAT-PTD has kept their original separately chemical property basically, and has their original activity, so they can each one does his duty in cell, shows what one's be.

It is relevant with the existence of protein transduction domain neutral and alkali amino acid (arginine and Methionin) that membrane process is worn in supposition at present, and these amino acid have strong positive charge, may be to interact to mediate by direct and positively charged cytolemma lipid to wear membrane process.PTD can import nearly all cell with the fusion rotein that other albumen form, and comprises that ordinary methods such as osteoclast, peripheral blood lymphocytes and primary cell are difficult to cells transfected, even can also pass hemato encephalic barrier.The principal feature that PTD pilot protein and polypeptide are gone into born of the same parents is that transduction speed is fast, efficient height, not damaging cells.Recently using this transduction technology can enter the liposome of medicine, compound, oligonucleotide, peptide section, full-length proteins (as antibody, enzyme, isolating protein), metal ion even 200nm in the cell.Operate also very easyly, a molecule that only needs to merge directly joins in the tissue culturing medium, can reach peak concentration in the born of the same parents about 15～30 minutes, and each intracellular protein concentration is close to identical.Like this, both can control intracellular protein concentration, again can the control action kou time, general extracellular protein concentration can produce biological action at 20-1000nmol/L.

6.PTD-fusion rotein Subcellular Localization

TAT albumen really has transduction is that a polypeptide fragment that is rich in basic aminoacids, has a positive charge is relevant with spanning transduction membrane in its sequence, thereby is called as nexin transduction domain (PTD, amino acid 47-57).Its core sequence is a YGRKKRRQRRR iso-electric point 12.7, is alkalescence.Can be divided into two portions: GRKKR according to function and play the localized signal of pyrenoids; And the arginine in the sequence is very important to the proteic film transduction of wearing.PTD is fixed on the PTD-fusion rotein on the nuclear by nuclear localization signal (NLS), and this may also be the reason of its reduced immunogenicity.Generally speaking, the target protein after merging with PTD can be located in by the nuclear localization signal of PTD in the target cell nuclear, so the PTD fusion rotein can distribute in the endochylema of cell and karyon simultaneously.

Other

The TAT-HOXB4 albumen that employings such as Krosl the are expressed hemopoietic stem cell that successfully increased, the Lck-HIV-Tat of employing enzyme defects such as Veldhoen are the Lck-PTD of the enzyme defect initial CD4 that transduces ⁺The T cell, it has successfully stoped CD4 as a result ⁺The interior biography of T cell activation signal has suppressed the T cell activation, causes the incapability of T cell.

Discovery such as American scholar Joon-Young Kim recently in addition: by retrovirus with ectogenic people hScurfin transfection to the Jurkat-T cell, can make its cell that changes the Treg sample into, can suppress the CD4 that educates altogether with it ⁺CD25 ^-T cell proliferation, and can regulate the Treg Expression of Related Genes.

8.PTD wear the film sequence in hematopoiesis and lymphoid application precedent

Treg is applied to inducing at home and abroad of transplantation immunity tolerance to begin, but natural Treg quantity seldom, the amplification difficulty, therefore many in recent years laboratories begin to adopt the induced t cell transplantation immunity tolerance that has adjusting sexual function after the Foxp3 gene transfection, and obtained zooperal success, but the shortcoming of transgenosis maximum is a transfection efficiency low (except that retroviral vector), expression regulation is difficulty relatively, potential danger is arranged, and especially retroviral vector has the Lck-PTD of the employing enzyme defects such as possible .2005 Veldhoen of the modificator gene sudden change initial CD4 that transduces ⁺The T cell, successful inducing T cell obtains regulatory function, does not see the report that adopts PTD and Foxp3 gene constructed expression fusion rotein to be applied to basis and clinical study at present both at home and abroad as yet.

Summary of the invention

The object of the invention be to provide people Scurfin albumen total length that band wears the film sequence and fragment, and preparation method thereof.The present invention is by making up the hScurfin total length and segmentally wear the synexin prokaryotic expression carrier, and expression plasmid is reconstructed, and obtains functional fusion rotein.

The invention provides following fusion rotein:

The PTD-hScurfin fusion rotein has the aminoacid sequence shown in the SEQ ID NO.1

PTD-Δ h FKH fusion rotein has the aminoacid sequence shown in the SEQ ID NO.2.

PTD-Δ hPRD fusion rotein has the aminoacid sequence shown in the SEQ IDNO.3.

PTD-hScurfin described herein, PTD-Δ hFKH and PTD-Δ hPRD fusion rotein are respectively the fusion roteins that the PTD peptide is formed in people Scurfin albumen total length, disappearance C end FKH and disappearance N end PRD fragment and the HIV-TAT albumen.

PTD-eGFP-hScurfin described herein, PTD-Δ hFKH-eGFP and PTD-Δ hPRD-eGFP fusion rotein are respectively that band is worn the people Scurfin total length of film sequence and eGFP fusion rotein, removed jaw architecture territory (Δ Forkhead domain, the people Scurfin fragment of people Scurfin fragment Δ FKH) and the fusion rotein of eGFP and removal proline rich structural domain (Δ Proline-rich domain, Δ PRD) and the fusion rotein of eGFP.

The present invention also provides the nucleic acid construct of the above-mentioned 6 kinds of fusion roteins of encoding respectively, said nucleic acid construct, it contains the aminoacid sequence of coding PTD-hScurfin, PTD-Δ hFKH, PTD-Δ hPRD, PTD-eGFP-hScurfin, PTD-Δ hFKH-eGFP or PTD-Δ hPRD-eGFP fusion rotein.

The invention provides the method for utilizing gene engineering method to prepare PTD-hScurfin, PTD-Δ hFKH and PTD-Δ hPRD fusion rotein, may further comprise the steps: the gene order that 1. obtains encoding fusion protein; 2. the sequence that 1. step is obtained is inserted in the suitable carriers, obtains the corresponding nucleic acids construct; 3. the nucleic acid construct that 2. step is obtained transforms suitable expression bacterium; 4. under the suitable culture condition, culturing step expression bacterium 3., and add an amount of isopropyl-(Isopropyl-β-D-thiogalactoside, IPTG), 0.2mM-1mM the final concentration abduction delivering is therefrom isolated the fusion rotein of expression.5. if step fusion rotein 4. is that the inclusion body formal representation is then by nickel post affinity purification under the denaturing agent existence condition.6. purified product is frozen standby after by the post that desalts.

Our experiments show that, after fusion rotein disclosed in this invention penetrates Jurkat T cell and periphery T cell, inhibition Jurkat T cell and periphery T cell activation and proliferation and regulation and control periphery T cell secretion Treg function relevant cell factor.Provide new method for preparing the Treg like cell or directly suppress the T lymphopoiesis in a large number, for inducing of immunological tolerance in the body laid a good foundation.

Above-mentioned fusion rotein can be made pharmaceutical composition with other medicinal thinners, carrier or vehicle etc. as activeconstituents.

Above-mentioned fusion rotein is in preparation Treg like cell, application in the medicine of preparation prevention and treatment treatment inflammatory immune related diseases and tolerance induction or tumour.

The present invention adopts first and makes up the PTD-hScurfin mode and obtain fusion rotein, directly merges target protein and transduces into Jurkat T and periphery CD4 by wearing the membrane structure territory ⁺CD25 ^-The T cell to obtain Treg in a large number, is broken through the bottleneck that restriction Treg uses, in the hope of finally being applied to clinical organ transplantation antirejection therapy.

Description of drawings

Fig. 1. be that band is worn the carrier pET28a-PTD plasmid structural representation of film sequence.

Fig. 2. for fusion rotein makes up mode chart.

Fig. 3. be hScurfin, Δ hFKH and Δ hPRD PCR product electrophoresis evaluation figure.

M:DL2000Marker; 1 swimming lane: hScurfin PCR product (1313bp); 2 swimming lanes: Δ hFKH PCR product (1008bp); 3 swimming lanes: Δ hPRD PCR product (705bp).

Fig. 4. shown the SDS-PAGE electrophorogram result of PTD-hScurfin (A, B and C), PTD-Δ hFKH, PTD-Δ hPRD, PTD-eGFP-hScurfin, PTD-Δ hFKH-eGFP, PTD-Δ hPRD-eGFP fusion rotein abduction delivering and purifying.

A:PTD-hScurfin is abduction delivering in BL21 and Rosetta (DE3), and two kinds of engineering bacterias are induced 2h～8h:M respectively at the IPTG of 0.2mM: albumen lower molecular weight standard Fermentas SM0441.

B:PTD-hScurfin is abduction delivering M in different strain: albumen lower molecular weight standard Fermentas SM0441; 1,2 swimming lanes: ultrasonic degradation product precipitation and supernatant.

C:1 swimming lane: total tropina; 2-7 swimming lane: PTD-hScurfin ni-sepharose purification eluted product (51kDa); M: albumen lower molecular weight standard Fermentas SM0441.

D:1-3 swimming lane: PTD-Δ hFKH ni-sepharose purification eluted product (43kDa); M: albumen lower molecular weight standard FermentasSM0441.

E:1-5 swimming lane: PTD-Δ hPRD ni-sepharose purification eluted product (33kDa); M: albumen lower molecular weight standard FermentasSM0441.

F:1 swimming lane: PTD-hScurfin-eGFP ni-sepharose purification eluted product (80kDa); M: albumen lower molecular weight standard Fermentas SM0441.

G:1 swimming lane: PTD-eGFP-Δ hPRD ni-sepharose purification eluted product (70kDa); M: albumen lower molecular weight standard Fermentas SM0441.

H:1 swimming lane: PTD-eGFP-Δ hFKH ni-sepharose purification eluted product (61kDa); M: albumen lower molecular weight standard Fermentas SM0441.

Fig. 5. the Western blots figure that has shown PTD-hScurfin, PTD-Δ hFKH and PTD-Δ hPRD expressing fusion protein identifies the result.

Wherein, 1 swimming lane: PTD-hScurfin (51kDa); 2 and 4 swimming lane: Fermentas dye Marker in advance; 3 swimming lanes: PTD-eGFP-hScurfin (80kDa); 5 swimming lanes: PTD-Δ hPRD (33kDa); 6 swimming lanes: PTD-Δ hFKH (43kDa); 7 swimming lanes: PTD-Δ hPRD-eGFP (60kDa); 8 swimming lanes: PTD-Δ hFKH-eGFP (70kDa).In this experiment: one of use anti-is: 1: 1000 anti-6 * His-Tag; Two is anti-: 1: the 5000HRP-goat anti-mouse igg.

Fig. 6. shown laser co-focusing photo (Hoechst33342 and the PI difference transfect cell nuclear of the Subcellular Localization analysis of fusion rotein, green fluorescence is the fluorescence for eGFP among fusion rotein PTD-hScurfin-eGFP, PTD-Δ hFKH-eGFP that observes the Subcellular Localization construction expression and the PTD-Δ hPRD-eGFP) and Western-blot figure result (target protein identify one anti-ly be: 1: 1000 anti-6 * His-Tag, two is anti-: 1: it is anti-GAPDH antibody that 5000HRP-goat anti-mouse igg, confidential reference items proteic resist).

(uv excitation light detects the nucleus that is blue-fluorescence for A:PTD-eGFP-hScurfin, PTD-eGFP-Δ hPRD and PTD-eGFP-Δ hFKH laser confocal microscope analysis chart, green color filter detects the nucleus of the fluorescence that takes on a red color, and blue color filter detects eGFP green fluorescence in the fusion rotein).

B: left figure: PTD-hScurfin wears film 2h and 24h immunoblotting assay; Right figure: the Western-blot of PTD-hScurfin, PTD-Δ hFKH and PTD-Δ hPRD figure in endochylema and the karyon protein extract.

Fig. 7. shown and used PHA 0.5ug/mL, 1ug/mL, 5ug/mL respectively, PHA 1ug/mL+PMA 25ng/mL, PHA 1ug/mL+PMA 50ng/mL, PHA 1ug/mL+PMA 100ng/mL stimulate Jurkat T cell 24h, the experimental result that incentive condition is groped.

A:PHA 1ug/mL+PMA 50ng/mL stimulate Jurkat T cell 12h microscope figure below (10 *, 20 *, 40 *) projection appears in cell surface, disperseed than being easier to.

B: do not stimulate Jurkat T groups of cells, 12h microscope figure below (10 *, 20 *, 40 *) the original poly-easily group of a cell maintenance growth characteristic, projection is not seen on the surface.

C:PHA 1ug/mL+PMA 50ng/mL stimulates Jurkat T cell 24h, and the cck-8 method is measured 490nm absorbancy analysis of cells propagation.

Fig. 8. shown PTD-hScurfin, PTD-Δ hFKH and the PTD-Δ hPRD fusion rotein 640nM experimental result to measuring respectively through the CCK-8 method of the Jurkat of PHA+PMA combined stimulation 24h and 48h T cell proliferation level affects.

A:PHA 1ug/mL+PMA 50ng/mL stimulates each fusion rotein treatment group of Jurkat T cell 24h (640nM, 1h adds before stimulating) and each control group cck-8 method to measure the relatively cell proliferation of 490nm absorbancy.

B:PHA 1ug/mL+PMA 50ng/mL stimulate each fusion rotein treatment group of Jurkat T cell 48h (640nM, 1h adds before stimulating) and each control group cck-8 method measure the relatively cell proliferation of 490nm absorbancy ( ^*P＜0.05).

Fig. 9. shown that PTD-hScurfin, PTD-Δ hFKH and PTD-Δ hPRD fusion rotein 640nM are respectively to the influence through PHA+PMA combined stimulation activatory periphery T cell propagation.

A: different concns PHA+PMA or PMA+Ionomycin stimulate back periphery blood T cell propagation situation 1:Cells Control; 2:PHA 0.5 μ g/mL; 3:PHA 1 μ g/mL; 4:PHA 2 μ g/mL; 5:PHA 1 μ g/mL+PMA 50ng/mL; 6:PHA 2 μ g/mL+PMA 30ng/mL; 7:PHA0.5 μ g/mL+PMA 100ng/mL; 8:PMA30ng/mL+Ionomycin1.2 μ g/ml; 9:PMA50ng/mL+Ionomycin 0.8 μ g/mL; 10:PMA70ng/mL+Ionomycin 0.4 μ g/mL.

B: different fusion roteins (640nm/L) and CsA (3 μ M) are to the influence of the propagation of peripheral blood lymphocyte behind PHA 1 μ g/mL+PMA 50ng/mL.[96 orifice plates every hole inoculation 1 * 105, CsA, PTD-eGFP, PTD-hScurfin, PTD-Δ hPRD and PTD-Δ hFKH fusion rotein are educated 1h altogether with cell respectively, do 3 multiple holes simultaneously for every group.The PHA/PMA (1 μ g/mL/50ng/mL) that then adds the dose,optimum that above-mentioned experiment draws stimulates] ( ^*P＜0.05).

Figure 10. shown Realtime pcr analysis fusion rotein produces IL-2 after PHA+PMA stimulates to periphery T cell influence.

After A:PHA 1 μ g/mL+PMA 50ng/mL stimulates periphery blood T cell, the transcriptional level of different time points IL-2.( ^*p＜0.05)

B: different fusion roteins (640nm/L) and CsA (3 μ M) are to the influence of 6 hours periphery blood T lymphocyte generation IL-2 behind PHA 1 μ g/mL+PMA 50ng/mL.[the every hole inoculation of 6 orifice plates 1 * 106/ml, CsA, PTD-eGFP, PTD-hScurfin, PTD-Δ hPRD and PTD-Δ hFKH fusion rotein are educated 1h altogether with cell respectively.The PHA/PMA (1 μ g/mL/50ng/mL) that then adds the dose,optimum that above-mentioned experiment draws stimulates, the experiment triplicate] ( ^*P＜0.05)

Figure 11. shown Realtime pcr analysis fusion rotein produces IL-10 after PHA+PMA stimulates to periphery T cell influence.

After A:PHA 1 μ g/mL+PMA 50ng/mL stimulates periphery blood T cell, the transcriptional level of different time points IL-10.( ^*p＜0.05)

B: different fusion roteins (640nm/L) and CsA (3 μ M) are to the influence of 2 hours periphery blood T lymphocyte generation IL-10 behind PHA 1 μ g/mL+PMA 50ng/mL.[the every hole inoculation of 6 orifice plates 1 * 106/ml, CsA, PTD-eGFP, PTD-hScurfin, PTD-Δ hPRD and PTD-Δ hFKH fusion rotein are educated 1h altogether with cell respectively.The PHA/PMA (1 μ g/mL/50ng/mL) that then adds the dose,optimum that above-mentioned experiment draws stimulates, the experiment triplicate] ( ^*P＜0.05)

Embodiment

Has the aminoacid sequence shown in the SEQID NO.1-3 at the PTD-hScurfin described in the embodiment, PTD-Δ hFKH and PTD-Δ hPRD fusion rotein.

The invention provides the method for producing PTD-hScurfin, PTD-Δ hFKH and PTD-Δ hPRD fusion rotein, this method comprises the following steps:

1. obtain the gene order of coding PTD-hScurfin, PTD-Δ hFKH and PTD-Δ hPRD fusion rotein;

2. the sequence that step 1 is obtained is inserted in the suitable carriers, obtains the corresponding nucleic acids construct;

3. the nucleic acid construct that obtains with step 2 transforms suitable engineering and expresses bacterium;

4. under the suitable culture condition, induce the engineering after the conversion of step 3 to express bacterium, and therefrom be purified into PTD-hScurfin, PTD-Δ hFKH and PTD-Δ hPRD fusion rotein.

5.PTD-hScurfin, PTD-Δ hFKH and PTD-Δ hPRD fusion rotein or carry eGFP observes fusion rotein as the laser co-focusing of the fusion rotein of spike Subcellular Localization experiment.

6.PTD-hScurfin, PTD-Δ hFKH and PTD-Δ hPRD fusion rotein Western-blot identify and the Subcellular Localization experiment.

7.PTD-hScurfin, PTD-Δ hFKH and PTD-Δ hPRD fusion rotein or carry eGFP and detect as the flow cytometer of the fusion rotein of spike and wear the membrane efficiency experiment.

8.Jurkat T cell and periphery CD4 ⁺CD25 ^-The PHA+PMA activation and proliferation condition of T cell is groped experiment

9.PTD-hScurfin, PTD-Δ hFKH and PTD-Δ hPRD fusion rotein be to the proliferation inhibition test of Jurkat T cell.

10.PTD-hScurfin, PTD-Δ hFKH and PTD-Δ hPRD fusion rotein be to the proliferation inhibition test of periphery T cell.

11.PTD-hScurfin, PTD-Δ hFKH and PTD-Δ hPRD fusion rotein suppress the experiment of periphery T cell secretion IL-2.

12.PTD-hScurfin, PTD-Δ hFKH and PTD-Δ hPRD fusion rotein promote the experiment of periphery T cell secretion IL-10.

More than test related material source:

PGEM T-Easy/hFOXP3 plasmid (Amp resistance) is so kind as to give for professor S.Sakaguchi of Kyoto Univ Japan.PET28a (having His-Tag) plasmid is available from Novagen company product, and the peGFP-N1 plasmid is available from invitrogen company product; Coli strain DH5 α Japan TAKALA company product; It is oldly to console the peak academician and give by Department Of Medicine, Peking University's immunology that BL-21 and Rosetta (DE3) are Novagen company product.PET28a-PTD plasmid (having His-tag), this laboratory of pET28a-PTD-eGFP (having His-tag) carrier makes up voluntarily.Ex TaqDNA polysaccharase, T4DNA ligase enzyme, nucleic acid electrophoresis molecular weight standard DL-2000 (TaKaRa company); Plasmid is extraction agent box (Axygen company) in a small amount; Plasmid purification test kit (Shanghai JaRa company); Restriction enzyme (Sac I, Xho I, HindIII) and pre-staining low molecular weight protein (LMWP) (Fermentas company); Bradford protein quantification test kit (Beijing Puli's Lay company); Cell proliferation reagent box CCK8 test kit (colleague of Amada Co., Ltd. chemistry institute).Purifying resin Profinity I MAC nickel metal-chelating filler (U.S. Bio-Rad company product), mouse anti-6 * His-Tag monoclonal antibody (Cell Signaling company), mouse anti human FOXP3 antibody (clone number: 236A/E7, eBioscience company), HRP mark goat anti-mouse IgG two anti-(company of China fir Golden Bridge in Beijing), ECL-plus colouring reagents box, the PD-10 prepacked column (U.S. GE company) that desalts, E.coli Rosetta (DE3), pET28a (+) plasmid and protein renaturation test kit (Novagen company product), molecular weight of albumen standard (Fermentas company product), reduced glutathion (GSH), Sleep-promoting factor B (GSSG), isopropyl-(IPTG Dioxane Free) (German Merck company product), PHA-P, NP-40, PMA, Hoechst 33342, penicillin, Streptomycin sulphate, kantlex, propidium iodide (PI) and PMSF are available from U.S. Sigma company, dialysis tubing (molecular weight cut-off 10,000, U.S. Pierce company), calf serum (Hangzhou folium ilicis chinensis company), Trizol, PMRI-1640, DMEM, calf serum and foetal calf serum are available from American I nvitrogen company, 2 * Brilliant

Green QPCR Master Mix is available from U.S. Stratagene company, and ReverTraAce is available from Japanese TOYOBO company.Chemical reagent such as all the other various salt commonly used, acid, alkali are import or homemade analytical pure.

Jurkat T cell strain is consoled the peak academician and is given by T cell research chamber, Department Of Medicine, Peking University is old.

Primer sees Table 1

Table 1. is used to make up the primer of various expression vectors

Above step specifically describes as follows:

1. prepare and evaluation purpose fusion rotein, comprise step (1)-(4):

(1) structure of pET28a-PTD-eGFP (N) plasmid (eGFP is at the N of fusion rotein end): with the peGFP-N1 plasmid is template, primer 1 (the forward direction primer has the PTD sequence) is obtained the sequence of the eGFP of band PTD with primer by pcr amplification, the PCR product inserts the structure of just finishing plasmid through the pET28a of BamH I and Sac I double digestion after using GenClean agarose dna gel recovery test kit (Shanghai JaRa company) to reclaim then.

(2) structure of pET28a-PTD plasmid: on the basis of pET28a-PTD-eGFP (N) plasmid construction, that promptly obtain with Sac I single endonuclease digestion excision intermediary eGFP is pET28a-PTD (Fig. 1)

(3) structure of pET28a-PTD-eGFP (C) plasmid (eGFP is at the C of fusion rotein end): with the peGFP-N1 plasmid is template, with the primer eGFP sequence that 2 amplifications obtain to primer, after the PCR product uses GenClean agarose dna gel to reclaim, insertion is promptly finished structure through the pET28a-PTD plasmid of Hind III and Xho I double digestion.

(3) structure of pET28a-PTD-hFOXP3 and pET28a-PTD-hFOXP3-eGFP expression plasmid: with the pGEMT-Easy/hFOXP3 plasmid is template, adopt primer primer 3 to be obtained the gene order of h FOXP3 by pcr amplification, after the PCR product uses GenClean agarose dna gel to reclaim, insert pET28a-PTD plasmid and pET28a-PTD-eGFP (N) plasmid respectively, promptly finish structure through Hind III and Xho I double digestion.

(4) structure of pET28a-PTD-Δ hFKH and pET28a-PTD-eGFP-Δ hFKH expression plasmid: with the pGEMT-Easy/hFOXP3 plasmid is template, adopt primer primer 4 to be obtained the sequence of Δ hFKH by pcr amplification, after the PCR product uses GenClean agarose dna gel to reclaim, insert pET28a-PTD plasmid and pET28a-PTD-eGFP (C) plasmid respectively, promptly finish structure through Sac I and Hind III double digestion.

(5) structure of pET28a-PTD-Δ hPRD and pET28a-PTD-eGFP-Δ hPRD expression plasmid: with the pGEMT-Easy/hFOXP3 plasmid is template, adopt primer to primer 5, obtain the sequence of Δ hPRD by pcr amplification, after the PCR product uses GenClean agarose dna gel to reclaim, insert pET28a-PTD plasmid and pET28a-PTD-eGFP (C) plasmid respectively, promptly finish structure through Sac I and Hind III double digestion.

(6) plasmid of structure difference transform bacteria extracts plasmid, adopts the T7 universal primer, and through the order-checking of Shanghai Ying Jun Bioisystech Co., Ltd, confirmation PCR product accurately is cloned in the expression vector.The result has successfully made up prokaryotic expression carrier pET28a-PTD-hScurfin, pET28a-PTD-Δ hFKH, pET28a-PTD-Δ hPRD, pET28a-PTD-eGFP-hScurfin, pET28a-PTD-Δ hFKH-eGFP, the pET28a-PTD-Δ hPRD-eGFP (accompanying drawing 2) of fusion rotein.To identify that the expression vector behind the correct clone is transformed in engineering expression bacterium Rosetta (DE3) escherichia coli by the thermal shock method, and by microbiotic Kana ⁺(final concentration is 50 μ g/ml) screens positive bacterium colony.With positive bacterium colony extracting plasmid, carry out PCR and enzyme and cut evaluation.(accompanying drawing 3)

Increase bacterium in the LB pipe (Kana concentration is 50 μ g/ml) of PH7.0 and cultivate identifying that correct positive bacteria drops on, when OD value=0.4～0.6, adding IPTG (Sigma company) induces.Through to inducing the experiment of time gradient in the process, thermograde, IPTG concentration gradient, finally determine the IPTG inductive condition, PTD-hScurfin and PTD-hScurfin-eGFP induce 8h to be the suitableeest inductive condition with 30 ℃, 0.2mmol/L; PTD-Δ hFKH, PTD-Δ hPRD, PTD-Δ hFKH-eGFP and PTD-Δ hPRD-eGFP induce 8h to be the suitableeest inductive condition with 30 ℃, 0.5mmol/L.Determine the expression amount maximum (accompanying drawing 4) of target protein by the SDS-PAGE electrophoretic analysis.

Collect inductive part thalline under these conditions, add an amount of PBS, ultrasonication in the ice bath, centrifugal 10 minutes of 12000rpm/min gets cleer and peaceful precipitation respectively through the SDS-PAGE electrophoretic analysis, confirms that 6 kinds of fusion roteins are mainly the inclusion body formal representation.Get supernatant then through Ni ⁺-IMAC post (Bio-Red company) carries out affinitive layer purification. go up the preceding Ni of sample ⁺-IMAC post is washed post through the sample-loading buffer of 10 times of volumes earlier.With ultrasonic degradation completely inclusion body with binding buffer liquid (50mMNaH2PO42H2O, 300mM NaCl, 5mM imidazoles, 8M urea, adjust pH to 8.0) the dissolving after-filtration, collect filtrate, according to Bio-Rad company process specifications in centrifuge tube with filtrate and Profinity IMAC Ni-Charged Resin combination, 4 ℃ of jog 1h adorn post then, and effluent liquid is washed centrifuge tube repeatedly, upper prop again is in order to avoid PD.Effluent liquid is crossed post 3-4 time repeatedly.Add lavation buffer solution (50mM NaH2PO42H2O, 300mM NaCl, 100mM imidazoles, 8M urea, adjust pH to 8.0), measure the A280 value of effluent liquid, treat that A280 leveled off at 0 o'clock, preparation adds elution buffer (50mMNaH2PO42H2O, 300mM NaCl, 300mM imidazoles, 8M urea are adjusted pH to 8.0).After waiting to wash the whole outflows of damping fluid, add elution buffer.The EP pipe is collected effluent liquid, and measures the A280 value.Below all carry out under the pH=8.0 environment, each component sample that gets collection is carried out the SDS-PAGE electrophoresis, proteic purification effect of analysis purposes and efficient (accompanying drawing 3).Ni ^{2 +}Albumen behind the-IMAC purifying is crossed the PD-10 of GE company prepacked column desalination and urea, then-20 ℃ be stored in 10% glycerine solution standby, time spent Bradford standard measure fusion rotein.By above step success abduction delivering and separation and purification fusion rotein PTD-hScurfin, PTD-Δ hFKH, PTD-Δ hPRD and PTD-eGFP-hScurfin, PTD-Δ hFKH-eGFP, PTD-Δ hPRD-eGFP (accompanying drawing 4).The albumen of purifying carries out Western-blot subsequently and identifies that (one is anti-: 1: 1000 anti-6 * His-Tag, two is anti-: 1: the target protein (accompanying drawing 5) of the 5000HRP-goat anti-mouse igg) confirming as our preparation.

2. fusion rotein carries out the following film functional experiment of wearing:

(1) cell cultures and the flow cytometer of wearing the film effect detect:

Jurkat T cell, routine are incubated at the RPMI-1640 nutrient solution that contains 10% calf serum, 100U/mL penicillin, 100g/L Streptomycin sulphate.With 2 * 10 ⁵/ porocyte concentration kind is in 24 orifice plates, with different concns (40nM, 160nM, 640nM, PTD-eGFP-target protein 1280nM) is educated 2h, 4h, 12h, 24h altogether.The centrifugal 10min of 1000 * g collects each porocyte respectively and washes 3 times with PBS, the FASCaliber flow cytometer is collected and the CellQuest software analysis shows: it is 640nM that fusion rotein the best is worn film concentration, the peak of wearing film is at 2h, wears membrane efficiency and descends gradually along with the prolongation of time subsequently.

(2) laser confocal microscope observation membrane-spanning protein is in intracellular location:

Collect respectively the cell of educating 2h altogether, the centrifugal 10min of 1000 * g with PTD-eGFP-hScurfin, PTD-Δ hFKH-eGFP and the PTD-Δ hPRD-eGFP fusion rotein of 640nM.Precooling PBS washes 2 times, and 1000 * g is centrifugal, 5min.Add 0.5mL 4% Paraformaldehyde 96 stationary liquid, slowly hanged cell, fix 30 minutes.The centrifugal stationary liquid that goes is washed 2 times with PBS, manually rocks during the washing.Add PI staining fluid (final concentration 50 μ g/mL) and slowly hanged cell, 4 ℃ of lucifuges dyeed 30 minutes, the PBS washing.Add 25 μ L PBS re-suspended cells and drop on the slide glass, cover clean cover glass, the nail varnish mounting.Laser Scanning Confocal Microscope uses green color filter to detect the nucleus of the fluorescence that takes on a red color down, uses blue color filter to detect eGFP green fluorescence in the fusion rotein.Add in addition Hoechst33342 (final concentration: 2ug/mL) to educate altogether in the viable cell system of 2h with 640nM albumen (in 24 orifice plates, 2 * 10 ⁵/ hole) 10min that dyes in 37 ℃ of incubators, Laser Scanning Confocal Microscope uses uv excitation light to detect the nucleus that is blue-fluorescence down, uses blue color filter to detect eGFP green fluorescence in the fusion rotein.The result from image no matter confirm viable cell still be after the cell fixation pair cell nuclear dye after the analysis of laser co-focusing tomography three-dimensional localization sees that all most of cell born of the same parents include the purpose fusion rotein that has the eGFP green fluorescence, and this fusion rotein can penetrate in the nucleus.(accompanying drawing 6A)

(3) endochylema and karyon protein extraction and immunoblotting detect:

The centrifugal 10min of 1000 * g collects respectively the Jurkat T cell of educating 2h and 24h altogether with PTD-hScurfin, PTD-Δ hFKH and the PTD-Δ hPRD fusion rotein of 640nM, PBS with 4 ℃ of precoolings washes 3 times, add the cold Buffer A of 400 μ l (10mMHEPEs, 10mM KCl, 0.1mM EDTA, 0.1mM EGTA, 0.5mM PMSF, pH7.9), ice bath 10min; Add 25 μ l 10%NP-40, Vortex 10s, ice bath 10min, the centrifugal 15s of 12000 * g collects supernatant, is plasmosin; (20mM HEPEs, 0.4M NaCl, 1mM EDTA, 1mM EGTA, 1mM PMSF, 1mMDTT pH7.9) to precipitation, put 30min on ice to add the cold Buffer B of 50 μ l; The centrifugal 10min of 12000 * g collects supernatant, is karyon albumen; Add 2 * SDS sample-loading buffer of equivalent, boil 5min ,-80 ℃ frozen standby.The endochylema karyon albumen that extracts separates with 12%SDS-PAGE glue; 350mA 2h is transferred to pvdf membrane with albumen; With 5% skimmed milk (being dissolved in TBS/T liquid) sealing 1h; Add anti-6 * His-Tag (1: 1000), 4 ℃ are spent the night, with TBS/T washing 3 times; Add HRP-goat anti-mouse igg (1: 5000), room temperature 1h, TBS/T washing 3 times; Add ECL-Plus chemoluminescence agent, effect 5min sweeps film, takes pictures; One anti-two anti-ly strips off liquid and washes film 5-10min, sealing 1h, 4 ℃ of anti-GAPDH antibody of night incubation (1: 1000), the TBS/T washing, HRP-goat anti-mouse igg (1: 5000) room temperature leaves standstill 1h, the ECL-Plus colour developing of TBS/T washing back, scanning detects GAPDH confidential reference items albumen, the result has further confirmed the result that laser co-focusing is analyzed from another point of view: the fusion rotein of preparation all can permeates cell membranes enter in the born of the same parents and be positioned in the nuclear in 2h-24h, and in wearing film 24h nucleus, still have the purpose fusion rotein, and can't with Western-blot detect target protein (accompanying drawing 6B) in the tenuigenin extract this moment.

3. cell inhibitory effect experiment:

(1) groping of Jurkat T cell and periphery blood T cell incentive condition:

Use PHA 0.5ug/mL, 1ug/mL, 5ug/mL respectively, PHA 1ug/mL+PMA 25ng/mL, PHA1ug/mL+PMA 50ng/mL, PHA 1ug/mL+PMA 100ng/mL stimulate Jurkat T cell observation 6h, 12h, the 24h effect of stimulation, through testing the optimal stimulation dosage of finally choosing be: PHA 1ug/mL+PMA 50ng/mL, stimulation time choose 24h (accompanying drawing 7).

(2) the CCK-8 method detects cell proliferation:

Jurkat T cell inhibitory effect test: cell is used an amount of concentration of RPMI-1640 adjustment that contains 10% calf serum.With Jurkat T cell respectively with 2 * 10 ⁴Every hole kind is in 96 orifice plates.With Ciclosporin A (CsA), PTD-eGFP fusion rotein and hScurfin albumen are done control group, and experimental group adds each fusion rotein and the cell of 640nM respectively and educates 1h altogether, do 3 multiple holes simultaneously for every group.PHA (1 μ the g/mL)+PMA (50ng/mL) that then adds dose,optimum stimulates, cell is placed 37 ℃, cultivate in the 5%CO2 incubator, stimulate 18h and 42h to add CCK-8 reagent 10 μ l/ holes (cumulative volume 100uL/ hole) respectively at PHA+PMA, continue to cultivate 6 hours, absorbancy when detecting 490nm with microplate reader, analytical data shows: PTD-hScurfin behind PHA+PMA combined stimulation 24h and the 48h, PTD-Δ hPRD (final concentration 640nM) has the obvious suppression effect with the propagation that control group compares the JurkatT cell, and PTD-Δ hFKH (final concentration 640nM) also presents obvious restraining effect after stimulating 48h, wherein the inhibition effect of PTD-hScurfin and CsA positive controls suitable (accompanying drawing 8).

Periphery T cell propagation inhibition test: periphery T cell is used an amount of concentration of RPMI-1640 adjustment that contains 10% calf serum.With periphery blood T cell respectively with 1 * 10 ⁵Every hole kind is in 96 orifice plates. with the positive contrast of CsA (final concentration is 3 μ M), with PTD-eGFP and hScurfin fusion rotein (640nM) as negative control group, experimental group PTD-hScurfin, PTD-Δ hPRD and PTD-Δ hFKH fusion rotein concentration are educated 1h with cell respectively altogether with 640nM respectively, doing 3 multiple holes simultaneously for every group. PHA (1 μ the g/mL)+PMA (50ng/mL) that then adds the dose,optimum that above-mentioned experiment draws stimulates, cell is placed 37 ℃, cultivate in the 5%CO2 incubator, stimulate 20h to add CCK-8 reagent 10 μ l/ holes (cumulative volume 100 μ L/ holes) in PHA+PMA, continue to cultivate 4h, absorbancy when detecting 490nm with microplate reader, analytical data. found that: the blank group PBMC that not adding albumen stimulates breeds obviously; , the propagation of PBMC there is not obvious influence with the hFOXP3 albumen of wearing the film sequence; And band is worn the propagation that the total length PTD-hScurfin of film sequence significantly suppresses PBMC; The segmental inhibition ability of PTD-Δ hPRD that band is worn the film sequence is higher than PTD-Δ hFKH fragment, but all be lower than the PTD-hScurfin of total length, illustrate that the FKH zone plays an important role in the inhibit feature of Foxp3, and PRD fragment (N-terminal proline rich zone) also has certain inhibit feature. test explanation, the biological function that our constructed PTD-hScurfin, PTD-Δ hPRD and PTD-Δ hFKH fusion rotein have the propagation that suppresses periphery T cell. ( ^*P＜0.05).(accompanying drawing 9).

3. the expression experiment of regulation and control Treg function relevant cell factor:

The RT-PCR of cytokine IL-2 and IL-10 detects: gather healthy people's anticoagulant heparin peripheral blood, separate acquisition periphery mononuclearcell through lymphocyte separation medium, through adherent removal scavenger cell and monocyte, adopt CD19 and CD8 monoclonal antibody and complement to kill and wound then and remove B cell and CD8 ⁺The T cell, after the washing with cell inoculation in 6 orifice plates, every hole 2 * 10 ⁶, educate 1h altogether with fusion roteins (640nM) such as CsA (3 μ M), PTD-eGFP, PTD-hScurfin, PTD-Δ hPRD and PTD-Δ hFKH respectively, then adding PHA (1 μ g/mL)+PMA (50ng/mL) stimulates active cells 0h, 2h, 4h, 6h, 8h, collecting cell.Collect each sample 2 * 10 ⁶Individual cell, the Trizol cracking process extracts total RNA routinely.

Carry out reverse transcription according to following embodiment:

Reagent volume

5×reaction?buffer 4.0μl

dNTP?mix(10mM?each) 1.0μl

Recombinant?RNase?inhibitor 0.5μl

M-MLV?reverse?transcriptase 1.0μl

Total 6.5μl

42 ℃ of reactions are 20 minutes behind the mixing, and 94 ℃ of heating deactivation in 5 minutes is synthetic to stop cDNA, 4 ℃ 5 minutes.Carry out the grads PCR amplification according to following system again: set up 10 μ lPCR reaction systems in 0.2ml PCR pipe, each composition of adding is respectively:

10×PCR?buffer 1μl

dNTPs(10mM?each) 1μl

Forward?primer 0.2μl

Reverse?primer 0.2μl

cDNA 0.2μl

Taq archaeal dna polymerase 0.06 μ l

Sterilized water 7.34 μ l

Cumulative volume 10 μ l

94 ℃ of pre-sex change are 2 minutes behind the mixing, carry out the grads PCR amplification.

Each gene amplification the primer and PCR react selected temperature:

1.β-actin?Forward?primer：CAC?GAA?ACT?ACC?TTC?AAC?TCC；

Reverse?primer：CAT?ACT?CCT?GCT?TGC?TGA?TC；

2.IL-2Forward?primer：CAA?AGA?AAA?CAC?AGC?TAC?AACT；

Reverse?primer：TCT?CAT?CAG?CAT?ATT?CAC?ACAT；

3.IL-10Forward?primer：AGG?GCA?CCC?AGT?CTG?AGAACA；

Reverse?primer：CGG?CCT?TGC?TCT?TGT?TTT?CAC

4. selected temperature is: 53.3 ℃, and 54.2 ℃, 55.2 ℃, 56.4 ℃, 57.5 ℃, 58.5 ℃, 59.4 ℃, 60.0 ℃, 60.4 ℃, grope suitable annealing temperature, determine to adopt 56 ℃ at last as annealing temperature.

Above-mentioned amplification PCR products is reclaimed, connect the T carrier, make up standard plasmid, and order-checking is identified.Adopt realtimeRT-PCR to measure the expression level of cytokine IL-2 and IL-10.With above-mentioned cDNA is template, and 2 * SYBRGreen QPCRMaster MIX is placing 0.2ml PCR pipe on ice to add following ingredients successively:

2×MIX 7.5μl

ROX(1∶500) 0.3μl

Forward?primer 0.2μl

Reverse?primer 0.2μl

cDNA 1.0μl

ddH ₂O 5.8μl

Cumulative volume 15 μ l

Mixing instantaneous centrifugal 5 seconds, makes each reacted constituent concentrate on the PCR pipe end.Reaction conditions is as follows: 95 ℃ of 10min, 95 ℃ of 30sec, 56 ℃ of 30sec, 72 ℃ of 1min (acquire fluorescence), 40 circulations.

Periphery blood T cell is after stimulation oversaturation PHA+PMA stimulates, and the IL-2 transcriptional level is the highest when 6～8h, and therefore adopting stimulates 6h to test as only time point.Found that: PTD-hScurfin, PTD-Δ hPRD and PTD-Δ hFKH can suppress periphery blood T cell secretion IL-2, wherein PTD-hScurfin, PTD-Δ hPRD have the significance meaning, and PTD-Δ hFKH does not see statistical significance (Figure 10).Periphery blood T cell stimulates the secretion level of back IL-10 progressively to rise, and level is lower when 2h, therefore relatively is fit to observe fusion rotein and improves the effect that it produces IL-10, therefore selects 2h to stimulate as the experimental period point.Found that: PTD-hScurfin, PTD-Δ hPRD and PTD-Δ hFKH can promote periphery blood T cell secretion IL-10, wherein PTD-hScurfin, PTD-Δ hPRD have the significance meaning, and PTD-Δ hFKH does not see statistical significance (Figure 11).

SEQUENCE?LISTING

＜110〉Jiangsu University

＜120〉band is worn people Scurfin albumen total length and the fragment and the preparation method of film sequence

<130>

<160>3

<170>PatentIn?version?3.3

<210>1

<211>482

<212>PRT

＜213〉people

<400>1

Met?Gly?Ser?Ser?His?His?His?His?His?His?Ser?Ser?Gly?Leu?Val?Pro

1 5 10 15

Arg?Gly?Ser?His?Met?Ala?Ser?Met?Thr?Gly?Gly?Gln?Gln?Met?Gly?Arg

20 25 30

Gly?Ser?Tyr?Gly?Arg?Lys?Lys?Arg?Arg?Gln?Arg?Arg?Arg?Glu?Leu?Arg

35 40 45

Arg?Gln?Ala?Leu?Pro?Asn?Pro?Arg?Pro?Gly?Lys?Pro?Ser?Ala?Pro?Ser

50 55 60

Leu?Ala?Leu?Gly?Pro?Ser?Pro?Gly?Ala?Ser?Pro?Ser?Trp?Arg?Ala?Ala

65 70 75 80

Pro?Lys?Ala?Ser?Asp?Leu?Leu?Gly?Ala?Arg?Gly?Pro?Gly?Gly?Thr?Phe

85 90 95

Gln?Gly?Arg?Asp?Leu?Arg?Gly?Gly?Ala?His?Ala?Ser?Ser?Ser?Ser?Leu

100 105 110

Asn?Pro?Met?Pro?Pro?Ser?Gln?Leu?Gln?Leu?Pro?Thr?Leu?Pro?Leu?Val

115 120 125

Met?Val?Ala?Pro?Ser?Gly?Ala?Arg?Leu?Gly?Pro?Leu?Pro?His?Leu?Gln

130 135 140

Ala?Leu?Leu?Gln?Asp?Arg?Pro?His?Phe?Met?His?Gln?Leu?Ser?Thr?Val

145 150 155 160

Asp?Ala?His?Ala?Arg?Thr?Pro?Val?Leu?Gln?Val?His?Pro?Leu?Glu?Ser

165 170 175

Pro?Ala?Met?Ile?Ser?Leu?Thr?Pro?Pro?Thr?Thr?Ala?Thr?Gly?Val?Phe

180 185 190

Ser?Leu?Lys?Ala?Arg?Pro?Gly?Leu?Pro?Pro?GlyIle?Asn?Val?Ala?Ser

195 200 205

Leu?Glu?Trp?Val?Ser?Arg?Glu?Pro?Ala?Leu?Leu?Cys?Thr?Phe?Pro?Asn

210 215 220

Pro?Ser?Ala?Pro?Arg?Lys?Asp?Ser?Thr?Leu?Ser?Ala?Val?Pro?Gln?Ser

225 230 235 240

Ser?Tyr?Pro?Leu?Leu?Ala?Asn?Gly?Val?Cys?Lys?Trp?Pro?Gly?Cys?Glu

245 250 255

Lys?Val?Phe?Glu?Glu?Pro?Glu?Asp?Phe?Leu?Lys?His?Cys?Gln?Ala?Asp

260 265 270

His?Leu?Leu?Asp?Glu?Lys?Gly?Arg?Ala?Gln?Cys?Leu?Leu?Gln?Arg?Glu

275 280 285

Met?Val?Gln?Ser?Leu?Glu?Gln?Gln?Leu?Val?Leu?Glu?Lys?Glu?Lys?Leu

290 295 300

Ser?Ala?Met?Gln?Ala?His?Leu?Ala?Gly?Lys?Met?Ala?Leu?Thr?Lys?Ala

305 310 315 320

Ser?Ser?Val?Ala?Ser?Ser?Asp?Lys?Gly?Ser?Cys?CysIle?Val?Ala?Ala

325 330 335

Gly?Ser?Gln?Gly?Pro?Val?Val?Pro?Ala?Trp?Ser?Gly?Pro?Arg?Glu?Ala

340 345 350

Pro?Asp?Ser?Leu?Phe?Ala?Val?Arg?Arg?His?Leu?Trp?Gly?Ser?His?Gly

355 360 365

Asn?Ser?Thr?Phe?Pro?Glu?Phe?Leu?His?Asn?Met?Asp?Tyr?Phe?Lys?Phe

370 375 380

His?Asn?Met?Arg?Pro?Pro?Phe?Thr?Tyr?Ala?Thr?Leu?Ile?Arg?Trp?Ala

385 390 395 400

Ile?Leu?Glu?Ala?Pro?Glu?Lys?Gln?Arg?Thr?Leu?Asn?Glu?Ile?Tyr?His

405 410 415

Trp?Phe?Thr?Arg?Met?Phe?Ala?Phe?Phe?Arg?Asn?His?Pro?Ala?Thr?Trp

420 425 430

Lys?Asn?Ala?Ile?Arg?His?Asn?Leu?Ser?Leu?His?Lys?Cys?Phe?Val?Arg

435 440 445

Val?Glu?Ser?Glu?Lys?Gly?Ala?Val?Trp?Thr?Val?Asp?Glu?Leu?Glu?Phe

450 455 460

Arg?Lys?Lys?Arg?Ser?Gln?Arg?Pro?Ser?Arg?Cys?Ser?Asn?Pro?Thr?Pro

465 470 475 480

Gly?Pro

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Met?Gly?Ser?Ser?His?His?His?His?His?His?Ser?Ser?Gly?Leu?Val?Pro

1 5 10 15

Arg?Gly?Ser?His?Met?Ala?Ser?Met?Thr?Gly?Gly?Gln?Gln?Met?Gly?Arg

20 25 30

Gly?Ser?Tyr?Gly?Arg?Lys?Lys?Arg?Arg?Gln?Arg?Arg?Arg?Glu?Leu?Arg

35 40 45

Arg?Gln?Ala?Ser?Met?Pro?Asn?Pro?Arg?Pro?Gly?Lys?Pro?Ser?Ala?Pro

50 55 60

Ser?Leu?Ala?Leu?Gly?Pro?Ser?Pro?Gly?Ala?Ser?Pro?Ser?Trp?Arg?Ala

65 70 75 80

Ala?Pro?Lys?Ala?Ser?Asp?Leu?Leu?Gly?Ala?Arg?Gly?Pro?Gly?Gly?Thr

85 90 95

Phe?Gln?Gly?Arg?Asp?Leu?Arg?Gly?Gly?Ala?His?Ala?Ser?Ser?Ser?Ser

100 105 110

Leu?Asn?Pro?Met?Pro?Pro?Ser?Gln?Leu?Gln?Leu?Pro?Thr?Leu?Pro?Leu

115 120 125

Val?Met?Val?Ala?Pro?Ser?Gly?Ala?Arg?Leu?Gly?Pro?Leu?Pro?His?Leu

130 135 140

Gln?Ala?Leu?Leu?Gln?Asp?Arg?Pro?His?Phe?Met?His?Gln?Leu?Ser?Thr

145 150 155 160

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

165 170 175

Ser?Pro?Ala?Met?Ile?Ser?Leu?Thr?Pro?Pro?Thr?Thr?Ala?Thr?Gly?Val

180 185 190

Phe?Ser?Leu?Lys?Ala?Arg?Pro?Gly?Leu?Pro?Pro?Gly?Ile?Asn?Val?Ala

195 200 205

Ser?Leu?Glu?Trp?Val?Ser?Arg?Glu?Pro?Ala?Leu?Leu?Cys?Thr?Phe?Pro

210 215 220

Asn?Pro?Ser?Ala?Pro?Arg?Lys?Asp?Ser?Thr?Leu?Ser?Ala?Val?Pro?Gln

225 230 235 240

Ser?Ser?Tyr?Pro?Leu?Leu?Ala?Asn?Gly?Val?Cys?Lys?Trp?Pro?Gly?Cys

245 250 255

Glu?Lys?Val?Phe?Glu?Glu?Pro?Glu?Asp?Phe?Leu?Lys?His?Cys?Gln?Ala

260 265 270

Asp?His?Leu?Leu?Asp?Glu?Lys?Gly?Arg?Ala?Gln?Cys?Leu?Leu?Gln?Arg

275 280 285

Glu?Met?Val?Gln?Ser?Leu?Glu?Gln?Gln?Leu?Val?Leu?Glu?Lys?Glu?Lys

290 295 300

Leu?Ser?Ala?Met?Gln?Ala?His?Leu?Ala?Gly?Lys?Met?Ala?Leu?Thr?Lys

305 310 315 320

Ala?Ser?Ser?Val?Ala?Ser?Ser?Asp?Lys?Gly?Ser?Cys?Cys?Ile?Val?Ala

325 330 335

Ala?Gly?Ser?Gln?Gly?Pro?Val?Val?Pro?Ala?Trp?Ser?Gly?Pro?Arg?Glu

340 345 350

Ala?Pro?Asp?Ser?Leu?Phe?Ala?Val?Arg?Arg?His?Leu?Trp?Gly?Ser?His

355 360 365

Gly?Asn?Ser?Thr?Phe?Pro?Glu?Phe?Leu?His?Asn?Met?Asp?Tyr?Phe?Lys

370 375 380

Phe?His?Asn?Met

385

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Met?Gly?Ser?Ser?His?His?His?His?His?His?Ser?Ser?Gly?Leu?Val?Pro

1 5 10 15

Arg?Gly?Ser?His?Met?Ala?Ser?Met?Thr?Gly?Gly?Gln?Gln?Met?Gly?Arg

20 25 30

Gly?Ser?Tyr?Gly?Arg?Lys?Lys?Arg?Arg?Gln?Arg?Arg?Arg?Glu?Leu?Arg

35 40 45

Arg?Gln?Ala?Ser?Gly?Val?Cys?Lys?Trp?Pro?Gly?Cys?Glu?Lys?Val?Phe

50 55 60

Glu?Glu?Pro?Glu?Asp?Phe?Leu?Lys?His?Cys?Gln?Ala?Asp?His?Leu?Leu

65 70 75 80

Asp?Glu?Lys?Gly?Arg?Ala?Gln?Cys?Leu?Leu?Gln?Arg?Glu?Met?Val?Gln

85 90 95

Ser?Leu?Glu?Gln?Gln?Leu?Val?Leu?Glu?Lys?Glu?Lys?Leu?Ser?Ala?Met

100 105 110

Gln?Ala?His?Leu?Ala?Gly?Lys?Met?Ala?Leu?Thr?Lys?Ala?Ser?Ser?Val

115 120 125

Ala?Ser?Ser?Asp?Lys?Gly?Ser?Cys?Cys?Ile?Val?Ala?Ala?Gly?Ser?Gln

130 135 140

Gly?Pro?Val?Val?Pro?Ala?Trp?Ser?Gly?Pro?Arg?Glu?Ala?Pro?Asp?Ser

145 150 155 160

Leu?Phe?Ala?Val?Arg?Arg?His?Leu?Trp?Gly?Ser?His?Gly?Asn?Ser?Thr

165 170 175

Phe?Pro?Glu?Phe?Leu?His?Asn?Met?Asp?Tyr?Phe?Lys?Phe?His?Asn?Met

180 185 190

Arg?Pro?Pro?Phe?Thr?Tyr?Ala?Thr?Leu?Ile?Arg?Trp?Ala?Ile?Leu?Glu

195 200 205

Ala?Pro?Glu?Lys?Gln?Arg?Thr?Leu?Asn?Glu?Ile?Tyr?His?Trp?Phe?Thr

210 215 220

Arg?Met?Phe?Ala?Phe?Phe?Arg?Asn?His?Pro?Ala?Thr?Trp?Lys?Asn?Ala

225 230 235 240

Ile?Arg?His?Asn?Leu?Ser?Leu?His?Lys?Cys?Phe?Val?Arg?Val?Glu?Ser

245 250 255

Glu?Lys?Gly?Ala?Val?Trp?Thr?Val?Asp?Glu?Leu?Glu?Phe?Arg?Lys?Lys

260 265 270

Arg?Ser?Gln?Arg?Pro?Ser?Arg?Cys?Ser?Asn?Pro?Thr?Pro?Gly?Pro

275 280 285

Claims (6)

1. a PTD-hScurfin fusion rotein is characterized in that, has the aminoacid sequence shown in the SEQ ID NO.1

2. a PTD-Δ h FKH fusion rotein is characterized in that having the aminoacid sequence shown in the SEQ ID NO.2.

3. a PTD-Δ h PRD fusion rotein is characterized in that having the aminoacid sequence shown in the SEQ ID NO.3.

4. a nucleic acid construct is characterized in that, it contains the aminoacid sequence of any one described fusion rotein among the coding claim 1-3.

5. a method for preparing each described fusion rotein among the claim 1-3 is characterized in that, this method comprises the following steps: that (1) obtains the gene order of encoding fusion protein; (2) sequence that step (1) is obtained is inserted in the suitable carriers, obtains the corresponding nucleic acids construct; (3) nucleic acid construct that step (2) is obtained transforms suitable expression bacterium; (4) under the suitable culture condition, the expression bacterium of culturing step (3), and add an amount of isopropyl-, 0.2mM-1mM final concentration abduction delivering is therefrom isolated the fusion rotein of expression.

6. preparation method according to claim 5, it is characterized in that, when the fusion rotein of step (4) is the inclusion body formal representation, also have in step (4) back: by nickel post affinity purification under the denaturing agent existence condition, purified product is frozen standby after by the post that desalts with inclusion body.

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