CN1225682A - Method and compositions for regulating T cell subsets by modulating transcription facter activity - Google Patents

Method and compositions for regulating T cell subsets by modulating transcription facter activity Download PDF

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CN1225682A
CN1225682A CN97195740A CN97195740A CN1225682A CN 1225682 A CN1225682 A CN 1225682A CN 97195740 A CN97195740 A CN 97195740A CN 97195740 A CN97195740 A CN 97195740A CN 1225682 A CN1225682 A CN 1225682A
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cell
nip45
maf
transcription factor
family protein
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L·H·格里姆彻尔
M·R·霍德格
I·C·霍
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Harvard College
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Priority claimed from US08/755,592 external-priority patent/US6537810B1/en
Priority claimed from US08/755,584 external-priority patent/US5858711A/en
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Abstract

Methods for modulating production of a T helper type 2 (Th2)-associated cytokine, in particular interleukin-4, by modulating the activity of one or more transcription factors that cooperate with NF-AT family proteins to regulate expression of a Th2-associated cytokine gene are disclosed. In one embodiment, the activity of a maf family protein (e.g., c-Maf or a small maf protein, such as p18) is modulated. In another embodiment, the activity of a protein that interacts with an NF-AT family protein (e.g., NIP45) is modulated. Combination methods, for example wherein the activities of a maf family protein and an NF-AT protein are modulated or the activities of a maf protein and an NF-AT-interacting protein are modulated, are also encompassed by the invention. Methods for modulating development of T helper type 1 (Th1) or T helper type 2 (Th2) subsets in a subject using agents that modulate transcription factor activity are also disclosed. NIP45 compositions, including isolated nucleic acid molecules encoding NIP45, antisense nucleic acid molecules, recombinant expression vectors containing a NIP45 nucleic acid molecule, host cells into which such expression vectors have been introduced and non-human transgenic animals carrying a NIP45 transgene, are also provided by the invention. The invention further provides isolated NIP45 proteins and peptides, NIP45 fusion proteins and anti-NIP45 antibodies. Methods of using the NIP45 compositions of the invention are also disclosed.

Description

Regulate the T cell subsets by regulating transcription factor activity
Background of invention
The CD4+T helper is not the colony of homogeneity, but can based on cytokine secretion be divided at least two be called the subgroup that auxiliary 1 type (Th1) of T and T assist 2 types (Th2) (referring to for example Mosmann, T.R. etc. (1986) J.Immunol.136:2348-2357; Paul, WE and Seder, R.A. (1994) Cell76:241-251; Seder, R.A. and Paul, W.E. (1994) AnnRev.Immunol.12:635-673).Th1 emiocytosis interleukin-2 (IL-2) and interferon-(IFN-γ) and Th2 cell produce interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-10 (IL-10) and interleukin-13 (IL-13).These two subgroups all produce the cytokine such as tumour necrosis factor (TNF) and granulocyte/macrophage colony stimulating factor (GM-CSF).Except the pattern difference of their cytokine-expressings, it is believed that Th1 has different functionally activies with the Th2 cell.For example, the Th1 cell participates in inducing delayed type hypersensitivity to reply, and the Th2 cell participates in bone-marrow-derived lymphocyte is provided effectively " help " and stimulates generation IgG1 and IgE antibody.
Existing enough evidences show, the ratio of Th1 and Th2 cell and the height correlation as a result that comprises the large quantities of immune-mediated clinical disease of autoimmune disease, allergic disease and transmissible disease.For example, in the experimental leishmania mouse of mouse infects, there is the animal of resistance mainly to increase Th1 to infection and replys, the animal of progressive infection susceptible is then mainly increased Th2 reply that (Heinzel, F.P. wait (1989) J.Exp.Med.169:59-72; Locksley, R.M. and Scott, P. (1992) Immunoparasitology Today1:A58-A61).In the mouse schistosomicide, observe Th1 and take place simultaneously to Th2 conversion and the ovulation in tissue of female parasite, and be associated with the deterioration of this disease condition (Pearce, E.J. etc. (1991) J.Exp.Med.173:159-166; Grzych, J-M. etc., (1991) J.Immunol.141:1322-1327; Kullberg, M.C. waits (1992) J.Immunol.148:3264-3270).The feature that comprises many human diseasess of chronic infection (for example human immunodeficiency virus (HIV) and tuberculosis) and some metastatic carcinoma is also changed (referring to for example Shearer to Th2 for Thl, G.M. and Clerici, M. (1992) Prog.Chem.Immunol.54:21-43; Clerici, M and Shearer, G.M. (1993) ImmunologyToday 14:107-111; Yamamura, M waits (1993) J.Clin.Invest.91:1005-1010; Pisa, P. waits (1992) Proc.Natl.Acad.Sci.USA89:7708-7712; Fauci, A.S. (1988) Science239:617-623).In addition, having shown that some autoimmune disease is replied with dominant Th1 is associated.For example, patient with rheumatoid arthritis has dominant Th1 cell (Simon in synovial tissue, AK., Deng (1994) proc.Natl.Acad Sci.USA91:8562-8566), can induce experimental from immune encephalomyelitis (EAE) (Kuchroo with autoreaction Th1 cell, V.K., wait (1993) J.Immunol.151:4371-4381).
Change or handle the mechanism of auxiliary precursor cell (Thp) differentiation of Capability Requirement understanding CD4 T of Th1 and Th2 subgroup ratio, described precursor cell is only secreted IL-2, is selected to Th1 or Th2 effector cell.Know very much cytokine self be effective Th cell induction thing and form an inherent regulation ring (referring to for example Paul, W.E. and Seder, R.A. (1994) Cell76:241-251; Seder, R.A. and Paul, W.E. (1994) Ann.Rev.Immunol.12:635-673).Therefore, IL-4 promotes the Th2 cytodifferentiation and prevents that the precursor differentiation from becoming the Th1 cell that IL-12 and IFN-γ then have opposite effect.Therefore a kind of method of possible change Th1:Th2 ratio is the level that increases or reduce the cytokine of selecting.Shown the antibody that directly gives cytokine or cytokine to some by or the cell-mediated disease of Th1 or Th2 effective.For example, recombinate IL-4 or IL-12 antibody has improved autoimmune disease EAE that Th1 drives (referring to Racke; (1994) J.Exp.Med.180:1961-1966 such as M.K.; And Leonard, J.P. etc. (1995) J.Exp.Med.181:381-386), anti-IL-4 antibody has then been cured the large-scale leishmania of parasitosis (Sadick, M.D. etc. (1990) J.Exp.Med.171:115-127) of Th2 mediation.Yet as the optional means of treatment, system gives cytokine or antibody may have adverse side effect, therefore, still needs to handle the method for replacing of Th1/Th2 subgroup.
Tissue specific expression or the molecular basis of any T cell cytokine always indigestion of IL-4 in the Th2 cell.A kind of may be to have the aporepressor that optionally makes the cytokine silence.The Transcriptional Silencing of existing a lot of document description bacteriums, yeast and mammalian genes.Example comprises coli heat regulatory gene (Groansson, (1990) Nature344:682-685 such as M.), yeast α 2 mating type gene (Keleher, (1988) Cell53:927-936 such as C.A.) and Mammals MHCI type and TcR α gene (Weisman, J.D. and Singer, D.S. (1991) Mol.Cell.Biol.11:4228-4234; Winoto, A. and Blatimore, D. (1989) Cell59:649-655).Really, relate to the early stage experiment prompting that the IL-2 genomic dna is injected into the xenopus ovocyte, with activate the aporepressor (Mouzaki, A. etc. (1991) EMBO are J.10:1399-1406) that has IL-2 in the relative tranquillization T cell extract.These researchs are pointed out, shortage IL-2 produces to interact by the negative element with this IL-2 promotor due to and makes the albumen of IL-2 Transcriptional Silencing in tranquillization T cell.
Second kind of possibility is to have Th selectivity trans-activator.The research that cytokine gene is transcribed element provides the understanding that these cytokine genes are regulated.For example, the analysis of IL-4 cytokine promotor function critical sites (Rooney, J.W. etc. (1995) Immunity2:473-483 to the several transcription factors that comprise NF-AT and AP-1 family member have been disclosed; Szabo, S.J. etc. (1993) Mol.Cell.Biol.13:4793-4805).NF-AT contains cyclosporin A susceptibility kytoplasm phosphorprotein and many subunits transcription complex (Flanagan, W.M. etc. (1991) Nature352:803-807 of the induced nuclear consitution be made up of AP-1 family member albumen; Jain, J. etc. (1992) Nature356:801-804).The purifying of NF-ATp and clone have disclosed the identical district (McCaffey, P.G. etc. (1993) Science262:750-754) of Rel homeodomain (RHD) finite sequence with the NF κ B family of transcription factor.Follow-up clone and the order-checking of three genes involved NF-ATc, NF-AT4/x/c3 and NF-AT3/c4 have disclosed similar structural domain.The NF-AT family member in this territory total about 70% sequence similarity and with the RHD of the Rel/NF κ B family of the transcription factor 18% sequence similarity of having an appointment.Consistent with its very limited sequence similarity in RHD, in NF κ B and the proteic behavior of NF-AT, significant difference is arranged, the approach of mediation NF-AT target gene transcriptional regulatory is known little about it.Yet, consider the NF-AT family member can in conjunction with and trans-activation comprise promotor (Rooney, J. etc. (1995) Immunity2:545-553 of a plurality of cytokine genes of IL-2 and IL-4; Szabo, S.J. etc. (1993) Mol.Cell.Biol.13:4793-4805; Flanagan, W.M. etc. (1991) Nature352:803-807; Northrop, J.P. etc. (1994) Nature369:497), NF-AT albumen can not directly be transcribed responsible to the mediation Th1 or the Th2 specific cell factor.
Great majority (even not being whole) NF-AT binding site in the cytokine promotor regulatory region is attended by in conjunction with near the site that is generally AP-1 family member's ancillary transcription factor.Shown the collaborative and combination collaboratively of NF-AT and AP-1 albumen, and be essential the complete activity of IL-2 and IL-4 promotor.Having shown different AP-1 albumen, specifically is c-Jun, c-Fos, Fra-1, Fra-2, Jun B and Jun D, combines (Rao, A etc. (1994) Immunol.Today15:274-281 with these sites; Jain, J. etc. (1993) Nature365:352-355; Boise, L.H. etc. (1993) Mol.Cell.Biol.13:1911-1919; Rooney, J. etc. (1995) Immunity 2:545-553; Rooney, J. etc. (1995) Mol.Cell.Biol.15:6299-6310).Yet all these AP-1 albumen are not all expressed with Th1 or Th2 specificity mode, and do not have AP-1 family member difference to raise evidence (Roorney, J. etc. (1995) Mol.Cell.Biol.15:6299-6310) to described IL-2 or IL-4 complex loci.Therefore, perhaps NF-AT albumen or AP-1 family member c-Jun, c-Fos, Fra-1, Fra-2, Jun B and JunD all can not be the reasons of the tissue-specific transcription of IL-4 in the Th2 cell.
Summary of the invention
The present invention relates to regulate the method for the generation of Th2 relevant cell factor and adjusting Th1 or Th2 subgroup, relate to the composition that can be used for this adjusting by regulating one or more activity of regulating the transcription factor of Th2 specific cell factor gene expression.As further described herein, the tissue specific expression that has now found that IL-4 in the Th2 cell is not because aporepressor but because Th2 specificity trans-activation.Confirm that now proto-oncogene c-Maf is responsible for the tissue specific expression of Th2 relevant cell factor interleukin-4.In addition, c-Maf is ectopic expression in the cell (for example Th1 cell, B cell and non-lymphoidocyte) of non-Th2 cell, causes this IL-4 promotor to activate and produce under proper condition endogenous IL-4.C-Maf and the genetic expression of NF-AT synergistic activation Th2 relevant cell factor have been found in addition.
In addition, now separated with characterized be called NIP45 (being NF-AT interaction protein 45 (NF-AT Interacting Protein45)) with the interactional 45kDa albumen of NF-AT protein family member.Separate NIP45 based on it with the interactional ability of Rel homeodomain (RHD).Shown that NIP45 and NF-AT and c-Maf work in coordination with stimulating cytokine genetic expression.NIP45 strengthens the genetic expression by c-Maf and NF-AT mediation, makes when these three factors (c-Maf, NF-AT and NIP45) all have activity in cell, stimulates high-level endogenous IL-4 to produce.The little maf albumen of finding the trans-activation domain of shortage such as p18 again can check the genetic expression of Th2 relevant cell factor in addition, for example the expression that is mediated by c-Maf.
Therefore, the present invention relates to cooperate with the NF-AT family protein with the expression of the transcription factor of regulating the genetic expression of Th2 relevant cell factor or the method that the active Th2 of adjusting relevant cell factor is expressed by regulating one or more.In one embodiment, cooperate with the NF-AT family protein and regulate the genetic expression of Th2 relevant cell factor and therefore its expression or active transcription factor of being regulated are Th2 idiosyncratic transcription factor (for example Th2 specificity maf family proteins).In another embodiment, cooperate with the NF-AT family protein and regulate the genetic expression of Th2 relevant cell factor and therefore its expression or active transcription factor of being regulated are maf family protein, for example c-Maf.In an embodiment again, cooperate with the NF-AT family protein and to regulate the genetic expression of Th2 relevant cell factor and therefore its expression or active transcription factor of being regulated are and the interactional albumen of NF-AT family protein, for example NIP45.In an embodiment again, regulate proteic statement of little maf or activity such as p18.Method of the present invention can relate to the expression or the activity of the combination (for example c-Maf+NF-AT or NF-AT+NIP45 or c-Maf+NF-AT+NIP45) of regulating a transcription factor (for example c-Maf or p18) or transcription factor.
Control method of the present invention relates generally to the generation of regulating transcription factor expression or active medicine exposing cell, make the Th2 relevant cell factor of regulating cell.Specifically, recommendation medicine of the present invention is done in cell in order to regulate the activity of this transcription factor.In one embodiment, control method of the present invention stimulates the generation of Th2 relevant cell factor.For example, can be in the generation of Th1 cell, B cell or non-lymphoidocyte moderate stimulation Th2 relevant cell factor.In another embodiment, control method of the present invention suppresses the generation of Th2 relevant cell factor.The Th2 relevant cell factor of being regulated in the method is preferably interleukin-4.
Can use various medicine irritations to regulate the expression or the activity of the transcription factor of Th2 relevant cell factor genetic expression.For example, pungency medicine of the present invention can be that coding imports this cell and the nucleic acid molecule of the transcription factor expressed therein.Perhaps, can use the expression that strengthens this transcription factor or active chemicals as the pungency medicine.
Can use various medicines to suppress the expression or the activity of the transcription factor of adjusting Th2 relevant cell factor genetic expression.The example of suitable suppressive drug comprises with the antisense nucleic acid molecule of the gene complementation of this transcription factor of coding, combines the intracellular antibody of this transcription factor (for example in nucleus), the inhibition form of this transcription factor (for example dominant form) and this transcription factor expression of inhibition or active chemicals.
The present invention also comprises the expression or the active integrated processes of the transcription factor that relates to two kinds, three kinds of adjustings or the genetic expression of multiple adjusting Th2 relevant cell factor.Therefore, in other embodiments of the present invention, at least a to regulating the active medicine exposing cell of other transcription factor that Th2 relevant cell factor gene works with at least a adjusting.Best, this at least a its expression or active other transcription factor of being regulated are selected from NF-AT family protein, NF-AT interaction protein, maf family protein and AP-1 family protein.
Can in external or body, regulate the cytokine generation of cell according to method of the present invention.In one embodiment, with regulating medicine, then this cell is given the curee to regulate the growth of Th1 among this curee and/or Th2 subgroup thus at external exposing cell.Therefore, on the other hand, the invention provides the method for in the curee, regulating Th1 or the growth of Th2 subgroup.Except the cell that wherein will in vitro modify gives this curee's embodiment, in another embodiment, these methods relate to and directly give this curee and regulate the medicine that one or more regulate the transcription factor activity of Th2 relevant cell factor genetic expression, make that Th1 or the exhibition of Th2 cells whose development among this curee regulated.
Can in various clinical settings, handle Th1 with control method of the present invention: the Th2 ratio.For example, suppress Th2 and form and to be used for allergic disease, malignant tumour and transmissible disease, form and to be used for autoimmune disease and organ transplantation and strengthen Th2.
The NIP45 composition that relates in one aspect to again of the present invention.The invention provides the composition isolated of the nucleotide sequence of NIP45 albumen and separated coding NIP45, relative other composition and its using method.Determined the proteic aminoacid sequence of NIP45 (being shown in SEQ ID NO:6), (its nucleotides sequence is shown in SEQ ID NO to have separated the proteic cDNA of coding NIP45; 5).In one aspect, the invention provides isolated nucleic acid molecule or its fragment of coding NIP45.In one embodiment, the invention provides the isolated nucleic acid molecule that comprises the proteic nucleotide sequence of coding NIP45.In another embodiment, the invention provides the isolated nucleic acid molecule of the nucleotide sequence that comprises proteins encoded, wherein this albumen comprises with the aminoacid sequence of the amino acid sequence homologous of SEQ ID NO:6 and with the Rel homeodomain of NF-AT family protein and interacts.In an embodiment again, the invention provides under stringent condition isolated nucleic acid molecule with the making nucleic acid molecular hybridization of the nucleotide sequence that comprises SEQ ID NO:5.In an embodiment again, the invention provides the isolated nucleic acid molecule of the nucleotide sequence that comprises SEQ ID NO:5.In other embodiments, the invention provides the isolated nucleic acid molecule of the aminoacid sequence of coding SEQ ID NO:6.The isolated nucleic acid molecule that the present invention also comprises coding NIP45 fusion rotein with separate antisense nucleic acid molecule.
Another aspect of the present invention relates to carrier (for example recombinant expression vector) that contains NIP45 nucleic acid molecule of the present invention and the host cell that has imported this carrier.In one embodiment, by in suitable medium, cultivating this host cell, use this host cell to produce NIP45 albumen.If desired, can separate NIP45 albumen from this host cell or this substratum then.
Isolating NIP45 albumen or its part of relating in one aspect to again of the present invention.In one embodiment, the invention provides and the interactional isolating NIP45 albumen of NF-AT family protein or its part.In an embodiment again, the invention provides comprise with the aminoacid sequence of the amino acid sequence homologous of SEQ ID NO:6 and with the interactional protein isolate of NF-AT family protein.The present invention also comprises the NIP45 fusion rotein.
Can use the anti-NIP45 antibody of NIP45 albumen of the present invention or its produced in fragments.Therefore, the present invention also provides specificity in conjunction with the proteic antibody of NIP45.In one embodiment, this antibody is monoclonal.In another embodiment, with this antibody of detectable substance mark.
Can use NIP45 coding nucleic acid molecule of the present invention to prepare non-human transgenic animal, this animal contains the genetically modified cell that carries coding NIP45 albumen or NIP45 protein part.Therefore, the present invention also provides this transgenic animal.In one embodiment, this NIP45 transgenosis of being carried by these transgenic animal has changed the coding proteic native gene of endogenous NIP45 (for example homologous recombination animal).
Another aspect of the present invention relates to the method that NIP45 albumen or NIPmRNA exist that detects in biological sample.For detecting NIP45 albumen or NIPmRNA, contact this biological sample with the medicine that can detect NIP45 albumen (for example anti-NIP45 antibody of mark) or NIP45mRNA (for example can with the labeling nucleic acid probe of NIP45mRNA hybridization), make to detect NIP45 albumen or the existence of NIPmRNA in this biological sample.
The method of the compound of differentiating the active or expression of regulating NIP45 and the method that interactional compound between NIP45 and the NF-AT family protein is regulated in discriminating of relating in one aspect to again of the present invention.The present invention also comprises discriminating and the interactional proteic screening method of NIP45.
Of the present invention relating in one aspect to again to express the proteic composition of maf in host cell, this host cell is such as being immunocyte and expressing the proteic host cell of maf.In one embodiment, these compositions comprise the recombinant expression vector of coding maf family protein, and wherein said maf encoding sequence functionally is connected to the adjusting sequence that instructs this maf family protein to express in particular cell types such as lymphoidocyte (for example T cell or B cell) or hemopoietic stem cell.In another embodiment, these compositions comprise host cell, such as host's lymphoidocyte (for example host T cell or host B cell) or host's hemopoietic stem cell, have imported the recombinant expression vector of coding maf family protein in the described host cell.
The present invention also provides and expresses the proteic transgenic animal of c-Maf.In an optimum implementation, these transgenic animal are mouse, and this mouse overexpression c-Maf in the T cell, preferably use the transgenosis that comprises the CD4 promotor/enhanser that functionally is connected to c-mafcDNA.
The accompanying drawing summary
Figure 1A is the diagram of employed cell fusion method, does not cause because of repressor with the proof cytokine-expressing.
Figure 1B be reversed transcriptive enzyme-polymerase chain reaction (RT-PCR) analyze by do not merge Th1 clone (D1.1), do not merge IL-2 that Th2 clone (D10), Th1 and Th2 homokaryons and Th1-Th2 heterokaryon express and IL-4 cytokine, contrast beta-actin, mRNA.
Fig. 2 A is the Northern engram analysis, is depicted in the expression of isolating cDNA clone in Th1 cell, Th2 cell or the B lymphoma cell.Use equates the RNA applied sample amount to the specific contrast probe of GAPDH to show.
Fig. 2 B is the Northern engram analysis, and being depicted in the external normal differentiation of splenocyte originally becomes and separate the expression of cDNA clone to adjusted between the Th2 cell stage.From the total RNA of cellular segregation in the time point results of indicating.Measure the generation of the cytokine (IL-10) of suitable dilution culture supernatant by ELISA, be divided into Th1 or Th2 pedigree with mensuration.
Fig. 3 A is a histogram, is depicted among the Th1 clone (AE7) by c-Maf trans-activation IL-4 promotor.With wild-type IL-4 CAT report construction and or control plasmid (pMEX-Neo I), c-Maf expression plasmid (pMEX-Maf) or c-Fos expression plasmid (pMEX-c-Fos) cotransfection AE7 cell.After transfection, stimulated half of each sample in 24 hours with anti-cd 3 antibodies.48 hours results all samples after transfection, and measure relative CAT activity.
Fig. 3 B is the photo of thin layer chromatography board, describe the relative CAT activity in the M12B lymphoma cell, this cell with wild-type IL-4CAT report construction and or two kinds of control plasmids (pMEX-Neo I and pREP4), c-Maf expression plasmid and control plasmid (pMEX-Maf and pREP4), c-Fos expression plasmid and control plasmid (pMEX-c-Fos and pREP4), c-Jun expression plasmid and control plasmid (pMEX-c-Jun and pREP4), control plasmid and NF-ATp expression plasmid (pMEX-Neo I and pREP-NF-ATp), c-Maf expression plasmid and NF-ATp expression plasmid (pMEX-Maf and pREP-NF-ATp) or c-Fos expression plasmid and NF-ATp expression plasmid (pMEX-c-Fos and pREP-NF-ATp) cotransfection.After transfection, stimulated half of each sample in 24 hours with PMA and ionomycin.After transfection, gather in the crops all samples in 48 hours and measure relative CAT activity.
Fig. 4 is a histogram, describes the endogenous generation IL-4 in the M12 cell by c-Maf and NF-ATp ectopic expression.To or not stimulate or stimulate 24 hours with the cell of control plasmid of indicating or expression plasmid stable transfection with PMA and ionomycin.It is quantitative to carry out cytokine that 200 μ l supernatant liquors of each sample are carried out ELISA.
Fig. 5 A is to use in the post-stimulatory Th2 (D10 that indicates time point results of anti-cd 3 antibodies, CDC35) or Th1 (AE7, S53) the DNA enzyme I footprint gel photograph of Ke Long nuclear extract this IL-4 promotor of carrying out, it is depicted in this IL-4 promotor the and then Th2 specificity footprint in downstream, this supposition MARE site.With two the Th2 specificitys super quick residue of * indication on the noncoding strand of this II-4 promotor.The IL-4 promoter probe (no nuclear extract) of five swimming lanes and the DNA enzyme I digest of Maxam-Gilbert A/G ladder have been carried out on the sample side that this DNA enzyme I is handled.
The near-end regulatory region of Fig. 5 B graphic representation mouse IL-4 promotor.Top shows the primary sequence of this mouse IL-4 promotor.The numeral of indicating is with respect to for+1 the transcription initiation site.Asterisk indication super quick residue of observed Th2 specificity on DNA enzyme I footprint.The bottom is presented at wild-type (59 to-28) oligonucleotide that uses in EMSA shown in Fig. 6 and 7 and the Function detection and the sequence of described 4bp mutant.The Nucleotide that changes is with the demonstration of small letter runic and corresponding to the numbering system that shows in the top.
Fig. 6 is that the electrophoretic mobility change detects the photo of (EMSA), and confirmation is that c-Maf rather than c-Jun are bonded to near-end IL-4 promotor and form complex body with NF-ATp.Double chain oligonucleotide with albumen of indicating and mark carries out EMSA.
Fig. 7 A is the photo of histogram (top) and thin layer chromatography board (bottom), describe relative CAT activity in the M12 cell, described M12 cell with the c-Maf expression vector and or wild-type IL-4CAT report construction or be shown in a kind of 4bp mutant cotransfection among Fig. 5 B, prove that c-Maf is reflected on this MARE and the Th2 specificity footprint the trans-activation of this IL-4 promotor.The average of three independent experiments and a representative experiment are shown in top and bottom respectively.
Fig. 7 B is to use reorganization c-Maf, IL-4 promotor (59 to-27) probe and the photo of the EMSA that the unmarked double chain oligonucleotide indicated carries out as the competition thing, prove that the c-Maf that recombinates is reflected on this MARE and the Th2 specificity footprint with combining of IL-4 promotor.
Fig. 8 is three parts a yeast colony photo, they with the NIP45 plasmid and or NF-ATp-RHD transform with LacZ report plasmid pSH18 as " bait " or contrast bait Max, CDK2 or pEG202, show that only those contain bacterium colonies expression LacZ reporter gene of NIP45 plasmid and NF-ATp-RHD bait.
Fig. 9 is the photo of immunoprecipitation/Western Blot experiment, proves that NIP45 and NF-ATp interact in the HepG2 cell.
Figure 10 relatively separates from the original NIP45cDNA of the two hybrid screenings of yeast to clone (top) and separate the synoptic diagram of cloning the structure of (bottom) from the longest NIP45cDNA in D10.G4 λ zap II library.
Figure 11 describes the nucleotide sequence of original NIP cDNA clone and separate thing and the aminoacid sequence of supposition.
Figure 12 describes the hydrophobicity graph of NIP45cDNA.
Figure 13 is the photo of rna blot analysis NIP45 transcript level in various tissues.
Figure 14 A is the photo of the immunofluorescence analysis of bhk cell, and described cell detects as one-level antibody and the anti-mouse second class grade chemical of indocarbocyanine labelled goat with the proteic expression constructs transfection of NIP45 of coding HA epi-position mark and with HA peptide specific monoclonal antibody.
Figure 14 B is an isocellular photo described in Fig. 7 A that redyes with DNA dye Hoechst33258.
Figure 14 C is the photo of the immunofluorescence analysis of the bhk cell that do not stimulate, described cell with coding NF-AT4 expression constructs transfection and with anti-NF-AT4 specific antibody as one-level antibody and the anti-mouse second class grade chemical detection of indocarbocyanine labelled goat.
Figure 14 D is an isocellular photo described in Fig. 7 C that redyes with DNA dye Hoechst33258.
Figure 14 E is the photo of the immunofluorescence analysis of the bhk cell handled of ionomycin, described cell with coding NF-AT4 expression constructs transfection and with anti-NF-AT4 specific antibody as one-level antibody and the anti-mouse second class grade chemical detection of indocarbocyanine labelled goat.
Figure 14 F is an isocellular photo described in Fig. 7 D that redyes with DNA dye Hoechst33258.
Figure 15 is the histogram (right side) that the active relative multiple of photo (left side) and the quantitative assay CAT of CAT detected result in the HepG2 cell is induced (fold induction), described cell used 3X NF-AT-CAT reporter gene construction (containing three NF-AT binding sites) and or contrast expression plasmid or only NF-AT family expression plasmid (NF-ATp, NF-ATc, NF-AT3 or NF-AT4) itself (-) or with composition (+) transfection of NIP45 expression plasmid.
Figure 16 is the photo (left side) and the active relative multiple inductive histogram (right side) of quantitative assay CAT of CAT detected result in the HepG2 cell, described cell used IL-4-CAT reporter gene construction (extend to the IL-4 promotor-732bp) and the composition transfection of NF-ATp, NIP45 and/or c-Maf expression constructs, as indicating.
Figure 17 is a histogram, be depicted in IL-4 (in pg/ml) level in the M12B lymphoma cell supernatant liquor, described cell has been used the expression plasmid of NF-ATp, c-Maf and expression plasmid (bottom band) transient cotransfection of pCI vehicle Control (upper strap portion) or NF-ATp, c-Maf and NIP45.
Figure 18 is the Northern engram analysis that becomes the transcript of the expression in the 0th, 1,3,5 or 7 day between the Th2 cell stage in external normally splenocyte differentiation originally, and upwards adjusting expression and p18 regulate expression in time downwards in time to describe c-maf.
Figure 19 is the photo of thin layer chromatography board, describe relative CAT activity in the M12 cell, this cell used IL-4 promotor reporter gene construction and or the p18 expression vector of the c-Maf expression vector (5 μ g) of independent c-Maf expression vector (5 μ g), independent p18 expression vector (10 μ g) or constant basis and increasing amount (2.5,5 or μ g) transfection, describe p18 and check the IL-4 promoter activity.
Figure 20 is to use the synoptic diagram of CD4 promotor/enhanser with c-maf transgenic over expression in the T cell of regulating c-maf cDNA expression.
Figure 21 is a histogram, describe or lymphoglandula (LN), spleen or the thymus gland of wild-type mice or c-maf transgenic mice in total cell count (x10 6), confirm that the c-maf transgenic mice shows cell count reduction in lymphoid organ.
Fig. 2 is a histogram, describes the basal level (ng/ml) of SERUM IgE in wild-type mice or the c-maf transgenic mice, confirms that the c-maf transgenic mice shows SERUM IgE basal level and improves.
Detailed Description Of The Invention
In one aspect, the present invention relates to regulate the method and composition of cytokine-expressing and T subgroup by the activity of regulating transcription factor. The present invention is at least part of not to be because the effect of specific aporepressor (as showing among the embodiment 1) based on the specific expressed IL-4 gene of following discovery: Th2, but because the effect of specific trans-activation. As further described herein, now having identified the trans-activator that the specific expressed IL-4 gene of Th2 is responsible for is the c-Maf proto-protein, it optionally in differentiation with mature T h2 cells, and be not present in (referring to embodiment 2) in the Th1 cell. C-Maf is ectopic expression in the cell (for example Th1 cell and B cell) of usually not expressing c-Maf, causes this IL-4 promoter trans-activation (referring to embodiment 3) and produces under proper condition endogenous IL-4 (referring to embodiment 4). In addition, a kind of protein blot that in the nuclear extract of Th2 cell rather than Th1 cell, exists the IL-4 promoter (referring to embodiment 5) in maf response element (MARE) district, and restructuring c-Maf is bonded to (referring to embodiment 6) on this IL-4 promoter in external. The ability of c-Maf trans-activation IL-4 is reflected on the interior MARE of this IL-4 promoter and the Th2 specificity footprint (referring to embodiment 7).
The present invention is also at least part of based on finding interactional with NF-AT and strengthening the albumen of the transcriptional activation that is caused by c-Maf and NF-AT. This albumen NIP45 (referring to embodiment 8) is differentiated in interaction based on the Rel homeodomain (RHD) of it and NF-AT. Coimmunoprecipitation experiment confirm, NIP45 and NF-AT be interaction (referring to embodiment 9) in mammalian cell in body. The cDNA of coding NIP45 is sequenced and characterizes (referring to embodiment 10). Inspection to the tissue expression pattern of NIP45mRNA discloses, and the NIP45 transcript is preferentially expressed (referring to embodiment 11) in spleen, thymus gland and testis. Subcellular Localization studies confirm that NIP45 albumen is evenly distribution (referring to embodiment 12) in nucleus. Functional study shows that the collaborative stimulation of NIP45 and NF-AT is transcribed from the promoter that contains the NF-AT binding site, and works in coordination with transcribe (referring to the embodiment 13) that stimulates from the IL-4 promoter with NF-AT and c-Maf. In addition, NIP45, NF-AT can unanimously act on c-Maf, induce endogenous IL-4 gene expression (referring to embodiment 14) in the cell (for example B cell) of usually not expressing IL-4.
The present invention is more at least part of based on following discovery: the little maf albumen p18 that lacks activation domain can check the cytokine gene expression that is mediated by c-Maf. The downward adjusting to adjusted and p18 gene expression of the differentiation of external t helper cell precursor and c-maf gene expression interrelate (referring to embodiment 15). In addition, compare with the IL-4 promoter activity when only having c-Maf, p18 and c-Maf coexpression have checked IL-4 promoter activity (referring to embodiment 16).
The present invention is again based on the generations (referring to embodiment 17) of the c-maf transgenic animals of overexpression c-Maf albumen in the T cell. These animals show and the very similar phenotype of the transgenic animals of overexpression IL-4, namely little Thymus and spleen, CD+/CD8 +Thymocyte quantity significantly descends (two positive), CD4+Positive T cell descends and SERUM IgE foundation level increases.
Be more readily understood the present invention for making, at first define some term.
The cell factor that term used herein " Th2 relevant cell factor " refers to preferentially or only given birth to by Th2 cell rather than Th1 cell special product. The example of Th2 relevant cell factor comprises IL4, IL-5, IL-6 and IL-13. The preferred Th2 relevant cell factor of regulating its generation according to method of the present invention is interleukin-4.
Term used herein " transcription factor " refers to work in nucleus with the factor (for example albumen) of the transcriptional expression of regulatory gene. Term " transcription factor " comprises the factor that the factor that direct adjusting transcribes (for example have intrinsic (mstrinsic) transcriptional activation or suppress active) and indirect regulation transcribe (for example by with have intrinsic transcriptional activation or suppress active other factors interaction).
The transcription factor of " regulating the gene expression of Th2 relevant cell factor with the cooperation of nuclear factor of activated T cells family protein " used herein refers to the transcription factor with the gene expression of the collaborative or consistent effect adjusting of NF-AT albumen Th2 relevant cell factor. That is to say, compare during with individualism that Th2 relevant cell factor gene (for example IL-4) was expressed higher when NF-AT and this collaborative transcription factor all existed. This collaborative transcription factor can be with or without physics with NF-AT and contact. The transcription factor of regulating the gene expression of Th2 relevant cell factor that cooperates with the NF-AT family protein comprises maf family protein (for example c-Maf) and NF-AT interaction protein (for example NIP45).
The transcription factor of " adjusting to Th2 relevant cell factor gene is worked " used herein refers to participate in the transcription factor that the genetic transcription of Th2 relevant cell factor is regulated, no matter and whether it cooperates with the NF-AT family protein. The transcription factor of regulating the gene expression of Th2 relevant cell factor that cooperates with NF-AT also is the factor that the adjusting of Th2 relevant cell factor gene is worked. Yet other transcription factor that does not cooperate with NF-AT also can work to the adjusting of Th2 relevant cell factor gene. The example that it is believed that the transcription factor that the adjusting of Th2 relevant cell factor gene is worked comprises NF-AT family protein, NF-AT interaction protein, maf family protein, AP-1 family protein and Stat6 (Lederer, J. etc. (1996) J.Exp.Med. 184:397-406).
Term used herein " Th2 idiosyncratic transcription factor " refers in the Th2 cell rather than preferentially or only in the transcription factor of Th1 cells.
Term used herein " contact " (namely using the medicine exposing cell) is included in external with this medicine and described cell incubation (for example this medicine being added the cell in cultivating) and give the curee this medicine altogether, so that this medicine and this curee's cell contacts in body.
The various forms of term used herein " adjusting " comprises stimulation (for example strengthen or to adjusted particular responses or activity) and suppresses (for example reduction or regulate particular responses or activity downwards).
Term used herein " maf family protein " refers to comprise the member of the AP-1/CREB/ATF subfamily of v-Maf, c-Maf, mafB, Nrl, mafK, mafF, mafG and p18. Referring to for example Nishizawa, M etc. (1989) Proc.Natl.Acad. Sci.USA 86:7711-7715; Kataoka, K. etc. (1993) J.Virol.67:2133-2141; Swaroop, A etc. (1992) proc.Natl.Acad.Sci.USA89:266-270; Fujiwara, K.T. etc. (1993) Oncogene8:2371-2380; Igarashi, K. etc. (1995) J.Biol.Chem.270:7615-7624; Andrews, N.C. etc. (1993) Proc.Natl.Acad.Sci.USA90:11488-11492; And Kataoka, K. etc. (1995) Mol.Cell.Biol.15:2180-2190.
Term used herein " little maf albumen " refers to lack the maf family protein corresponding to the domain of the amino terminal activation domain of c-Maf. The example of little maf albumen comprises mafK, mafF, mafG and p18.
Term used herein " NF-AT family protein " (also interchangeable be called simply " NF-AT ") refers to the member of the nuclear factor family of activated T cell transcription factor, comprises NF-ATp, NF-ATc, NF-AT4/x/c3 and NF-AT3/c4.
Term used herein " the Rel homeodomain of NF-AT family protein " (being abbreviated as the RHD territory) refers to have the territory of about 70% sequence similarity in the RHD with transcription factor Rel/NF κ B family in the NF-AT family protein.
Term used herein " NF-AT interaction protein " (can use with " with the interactional albumen of NF-AT family protein " is mutual) refers to form the factor (for example with NF-AT family protein coimmunoprecipitation) that physics contacts with the NF-AT family protein. This NF-AT interaction protein RHD best and the NF-AT family protein interacts. The example of NF-AT interaction protein is NIP45.
Term used herein " NIP45 " comprises the albumen with the amino acid sequence (or by being shown in the nucleotide sequence coded of SEQ ID NO:5) that is shown in SEQ ID NO:6, with and mammal homologue (for example human NIP45) or its keep modified forms (for example sudden change or clipped form) with the RHD interaction ability of NF-AT.
Term used herein " AP-1 family protein " refers to it is transcription factor AP-1 family member's albumen, and the example comprises c-Jun, c-Fos, Fra-1, Fra-2, JunB and JunD.
Term used herein " nucleic acid molecule " comprises dna molecular (for example cDNA or genomic dna) and RNA molecule (for example mRNA).This nucleic acid molecule can be strand or two strands, but best double-stranded DNA.
" isolated nucleic acid molecule " used herein refers to not contain the natural nucleic acid molecule that is positioned at the gene order (that is, being positioned at the contiguous gene order of this isolated nucleic acid molecule in this nucleic acid source organism genomic dna certainly) of this nucleic acid flank in this nucleic acid source organism genomic dna certainly.For example, in various embodiments, separation NIP45 nucleic acid molecule can contain the natural nucleotide sequence that is positioned at this nucleic acid molecule flank in the genomic dna of this nucleic acid source cell certainly less than about 5kb, 4kb, 3kb, 2kb, 1kb, 0.5kb or 0.1kb.In addition, " separation " nucleic acid molecule such as the cDNA molecule can not contain other cellular material.
Hybridization and cleaning condition described in term used herein " hybridize under stringent condition ", and at least 60% homologous nucleotide sequence keeps the phase mutual cross usually each other under this condition.This condition preferably makes mutual homology be at least at least 65%, more preferably at least 70% and most preferably be at least 75% sequence and keep the phase mutual cross usually.This strict hybridization is those skilled in the art's known conditions, and is stated from the molecular biology current methods, John Wiley﹠amp; Sons, N.Y. (1989), 6.3.1-6.3.6.The limiting examples of the recommendation of stringent hybridization condition is in about 45 ℃ of hybridization in 6X sodium chloride/sodium citrate (SSC), then cleans one or many in 50-65 ℃ in 0.2X SSC, 0.1%SDS.
" naturally occurring " used herein nucleic acid molecule refers to have the RNA or the DNA (native protein of for example encoding) of the nucleotide sequence that takes place in the Nature.
" antisense " used herein nucleic acid comprises " justice is arranged " nucleic acid complementary nucleotide sequence with proteins encoded, for example, with the coding strand complementation of double-stranded cDNA molecule, with the mRNA sequence complementary or with the coding strand complementary nucleotide sequence of gene.Therefore, antisense nucleic acid can be connected with phosphorothioate odn is arranged by hydrogen bond.
Term used herein " coding region " refers to comprise the nucleotide sequence district that translation becomes the codon of amino-acid residue, and " non-coding region " refers to not translate and become amino acid whose nucleotide sequence district (for example 5 ' and 3 ' non-translational region).
Term used herein " carrier " refers to transport the nucleic acid molecule of another nucleic acid of its connection.A kind of bearer type is " plasmid ", and it refers to wherein can connect the circular double-stranded DNA ring of other DNA section.Another type of carrier is a virus vector, wherein other DNA section can be connected into this viral genome.Some carrier can be in importing its host cell self-replicating (bacteria carrier and the additive type Mammals carrier that for example have the bacterium replication orgin).Other carrier (for example non-add type Mammals carrier) is integrated into the genome of host cell when importing host cell, and therefore duplicates with host genome.In addition, some carrier can instruct its expression of gene that can be operatively connected.This carrier is referred to herein as " recombinant expression vector " or is called for short " expression vector ".Generally speaking, the expression vector that utilizes in the recombinant DNA technology often is the plasmid form.In this manual, can exchange use " plasmid " and " carrier ", because plasmid is the carrier format of normal use.Yet, the present invention includes the expression vector that those play other form of same function, for example virus vector (for example replication defect type retrovirus, adenovirus and adeno-associated virus).
Term used herein " host cell " refers to import the cell such as the nucleic acid of the present invention of recombinant expression vector of the present invention.This paper exchanges and uses term " host cell " and " recombinant host cell ".Should understand this term and not only refer to the specific cell that is subject to processing, and refer to the filial generation or the potential filial generation of this cell.Because because or sudden change or environmental influence, some modification may take place in the follow-up generation, this filial generation in fact may be different with parental cell, but still belong to the scope of term used herein.
The nucleic acid molecule of " being suitable for expressing the form of this nucleic acid molecule in cell " used herein means that this nucleic acid molecule comprises one or more adjusting sequences, this nucleic acid molecule is selected based on host cell that is used to express and required expression level, and functionally be connected to this nucleic acid molecule that desire is expressed, make and in this host cell, express by the albumen of described nucleic acid molecule encoding.The example of this nucleic acid molecule comprises that containing coding desires the recombinant expression vector of the nucleotide sequence of expressed proteins in host cell.
" transgenic animal " used herein refer to the non-human animal be preferably Mammals, mouse more preferably, and wherein one or more cells of this animal comprise " transgenosis ".Term " transgenosis " refers to foreign DNA, this foreign DNA is integrated into by it grows in the cellular genome of transgenic animal and is retained in the genome of mature animal, for example instructs in the one or more cell types of this transgenic animal or tissue and expresses the encoding gene product.
" homologous recombination animal " used herein refers to transgenic nonhuman's type of animal, be preferably Mammals, mouse more preferably, wherein before this animal development, change this native gene by the homologous recombination acetonitrile between the foreign DNA of native gene and this zooblast of importing (for example protoblast of this animal).
" protein isolate " used herein finger protein roughly do not contain cellular material or substratum when it produces from cellular segregation or by recombinant DNA technology, do not contain precursor or other pharmaceutical chemicals during when chemosynthesis.
The immunocompetence part that term used herein " antibody " comprises immunoglobulin molecules and immunoglobulin molecules is promptly such as Fab and F (ab ') 2The segmental molecule that contains specificity in conjunction with (immune response with it) antigenic antigen binding site.Term used herein " monoclonal antibody " and " monoclonal antibody combination " refer to only contain a kind of can with a group antibody molecule of the immunoreactive antigen binding site of specific antigen epi-position.Therefore monoclonal antibody combination is showed single binding affinity to specific antigen immunoreactive with it usually.
The medicine of " transcription factor expression or activity are regulated in effect in the born of the same parents " used herein refers to work to regulate transcription factor expression or active medicine in the cell intracellular region territory of for example tenuigenin or nuclear.Therefore, the medicine that is bonded to cell surface such as antibody " made in order to regulate transcription factor expression or active medicine " not comprise in term in the born of the same parents.Work comprises nucleic acid molecule, antisense nucleic acid molecule, intracellular antibody, the dominant inhibitor of this transcription factor of encoding in order to the example of regulating transcription factor expression or active medicine and enters cell and adjusting (promptly stimulating or inhibition) transcription factor expression or active chemicals in the born of the same parents.
Term used herein " born of the same parents in binding molecule " comprises by being bonded to albumen self or coding this proteic nucleic acid (for example mRNA molecule) and works in born of the same parents and suppress purpose target protein (for example transcription factor) expression or active medicine.The example of binding molecule comprises antisense nucleic acid, intracellular antibody and dominant inhibitor in the born of the same parents.
All respects of the present invention are described in following trifle in more detail.The adjusting that the I.Th2 relevant cell factor produces
Having identified the transcription factor that the specific expressed interleukin-4 gene of Th2 is responsible for is the c-Maf proto-oncogene.Therefore, expression and/or the active means that interleukin-4 produces of regulating that provide of c-Maf are provided.Because IL-4 from self-control effect in the Th2 cell development (referring to for example Paul, W.E. and Seder, R.A. (1994) Cell76:241-251; Seder, R.A. and Paul, W.E. (1994) Ann.Rev.Immunol.12:635-673), and the generation of IL-4 can cause other Th2 relevant cell factor by further Th2 differentiation generation such as IL-5, IL-6, IL-10 and IL-13 thus, and c-Maf expression and/or the active general method of regulating the generation of Th2 relevant cell factor that provides are provided.
The maf protein family is the AP-1/CREB/ATF albumen subfamily that comprises v-Maf, c-Maf, mafB, Nrl, mafK, mafF, mafG and p18.V-maf oncogene initial separation is from the spontaneous aponeurosis fibrosarcoma (musculoaponeuroticfibroscarcoma) of chicken, and differentiate to be the retroviral transforming gene AS42 of bird (Nishizawa, M. etc. (1989) Proc.Natl.Acad.Sci.USA86:7711-7715).V-maf coding and c-fos and c-jun oncogene homologous 42kd basic region/leucine zipper (b-zip) albumen.Its cell homologue c-maf proto-oncogene only has two structural changess (Kataoka, K. etc. (1993) J.Virol.67:2133-2141) in the coding region of v-maf.Maf family comprises c-Maf, mafB, human retina specific proteins Nrl (Swaroop, A. etc. (1992) Proc.Natl.Acad Sci.USA89:266-270), mafK, mafF, mafG and p18.Four (mafK, mafF, mafG and p18) each the coding disappearances in back contain albumen (" little maf albumen ") (Fujiwara, K.T. etc. (1993) Oncogene8:2371-2380 of the c-Maf N-terminal 2/3rds of trans-activation domain; Igarashi, K. etc. (1995) J.Biol.Chem270:7615-7624; Andrews, N.C. etc. (1993) Proc.Natl.Acad.Sci.USA90:11488-11492; Kataoka, K. etc. (1995) Mol.Cell.Biol.15:2180-2190).C-maf and other maf family member be one-level and Fos and Jun formation homodimer and heterodimer each other, with AP-1 albumen consistent (Kerppola, T.K. and Curran, T. (1994) Oncogene9:675-684 of paired known capabilities each other; Kataoka, K. etc. (1994) Mol.Cell.Biol.14:700-712).The c-Maf homodimer bonded DNA target sequence of called after c-Maf response element (MARE) is 13 or the 14bp element that contains core TRE (T-MARE) or CRE (C-MARE) palindromic sequence respectively.Before the present invention, function to the maf family member is known little about it, although c-Maf has shown the transcribe (Kurscher of stimulation from Purkinje neuronal specificity promoter L 7, C. and Morgan, J.I. (1994) Mol.Cell.Biol.15:246-254), and Nrl is display driver QRl retina expression of specific gene (Swaroop, A. etc. (1992) Proc.Natl.Acad.Sci.USA89:266-270).Yet, before the present invention, do not have any report to refer to that c-Maf or other maf family member relate to the generegulation expressed in lymphoidocyte or the expression of cytokine gene in any tissue.
Shown little maf when for homodimer in conjunction with the time work as α and beta globin transcription repressor, but when as the time with the heterodimer partner of red corpuscle class atopen p45NF-E2, transcribing for the activation globin gene is essential (Kataoka, K. etc. (1995) Mol.Cell.Biol.15:2180-2190; Igarashi, K. etc. (1994) Nature367:568-572).Shown that the MafK overexpression induces erythroleukemia cell differentiation (Igarahsi, K. etc. (1995) proc.Natl.Acad.Sci.USA92:7445-7449).The invention provides little maf albumen (for example p18) and can regulate the evidence of Th2 relevant cell factor genetic expression.Therefore, regulate proteic expression of little maf and/or the active means of regulating Th2 relevant cell factor genes produce that also provide.
The present invention provides NF-AT interaction protein NIP45 in addition, and it combines with NF-AT and the genetic expression of the collaborative Th2 of adjusting relevant cell factor.NIP45 is differentiated in interaction based on the Rel homeodomain of itself and NF-ATp.Following more detailed description NIP45.Therefore expression and/or the active means of regulating Th2 relevant cell factor genes produce that also provide such as the NF-AT interaction protein of NIP45 are provided.
Therefore, the invention provides expression or the active method of regulating the Th2 relevant cell factor production of cell that relates to one or more transcription factors of Th2 relevant cell factor genetic expression by adjusting.In an embodiment of the inventive method,, make that the generation of Th2 relevant cell factor of cell is regulated with regulating transcription factor expression or active medicine exposing cell.In one embodiment, the transcription factor that is conditioned is accredited as to cooperate with the NF-AT family protein regulates the transcription factor (for example c-Maf or NIP45) of Th2 relevant cell factor genetic expression.In another embodiment, the transcription factor that is conditioned is maf family protein (for example c-Maf or such as the little maf albumen of p18).In an embodiment again, described transcribing (transcnption) is NF-AT interaction protein (for example NIP45).In the suitableeest embodiment, the activity that is characterized as effect adjusting transcription factor in born of the same parents of adjusting medicine of the present invention.In one embodiment, stimulate transcription factor expression and/or active pungency medicine exposing cell by using, the Th2 relevant cell factor of irritation cell produces.In another embodiment of the inventive method,, suppress the generation of the Th2 relevant cell factor of cell by with suppressing transcription factor expression and/or active suppressive drug exposing cell.
As confirming that in an embodiment although c-Maf is responsible for the tissue specificity of IL-4 genetic expression, c-Maf and one or more other transcription factor synergies activate the IL-4 genetic transcription.Specifically, c-Maf and NF-AT albumen synergy stimulate IL-4 genetic expression.In addition, other member who has confirmed NF-AT albumen and transcription factor AP-1/CREB/TF family participates in regulating Th1 and Th2 relevant cell factor expression of gene.As further confirming among the embodiment,, stimulate expression from the promotor that contains the NF-AT site with interactional albumen NIP45 of NF-AT and NF-AT synergy.In addition, when having all three kinds of factor c-Maf, NF-AT and NIP45, strengthened Th2 relevant cell factor expression of gene.Therefore, in another embodiment, the method for the present invention of regulating the generation of cell Th2 relevant cell factor can comprise with regulating transcription factor expression or active multiple medicine exposing cell.Therefore, in the inventive method with first kind of medicine exposing cell, this method can comprise with one or more other medicines of regulating regulating one or more other transcription factor activities that Th2 relevant cell factor gene works again and contacts this cell.Other transcription factor expression of described adjusting or active other medicines preferably are selected from NF-AT family protein, NF-AT interaction protein, maf family protein and AP-1 family protein.
As proving in an embodiment, little maf albumen (for example p18) can check the Th2 relevant cell factor expression of gene by pros-and-cons type activator (for example c-Maf) mediation again.Therefore, in an embodiment again, regulating the inventive method that cell Th2 relevant cell factor produces comprises with regulating (that is, stimulate or suppress) little maf protein expression or active medicine self or uniting with the medicine (for example other maf family protein, NF-AT family protein or NF-AT interaction protein) of regulating other transcription factor activity and contacts described cell.This little maf albumen is preferably p18.Proteic other example of little maf comprises mafK, mafF and mafG.
A. pungency medicine
According to method of the present invention, be the generation of irritation cell Th2 relevant cell factor, with stimulating transcription factor (for example c-Maf, NIP45, p18) expression and/or the active pungency medicine of regulating the genetic expression of Th2 relevant cell factor to contact this cell.Can produce the Th2 relevant cell factor at cell type (for example Th1 cell, B cell or the non-lymphoidocyte) moderate stimulation of not expressing this cytokine usually.In addition, can produce the Th2 relevant cell factor, break up along the Th2 approach to impel them, rather than break up along the Th1 approach at auxiliary precursor cell (Thp) moderate stimulation.
The pungency medicine of recommending is the nucleic acid molecule of the transcription factor of coding and regulating Th2 relevant cell factor genetic expression, wherein with this nucleic acid molecule to be suitable in cell, expressing the form transfered cell of this transcription factor.For example, c-Maf cDNA is cloned into recombinant expression vector, and this carrier is transfected into cell.As confirming that in embodiment 3 c-maf recombinant expression vector ectopic expression in Th1 cell, B cell or non-lymphoidocyte causes the IL-4 promotor to activate.In addition, (following more go through) under proper condition stimulates endogenous IL-4 genetic transcription, and the cell that causes not expressing this cytokine usually produces IL-4 (referring to embodiment 4).
For in cell, expressing the maf family protein, generally at first the cDNA of maf family is imported recombinant expression vector with standard molecular biological technique.Can be by for example using polymerase chain reaction (PCR) amplification or obtaining the cDNA of maf family by screening suitable cDNA library.The nucleotide sequence of the maf cDNA of family known in the art can be used it for design PCR primer with by standard pcr amplification cDNA or be used to design hybridization probe, and this probe can be used for the standard hybridizing method and screen the cDNA library.The cDNA of this maf family is preferably the c-maf proto-oncogene.Kurscher C. and Morgan, J.I. (1995) Mol.Cell.Biol.15:246-254 disclose the nucleotide sequence of Mammals (mouse) c-maf cDNA and the aminoacid sequence of supposition, and are stored in the GenBank database with retrieval number S74567.This Mammals c-maf and bird v-maf sequence (are disclosed in Nishizawa, M.K. etc. (1989) Proc.Natl.Acad.Sci.USA86:7711-7715 and GenBank retrieve number D28598 and D28596) the height homology, show that c-maf is quite conservative between planting.Can use standard molecular biological technique (for example PCR or cDNA library screening) and, separate the c-mafcDNA that comprises other human Mammals kind based on the primer or the probe of mouse or bird sequences Design.Also be stored in the GenBank database with the human Partial cDNA Sequence of mouse c-mafcDNA homologous with retrieval number H24189 and N75504.Also known other maf family member's in this area sequence, for example MafB (Kataoka, K. etc. (1994) Mol.Cell Biol.14:7581-91; GenBank retrieval number D28600), MafG (Kataoka etc. (1994) Mol.Cell Biol.14:7581-91; GenBank retrieval number D28601 and D28602), MafF (GenBank retrieval number D16184) and MafK (Igarashi, K. etc. (1995) J.Biol.Chem.270:7615-7624; GenBank retrieval number D16187 and D42124).
Behind separation or the amplification maf cDNA of family, this dna fragmentation is imported expression vector.Term used herein " carrier " refers to transport the nucleic acid molecule of another nucleic acid that its connects.A kind of bearer type is " plasmid ", and it refers to wherein can connect the circular double stranded DNA ring of other DNA section.Another type of carrier is a virus vector, wherein other DNA section can be connected into this viral genome.Some carrier can be in importing its host cell self-replicating (bacteria carrier and the additive type Mammals carrier that for example have the bacterium replication orgin).Other carrier (for example non-add type Mammals carrier) is integrated into the genome of host cell when importing host cell, and therefore duplicates with host genome.In addition, some carrier can instruct the expression of gene that it can be operatively connected.This carrier is referred to herein as " recombinant expression vector " or is called for short " expression vector ".Generally speaking, the expression vector that utilizes in the recombinant DNA technology often is the plasmid form.In this manual, can exchange use " plasmid " and " carrier ", because plasmid is the carrier format of normal use.Yet, the present invention includes the expression vector that those play this other form of same function, for example virus vector (for example replication defect type retrovirus, adenovirus and adeno-associated virus).
Recombinant expression vector of the present invention comprises the nucleic acid molecule that is suitable for expressing the form of this nucleic acid molecule in host cell, mean that described recombinant expression vector comprises one or more adjusting sequences, this nucleic acid molecule that desire is expressed is selected and functionally be connected to described adjusting sequence based on host cell that is used to express and required expression level.In recombinant expression vector, " functionally connect " means that the purpose nucleotide sequence is connected to this adjustings sequence (for example in-vitro transcription/translation system during maybe when this carrier importing host cell in host cell) in the mode that allows to express this nucleotide sequence.Term " adjusting sequence " comprises promotor, enhanser and other expression controlling elements (for example polyadenylation signal).This adjusting sequence description is in for example Goeddel; Gene Expression Technology:Methods in Enzymology185, AcademicPress, San Diego, CA (1990).Regulate sequence and comprise those sequences that in many host cell types, instruct the constitutive expression nucleotide sequence, those sequence (for example tissue specificity adjusting sequence) or those sequences (for example induction type adjusting sequence) that only instruct nucleotide sequence to express under certain conditions that only in some host cell, instruct nucleotide sequence to express.
The design that it will be understood to those of skill in the art that expression vector can be depended on selection that will transformed host cells, required protein expression level etc.When being used for mammalian cell, provide the controlled function of described expression vector usually by viral regulatory element.For example, Chang Yong promotor is derived from polyoma virus, adenovirus, cytomegalovirus and simian virus 40.The limiting examples of mammalian expression vector comprises pCDM8 (Seed, B., (1987) Nature329:840) and pMT2PC (Kaufman etc. (1987), EMBO J.6:187-195).Commercially available have various mammalian expression vectors that carry the different adjustment sequence.Be the described nucleic acid of constitutive expression in mammalian host cell, the adjusting sequence of recommendation is cytomegalovirus promotor/enhanser.In addition, the induction type regulation system that is used for mammalian cell known in the art, for example the system of wherein being expressed by the heavy metal ion regulatory gene is (referring to (1982) Cell29:99-108 such as for example Mayo; Brinster etc. (1982) Nature296:39-42; Searle etc. (1985) Mol.Cell.Biol.5:1480-1489), (be stated from HeatShock Response by what heat shock was regulated referring to (1991) such as for example Nouer, editor Nouer, L., CRC, Boca Raton, FL, 167-220 page or leaf), by hormone regulation (referring to (1981) Nature294:228-232 such as for example Lee; Hynes etc. (1981) Proc.Natl.Acad.Sci.USA78:2038-2042; Klock etc. (1987) Nature329:734-736; Israel﹠amp; Kaufman (1989) Nucl.Acids Res.17:2589-2604; Announce WO93/23431 with PCT), FK506 associated molecule (the announcing WO94/18317) of regulating or tsiklomitsin (Gossen, M. and Bujard, H. (1992) Proc.Natl.Acad.Sci.USA89:5547-5551 that regulate referring to for example PCT; Gossen, M. etc. (1995) Science 268:1766-1769; PCT announces WO94/29442; Announce WO96/01313 with PCT).Have, many tissue specificities known in the art are regulated sequence, comprise albumin promoter (liver specificity again; Pinkert etc. (1987) Genes Dev.1:268-277), lymph specificity promoter (Calame and Eaton (1988) Adv.Immunol.43:235-275), particularly TXi Baoshouti promotor (Winoto and Baltimore (1989) EMBO J.8:729-733) and immune protein promotor (Banerji etc. (1983) Cell33:729-740; Queen and Baltimore (1983) Cell33:741-748) lymph specificity promoter (Calame and Eaton (1988) Adv.Immunol.43:235-277), neuronal specificity promotor (neurofilament promotor for example; Byrne and Ruddle (1989) Proc.Natl.Acad.Sci.USA86:5473-5477), pancreas specificity promoter (Edlund etc. (1985) Science230:912-916) and mammal galactophore specificity promoter (whey promotor for example; United States Patent (USP) 4,873, No. 316 and European application publication No. 264,166).Also comprise growing and regulate promotor, for example mouse hox promotor (Kessel and Gruss (1990) Science249:374-379) and afp promoter (Campes and Tilghman (1989) Genes Dev.3:537-546).
Can carrier DNA be imported mammalian cell by conventional rotaring dyeing technology.Various forms of term used herein " transfection " refers to the various technology that exogenous nucleic acid (for example DNA) imported mammalian host cell of this area approval, comprises coprecipitation of calcium phosphate, deae dextran mediation transfection, fat transfection or electroporation.Can in (molecular clonings: laboratory manual, second edition, Cold Spnng Harbor Laboratory press (1989)) such as Sambrook and other laboratory manual, find the appropriate method of transfection host cell.
Be the stable transfection mammalian cell, known to the expression vector and the rotaring dyeing technology that use, only there is the small portion cell foreign DNA may be integrated into its genome.For differentiating and select these intasomies, but the gene of the selective marker (for example to antibiotics resistance) of generally will encoding imports host cell with goal gene.The marks packets selected of recommending is drawn together those and is given mark to medicine (for example G418, Totomycin and methotrexate) resistance.But the nucleic acid importing host cell of the selective marker of can on the carrier that is independent of coding maf family protein, will encode, or be preferably on the identical carrier and import.Can by medicament selection differentiate with importing nucleic acid stability cells transfected (but the cell of for example having integrated this selectable marker gene will survive, and other necrocytosis).
Use nucleotide sequence known in the art or disclosed herein, can prepare the nucleic acid molecule that is suitable for other transcription factor of the coding and regulating Th2 relevant cell factor genetic expression of the form of expression transcription factor in host cell according to above-mentioned.This nucleotide sequence can be used to design the PCR primer of permission by standard method amplification cDNA, or is used to design use standard hybridizing method and is used to screen the hybridization probe in cDNA library.The nucleotide sequence of NIP45 and the aminoacid sequence of supposition are disclosed in SEQ IDNO:5 and 6 respectively.Proteic nucleotide sequence of little maf that comprises p18, mafK, mafF and mafG known in the art and speculating acid sequence (referring to for example Fujiwara, K.T. etc. (1993) Oncogene8:2371-2380; Igarashi, K. etc. (1995) J.Biol.Chem.270:7615-7624; Andrews, N.C. etc. (1993) Proc.Natl.Acad.Sci.USA90:11488-11492; Kataoka, K. etc. (1995) Mol.Cell.Biol.15:2180-2190).Nucleotide sequence and the speculating acid sequence that comprises the NF-AT family protein of NF-ATp, NF-ATc, NF-AT4/x/c3 and NF-AT3/c4 known in the art.Identified four NF-AT family members (referring to for example Emmel, E.A. etc. (1989) Science246:1617-1620; Flanagan, W.M. etc. (1991) Nature352:803-807; Jain, J. etc. (1993) Nature365:352-355; McCaffrey, P.G. etc. (1993) Science262:750-754; Rao, A. (1994) Immunol.Today15:274-281; Norhrop, J.P. etc. (1994) Nature369:497).This NF-AT cDNA is preferably the cDNA of NF-ATp.The nucleotide sequence of Mammals NF-ATpcDNA and the aminoacid sequence of supposition are disclosed in McCaffrey, P.G. etc. (1993) Science262:750-754.The nucleotide sequence of Mammals NF-ATc cDNA and the aminoacid sequence of supposition are disclosed in Northrop, J.P. etc. (1994) Nature369:497, and be stored in the GenBank database with retrieval number U08015.The nucleotide sequence of Mammals NF-AT3 cDNA and NF-AT4cDNA and the aminoacid sequence of supposition are disclosed in Hoey, T. etc. (1995) Immunity2:461-472.The nucleotide sequence of AP-1 family protein known in the art and the aminoacid sequence of supposition.For example, the nucleotide sequence of human c-fos and the aminoacid sequence of supposition are disclosed in van Straaten, F. etc. (1983) Proc.Natl.Acad.Sci.USA80:3183-3187.The nucleotide sequence of human c-jun and the aminoacid sequence of supposition are disclosed in Bohmann, D. etc. (1987) Science238:1386-1392.Human jun-B and the nucleotide sequence of jun-D and the aminoacid sequence of supposition are disclosed in Nomura, N. etc. (1990) Nucl.AcidsRes.18:3047-3048.Human fra-1 and the nucleotide sequence of fra-2 and the aminoacid sequence of supposition are disclosed in Matsui, M. etc. (1990) Oncogene5:249-255.
The stimulating drug that stimulates another form that the Th2 relevant cell factor expresses in cell be stimulate the endogenous transcription factor of regulating Th2 relevant cell factor genetic expression in the cell (for example, such as the maf family protein of c-Maf or p18 or such as NIP45 with the interactional albumen of NF-AT) expression or active chemical compound.Use and select to stimulate the screening of this transcription factor expression or active compound to detect this compound of discriminating.The example that suitable screening detects is described in greater detail in following V trifle.
Except expression or active first medicine with first transcription factor of stimulate regulating the genetic expression of Th2 relevant cell factor, stimulating method of the present invention can comprise using to stimulate regulating expression or active one or more other medicines of one or more other transcription factors that Th1 or the genetic expression of Th2 relevant cell factor works.In embodiment 4, be presented at M12B lymphoma cell moderate stimulation and express endogenous IL-4 and need import c-Maf expression vector and NF-AT expression vector, prove that therefore c-Maf and NF-AT synergy activates IL-4 and transcribes, and c-maf is responsible for to the tissue specificity of expressing.In embodiment 14, show again by coexpression c-Maf, NF-AT and NIP45 to strengthen the expression that stimulates endogenous IL-4 in the M12 B lymphoma cell.Though those of skill in the art will appreciate that some cell and can express endogenous c-Maf, NF-AT and/or the NIP45 of capacity, making only uses single medicine promptly may be enough to stimulate Th2 relevant cell factor expression of gene, but may need to stimulate a plurality of transcription factors to stimulate the Th2 relevant cell factor to produce with some cell type in some cases to obtain purpose, described a plurality of transcription factors for example be c-Maf and NF-AT together, c-Maf and NIP45 together or all these three kinds of albumen (c-Maf, NF-AT and NIP45).
Therefore, in with the inventive method that stimulates first kind of transcription factor expression or active first kind of medicine exposing cell, this method can comprise with at least a other medicines again and contacts this cell, and described at least a other medicines stimulate regulating the expression or the activity of at least a other transcription factor that Th1 or the genetic expression of Th2 relevant cell factor works.Described expression or active at least a other transcription factor of being regulated preferably are selected from NF-AT family protein, NF-AT interaction protein, maf family protein and AP-1 family protein.For example, stimulating method of the present invention can relate to expression or the activity of using first kind of medicine irritation c-Maf, use second kind of medicine irritation or NF-AT family protein or with expression or the activity of the interactional albumen of NF-AT family protein (for example NIP45).In another embodiment, stimulating method of the present invention relates to expression or the activity of using first kind of medicine irritation c-Maf, uses expression or the expression or the activity active and the third medicine irritation of use and the interactional albumen of NF-AT family protein (for example NIP45) of second kind of medicine irritation NF-AT family protein.At cell moderate stimulation NF-AT or the active recommendation medicine of NIP45 is the recombinant expressed of NF-AT or NIP45 of encoding respectively, wherein this recombinant expression vector is imported this cell, and at this cell inner expression NF-AT or NIP45.The coding expression vector and the transfered cell that can as above-mentioned c-Maf expression vector, prepare NF-AT and NIP45.
Be to use interior NF-AT of one or more irritation cells or the active chemical compound of NIP45 second kind of (or additional) medicine with NF-AT in the cDNA irritation cell of NF-AT or NIP45 or the active method of replacing of NIP45 as stimulating method of the present invention.The active compound of NF-AT in the irritation cell known in the art (about summarizing referring to Rao A. (1994) Immunol.Today15:274-281).For example, stimulate some cell with phorbol ester phorbol tetradecanoic acid acetic ester (PMA) and Calcium ionophore (for example ionomycin), cause the NF-AT transposition to nucleus (referring to for example Flanagan, W.M. etc. (1991) Nature352:803-807; Jam, J. etc. (1993) Nature365:352-355).In addition, for example stimulate the T cell to cause NF-AT activation in the T cell by TXi Baoshouti (TcR) with anti-cd 3 antibodies.
Except NF-AT albumen, shown the AP-1 family member who comprises c-Jun, c-Fos, Fra-1, Fra-2, Jun B and Jun D participate in regulating the expression of Th1 and Th2 relevant cell factor gene (for example IL-2 and IL-4) (referring to for example Rao, A. etc. (1994) Immunol.Today15:274-281; Jam, J. etc. (1993) Nature365:352-355; Boise, L.H. etc. (1993) Mol.Cell.Biol.13:1911-1919; Rooney, J. etc. (1995) Immunity2:545-553; Rooney, J. etc. (1995) Mol.Cell.Biol.15:6299-6310).Although these factors are not responsible for the Th1/Th2 specificity of described cytokine gene expression, and these factors be it seems not work in coordination with c-Maf and are regulated IL-4 expression of gene (referring to embodiment), but shown that the AP-1 family member strengthens the expression (referring to for example Rooney, J. etc. (1995) Immunity2:545-553) of IL-4 in the Th2 cell.Therefore, in some cases, except stimulating c-Maf activity (with stimulating the NF-AT activity), it may be useful also stimulating AP-1 family protein activity.Therefore, in one embodiment, stimulating method of the present invention relates to first kind of medicine irritation c-Maf of use expresses or activity, uses second kind of proteic expression of medicine irritation AP-1 or activity.In another embodiment, the present invention relates to use expression or the activity of first kind of medicine irritation c-Maf, use second kind of proteic expression of medicine irritation NF-AT or active proteic expression of the third medicine irritation AP-1 or the activity used.Also can unite with maf, AP-1 and/or NF-AT family protein and regulate the NIP45 activity.
At the active recommendation medicine of cell moderate stimulation AP-1 is that coding AP-1 is proteic recombinant expressed, and wherein this recombinant expression vector is imported into this cell, and at this cell inner expression AP-1 albumen.Can as above-mentioned c-Maf expression vector, prepare AP-1 coding expression vector and transfered cell.Perhaps, can use the interior active chemical compound of AP-1 of one or more irritation cells as the other medicines in the stimulating method of the present invention.The active compound of AP-1 in the irritation cell known in the art comprises PMA/ Calcium ionophore (for example ionomycin) and anti-cd 3 antibodies.
B. suppressive drug
According to method of the present invention,, contact this cell with transcription factor (for example c-Maf, NIP45, the p18) expression and/or the active suppressive drug that suppress the genetic expression of adjusting Th2 relevant cell factor for suppressing the generation of cell Th2 relevant cell factor.In one embodiment, regulate the expression of transcription factor of Th2 relevant cell factor genetic expression and/or the generation that active medicine contacts this cell inhibition cell Th2 relevant cell factor with regulating with the cooperation of NF-AT family protein.In another embodiment, by with expression or the active medicine exposing cell of regulating Th2 idiosyncratic transcription factor (being preferably c-Mar), suppress the Th2 relevant cell factor generation of this cell.In another embodiment, by with expression or the active medicine exposing cell of regulating the interactional albumen of surplus NF-AT family protein (being preferably NIP45), suppress the Th2 relevant cell factor generation of this cell.In an embodiment again,, suppress the generation of this cell Th2 relevant cell factor by with regulating little maf protein expression or active medicine exposing cell.Discuss as above-mentioned stimulating method, inhibition method of the present invention can comprise with two or more regulates transcription factor expression or the active medicine exposing cell that two or more regulate the genetic expression of Th2 relevant cell factor, and described transcription factor comprises maf family protein, NF-AT family protein, NF-AT interaction protein and AP-1 family protein.
Can or assist the generation that suppresses the Th2 relevant cell factor in the precursor cell (Thp) at for example Th2 cell, to impel them along Th1 approach rather than the differentiation of Th2 approach.Suppressive drug of the present invention can be for example to work to suppress binding molecule in this transcription factor expression or the active born of the same parents.Term used herein " binding molecule in the born of the same parents " is included in effect nucleic acid (for example mRNA molecule) inhibition this proteic expression or activity by being bonded to albumen or proteins encoded in the born of the same parents.Below the example of the interior binding molecule of born of the same parents comprises antisense nucleic acid, intracellular antibody and dominant inhibitor in greater detail.
In one embodiment, suppressive drug of the present invention is gene or the part complementary antisense nucleic acid molecule of described gene or the recombinant expression vector of the described antisense nucleic acid molecule of encoding with the encoding transcription factor (for example, such as the maf family protein of c-Maf or p18 or such as the NF-AT interaction protein of NIP45).Use antisense nucleic acid regulate downwards being expressed in of specific protein in the cell well-known this area in (referring to for example Weintraub, H. etc., as the sense-rna of genetic analysis molecular tool, Reviews-Trends in Genetics, the 1st (1) rolls up 1986; Askari, F.K. and McDonell, W.M. (1996) N.Eng.J.Med.334:316-318; Bennett, M.R. and Schwartz, S.M. (1995) Circulation92:1981-1993; Mercola, D. and Cohen, J.S. (1995) Cancer Gene Ther.2:47-59; Rossi, J.J. (1995) Br.Med.Bull.51:217-225; Wagner, R.W. (1994) Nature372:333-335).Antisense nucleic acid molecule comprises coding strand (for example mRNA sequence) the complementary nucleotide sequence with another nucleic acid molecule, and thus can hydrogen bond be connected to the coding strand of described another nucleic acid molecule.With the sequence complementary antisense sequences of mRNA can with the coding region of this mRNA in, 5 ' or the 3 ' non-translational region of this mRNA or the sequence complementation that the joining region between coding region and the non-translational region (for example being arranged in being connected of 5 ' non-translational region and coding region) found.In addition, antisense nucleic acid can with for example sequence complementation of transcriptional initiation sequence or regulatory element of regulatory region of the gene of this mRNA of coding.Antisense nucleic acid be preferably designed as with this coding strand on before the initiator codon or cross over district's complementation in the 3 ' non-translational region of the district of initiator codon or mRNA.Can suppress the antisense nucleic acid of the transcription factor of this paper discussion based on the nucleotide sequence design of disclosed herein or transcription factor known in the art, and make up according to Wo Sen and Ke Like basepairing rule at cell inner expression.
Antisense nucleic acid can be with various multi-form existence.For example, this antisense nucleic acid can be only with the part complementary oligonucleotide of maf family gene.Can be with chemosynthesis program construction antisense oligonucleotide known in the art.Can use the Nucleotide chemistry synthesising antisense scant nucleotide of naturally occurring Nucleotide or various modifications, this modification is design in order to the biological stability that strengthens this molecule or strengthens this antisense nucleic acid and the physical stability of the duplex that forms between the phosphorothioate odn is arranged, for example can use thiophosphoric acid derivative and acridine substituted nucleotide.For suppressing the expression of the transcription factor of cell in the culture, one or more antisense oligonucleotides can be added the cell in the substratum, typical concn is 200 μ g oligonucleotide/ml.
Perhaps, can use wherein subclone with the localized nucleic acid of antisense (that is, and from the nucleic acid that inserts transcribed nucleic acid will with purpose target nucleic acid antisense) expression vector, produce antisense nucleic acid biologically.Can be connected to the adjusting sequence of the nucleic acid of antisense positional cloning selection operation, this regulates sequence-directed this antisense rna molecule at the purpose cell inner expression, for example can select promotor and/or enhanser or other to regulate sequence, they instruct composing type, tissue specificity or the inducible expression of sense-rna.Carrier antisense expression vector as above-mentioned recombinant expression vector is except going into this carrier with this cDNA (or its part) with the antisense positional cloning.This antisense expression vector can be the form of recombinant plasmid, phagemid or attenuated virus for example.As above-mentioned recombinant expression vector, use the standard rotaring dyeing technology with this antisense expression vector transfered cell.
In another embodiment, the antisense nucleic acid as suppressive drug is a ribozyme.Ribozyme is to have can cutting of ribonuclease activity to have the catalytic RNA molecule of the single-chain nucleic acid (for example mRNA) in the district of complementation with it (about ribozyme is summarized referring to for example Ohkawa J. etc. (1995) J.Biochem.118:251-258; Sigurdsson, S.T. and Eckstem, F. (1995) TrendsBiotechnol.13:286-289; Rossi, J.J. (1995) Trends Biotechnol.13:301-306:Kiehmtopf, M. etc. (1995) J.Mol.Med.73:65-71).There is the specific ribozyme can be to the mRNA of the transcription factor of coding this paper discussion based on the nucleotide sequence design of this transcription factor.For example, can make up tetrahymena L-19IVS RNA derivative, wherein the base sequence complementation of the cutting of the desire among the base sequence of avtive spot and c-mafmRNA or other transcription factor mRNA.Referring to the United States Patent (USP) of for example Cech etc. 4,987, No. 071 and 5,116, No. 742.Perhaps, can use c-maf mRNA (or other transcription factor mRNA) from the RNA library of molecules, to select catalytic RNA with specific ribonucleic acid enzymic activity.Referring to for example Bartel, D. and Szostak, J.W. (1993) Science261:1411-1418.
Can be the specific intracellular antibody of transcription factor that this paper is discussed in order to Maf protein expression and/or active another type suppressive drug in the inhibition cell.Use intracellular antibody suppress the intracellular protein function be known in the art (referring to for example Carlson, J.R. (1988) Mol.Cell.Biol.8:2638-2646; Biocca, S. etc. (1990) EMBO is J.9:101-108; Werge, T.M. etc. (1990) FEBS Letters274:193-198; Carlson, J.R. (1993) Proc.Natl.Acad.Sci.USA90:7427-7428; Marasco, W.A. etc. (1993) Proc.Natl.Acad.Sci.USA90:7889-7893; Biocca, S. etc. (1994) Bio/Technology12:396-399; Chen, S-Y etc. (1994) Human Gene Therapy5:595-601; Duan, L etc. (1994) Proc.Natl.Acad.Sci.USA91:5075-5079; Chen, S-Y. etc. (1994) Proc.Natl.Acad.Sci.USA9l:5932-5936; Beerli, R.R. etc. (1994) J.Biol.Chem.269:23931-23936; Beerli, R.R. etc. (1994) Biochem.Biophys.Res.Commun.204:666-672; Mhashilkar, A.M. etc. (1995) EMBO is J.14:1542-1551; Richardson, J.H. etc. (1995) Proc.Natl Acad.Sci. USA92:3137-3141; The PCT publication No. WO94/02610 of Marasco etc.; Announce WO95/03832 with the PCT of Duan etc.
For using intracellular antibody arrestin activity, prepare recombinant expression vector, the form of the described antibody chain of this vector encoded makes that when this carrier transfered cell part is expressed this antibody chain as function antibody in the born of the same parents of this cell.For suppressing transcription factor activity, be preferably in the intracellular antibody of the interior expression specificity of nuclear of this cell in conjunction with this transcription factor according to inhibition method of the present invention.Can be by removing those nucleotide sequences of coding N-terminal hydrophobicity leader sequence from this light chain of antibody and heavy chain gene, and at nucleotide sequence described light chain and heavy chain gene or N-terminal or C-terminal adding coding nuclear localization signal, (referring to for example Biocca, S. etc. (1990) EMBO J.9:101-108 to reach the nuclear expression of intracellular antibody; Mhashilkar, A.M. etc. (1995) EMBO are J.14:1542-1551).The nuclear localization signal of the nuclear guiding that is used for described intracellular antibody chain of recommending be the SV40 large T antigen nuclear localization signal (referring to Biocca, S. etc. (1990) EMBOJ.9:101-108; Mhashilkar, A.M. etc. (1995) EMBO are J.14:1542-1551).
Be preparation intracellular antibody expression vector, from secretion the hybridoma separation of the monoclonal antibody of maf protein-specific being encoded usually has the light chain of antibody of specific antibody chain and the cDNA of heavy chain to the Maf family protein of for example this paper discussion or the purpose target protein of other transcription factor.This area described anti-Maf family protein Antiserum Preparation (referring to for example Fujiwara, K.T. etc. (1993) Oncogene8:2371-2380; Kataoka, K. etc. (1993) J.Virol.67:2133-2141; Kerppola, T.K. and Curran, T. (1994) Oncogene9:675-684; Igarashi, K etc. (1995) Proc.Natl.Acad.Sci USA92:7445-7449).Can prepare anti-Maf protein antibodies by with the suitable object (for example rabbit, goat, mouse or other Mammals) of Maf protein immunogen immunity.Suitable immunogen preparation can contain for example recombinant expressed Maf albumen or the Maf peptide of chemosynthesis.Said preparation can also comprise the adjuvant or the similar immunostimulation medicine of or Freund complete such as Fu Shi.Can obtain producing the cell of antibody from this object, and be used for preparing monoclonal antibody by standard technique, described technology such as the hybridoma technology of at first describing by Kohler and Milstein (1975, Nature256:495-497) (also referring to (1981) J.Immunol 127:539-46 such as Brwon; Brown etc. (1980) J Biol Chem255:4980-83; Yeh etc. (1976) PNAS76:2927-31; With (1982) Int.J.Cancer29:269-75 such as Yeh).The technology that produces monoclonal antibody hybridoma is well-known (generally referring to R.H.Kenneth, be stated from Monoclonal Antibodies:A New Dimension In BiologicalAnalyses, Plenum Publishing Corp.New York, New York (1980); E.A.Lerner (1981) Yale J.Biol.Med., 54:387-402; M.L.GeRer etc. (1977) Somatic Cell Genet., 3:231-36).In brief, merge with immortal cell line (being generally myelomatosis) and from mammiferous lymphocyte (being generally splenocyte) with the immunity of above-mentioned maf protein immunogen, and the culture supernatant of the hybridoma that screening obtains produces the hybridoma of specificity in conjunction with the proteic monoclonal antibody of this Maf to differentiate.Many well-known any of scheme that are used to merge lymphocyte and immortalized cell system all can be applicable to produce the purpose of anti-Maf protein monoclonal antibody (referring to (1977) Nature266:550-52 such as for example G.Galfre; Somatic Cell Genet. such as Gefter see above-mentioned quoting; Lerner, Yale J.Biol.Med. sees above-mentioned quoting; Kenneth, Monoclonal Antibodies sees above-mentioned quoting).In addition, those skilled in the art will appreciate that the variation that many this methods are arranged is also useful.Usually, this immortal cell line (for example myeloma cell line) is derived from the mammalian species identical with this lymphocyte.For example, the lymphocyte of the immunogen preparation mice immunized of the present invention of can using by oneself in the future and immortalization mouse cell lines merge, and produce murine hybridoma.The immortal cell line of recommending is the responsive mouse myeloma cell line of substratum (" HAT substratum ") to containing xanthoglobulin, aminopterin and thymidine.Any fusion object that all can be used as according to standard technique in any multiple myeloma cell line, for example P3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/O-Ag14 myelomatosis system.These myelomatosis systems can (Rockville Md.) obtains from American type culture collection (ATCC).Usually use polyoxyethylene glycol (" PEG "), HAT responsive type murine myeloma cell and mouse boosting cell are merged.The hybridoma that uses HAT substratum screening to obtain from fusion then, this substratum kill and do not merge and the myeloma cell of fusion not yet in effect (unproductively fused) (do not merge splenocyte because unconverted and will death after several days).Differentiate the hybridoma of generation specificity by the antibody that for example uses standard ELISA to detect screening hybridoma culture supernatant in conjunction with the proteic monoclonal antibody of this maf.
The method of replacing of the hybridoma of preparation secrete monoclonal antibody is, can be by making up immunoglobulin (Ig) library (for example antibody phage display libraries) with this albumen or the reorganization of its peptide screening, separate specificity thus in conjunction with this proteic immunoglobulin library member, and the monoclonal antibody of the transcription factor that discriminating and separation and combination this paper discuss.Commercially available test kit (the recombinant phages antibody system of Pharmacia (Recombinant PhageAntibody System) for example, the Directory Number 27-9400-01 that preparation and screening phage display library are arranged; With phage display test kit (the Phage Display Ki) t of Stratagene SurfZAPTM, Directory Number 240612).In addition, being particularly useful for making and screening the example that antibody shows the method in library and reagent can be referring to No. 5,223,409, United States Patent (USP) such as for example Ladener; International publication number WO92/18619 such as Kang; International publication WO91/17271 such as Dower; International publication WO92/20791 such as Winter; International publication number WO92/15679 such as Markland; International publication WO93/01288 such as Breitling; International publication number WO92/01047 such as McCafferty; International publication number WO92/09690 such as Garrard; Fuchs etc. (1991) Bio/Technology9:1370-1372; Hay etc. (1992) Hum Antibod Hybridomas 3:81-85; Huses etc. (1989) Science246:1275-1281; Griffiths etc. (1993) EMBO J 12:725-734; Hawkins etc. (1992) J Mol Biol226:889-896; Clarkson etc. (1991) Nature 352:624-628; Gram etc. (1992) PNAS 89:3576-3580; Garrad etc. (1991) Bio/Technology 9:1373-1377; Hoogenboom etc. (1991) Nuc Acid Res19:4133-4137; Barbas etc. (1991) PNAS 88:7978-7982; With Nature (1990) 348:552-554 such as McCafferty.
In case differentiate the specific monoclonal antibody of purpose transcription factor (for example or hybridoma deutero-monoclonal antibody or from the recombinant antibodies of combinatorial library), then separate the DNA of this monoclonal antibody light chain of coding and heavy chain by standard molecular biological technique.For hybridoma deutero-antibody, can obtain light chain and heavy chain cDNA by for example pcr amplification or cDNA library screening.For such as recombinant antibodies, can in the library screening process, show that from isolating displaying packing (for example phage) reclaims the cDNA of coding light chain and heavy chain from phage display library.Known in the art can be in order to the light chain of antibody and the heavy chain nucleotide sequence of preparation PCR primer or cDNA library probe.For example, many this sequences are disclosed in Kabat, E.A. etc. (1991) Sequences of Proteins of Immunological Interest, the 5th edition, U.S. Department of Health and Human Service, NIH Publication No.91-3242 and " Vbase " ethnic group are sequence library.
In case obtain light chain of antibody and sequence of heavy chain, then use standard method that it is cloned into recombinant expression vector.As discussed above, remove, will encode nuclear localization signal (for example from the SV40 large T antigen) is connected to encode light chain and heavy chain in framework the sequence of the hydrophobicity leader sequence of coding light chain and heavy chain or amino or C-terminal.This expression vector several one of multi-form intracellular antibodies of can encoding.For example, in one embodiment, this vector encoded full length antibody light chain and heavy chain make full length antibody express in born of the same parents.In another embodiment, this vector encoded full-length light chains and the VH/CH1 district of heavy chain only make the Fab fragment express in born of the same parents.In the most preferred embodiment, this vector encoded single-chain antibody (scFv), wherein the variable region of light chain and heavy chain is by pliable and tough peptide linker ((Gly for example 4Ser) 3) connect and express as single chain molecule.Be to suppress transit cell record factor active, the expression vector of this transcription factor specificity intracellular antibody of coding is imported this cell by all standard transfection methods as discussed above.
A form again of suppressive drug of the present invention is the inhibition form of the transcription factor (for example maf albumen) discussed of this paper, and this paper also is called the dominant inhibitor.Known maf protein family and other AP-1 family member such as Fos and Jun homodimerization and heterodimerization (referring to for example Kerppola, T.K. and Curran, T. (1994) Oncogene9:675-684; Kataoka, K. etc. (1994) Mol.Cell.Biol.14:700-712).A kind of means that suppress the dimeric transcription factor activity of formation are by using the dominant inhibitor, it has the ability Dimerized with functional transcription factor, but the ability (referring to for example Petrak, D. etc. (1994) J.Immunol.153:2046-2051) that lacks activated transcription.By Dimerized with functional transcription factor, this dominant inhibitor can suppress their functionally active.The means that this process can natural existence be expressed as regulatory gene.For example, some " little " maf albumen such as mafK, mafF, mafG and p18 are arranged, their disappearances contain N-terminal (Fujiwara, K.T. etc. (1993) Oncogene8:2371-2380 of the c-Maf 2/3rds of trans-activation domain; Igarashi, K. etc. (1995) J.Biol.Chem.270:7615-7624; Andrews, N.C. etc. (1993) Proc.Natl.Acad.Sci.USA90:11488-11492; Kataoka, K. etc. (1995) Mol.Cell.Biol.15:2180-2190).The proteic homodimer of little maf is as the negative transcriptional regulatory agent (Igarashi that works, (1994) Nature367:568-572 such as K.), and shown the trans-activation (Kataoka, K etc. (1996) Oncogene12:53-62) that three (MafK, MafF and MafG) competitive inhibition in the little maf albumen are mediated by the v-Maf cancer protein.In addition, having identified MafB is the interaction partner of Ets-1, and shows the trans-activation and the inhibition red corpuscle class differentiation (Sieweke, M.H. etc. (1996) Cell85:49-60) of the transferrin receptor of its system Ets-1 mediation.
Therefore, suppressive drug of the present invention can be to have with the Dimerized ability of c-Maf but lack the proteic form of Maf of activated transcription ability.The proteic this dominant form of Maf can be little Maf albumen (for example MafK, MafF, MafG), the MafB of for example natural shortage trans-activation domain or the c-Maf mutant form of wherein removing trans-activation domain.Use the proteic recombinant expression vector of coding Maf, can in this cell, express this dominant Maf albumen by standard transfection method transfered cell.Be the c-Maf mutant form of expression deletion trans-activation domain, remove the nucleotide sequence of the aminoterminal trans-activation domain of coding c-Maf by the standard recombinant dna technology from c-maf cDNA.Preferably remove amino acid/11-122 at least.More preferably remove amino acid/11-187 or amino acid/11-257.The nucleotide sequence that keeps coding alkalescence-leucine zipper district.The cDNA of this brachymemma is inserted recombinant expression vector, then this carrier transfered cell is expressed the c-maf of the disappearance trans-activation domain of this brachymemma with permission in this cell.
Can be expression or the active chemical compound that suppresses endogenous maf family protein in the cell in order to the suppressive drug of the proteic expression of maf and/or active another type in the inhibition cell.Can use the screening of selecting to suppress expression of maf family protein or active compound to detect, differentiate this compound.In following V trifle the example that suitable screening detects is described in more detail.
Discuss about the pungency medicine as above-mentioned, inhibition method of the present invention can comprise uses one or more other suppressive drugs, and described other suppressive drug suppresses regulating the expression or the activity of one or more other transcription factors that Th1 or the genetic expression of Th2 relevant cell factor works.For example, in one embodiment, inhibition method of the present invention comprises expression or the active second kind of medicine exposing cell with the expression that suppresses the maf family protein or active first kind of medicine and inhibition NF-AT family protein or NF-AT interaction protein (for example NIP45).In another embodiment, inhibition method of the present invention comprises expression or the active second kind of medicine exposing cell with the expression that suppresses the maf family protein or active first kind of medicine and inhibition AP-1 family protein.In an embodiment again, inhibition method of the present invention comprises with the expression that suppresses the maf family protein or active first kind of medicine, the expression that suppresses the NF-AT family protein or active second kind of medicine and suppresses expression or active the third medicine exposing cell of NF-AT interaction protein (for example NIP45).The example that suppresses NF-AT albumen or NF-AT interaction protein and the proteic suppressive drug type of AP-1 comprises the chemical compound of antisense nucleic acid, intracellular antibody, dominant inhibitor and above-mentioned inhibition intrinsic protein.About the latter, the nuclear transposition of NF-ATp known in the art suppressed by immunosuppressive drug cyclosporin A and FK506 (referring to for example Rao, A. (1994) Immunol.Today15:274-281; Rao, A. (1995) J.Leukoc.Biol.57:536-542).Therefore, in an embodiment of inhibition method, will express or active medication combined use with inhibition c-Maf such as the immunosuppressive drug of cyclosporin A or FK506 (or other suppresses the related drugs of calcineurin approach).
Can in external or body, (latter further discusses) regulate the method for the present invention that cell Th2 relevant cell factor produces in following trifle.For in the external present method of carrying out, can obtain cell from the curee by standard method, and at external use pungency of the present invention or suppressive drug incubation (promptly cultivating) to stimulate or to suppress the generation of Th2 relevant cell factor respectively.For example, can obtain peripheral blood lymphocytes (PBMC), and separate by for example density gradient centrifugation with Ficoll/Hypaque from the curee.Can be by standard method eliminating or enrichment specific cells colony.For example, can be by fractionation by adsorption monocyte/macrophage on the spear material.Can select by positive and/or negative, utilize the antibody enrichment of anti-T cell or B cell surface marker or get rid of T cell or B cell, for example, use the cell that wraps the magnetic beads separation and combination mAb that is finished the secondary antibody of unifying level mAb then by with specificity one-level monoclonal antibody (mAb) incubation cell.Can pass through similar techniques, use the hemopoietic stem cell of stem cell specificity mAb (for example anti-CD34mAb) separation source from peripheral blood or marrow.Can also separate specific cell colony by fluorescence-activated cell sorting according to standard method.The monoclonal antibody of anti-cell specific surfaces mark known in the art and many have commercially available.
When causing stimulating the Th2 relevant cell factor in external use pungency medicine particularly IL-4 produces, can reclaim these cytokines from this culture supernatant and do further use.For example, the T cell that the part of this culture supernatant or its purifying can be used for cultivating is to influence external Th1 or Th2 cell development.Perhaps, the part of this culture supernatant or its purifying can be given the curee with influence, the development that Th1 replys Th2 among this curee.
In addition, can give the curee with cell, to influence the development that Th1 replys Th2 among this curee in external use pungency or suppressive drug processing.Therefore, in another embodiment, regulate the inventive method that the Th2 relevant cell factor of cell produces and also comprise and give the curee, to regulate Th1 or Th2 cells whose development among the curee thus with this cell.The cell type of recommending of in vitro modifying and giving comprises T cell, B cell and hemopoietic stem cell.For giving the curee, may be preferably in cell is given at first to remove medicine residual the culture from cell before the curee.This can for example finish by Ficoll/Hypaque gradient centrifugation cell.About genetically modified cell in vitro and then give curee's further discussion again, also can be referring to No. 5,399,346, the United States Patent (USP) of W.F.Anderson etc.II. regulate Th1 or the cytocerastic method of Th2 among the curee
Another aspect of the present invention relates to Th1 or the cytocerastic method of Th2 among the curee of regulating.Term " curee " comprises the live organism that wherein can draw immunne response.The curee who recommends is a Mammals.Curee's example comprises the mankind, monkey, dog, cat, mouse, rat, milk cow, horse, goat and sheep.As above-mentioned discussion, a kind of method teacher who regulates Th1/Th2 ratio among the curee regulates drug treating cell (for example T cell, B cell or hemopoietic stem cell) with of the present invention one or more in vitro, make that the generation of Th2 relevant cell factor of described cell is regulated, then give this curee described cell.In another embodiment, by giving the medicine that this curee regulates transcription factor activity, described transcription factor is regulated the genetic expression of Th2 relevant cell factor, makes among this curee that Th1 or Th2 cells whose development are regulated, and regulates the ratio of Th1/Th2.In an optimum implementation, this transcription factor is the maf family protein, is preferably c-Maf albumen or little maf albumen (for example p18).In another optimum implementation, this transcription factor is and the interactional albumen of NF-AT family protein, preferably NIP45.This Th2 relevant cell factor is IL-4 preferably.Can promote the development that Th2 replys among the curee by giving one or more pungency medicines of the present invention, and can promote the development that Th1 replys among the curee by giving one or more suppressive drugs of the present invention.As discussed above, in some cases, may need the activity of a plurality of transcription factors of additive regulating (for example combination of maf family protein, NF-AT family protein, NF-AT interaction protein and/or AP-1 family protein).
About comprising the pungency or the suppressive drug of nucleic acid (recombinant expression vector that comprises the encoding transcription factor, sense-rna, intracellular antibody or dominant inhibitor), can use known in the artly, this medicine be imported curee's cell nucleic acid (for example DNA) method of transfered cell in vivo.The example of this method comprises:
Direct injection: can be by directly DNA being injected into cell, and with exposed DNA transfered cell (referring to (1991) Nature332:815-818 such as for example Acsadi; Wolff etc. (1990) Science247:1465-1468).For example, can use the transfer device (for example " particle gun ") that DNA is injected into cell.This device has commercially available (for example BioRad).
Receptor-mediated DNA takes in: can also be by DNA is compound to the positively charged ion such as polylysine, and this positively charged ion is coupled to the part of cell surface receptor, and in body with the naked DNA transfered cell (referring to for example Wu, G and Wu, C.H. (1988) J.Biol.Chem.263:14621; Wilson etc. (1992) J.Biol.Chem.267:963-967; With United States Patent (USP) 5,166,320).Being combined with of DNA-ligand complex and acceptor is beneficial to by receptor-mediated endocytosis absorption DNA.Can use the DNA-ligand complex that is connected to adenovirus capsid, the natural destruction endosome of described adenovirus capsid and material is released in the tenuigenin is degraded this mixture (referring to (1991) Proc.Natl.Acad.Sci.USA88:8850 such as for example Curiel to avoid in the born of the same parents lysosome; Cristiano etc. (1993) Proc.Natl.Acad.Sci.USA90:2122-2126).
Retrovirus: well characterization the defective type retrovirus with the transgenosis that is used for the gene therapy purpose (relevant summary referring to Miller, A.D. (1990) Blood76:271).Can make up recombinant retrovirus with the purpose nucleotide sequence that is integrated into this reverse transcription virus gene group.In addition, can remove the part of this reverse transcription virus gene group, make that this retrovirus is a replication defect type.Then this replication defect type retrovirus is packaged as and is used for by standard technique by using the virus particle of helper virus target cell infection.Produce recombinant retrovirus and can be referring to the molecular biology current methods with the scheme of this virus cells infected in external or body, Ausubel, F.M etc. (editor) Greene PublishingAssociates, (1989), 9.10-9.14 joint and other standard laboratory handbook.Suitable retroviral example comprises pLJ, pZIP, pWE and pEM, and they all are well-known in the art.The example of suitable packaging virus system comprises ψ Crip, ψ Cre, ψ 2 and ψ Am.Used retrovirus in external and/or body, range gene to be imported many cell types, comprise that epithelial cell, endotheliocyte, lymphocyte, sarcoplast, liver cell, medullary cell (, wait (1985) Science230:1395-1398 referring to for example Eglitis; Danos and Mulligan (1988) Proc.Natl.Acad.Sci.USA85:6460-6464; Wilson etc. (1988) Proc.Natl.Acad.Sci.USA85:3014-3018; Armentano etc. (1990) Proc.Natl.Acad.Sci.USA87:6141-6145; Huber etc. (1991) Proc.Natl.Acad.Sci.USA88:8039-8043; Ferry etc. (1991) Proc.Natl.Acad.Sci.USA88:8377-8381; Chowdhury etc. (1991) Science 254:1802-1805; (1992) Proc.Natl.Acad.Sci.USA89:7640-7644 such as van Beusechem; Kay etc. (1992) Human GeneTherapy 3:641-647; Dai etc. (1992) Proc.Natl.Acad.Sci.USA89:10892-10895; Hwu etc. (1993) J.Immunol.150:4104-4115; United States Patent (USP) 4,868, No. 116; United States Patent (USP) 4,980, No. 286; PCT applies for WO89/07136; PCT applies for WO89/02468; PCT applies for WO89/05345; With PCT application WO92/07573).Retroviral vector needs the target cell division, so that this reverse transcription virus gene group (with the heterologous nucleic acids that inserts wherein) is integrated into host genome stably nucleic acid is imported this cell.Therefore, might stimulate target cell to duplicate.
Adenovirus: the genome that can operate adenovirus makes its encode and expression goal gene product, but is unactivated aspect the ability of duplicating in its normal lytic viral life cycle.Referring to (1988) BioTechniques6:616 such as for example Berkner; Rosenfeld etc. (1991) Science252:431-434; With (1992) Cell68:143-155 such as Resenfeld.Those skilled in the art have known the suitable adenovirus carrier that is derived from adenovirus strain Ad5 type d1324 or other adenovirus strain (for example Ad2, Ad3, Ad7 etc.).The advantage of recombinant adenovirus is, they do not need the splitted cell can become efficient gene transmission carrier, and can be used for infecting cell type widely, comprise airway epithelia (Rosenfeld etc. (1992) see above-mentioned quoting), endotheliocyte (Lemarchand etc. (1992) Proc.Natl.Acad.Sci.USA89:6482-6486), liver cell (Herz and Gerard (1993) Proc.Natl.Acad.Sci.USA90:2812-2816) and muscle cell (Quantin etc. (1992) proc.Natl.Acad.Sci.USA89:2581-2584).In addition, adenovirus DNA (with the allogeneic dna sequence DNA that the wherein contains) unconformability that imports is gone into the genome of host cell, and remain additive type, therefore avoid contingent potential problems because when the DNA that imports is integrated into host genome (for example retrovirus DNA), insert sudden change.In addition, the adenoviral gene group ability of carrying allogeneic dna sequence DNA is compared greatly with other gene delivery vector, and (Berkner etc. see above-mentioned quoting (as many as 8 kilobase to); HajAhmand and Graham (1986) J.Virol.57:267).The most of replication-defective adenovirals that use have all lacked the viral E1 and the E3 gene of all or part at present, but keep nearly 80% adenovirus genetic material.
Adeno-associated virus: adeno-associated virus (AAV) is naturally occurring defective virus, it need another virus for example adenovirus or simplexvirus as effectively duplicating and the productivity helper virus of life cycle.(relevant summary is referring to Curr.Topics in Micro.AndImmunol. (1992) 158:97-129 such as Muzyczka).It also is one of minority kind virus that its DNA can be integrated into somatoblast not, and shows the high frequency stable integration (referring to (1992) Am.J.Respir.Cell.Mol.Biol.7:349-356 such as for example Flotte; Samulski etc. (1989) J.Virol.63:3822-3828; With (1989) J.Virol.62:1963-1973 such as McLaughlin).Containing the carrier of little AAV to 300 base pairs can be packaged and can integrate.The space constraint of foreign DNA is about 4.5kb.Can use the AAV carrier of describing among (1985) Mol.Cell.Biol.5:3251-3260 such as Tratschin with the DNA transfered cell.Used the AAV carrier that various nucleic acid have been imported different cell types (referring to (1984) Proc.Natl.Acad.Sci.USA 81:6466-6470 such as for example Hermonat; Tratschin etc. (1985) Mol.Cell.Biol.4:2072-2081; Wondisford etc. (1988) Mol.Endocrinol.2:32-39; Tratschin etc. (1984) J.Virol.51:611-619; With (1993) J.Biol.Chem.268:3781-3790 such as Flotte).
Can estimate the particular expression carrier system of nucleic acid transfered cell and the effectiveness of method by the conventional standard method of using in this area.For example; can detect the DNA of transfered cell by filter hybridization technology (for example Southern trace), and by utilizing Northern trace, RNA enzyme protection or reversed transcriptive enzyme-polymerase chain reaction (RT-PCR) detection to transcribe the RNA of generation by the DNA that imports.Can detect gene product by suitable detection, for example by albumen that produces such as immunodetection or the functionally active that detects this gene product by the functional detection that detects such as zymetology with specific antibody.
Can will give the curee as medicinal compositions such as the modulability medicine that stimulates or suppress the chemical compound of endogenous transcription factor activity.This composition comprises this modulability medicine and pharmaceutically acceptable carrier usually.Term used herein " pharmaceutically acceptable carrier " comprises any He all and the medicinal solvent that adapts, dispersion medium, dressing, antiseptic-germicide and anti-mycotic agent, isotonic agent and absorption delay agent or the like.It is well-known in the art that this medium and medicine are used for active medicinal matter.Except under any conventional media or medicine and the inconsistent situation of this active compound, considered its application in said composition.The active compound that replenishes can also be added in the described composition.
Preparing medicinal compositions of the present invention adapts its route of administration with plan.For example, the solution or the suspension that are used for non-enteron aisle, intracutaneous or subcutaneous use can comprise following component: sterile diluent, such as water for injection, salt brine solution, expressed oil, polyoxyethylene glycol, glycerine, propylene glycol or other synthetic; Antioxidant is such as the antibacterial agent of phenylcarbinol or methyl p-hydroxybenzoate (methyl parabens); Such as xitix or sodium bisulfite; Sequestrant is such as ethylenediamine tetraacetic acid (EDTA); Regulate the medicine of osmotic pressure, such as acetate, Citrate trianion or phosphatic damping fluid with such as sodium-chlor or glucose.Can regulate pH with the acid or the alkali of all example hydrochloric acids or sodium hydroxide.Disposable syringe or multiple doses cillin bottle that parenteral formulation can be packaged in peace bottle, make with glass or spear material.
The medicinal compositions that is suitable for injecting use comprises aseptic aqueous solution (if water-soluble) or dispersion and is used for preparing the sterilized powder of aseptic parenteral solution or dispersion temporarily.For intravenous administration, suitable carriers comprises physiological saline, bacteriostatic water, Cremophor EL TM(BASF, Parsippany, NJ) or phosphate-buffered saline (PBS).In all cases, said composition must be aseptic and should be that fluidic is to be easy to injection.It must be stablized under production and condition of storage and must carry out anticorrosion microbiological contamination effect with antagonism such as bacterium and fungi.This carrier can be to contain for example solvent or the dispersion medium of water, ethanol, polyvalent alcohol (for example glycerine, propylene glycol and liquid macrogol or the like) and their suitable mixture.Can be for example by use dressing such as Yelkin TTS, by the granular size that needing under the situation of dispersion, to keep with by using tensio-active agent to keep suitable fluidity.Can for example p-Hydroxybenzoate, trichloro-butyl alcohol, phenol, xitix, Thiomersalate or the like reach and prevent microbial process by various antiseptic-germicides and anti-mycotic agent.In many cases, be preferably in and comprise isotonic agent in the said composition, for example sucrose, polyvalent alcohol, sodium-chlor such as N.F,USP MANNITOL, sorbyl alcohol.Can be by the medicine that comprises that in said composition for example delay of aluminum monostearate and gelatin absorbs, the prolongation that reaches Injectable composition absorbs.
Can add in the appropriate solvent with a kind of above-mentioned composition of enumerating that needs or its combination by this active compound, then carry out filtration sterilization and prepare aseptic parenteral solution requirement.Generally speaking, this active compound can be added in the aseptic solvent from above-mentioned other composition of enumerating that contain basic dispersion medium and needs and prepare dispersion.Under the situation of the sterile powder that is used to prepare aseptic parenteral solution, the preparation method of recommendation is vacuum-drying and lyophilize, produces this activeconstituents and any other needs the pulvis of composition from the solution of previous Sterile Filtration.
Oral compositions generally comprises inert diluent or edible carrier.They can be wrapped in gelatine capsule or be pressed into tablet.Be the purpose of oral therapeutic administration, this active compound can be added with vehicle, and use with tablet, lozenge or capsular form.Can also use fluid carrier to prepare oral compositions, to use as mouth-washes, wherein this compound in this fluid carrier can be oral, rinse and wash and cough and tell or swallow.The tackiness agent and/or the subsidiary material of pharmaceutically compatible can be included as the part of said composition.Tablet, pill, capsule, lozenge etc. can contain following composition or have compound any of similarity: tackiness agent, such as Microcrystalline Cellulose, tragakanta or gelatin; Such as the vehicle of starch or lactose, such as the disintegrating agent of alginic acid, Primogel or W-Gum; Wetting agent is such as the lubricant of Magnesium Stearate or Sterotes; Glidant is such as colloid silica; Sweeting agent is such as sucrose or asccharin; Seasonings is such as peppermint, wintergreen oil or orange seasonings.
In one embodiment, avoid this active compound of preparing carriers that body is removed fast with this compound of protection, described carrier for example comprises the controlled release preparation of the transfer system of implant and micro encapsulation.Can use biodegradable, biocompatible polymkeric substance, for example ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen protein, poe, poly(lactic acid).The method for preparing this preparation it will be apparent to those skilled in the art that.Also can be from AlzaCorporation and Nova Pharmaceuticals, commercially available this material that obtains of Inc..Can also use liposome suspension (comprising liposome) as pharmaceutically acceptable carrier with antiviral antigenic monoclonal antibody guiding cells infected.These can prepare according to method known to those skilled in the art, for example, and as United States Patent (USP) 4,522, No. 811 descriptions.
Be particularly conducive to the oral or non-enteron aisle composition of dosage unit form preparation and be easy to administration and dosage consistence.Dosage unit form used herein refers to be suitable for the physical sepn unit as the unitary dose of desiring treatment target; Each unit contains the active compound of calculating with the predetermined amount of the required result of treatment of generation that combines with the pharmaceutical carrier of needs.The specification of dosage unit form of the present invention is subjected to following factor domination and directly depends on the peculiar property of this active compound of following factor (a) and particular treatment effect of desiring to reach and (b) this restriction that is used for the treatment of individual active compound of this area inherent preparation.III. the application of the inventive method
Differentiate the transcription factor that control IL-4 produces and form the Th2 cell thus continuously, make and under various clinical settings, use control method of the present invention optionally to handle the T cell subsets.Stimulating method of the present invention (that is, using the method for pungency medicine) causes producing the Th2 relevant cell factor, follows promotion Th2 to reply with the downward Th1 of adjusting and replys.In contrast, inhibition method of the present invention (that is, using the method for suppressive drug) suppresses to produce the Th2 relevant cell factor, follows downward adjusting Th2 to reply and promotes Th1 to reply.Therefore, be the favourable patient's condition for treating wherein that Th2 replys, select stimulating method of the present invention to make to promote Th2 to reply and regulate Th1 downwards to reply.Perhaps, be the favourable patient's condition for treating wherein that Th1 replys, select inhibition method of the present invention to make to regulate Th2 to reply downwards and promote Th1 to reply.The inventive method be applied to treat disease regulate can cause that this patient's condition is cured, the symptom type relevant or quantity reduces or for a long time or short-term (that is, improving this patient's condition) or the simple instantaneous effect useful to the curee with this condition.
Identified and manyly replied the relevant patient's condition with preponderate Th1 or Th2 type, these patient's condition can be benefited from the adjusting of the acknowledgement type of having set up in the individuality of suffering from this patient's condition.Following more detailed description is applied to this class disease with immunomodulatory method of the present invention.
A. transformation reactions
The IgE that transformation reactions is regulated by the activity of the cytokine of Th2 cell and generation thereof by its generation is antibody-mediated.In transformation reactions, produce IL-4 by the Th2 cell, this further stimulates generation IgE antibody and activates mediation allergic cell, i.e. mastocyte and basophilic leukocyte.IL-4 also plays an important role in the inflammatory reaction of eosinocyte mediation.Therefore, inhibition method of the present invention can be used for suppressing the particularly IL-4 generation of Th2 relevant cell factor in the means that the transformation reactions patient produces as the pathogenic IgE antibody of downward adjusting.Can directly give this curee with suppressive drug, perhaps can obtain cell (for example Thp cell or Th2 cell) and contact this cell with suppressive drug in vitro, give this curee again from this curee.In addition, in some cases, give this curee altogether, can be of value to this allergen specific of inhibitions (for example desensitizing) and reply allergen and suppressive drug or the cell handled with suppressive drug.With by giving this transformation reactions curee with the amount that is enough to further stimulate the Th1 type to reply, further strengthen this treatment such as other Th1 promotor of cytokine IL-12 or anti-Th2 relevant cell factor antibody (for example anti-IL-4 antibody).
B. cancer
It is reported that Th2 promotes that (referring to for example Yamamura, M. waits (1993) J.Clin.Invest.91:1005-1010 in the cytokine expression rising in the cancer patients; Pisa, P. waits (1992) Proc.Natl.Acad.Sci.USA89:7708-7712), and malignant disease along with the deterioration of disease course usually with reply from the Th1 type that to turn to the Th2 type to reply relevant.Therefore, can use inhibition method of the present invention to suppress the generation of Th2 relevant cell factor among the cancer patients, with as opposing Th1 to the Th2 conversion with promote the Th1 that carries out among the patient to reply thus to improve the means of disease process.This inhibition method can relate to or directly suppressive drug be suffered from the curee of cancer or in vitro handle the cell (for example Thp or Th2 cell) that obtains from this curee with suppressive drug, then gives this curee again with described cell.Can further strengthen this treatment by giving this recipient with the amount that is enough to further stimulate the Th1 type to reply such as other Th1 promotor of cytokine IL-12 or anti-Th2 relevant cell factor antibody (for example anti-IL-4 antibody).
C. transmissible disease
Also reported once that Th2 promoted cytokine expression to strengthen in various transmissible diseases, described transmissible disease comprises that HIV infection, tuberculosis, leishmaniasis, schistosomicide, filaria nematode infections and intestinal nematode infection are (referring to for example; Shearer, G.M. and Clerici, M. (1992) Prog.Chem.Immunol.54:21-43; Clerici, M and Shearer, G.M. (1993) Immunology Today14:107-111; Fauci, A.S. (1988) Science239:617-623; Locksley, R.M. and Scott, P. (1992) Immunoparasitology Today 1:A58-A61; Pearce, E.J. waits (1991) J.Exp.Med.173:159-166; Grzych, J-M. waits (1991) J.Immunol.141:1322-1327; Kullberg, M.C. waits (1992) J.Immunol.148:3264-3270; Bancroft, A.J. waits (1993) J.Immunol.150:1395-1402; Pearlman, E waits (1993) Infect.Immun.61:1105-1112; Else, K.J. waits (1994) J.Exp.Med.179:347-351) and this class transmissible disease also with immunne response in Th1 relevant to the Th2 conversion.Therefore, can use inhibition method inhibition of the present invention to suffer from the generation of Th2 relevant cell factor among the curee of transmissible disease, also promote the Th1 that carries out among this patient to reply means thus to the Th2 conversion with the process of improving this infection as opposing Th1.This inhibition method can comprise or directly suppressive drug be suffered from the curee of transmissible disease, perhaps in vitro handle the cell (for example Thp or Th2 cell) that obtains from this curee, then give this curee again described cell with suppressive drug.Can further strengthen this treatment by giving this recipient with the amount that is enough to further stimulate the Th1 type to reply such as other Th1 promotor of cytokine IL-12 or anti-Th2 relevant cell factor antibody (for example anti-IL-4 antibody).
D. self finishes eqpidemic disease
Stimulating method of the present invention can be used for the treatment of to therapeutic and the relevant autoimmune disease of Th2 type dysfunction.Many autoimmune diseases are to autologous tissue reaction and promote to participate in the cytokine of described nosopathology and the inappropriate activated result of the T cell that produces from antibody.Regulating the T auxiliary type replys and can effect be arranged to the process of autoimmune disease.For example, in experimental brain allergy myelitis (EAE), when inducing, this disease stimulate the Th2 type to reply the degree (Paul, W.E. wait (1994) Cell76:241-251) that has weakened this autoimmune disease by giving IL-4.In addition, shown animal from this disease recovery with increase the Th2 type confirmed by the Th2 relevant cell factor and reply and strengthen relevant (Koury, S.J. wait (1992) J.Exp.Med.176:1355-1364).The T emiocytosis Th2 specific cell factor (Chen, C. wait (1994) Immunity 1:147-154) that can suppress in addition, EAE.Therefore because EAE moderate stimulation Th2 type is replied the protective effect with this disease of antagonism, reply at the curee's moderate stimulation Th2 that suffers from multiple sclerosis (EAE is its model) and have the treatment benefit.
Similarly, reply the protective effect that this disease of antagonism is provided in mouse type i diabetes moderate stimulation Th2 type.The morbidity (Rapoport, M.J. wait (1993) J.Exp.Med.178:87-99) of the type i diabetes that really, prevents or postponed in these mouse, to develop usually with IL-4 (it promotes that Th2 replys) treatment NOD mouse.Therefore, reply at the curee's moderate stimulation Th2 that suffers from or easily suffer from diabetes and can improve this sick effect or suppress this disease morbidity.
It may a useful autoimmune disease again be rheumatoid arthritis (RA) that its moderate stimulation Th2 type is replied.Studies show that the patient who suffers from rheumatoid arthritis has dominant Th1 cell (Simon, A.K. wait (1994) Proc.Natl.Acad.Sci.USA91:8562-8566) in synovial tissue.By replying, can follow the deleterious Th1 of downward adjusting to reply to improve the influence of this disease thus at the curee's moderate stimulation Th2 that suffers from RA.
Therefore, suffering from or easily suffering from wherein the Th2 type and reply among the curee of the autoimmune disease useful, can use stimulating method of the present invention to stimulate the Th2 relevant cell factor to produce disease process.The cell (for example Thp, Th1 cell, B cell, non-lymphoidocyte) that this stimulating method can comprise or directly the pungency medicine be given the curee or in vitro obtains from this curee with the pungency drug treating then gives this curee again with described cell.Can further strengthen this treatment by giving this recipient with the amount that is enough to further stimulate the Th2 type to reply such as other Th2 promotor of cytokine IL-4 itself or anti-Th1 relevant cell factor antibody (for example anti-IL-4 antibody).
Replying with above-mentioned wherein Th2 is that the autoimmune disease that needs compares, and can reply by the Th1 type and improve other autoimmune disease.Can use suppressive drug of the present invention to treat this class disease (as above-mentioned about as described in cancer and the transmissible disease).Can promote cytokine (for example IFN-γ) further to strengthen this treatment by give this curee Th1 with the amount that is enough to further stimulate the Th1 type to reply.
Can be in the above-mentioned animal model (for example EAS is as the multiple sclerosis model, and the NOD mouse is as diabetes model) of human diseases or other effectiveness of the medicine of test treatment autoimmune disease in mankind itself's immunological disease animal model of characterization well.This animal model comprises mrl/lpr/lpr mouse as the lupus erythematosus model, as the mouse collagen protein inductive sacroiliitis of rheumatoid arthritis model and the experimental myasthenia gravis of mouse (the Fundamental Immunology that edits referring to Paul, Raven Press, New York, 1989, the 40-856 pages or leaves).Give test animal with adjusting of the present invention (that is, pungency or inhibition) agent, monitor this disease process in the described test animal by the standard method of the particular model that uses then.Do not compare with treating the animal animal of control drug treatment (or with), promptly confirmed the effectiveness of this conditioning agent with the improvement of this patient's condition in the animal of this pharmacological agent.
Autoimmune disease and disorderly limiting examples with the autoimmunization composition that can handle according to the present invention comprise diabetes, sacroiliitis (comprises rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, arthritic psoriasis), multiple sclerosis, myasthenia gravis, systemic lupus erythematous, autoimmune thyroiditis, dermatitis (comprising atopic dermatitis and eczematoid dermatitis), psoriasis, Si Yegelun syndromes (keratoconjunctivitis sicca that comprises Si Yegelun syndromes secondary), alopecia areata, the transformation reactions that the arthropod bite reaction causes is replied, regional ileitis, aphthous ulcer, iritis, conjunctivitis, keratoconjunctivitis, ulcerative colitis, asthma, atopic asthma, lupus erythematosus,cutaneous, scleroderma, vaginitis, rectitis, dermatitis medicamentosa, the leprosy reversible reaction, erythema nodosum leprosum, from immune uveitis, allergic encephalomyelitis, acute necrotizing hemorrhagic encephalopathy, sexy sound nervosa anaudia is carried out in the special property sent out both sides, aplastic anemia, the pure red cell anaemia, idiopathic thrombocytopenia, polychondritis, Wegner granulomatosis, chronic active hepatitis, Stevens-Johnson's syndromes, the special property sent out sprue, lichen planus, regional ileitis, graves' ophthalmopathy, sarcoidosis, primary biliary cirrhosis, posterior uveitis and interstitial pulmonary fibrosis.
E. transplant
Though transplant rejection or transplanting are accepted may be not only owing to the specific T cells subgroup in the transplant recipient (promptly, Th1 or Th2 cell) effect (the relevant discussion referring to Dallman, M.J. (1995) Curr.Opin.Immunol.7:632-638), many studies show that in the graft survival that prolongs that dominant Th2 replys or transplant rejection in dominant Th1 reply.For example, transplanting is accepted to be associated with the generation of Th2 cytokine pattern and/or transplant rejection is associated with the generation of Th1 cytokine pattern (referring to for example Takeuchi, T. etc. (1992) Transplantation53:1281-1291; Tzakis, A.G. etc. (1994) J.Pediatr.Surg.29:754-756; Thai, N.L. etc. (1995) Transplantation59:274-281).In addition, adoptive transfer with the phenotypic cell of Th2 cytokine has prolonged dermatoplasty survival (Maeda, (1994) Int.Immunol.6:855-862 such as H.) and reduce graft to versus-host disease (Fowler, D.H. etc. (1994) Blood84:3540-3549; Fowler, D.H. etc. (1994) Prog.Clin.Biol.Res.389:533-540).Have again, promote the IL-4 of Th2 differentiation to prolong the survival (Levy of cardiac allograft thing, A.E. and Alexander, J.W. (1995) Transplantation60:405-406), and strengthened skin homograft rejection (Gorczynski, R.M. etc. (1995) Transplantation60:1337-1341) with the antibody combined IL-12 of giving of anti-IL-10 that promotes the Th1 differentiation.
Therefore, can use stimulating method of the present invention to stimulate and produce the Th2 relevant cell factor in the transplant recipient, to prolong the survival of graft.This stimulating method can be used for entity (solid) organ transplantation and bone marrow transplantation (for example inhibition of transplant is to versus-host disease).This stimulating method can comprise or directly give this transplant recipient with the pungency medicine, perhaps the cell that in vitro obtains from this curee with the pungency drug treating (for example Thp, Th1 cell, B cell, non-lymphoidocyte) then gives this curee again with this cell.Can further strengthen this treatment by promoting that such as other Th2 of IL-4 self or anti-Th1 relevant cell factor antibody medicine gives this receptor with the amount that is enough to further stimulate the Th2 type to reply.
Except the aforementioned patient's condition, control method of the present invention can also be used for other purpose.For example, stimulating method of the present invention (promptly, the method of use pungency medicine) can be used for stimulated in vitro generation Th2 promotes cytokine (for example IL-4) (for example can use the external exposing cell of pungency medicine with these cytokines of industrial production, stimulate generation IL-4 with clothing, and can reclaim IL-4, and be further purified as required and packing is used for commercial use) from this culture supernatant.
In addition, control method of the present invention can be applied to vaccine inoculation, to promote among the curee that purpose is antigenic or Th1 replys or Th2 replys.Be that medicine of the present invention can be used as auxiliary, so that replying to Th1 of vaccine replied or Th2 replys.For example, for stimulating to the antigenic antibody response of purpose (for example being used for the vaccine inoculation purpose), can give the curee altogether with this antigen and pungency medicine of the present invention, among this curee this antigenic Th2 is replied promoting, provide effective B cell because Th2 replys, help and promotion IgG1 generation.Perhaps, for promoting to the antigenic cellullar immunologic response of purpose, can give the curee altogether with this antigen and suppressive drug of the present invention, to promote among the curee this antigenic Th1 being replied, because Th1 replys the development (for example delayed hypersensitivity is replied) that helps cell-mediated immune responses.Purpose antigen and this modulability medicine together can be prepared and be become single medicinal compositions or composition isolated.In optimum implementation, give this curee simultaneously with purpose antigen and this modulability medicine.Perhaps, may need at first to give this antigen in some cases, give this modulability medicine or opposite (for example under the situation that the natural Th1 of causing of antigen replys then, give this antigen at first separately and reply to stimulate Th1, it may be useful giving this pungency medicine self then or strengthen immunne response is converted to Th2 replys with antigen).IV .Maf composition
Another aspect of the present invention relates to the composition that the Th2 relevant cell factor produces or Th1/Th2 grows that can be used for regulating the curee according to method of the present invention cell.The invention provides the recombinant expression vector of the nucleotide sequence that comprises coding maf family protein, described nucleotide sequence functionally is connected to the maf family protein is expressed in guidance specifically in some cell type adjusting sequence.In optimum implementation, the sequence-directed maf family protein of described adjusting is specific expressed in lymphoidocyte (for example T cell or B cell).In one embodiment, described lymphocyte is the T cell.T cell-specific regulatory element known in the art, for example the promotor regulatory region of TXi Baoshouti gene is (referring to for example Winoto and Baltimore (1989) EMBO J.8:729-733; Leiden, J.M. (1994) Annu.Rev.Immunol.11:539-570; Hettman, T. and Cohen, A. (1994) Mol.Immunol.31:315-322; Redondo, J.M. etc. (1991) Mol.Cell.Biol.11:5671-5680).Other example of T cell-specific regulatory element is those regulatory elements that are derived from following gene: and the CD3 gene (referring to for example Clevers, H. etc. (1988) Proc.Natl.Acad.Sci.USA85:8623-8627; Clevers, H.C. etc. (1988) Proc.Natl.Acad.Sci.USA85:8156-8160; Georgopoulos, K. etc. (1988) EMBO are J.7:2401-2407), the CD4 gene (referring to for example Sawada, S. and Littman, D.R. (1991) Mol.Cell.Biol.11:5506-5515; Salmon, P. etc. (1993) Proc.Natl.Acad.Sci.USA90:7739-7743; Hanna, Z. etc. (1994) Mol.Cell.Biol.14:1084-1094; Also referring to the human CD4 promoter sequence of GenBank searching number U01066 and S68043) and CD2 gene (referring to for example Zhumabekov, T. etc. (1995) J.Immunol.Methods 185:133-140).Can obtain comprising dna fragmentation by the standard molecular biology method such as one or more T cell-specific regulatory elements of the promotor of TXi Baoshouti gene and enhanser, for example by use corresponding to the Oligonucleolide primers of 5 ' and the 3 ' end in required district or from the genomic dna of T cell as template, obtain by PCR.In case obtain comprising the dna fragmentation of T cell-specific regulatory element, then can use standard molecular biological technique that it functionally is connected to the proteic cDNA of coding maf (for example can couple together these two dna fragmentations, make this regulatory element be positioned at this maf sequence 5 '), and import carrier such as plasmid vector.
In another embodiment, this lymphoidocyte is B cell (that is, the nucleotide sequence of coding maf family protein functionally is connected to the maf family protein is expressed in guidance specifically in the B cell adjusting sequence in this recombinant expression vector).B cell-specific regulatory element known in the art, for example the promotor regulatory region of immunoglobulin gene is (referring to (1983) Cell 33:729-740 such as for example Banerji; Queen and Baltimore (1983) Cell33:741-748).Other example of B cell-specific regulatory element be those be derived from CD20 (B1) gene (referring to for example Thevenin, C. etc. (1993) J.Biol.Chem.268:5949-5956; Rieckmann, (1991) J.Immunol.147:3994-3999 such as P.), be derived from Fc ε RIIa gene (referring to for example Suter, (1989) J.Immunol.143:3087-3092 such as U.) and be derived from main histocompatibility II genoid (referring to for example Glimcher, L.H. and Kara, C.J. (1992) Annu.Rev.Immunol.10:13-49; Benoist, C. and Mathis, D. (1990) Annu.Rev.Immunol.8:681-715) regulatory element.Can obtain comprising dna fragmentation by the standard molecular biology method, for example obtain by PCR as template by using corresponding to the Oligonucleolide primers of 5 ' and the 3 ' end in required district or from the genomic dna of B cell such as the B cell-specific regulatory element of the promotor of immunoglobulin gene and enhanser.In case obtain comprising the dna fragmentation of B cell-specific regulatory element, then can use standard molecular biological technique that it functionally is connected to the proteic cDNA of coding maf (for example can couple together these two dna fragmentations, make this regulatory element be positioned at described maf sequence 5 '), and import carrier such as plasmid vector.
In an embodiment again, the invention provides the recombinant expression vector of the nucleotide sequence that comprises coding maf family protein, described nucleotide sequence functionally is connected to the maf family protein is expressed in guidance specifically in hemopoietic stem cell adjusting sequence.Hemopoietic stem cell specificity regulatory element known in the art.Preferably use the regulatory element be derived from the CD34 gene (referring to for example Satterthwaite, A.B. etc. (1992) Genomics 12:788-794; Burn, T.C. etc. (1992) Blood 80:3051-3059).
Another aspect of the present invention relates to the recombinant host cell of expressing the maf family protein.Can use this host cell to produce Th2 relevant cell factor (for example IL-4).Also can give the curee, in this curee, to produce the Th2 relevant cell factor: the means of Th2 ratio as in this curee, handling Th1 with this host cell.Term " host cell " and " recombinant host cell " are used interchangeably in this article, refer to wherein import the cell of recombinant expression vector.Should be understood that this term not only refers to the special object cell, also refers to the filial generation of this cell.Because because or sudden change or environmental influence some modification may take place in the follow-up generation, this filial generation in fact may be different with parental cell, but as long as these daughter cells keep this recombinant expression vector, these filial generations still are included within the scope of term used herein " host cell ".
In one embodiment, the invention provides host's lymphoidocyte of the recombinant expression vector that imports coding maf family protein.This host's lymphoidocyte can be T cell or B cell.The T cell clone (such as describing among the embodiment) that host T cell of the present invention can be a for example vitro culture, or from the curee isolating normal T cell (for example periphery blood T cell or splenic t-cell).External preparation known in the art with cultivate the T cell clone or separate the standard method of T cell (for example from peripheral blood), for example by the mAb of use in conjunction with the surface markers (for example CD4 or CD8) of T cell-specific cell surface marker (for example CD3) or the specific subgroup of T cell.Can this recombinant expression vector be imported the T cell by a kind of of the various known transfection methods that DNA imported mammalian cell, described method comprises calcium phosphate or calcium chloride co-precipitation, the mediation of DEAE-dextran transfection, lipofectin or electroporation.Can (molecular cloning: laboratory manual, second edition finds in Cold Spring HarborLaboratory press (1989) and other laboratory manual to transform or the appropriate method of transfection host cell at Sambrook etc.
In another embodiment, host's lymphoidocyte of the present invention is a host B cell, has wherein imported the recombinant expression vector of coding maf family protein.The B lymphoma cell that this B cell can be a for example vitro culture (such as the M12 cell of describing among the embodiment), or from the curee isolating normal B cell (for example peripheral blood B cell or spleen B cell).This area can obtain the standard method of various B lymphoma cell lines and this cell of known vitro culture.In addition, the standard method of the normal B cell of separation known in the art (for example from peripheral blood) is for example by using the mAb in conjunction with B cell-specific cell surface marker (for example membrane immunoglobulin B7-1, CD20).Can as above-mentioned T cell, this recombinant expression vector be imported this B cell by standard method.
In an embodiment again, the invention provides host's hemopoietic stem cell of the recombinant expression vector that imports coding maf family protein.The standard method that can use this stem cell of separation known in the art is from curee's (for example from this curee peripheral blood or marrow) isolating hematopoietic stem cells, mAb (further describing of relevant separate stem cells for example by using binding hematopoietic stem cells specific cell surface markers (being preferably CD34), referring to for example Wagner, J.E. etc. (1995) Blood86:512-523; Murray, L. etc. (1995) Blood85:368-378; Bemardi, A.C. etc. (1995) Science 267:104-108; Bemstein, I.D. etc. (1994) Blood Cells20:15-24; Angelini, A. etc. (1993) Int.J.Artif.Organs 16 supplementary issue 5:13-18; Kato, K. and Radburch, A. (1993) Cytometry14:384-392; Lebkowski, J.S. etc. (1992) Transplantation 53:1011-1019; Lebkowski, J. etc. (1993) J.Hematother.2:339-342).。Can as above-mentioned T cell, this recombinant expression vector be imported this hemopoietic stem cell by standard method.
Those of skill in the art will be understood that above-mentioned composition about the maf family protein can be various different maf family proteins such as c-Maf and little maf (for example p18) preparation.
The present invention provides the genetically modified transgenic animal of carrying coding maf family protein again.In optimum implementation, this maf family transgenosis preferentially or is only expressed in the T of this animal cell.Can reach the tissue specific expression of maf family protein by this maf encoding sequence in the transgenosis is connected to this proteins encoded is expressed in guidance in particular cell types adjusting sequence.This tissue specificity regulatory element known in the art also will further describe in this article.The regulatory element of the genetically modified T cell specific expression of recommending of maf family is CD4 promotor/enhanser.It is c-maf that recommendation is used for genetically modified maf family protein.Recommend illustrating in Figure 20 of c-maf transgenosis construct.In this construction, this CD4 promotor/enhanser functionally is connected to first intron of mouse c-maf gene, functionally is connected to mouse c-mafcDNA again, and the latter functionally is connected to SV40 polyadenylic acid sequence again.The standard program of the preparation transgenic animal that can describe in further detail according to this paper prepares transgenic animal by the ovocyte of c-maf transgenosis construct microinjection being gone into fertilization.The phenotype of the proteic c-maf transgenic mice of overexpression c-Maf further describes in embodiment 17 in the T cell.In brief, these animals show and the very similar phenotype of IL-4 overexpression transgenic mice, and for example little spleen and thymus gland, two positive thymocyte and single positive CD4+ thymocyte number reduce and SERUM IgE basal level increases.The transgenic animal of expressing the maf family protein can be used for for example active ability of evaluation test compound adjusting maf family protein.For example, can express the transgenic animal test compounds of maf family protein, the effect of this test compounds in these transgenic animal can be by relatively determining in the phenotypes of these transgenic animal under the situation that has this test compounds and the phenotype that lacking these transgenic animal under the situation of this test compounds.V .NIP45 compoistion and method of use
A. isolated nucleic acid molecule
One aspect of the present invention relates to coding NIP45 or its segmental isolated nucleic acid molecule.In optimum implementation, isolated nucleic acid molecule of the present invention comprises the nucleotide sequence that is shown in SEQ ID NO:5.The sequence of SEQ ID NO:5 is corresponding to mouse NIP45 cDNA.This cDNA comprises the coding proteic sequence of NIP45 (that is, " coding region " is from Nucleotide 13 to 1248) and 5 ' non-translated sequence (Nucleotide 1-12) and 3 ' non-translated sequence (Nucleotide 1249-1946).Perhaps, this nucleic acid molecule can only comprise the coding region (that is Nucleotide 13-1248) of SEQ ID NO:5.
In addition, nucleic acid molecule of the present invention can only comprise the part of the coding region of SEQ ID NO:5, for example, and the fragment of the biologic activity part of coding NIP45.Term " the biologic activity part of NIP45 " comprises the part that keeps with the NIP45 of the RHD interaction ability of NT-AT family protein.The part of NIP45 and the interactional ability of NF-AT RHD are determined in can interacting outside standard body and detecting, are for example used NF-AT RHD fusion rotein.Part that can be by separating SEQ ID NO:5, express the part (for example by recombinant expressed in host cell) and for example use glutathione s-transferase (GST)-NF-AT-RHD fusion rotein to estimate the particularly interactional ability of NF-AT RHD of this part and NF-AT of being encoded of NIP45 albumen or peptide, prepare encoding human and learn active NIP45 nucleic acid fragment partly.
The present invention comprises again because the degeneracy of genetic code is different with SEQ ID NO:5 (or its fragment) and coding and the SEQ ID NO:5 proteic nucleic acid molecule of identical NIP45 of encoding thus.Therefore, in another embodiment, isolated nucleic acid molecule of the present invention has the proteic nucleotide sequence that coding has the aminoacid sequence that is shown in SEQ ID NO:6.In addition, the part that the present invention includes coding SEQ ID NO:6 nucleic acid molecule of its biologic activity part for example.
Can use standard molecular biological technique and sequence information provided herein, separation has the nucleotide sequence of SEQ ID NO:5 or the nucleic acid molecule of its part.For example, use SEQ IDNO:5 all or part of as hybridization probe and the standard of use hybridization technique (for example as be described in Sambrook, J. etc., molecular cloning: laboratory manual, second edition, cold spring harbor laboratory, cold spring port, NY, 1989), can separate NIP45cDNA from the cDNA library.In addition, can separate the nucleic acid molecule that comprises all or part SEQ ID NO:5 by the polymerase chain reaction of use based on the Oligonucleolide primers of the sequences Design of SEQ ID NO:5.For example, can be from cellular segregation mRNA (for example by (the thiocyanic acid Guanidinium extraction procedures of 1979Biochemistry18:5294-5299) such as Chirgwin and can use the reversed transcriptive enzyme (for example can be from Gibco/BRL, the Moloney MLV reversed transcriptive enzyme that Bethesda, MD obtain; Maybe can be from Seikagaku America, Inc., the AMV reversed transcriptive enzyme that St.Petersburg, FL obtain) preparation cDNA.Can be designed for the synthetic oligonucleotide primer thing of pcr amplification based on the nucleotide sequence that is shown in SEQ ID NO:5.Can use cDNA or genomic dna as template and use suitable Oligonucleolide primers, according to the Standard PC R amplification technique nucleic acid of the present invention that increases.The nucleic acid clone that so increases can be gone into appropriate carriers, and identify by dna sequence analysis.In addition, can pass through the standard synthetic technology, for example use automatic dna synthesizer, preparation is corresponding to the oligonucleotide of NIP45 nucleotide sequence.
Except being shown in the NIP45 nucleotide sequence of SEQ ID NO:5, it will be understood to those of skill in the art that in population, to have the dna sequence polymorphism that causes the NIP45 aminoacid sequence to change.This genetic polymorphism possibility in the NIP45 gene is present among the interior individuality of population owing to natural allelic variation.This natural allelic variation can cause the variation of 1-5% in the nucleotide sequence of gene usually.As the result of natural allelic variation with do not change any and all this nucleotide diversities among the NIP45 of NIP45 functionally active and the amino acid polymorphism that causes all belongs to scope of the present invention.In addition, coding is from the nucleic acid molecule of the NIP45 of other species and therefore have the mouse sequence that is different from SEQ ID NO:5 but the nucleotide sequence relevant with this mouse sequence belongs to scope of the present invention.Can be corresponding to the nucleic acid molecule of the natural allelic variation of mouse NIP45cDNA of the present invention and human and other Mammals homologue based on the homology of itself and mouse NIP45 nucleic acid molecule disclosed herein, use this mouse cDNA or its fragment as hybridization probe, under stringent hybridization condition, separate according to the standard hybridization conditions.Therefore, in another embodiment, isolated nucleic acid molecule of the present invention under stringent condition with comprise the making nucleic acid molecular hybridization of the nucleotide sequence of SEQ IDNO:5.In certain embodiments, the length of this nucleic acid is at least 15,30,50,100,200,300,400,500,600,700,800,900,1000 or 1500 Nucleotide.Under stringent condition with the isolated nucleic acid molecule of the present invention of SEQ ID NO:5 sequence hybridization preferably corresponding to naturally occurring nucleic acid molecule.In one embodiment, the natural human NIP45 albumen of this nucleic acid encoding.In another embodiment, this nucleic acid molecule encoding is such as the proteic mouse NIP45 of mouse NIP45 albumen.
Except being present in the naturally occurring allelic variation of the NIP45 sequence in the population, those of skill in the art are further understood that, can will change the nucleotide sequence that imports SEQ ID NO:5 by sudden change, the proteic aminoacid sequence that causes thus being encoded changes, and does not change the proteic functionally active of this NIP45.For example, can in the sequence of SEQ ID NO:5, cause the nucleotide subsitution of the amino-acid substitution that results in " nonessential " amino-acid residue." nonessential " amino-acid residue is the residue that can or not the functionally active of NIP45 from NIP45 wild-type sequence (for example sequence of SEQ ID NO:6) change, this functionally active such as it and the interactional ability of NF-ATRHD or it and NF-AT and c-Maf are collaborative in stimulated gene is transcribed, and " essential " amino-acid residue needs for functionally active.
Therefore, the nucleic acid molecule that another aspect of the present invention relates to, it is coded in containing the NIP45 albumen that changes in the active nonessential amino-acid residue of NIP45.This NIP45 albumen is different with the aminoacid sequence of SEQ ID NO:6, but keeps the NIP45 activity.In one embodiment, this isolated nucleic acid molecule comprises the nucleotide sequence of proteins encoded, and wherein this albumen comprises with the aminoacid sequence 60% homologous aminoacid sequence of SEQ ID NO:6 and with the RHD of NF-AT family protein interact at least.By this albumen of this nucleic acid molecule encoding preferably with SEQ ID NO:6 at least 70% homology, more preferably with SEQ ID NO:6 at least 80% homology in addition more preferably with SEQ ID NO:6 at least 90% homology, most preferably with SEQ ID NO:6 at least 95% homology.
For determining the homology per-cent of two aminoacid sequences (for example SEQ ID NO:6 and its mutant form), these sequence alignments relatively (for example for contrasting with another albumen is best, can be imported the space) to carry out the best in protein sequence.Relatively be positioned at the amino-acid residue of corresponding amino acid position then.Position in a sequence (for example SEQ ID NO:6) is occupied by the same amino-acid residue with the middle correspondence position of another sequence (for example mutant form of NIP45), it in this position homologous (that is, amino acid used herein " homology " equates with amino acid " equality ") at described molecule so.Percent homology between two sequences is by the function of the total same position number of these sequences (that is homology %=location of equal number/total positional number * 100).
Can be by one or more nucleotide subsitutions, interpolation or disappearance being imported the nucleotide sequence of SEQ ID NO:5, make one or more amino-acid substitutions, interpolation or disappearance be imported into the albumen that is encoded, and the proteic isolated nucleic acid molecule of NIP45 of the albumen homology of preparation coding and SEQ ID NO:6.Can be by the importing SEQ ID NO:5 that will suddenly change such as site-directed mutagenesis and PCR mediation mutagenesis.The non-essential amino acid residue that is preferably in one or more predictions carries out conservative amino acid replacement." conservative amino acid replacement " is the displacement of wherein replacing this amino-acid residue with the amino-acid residue with similar side chain.This area has defined the amino-acid residue family with similar side chain, comprises basic side chain (Methionin for example, arginine, Histidine), acid side-chain (aspartic acid for example, L-glutamic acid), uncharged polar side chain (glycine for example, l-asparagine, glutamine, Serine, Threonine, tyrosine, halfcystine), non-polar sidechain (L-Ala for example, Xie Ansuan, leucine, Isoleucine, proline(Pro), phenylalanine, methionine(Met), tryptophane), β branch side chain (Threonine for example, Xie Ansuan, Isoleucine) and aromatic side chains (tyrosine for example, phenylalanine, tryptophane, Histidine).Therefore, preferably use from the amino-acid residue in another amino-acid residue replacement NIP45 albumen of the same side chain family.Perhaps, in another embodiment, can for example suddenly change along all or part of the importing at random of NIP45 encoding sequence by saturation mutagenesis, can just screen the mutant that obtains, to differentiate the mutant that keeps with NF-AT interaction ability with the interactional ability of NF-AT RHD (for example using the GST-NF-AT-RHD fusion rotein).Behind mutagenesis SEQ ID NO:5, can be in host cell recombinant expressed mutant protein that should coding, and can be with external this mutant protein of interaction detection assay and the interactional ability of NF-AT.For example, can radio-labeled reorganization NIP45 albumen (for example form of the sudden change of SEQ ID NO:6 or brachymemma) and with GST-NF-AT RHD fusion rotein incubation.If formed this mixture then, the glutathione agarose micelle is added this mixture, with precipitation NIP45-GST-NF-AT RHD mixture.Cleaning this micelle with after removing non-specific binding, measure the radioalbumin that combines with this micelle amount and with the amount that residues in the radioalbumin in the elutriant relatively, so that this determines that can this NIP45 albumen have the RHD interaction with NF-AT thus.
Another aspect of the present invention relates to the isolated nucleic acid molecule with NIP45mRNA coding strand or gene antisense.Antisense nucleic acid of the present invention can with whole NIP45 coding strand or only its part complementation.In one embodiment, coding region (the whole coding region that for example comprises the SEQ ID NO:5 of the Nucleotide 13-1248) antisense of the coding strand of the nucleotide sequence of antisense nucleic acid molecule and coding NIP45.In another embodiment, the non-coding region antisense of the coding strand of the nucleotide sequence of this antisense nucleic acid molecule and coding NIP45.In certain embodiments, the length of antisense nucleic acid of the present invention is at least 15,30,50,100,200,300,400,500,600,700,800,900,1000 or 1500 Nucleotide.
The coding strand sequence (for example SEQ ID NO:5) of known coding NIP45 disclosed herein can design antisense nucleic acid of the present invention according to Wo Sen and Ke Like basepairing rule.This antisense nucleic acid molecule can with the whole coding region complementation of NIP45mRNA, perhaps can be only with the oligonucleotide of a part of antisense of the coding region of NIP45mRNA or non-coding region.For example, this antisense oligonucleotide can with NIP45 mRNA translation initiation site around district's complementation.The length of antisense oligonucleotide can be for example about 15,20,25,30,35,40,45 or 50 Nucleotide.Can use methods known in the art to use chemosynthesis and enzyme ligation to make up antisense nucleic acid of the present invention.For example, can use naturally occurring Nucleotide or be designed for the biological stability that increases this molecule or increase antisense and the various modified nucleotides of the physical stability of the duplex that forms between the phosphorothioate odn are arranged, chemosynthesis antisense nucleic acid (for example antisense oligonucleotide), for example, the Nucleotide that can use thiophosphoric acid derivative and acridine to replace.Perhaps, can use wherein with the antisense location (that is, and from the RNA of the transcribed nucleic acid of this insertion will with purpose target nucleic acid antisense, in following trifle, further describe) subclone expression of nucleic acids carrier, produce antisense nucleic acid biologically.
In another embodiment, antisense nucleic acid of the present invention is a ribozyme.Ribozyme is the catalytic RNA molecule with ribonuclease activity, and it can cut the single-chain nucleic acid such as mRNA that complementary district is arranged with it by enzyme.Can be based on NIP45 cDNA nucleotide sequence disclosed herein (that is, SEQ ID NO:5), design has specific ribozyme to the NIP45 coding nucleic acid.For example, can make up the derivative of tetrahymena L-19IVS RNA, wherein desire the base sequence complementation that enzyme is cut among the base sequence of avtive spot and the NIP45 coding mRNA.Referring to No. 4,987,071, United States Patent (USP)s such as for example Cech; With No. 5,116,742, United States Patent (USP)s such as Cech.Perhaps, can use NIP45mRNA from the RNA library of molecules, to select catalytic RNA with specific ribonucleic acid enzymic activity.Referring to for example Bartel, D. and Szostak, J.W. (1993) Science261:1411-1418.
The isolated nucleic acid molecule that relates in one aspect to coding NIP45 fusion rotein more of the present invention.Can pass through the standard recombinant dna technology, prepare this nucleic acid molecule of first nucleotide sequence that comprises coding NIP45 albumen, polypeptide or the peptide of second nucleotide sequence that at least functionally is connected to coding non-NIP45 albumen, polypeptide or peptide.In following C trifle, the NIP45 fusion rotein is described in further detail.
B. recombinant expression vector and host cell
Another aspect of the present invention relates to the carrier of the nucleic acid that contains coding NIP45 (or its part), preferably recombinant expression vector.Expression vector of the present invention comprises the nucleic acid of the present invention that is suitable for expressing the form of this nucleic acid in host cell, this means that this recombinant expression vector comprises one or more adjusting sequences, described adjusting sequence selects and functionally is connected to the nucleotide sequence that desire is expressed based on the host cell that is used to express.In recombinant expression vector, the nucleotide sequence of " functionally connect " feeling the pulse with the finger-tip is connected to this adjusting sequence in the mode that allows to express this nucleotide sequence (for example in-vitro transcription/translation system during maybe when this carrier importing host cell in host cell).Term " adjusting sequence " comprises promotor, enhanser and other expression controlling elements (for example polyadenylation signal).This adjusting sequence description is in for example Goeddel; GeneExpression Technology:Methods in Enzymology185, Academic Press, SanDiego, CA (1990).Regulate sequence comprises that those sequences that instruct the constitutive expression nucleotide sequence in many host cell types and those only instruct this nucleotide sequence to express in some host cell sequence (for example tissue specificity adjusting sequence).The design that it will be understood to those of skill in the art that expression vector can be depended on selection, desirable proteins expression level that will transformed host cells etc.Expression vector of the present invention can be imported host cell, to produce albumen that comprises fusion rotein or peptide or peptide (for example NIP45 albumen, the proteic mutant form of NIP45, NIP45 fusion rotein or the like) thus by nucleic acid encoding described herein.
Can design recombinant expression vector of the present invention in protokaryon or eukaryotic cell, to express NIP45 albumen.For example, can in such as colibacillary bacterial cell, insect cell (use rhabdovirus expression vector), yeast cell or mammalian cell, express NIP45.At Goeddel; Gene Expression Technology:Methods in Enzymology185, Academic Press, San Diego further discusses proper host cell among the CA (1990).Perhaps, can for example use T7 promotor adjusting sequence and T7 polysaccharase at in-vitro transcription and this recombinant expression vector of translation.
The most frequently used composing type or the inducible promoter that contains guidance or fusion or non-expressing fusion protein carries out the expression of albumen in prokaryotic organism in intestinal bacteria.Fusion vector adds to some amino acid wherein on the encoded protein, generally adds to the N-terminal of this recombinant protein.This fusion vector is fit to three purposes usually: 1) increase Recombinant Protein Expression; 2) strengthen the solubleness of this recombinant protein; With 3) by this recombinant protein of help purifying that works as the part in the affinity purification.In the fusion expression vector of being everlasting, import a proteolytic cleavage site in this junction of merging part and this recombinant protein, after this fusion rotein of purifying, can separate this recombinant protein from branches such as this fusions.This kind of enzyme and cognate recognition sequence thereof comprise factor Xa, zymoplasm and enteropeptidase.Typical fusion expression vector comprises pGEX (PharmaciaBiotech Inc; Smith, D.B. and Johnson, K.S. pMAL (New England Biolabs (1988) Gene67:31-40),, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ), they respectively with glutathione S-transferase (GST), maltose E is conjugated protein or albumin A merges to the target recombinant protein.
The example of the non-fusion coli expression carrier of suitable induction type comprises pTrc (Amann etc., Gene69:301-315) and pET11d (Studier etc. (1988), Gene ExpressionTechnology:Methods in Enzymology185, Academic Press, San Diego, Califormia (1990) 60-89).Target gene depends on host RNA polysaccharase transcribing from hybrid trp-lac promoter, fusion from the expression of pTrc carrier.Target gene depends on from transcribing by the T7gn10-1ac promoter, fusion of coexpressed viral rna polymerase (T7gnl) mediation from the expression of pET 11d carrier.The host strain BL21 (DE3) that retains (resident) λ prophage or the HMS174 (DE3) of the self-contained T7gnl gene under the control of lacUV5 promoter transcription of this varial polymerases origin provide.
A kind of strategy of maximum express recombinant protein is to express this albumen (Gottesman in the host bacteria that the ability of this recombinant protein of proteolytic cleavage weakens in intestinal bacteria, S., GeneExpression Technology:Methods in Enzymology 185, Academic Press, SanDiego, Califomia (1990) 119-128).Another strategy is to change the nucleotide sequence that desire is inserted the nucleic acid of expression vector, makes that each amino acid whose single codon is those preferential use (Wada etc., (1992) Nuc.Acids Res.20:2111-2118) in intestinal bacteria.Can carry out this change of nucleotide sequence of the present invention by the standard DNA synthetic technology.
In another embodiment, this NIP45 expression vector is a Yeast expression carrier.The example of the carrier of expressing in cereuisiae fermentum comprises pYepSecl (Baldari etc., (1987) EMBO is J.6:229-234), pMFa (Kurjan and Herskowitz, (1982) pJRY88 (Schultz etc. Cell30:933-943),, Gene54:113-123) and pYES2 (InvitrogenCorporation (1987), San Diego, CA).
Perhaps, can use rhabdovirus expression vector at expressed in insect cells NIP45.Be used in the baculovirus vector of cultivating expressing protein in the insect cell (for example Sf9 cell) and comprise pAc series (Smith etc., Mol.Cell Biol.3:2156-2165) and pVL series (Lucklow (1983), V.A. and Summers, M.D., (1989) Virology 170:31-39).
In an embodiment again, use mammalian expression vector in mammalian cell, to express nucleic acid of the present invention.The example of mammalian expression vector comprise pMex-Neo I, pCDM8 (Seed, B., (1987) Nature329:840) and pMT2PC (Kaufman etc. (1987), EMBOJ.6:187-195).When being used for mammalian cell, provide the controlled function of expression vector usually by viral regulatory element.For example, Chang Yong promotor is derived from polyoma, adenovirus 2, cytomegalovirus and simian virus 40.
In another embodiment, this recombinant mammalian expression vector can preferentially instruct in particular cell types and express this nucleic acid (for example using-system specificity regulatory element is expressed this nucleic acid).Tissue specificity regulatory element known in the art.The limiting examples of suitable tissue-specific promoter comprises lymph specificity promoter (Calame and Eaton (1988) Adv.Immunol.43:235-275), particularly TXi Baoshouti promotor (Winoto and Baltimore (1989) EMBO J.8:729-733) and immune protein promotor (Banerji etc. (1983) Cell33:729-740; Queen and Baltimore (1983) Cell33:741-748), albumin promoter (liver specificity; Pinkert etc. (1987) Genes Dev.1:268-277), neuronal specificity promotor (neurofilament promotor for example; Byme and Ruddle (1989) Proc.Natl.Acad.Sci.USA86:5473-5477), pancreas specificity promoter (Edlund etc. (1985) Science230:912-916) and mammary gland-specific promotor (whey promotor for example; United States Patent (USP) 4,873, No. 316 and European application publication No. 264,166).Also comprise growing and regulate promotor, for example mouse hox promotor (Kessel and Gruss (1990) Science249:374-379) and afp promoter (Campes and Tilghman (1989) Genes Dev.3:537-546).
The present invention provides in addition and comprises the recombinant expression vector of going into the dna molecular of the present invention of this expression vector with the antisense positional cloning.That is, this dna molecular functionally is connected to the adjusting sequence, the feasible RNA molecule of expressing (by transcribing this dna molecular) and NIP45mRNA antisense of its mode of connection.Can be connected to adjusting sequence with the nucleic acid of antisense positional cloning selection operation, this sequence-directed this antisense rna molecule continuous expression in various cells, for example can select viral promotors and/or enhanser or regulate sequence, they instruct composing type, tissue specificity or the cell type specificity of sense-rna to express.This antisense expression vector can be the form of recombinant plasmid, phagemid or attenuated virus for example, and wherein antisense nucleic acid produces under the control of efficient regulatory region, and the activity in this district is determined by the cell type that imports this carrier.The discussion that relevant use inverted defined gene regulatory gene is expressed, referring to Weintraub, H. etc., as the sense-rna of genetic analysis molecular tool, Reviews-Trends in Genetics, the 1st (1) volume 1986.
Another aspect of the present invention relates to and imports carrier of the present invention, the recombinant host cell of recombinant expression vector preferably.Host cell can be protokaryon or eukaryotic cell.For example, can in such as colibacillary bacterial cell, insect cell, yeast or mammalian cell (for example Chinese hamster ovary cell (CHO) or COS cell), express NIP45 albumen.Known other proper host cell of those skilled in the art.Can carrier DNA be imported protokaryon or eukaryotic cell by routine conversion or rotaring dyeing technology.Term used herein " conversion " and " transfection " refer to the technology that heterologous nucleic acids (for example DNA) is imported host cell of various this areas approval, comprise transfection, lipofectin or the electroporation of calcium phosphate or calcium chloride co-precipitation, the mediation of DEAE-dextran.Can in Sambrook etc. (molecular cloning: laboratory manual, second edition, press of cold spring harbor laboratory (1989)) and other laboratory manual, find and transform or the appropriate method of transfection host cell.
Be the stable transfection mammalian cell, known to the expression vector and the rotaring dyeing technology that use, only there is the small portion cell foreign DNA may be integrated into its genome.For differentiating and select these intasomies, but the gene of the selective marker (for example to antibiotics resistance) of generally will encoding imports host cell with goal gene.The marks packets selected of recommending draw together those give to medicine (for example G418, Totomycin and methotrexate) resistance selected marker.But can be on the identical carrier of coding NIP45 or the nucleic acid of the selective marker of on separate carrier, will encode importing host cell.Can by medicament selection differentiate nucleic acid stability cells transfected with importing (but the cell of for example having integrated this selectable marker gene will survive, and other necrocytosis).
Host cell of the present invention such as protokaryon in cultivating or eukaryotic host cell can be used for producing (promptly expressing) NIP45 albumen.Therefore, the present invention provides and uses host cell of the present invention to produce the proteic method of NIP45.In one embodiment, this method is included in and cultivates host cell of the present invention (wherein having imported the recombinant expression vector of coding NIP45) in the suitable substratum until producing NIP45.In another embodiment, this method comprises from this substratum or this host cell again and separates NIP45.The NIP45 albumen of natural form is intracellular protein, therefore, can be in recombinant host cell express recombinant NIP45 albumen in the born of the same parents, for example reclaim this reorganization NIP45 albumen then, and separate reorganization NIP45 albumen from this host cell by this host cell of cracking and from this lysate.Perhaps, can make this albumen from this secretory host cell, and preparation be as the reorganization NIP45 albumen of extracellular protein by the allos signal sequence being connected to this proteic N-terminal.In this case, can from the substratum of culturing cell, reclaim reorganization NIP45 albumen.
Can also use some host cell of the present invention to produce non-human transgenic animal.Host cell of the present invention is fertilized oocyte or the embryonic stem cell that has imported the NIP45 encoding sequence.Then, non-human transgenic animal or the reformed homologous recombination animal of wherein endogenous NIP45 sequence that can use this host cell to produce to have imported external source NIP45 sequence in its genome.This animal can be used to study function and/or the activity of NIP45 and be used for differentiating and/or estimating the active instrumentality of NIP45.Therefore, another aspect of the present invention relates to the non-human transgenic animal that contains the genetically modified cell that carries coding NIP45 albumen or NIP45 protein part.In the branch embodiment (subembodiment) of transgenic animal of the present invention, this transgenosis has changed coding endogenous NIP45 proteic native gene (homologous recombination animal for example, wherein this endogenous NIP45 gene is by functional destruction or " rejecting ", and perhaps the nucleotide sequence of this endogenous NIP45 gene has suddenlyd change or this endogenous NIP45 gene transcription regulatory region is changed).
Can produce transgenic animal of the present invention by the NIP45 coding nucleic acid is for example imported the male pronucleus of fertilized oocyte and this ovocyte is grown by microinjection in the female replace-conceive of false pregnancy (foster) animal.The mouse NIP45cDNA sequence of SEQ ID NO:5 can be imported the genome of non-human animal (for example mouse) as transgenosis.Perhaps, can based on the hybridization of mouse NIP45cDNA, separate the similar thing of Mammals, and use it is done transgenosis such as the mouse NIP45 gene of people NIP45 gene.Intron sequences and polyadenylation signal can also be included in this transgenosis, to increase this genetically modified expression efficiency.Tissue specificity can be connected to with regulating series of operations this NIP45 transgenosis, to instruct the NIP45 protein expression of specific cells.Producing transgenic animal by embryo operation and microinjection has particularly become the ordinary method of this area such as the method for the animal of mouse, and is described in the United States Patent (USP) 4,736 of Leder for example etc., No. 866 and 4,870, the United States Patent (USP) 4 of No. 009, Wagner etc., 873, No. 191 and Hogan, B., Manipulatmg the Mouse Embryo, (press of cold spring harbor laboratory, the cold spring port, N.Y., 1986).Use similar method to produce other transgenic animal.Can be based on the expression of NIP45mRNA in the tissue of genetically modified existence of NIP45 and/or described animal in its genome or the cell, differentiate that transgenosis sets up animal (transgenic founderanimal).Can set up animal with transgenosis then breeds and more carries this genetically modified animal.In addition, the genetically modified transgenic animal of carrying coding NIP45 further can be bred to carrying other genetically modified other transgenic animal.
For producing the homologous recombination animal, prepare a carrier, it contains and imports disappearance, interpolation or metathetical at least a portion NIP45 gene, to change (for example functional destruction) endogenous NIP45 gene thus.This NIP45 gene is mouse NIP45 gene preferably.For example, the mouse NIP45cDNA that uses SEQ IDNO:5 can separate mouse NIP45 gene from the mouse gene group DNA library as probe.Then can be with the gene constructed homologous recombination vector that is suitable for changing endogenous NIP45 gene in the mouse genome of this mouse NIP45.In optimum implementation, design this carrier and make when homologous recombination, this endogenous NIP45 gene is functionally destroyed (that is encode functional protein no longer; Also be called " rejecting " carrier).Perhaps, can design this carrier and make when homologous recombination, this endogenous NIP45 gene is suddenlyd change or other change, but still encode functional protein (for example can change the upstream regulation district to change the proteic expression of endogenous NIP45 thus).In this homologous recombination vector, hold other nucleic acid to be positioned at its two flank in 5 ' and 3 ' of this NIP45 gene alteration part, between the endogenous NIP45 gene in external source NIP45 gene that carries by this carrier and embryonic stem cell homologous recombination to take place by the NIP45 gene.This another NIP45 nucleic acid that is positioned at two sides has enough length successfully to carry out homologous recombination with this native gene.Usually, the flanking DNA (5 ' and 3 ' all have) with several thousand base pairs is included in (referring to for example Thomas, K.R. and Capecchi, the description of the relevant homologous recombination vector of M.R. (1987) Cell 51:503) in this carrier.The NIP45 gene that this carrier is imported embryonic stem cell line (for example passing through electroporation) and select wherein to import with the cell (referring to for example Li, E. etc. (1992) Cell69:915) of endogenous NIP45 dna homolog reorganization.Then the injection cell of selecting is gone into the blastocyst of animal (for example mouse), to form aggregation chimera (referring to for example Bradley, A. be stated from Teratocarcmomas and Embryonic Stem Cells:A Practical Approach, E.J.Robertson edits (IRL, Oxford, 1987) the 113-152 page or leaf).Chimeric embryo can be implanted the female replace-conceive animal of suitable false pregnancy then, and make fetal development mature.Can use the filial generation that contains this homologous recombination DNA in its sexual cell is to transmit to breed animal by this genetically modified kind, and wherein all cells of this animal all contains this homologous recombination DNA.The method that makes up homologous recombination vector and homologous recombination animal is further described in Bradley, the PCT international publication number WO90/11354 of A. (1991) CurrentOpinion in Biotechnology2:823-829 and Le Mouellec etc.; The WO91/01140 of Smithies etc.; The WO92/0968 of Zijlstra etc.; WO93/04169 with Bems etc.
C. isolating NIP45 albumen and anti-NIP45 antibody
Another aspect of the present invention relates to isolating NIP45 albumen, such as the part of its biologic activity part and be suitable for as immunogen to produce the peptide fragment of anti-NIP45 antibody.In one embodiment, the invention provides isolating NIP45 protein formulation.This NIP45 albumen preferably has the aminoacid sequence that is shown in SEQ ID NO:6.In other embodiments, this NIP45 albumen basically with SEQ ID NO:6 homology, and the proteic functionally active of reservation SEQ ID NO:6, but because natural allelic variation or mutagenesis are different on aminoacid sequence, or the proteic Mammals homologue of SEQ IDNO:6 (for example human homology's thing), as describing in detail in the above-mentioned A trifle.Therefore, in another embodiment, this NIP45 albumen is an albumen, and it comprises with the aminoacid sequence 60% homologous aminoacid sequence of SEQ ID NO:6 and with the RHD of NF-AT family protein interact at least.This albumen preferably with SEQ ID NO:6 at least 70% homology, more preferably with SEQ ID NO:6 at least 80% homology in addition more preferably with SEQ ID NO:6 at least 90% homology, most preferably with SEQ ID NO:6 at least 95% homology.
In other embodiments, the invention provides the proteic part of isolating NIP45.For example, the present invention also comprises the proteic part with the interactional NIP45 of NF-AT.As confirming that in an embodiment the RHD of NIP45 albumen and NF-AT interacts.Can use external interaction to detect (the use GST-NF-AT RHD fusion rotein of for example in above-mentioned A trifle, describing), to measure NIP45 peptide fragment and the interactional ability of NF-AT Rel homeodomain, to differentiate and the interactional peptide fragment of NF-AT thus.
Preferably produce NIP45 albumen by recombinant DNA technology.For example, this proteic cloned nucleic acid molecule of will encoding is gone into expression vector (as above-mentioned), and this expression vector is imported host cell (as above-mentioned) and express this NIP45 albumen in this host cell.Can use the standard protein purification technology then, by suitable purifying flow process from this cellular segregation NIP45 albumen.Recombinant expressed alternative method is to use standard peptide synthetic technology chemosynthesis NIP45 polypeptide.In addition, can be from cell (for example from the T cell) for example by using the immunoprecipitation of anti-NIP45 antibody, separating natural NIP45 albumen.
The present invention also provides the NIP45 fusion rotein.NIP45 used herein " fusion rotein " comprises the NIP45 polypeptide that functionally is connected to non-NIP45 polypeptide." NIP45 polypeptide " refers to have the polypeptide corresponding to the aminoacid sequence of NIP45 albumen or its peptide fragment, and " non-NIP45 polypeptide " refers to have the polypeptide corresponding to another proteic aminoacid sequence.In this fusion rotein, term " functionally connects " and refer to that this NIP45 polypeptide and this non-NIP45 polypeptide merge mutually in framework.This non-NIP45 polypeptide can merge N-terminal or the C-terminal to this NIP45 polypeptide.For example, in one embodiment, this fusion rotein is the GST-NIP45 fusion rotein, and wherein this NIP45 sequence merges the C-terminal to this GST sequence.In another embodiment, this fusion rotein is the NIP45-HA fusion rotein, wherein this NIP45 nucleotide sequence insert pCEP4-HA carrier (Herrscher, R.F. etc. (1995) Genesd Dev.9:3067-3082) make this NIP45 sequence at the framework endomixis to influenza hemagglutinin epi-position mark.This fusion rotein can be so that purification of Recombinant NIP45.
The most handy standard recombinant dna technology of NIP45 fusion rotein of the present invention is produced.For example, according to routine techniques, the dna fragmentation of the different peptide sequences of coding is connected in framework, this routine techniques, for example use that flush end or staggered end connect, Restriction Enzyme digestion with suitable end be provided, fill up sticky end when suitable, alkaline phosphatase treatment is connected with enzyme to avoid unwanted connection.In another embodiment, can synthesize this fusion gene by the routine techniques that comprises automatic dna synthesizer.Perhaps, can use anchor primer to carry out the pcr amplification of gene fragment, this primer produces between two adjacent gene fragments and forms complementary overhang, this dangle can be annealed subsequently and increase again with produce chimeric gene sequence (referring to, for example, the molecular biology current methods, John Wiley﹠amp such as editor Ausubel; Sons:1992).In addition, commercially available have many coding to merge the expression vector of part (for example gst polypeptide or HA epi-position mark).The NIP45 coding nucleic acid can be cloned into this expression vector, make this fusion part in framework, be connected to this NIP45 albumen.
Use the standard technique of polyclone and Monoclonal Antibody, can use separate NIP45 albumen or its fragment as immunogen to produce antibody in conjunction with NIP45.Can use NIP45 albumen to produce antibody, the antigenic peptide fragment that perhaps can use NIP45 is as immunogen.The antigenic peptide fragment of NIP45 comprises at least 8 amino-acid residues of the aminoacid sequence that is shown in SEQ ID NO:6 usually and comprises the epi-position of NIP45, makes the antibody that resists this peptide and the NIP45 that produce form the specific immunity mixture.This antigen peptide preferably comprises at least 10 amino-acid residues, more preferably comprises at least 15 amino-acid residues even more preferably comprise at least 20 amino-acid residues, most preferably comprises at least 30 amino-acid residues.The preferred epi-position that this antigen peptide comprises is to be positioned at for example district of the NIP45 in wetting ability district of this protein surface.The hydrophobicity analysis of the NIP45 protein sequence of SEQ ID NO:6 is shown in Figure 12.
Usually use the NIP45 immunogen, by preparing antibody with the suitable object of this immunogen immune (for example, rabbit, goat, mouse or other Mammals).Suitable immunogen preparation can comprise for example recombinant expressed NIP45 albumen or the NIP45 peptide of chemosynthesis.Said preparation can comprise the adjuvant or the similar immunostimulation medicine of or Freund complete such as Dai Shi again.With the suitable object of immunogenicity NIP45 preparation immunity, induce the anti-NIP45 antibody response of polyclone.
Therefore, the relevant anti-NIP45 antibody of another aspect of the present invention.Can prepare the anti-NIP45 antibody of polyclone as above-mentioned by with the suitable object of NIP45 immunogen immune.Can pass through standard technique, detect (ELISA) such as the enzyme linked immunological absorption of using immobilization NIP45 and detect tiring in time by the anti-NIP45 antibody in the object of inoculation.If desired, can separate the antibody molecule of this guiding NIP45, and be further purified, to obtain the IgG part by the well-known technology of for example albumin A chromatography from this Mammals (for example) from blood.Appropriate time after immunity, for example when anti-NIP45 antibody titer is the highest, can obtain antibody produced cell and be used for preparing monoclonal antibody from this object by standard technique, the hybridoma technology that described technology is for example at first described for Kohler and Milstein (1975, Nature256:495-497) (also referring to (1981) J.Immunol 127:539-46 such as Brown; Brown etc. (1980) J Biol Chem255:4980-83; Yeh etc. (1976) PNAS 76:2927-31; With (1982) Int.J.Cancer29:269-75 such as Yeh), recent human B cell hybridoma technology (Kozbor etc. (1983) ImmunolToday4:72), EBV hybridoma technology (Cole etc. (1985), Monoclonal Antibodies andCancer Therapy, Alan R.Liss, 77-96 page or leaf) or the trioma technology Inc..The technology that produces monoclonal antibody hybridoma is well-known (generally referring to R.H.Kenneth, be stated from Monoclonal Antibodies:A New Dimension In Biological Analyses, PlenumPublishing Corp., New YorK, New York (1980); E.A.Lerner (1981) Yale J.Biol.Med., 54:387-402; M.L.Gefter etc. (1977) Somauc Cell Genet., 3:231-36).In brief, with immortal cell line (being generally myelomatosis) with merge with the mammiferous lymphocyte (being generally splenocyte) of NIP45 immunogen such as above-mentioned immunity, the culture supernatant of the hybridoma that screening obtains is to differentiate the hybridoma of generation in conjunction with the monoclonal antibody of NIP45.
Any many well-known method that is used to merge lymphocyte and immortalized cell system all can be used for producing anti-NIP45 monoclonal antibody purpose (referring to, for example, G.Galfre etc. (1977) Nature266:55052; Somatic Cell Genet. such as Gefter see above-mentioned quoting; Lerner, Yale J.Biol.Med. sees above-mentioned quoting; Kenneth, Monoclonal Antibodies sees above-mentioned quoting).In addition, those skilled in the art will be understood that many variations of this method are also useful.Usually, this immortal cell line (for example, myeloma cell line) is derived from the mammalian species identical with described lymphocyte.For example, can use by oneself in the future immunogen preparation mice immunized lymphocyte of the present invention and immortalization mouse cell lines merges the preparation murine hybridoma.The immortal cell line of recommending is the responsive mouse myeloma cell line of substratum (" HAT substratum ") to containing xanthoglobulin, aminopterin and thymidine.Any fusion object that all can be used as according to standard technique of multiple myeloma cell line, for example P3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/O-Ag14 myelomatosis system.These myelomatosis systems can be from American type culture collection (ATCC), Rockville, and Md. obtains.Usually use polyoxyethylene glycol (" PEG ") that responsive murine myeloma cell of HAT and mouse boosting cell are merged.The hybridoma that uses the screening of HAT substratum to obtain from fusion then, this substratum kill the myeloma cell (not merging splenocyte because will be dead after several days by conversion) of not fusion and fusion not yet in effect.By for example using standard ELISA,, produce the hybridoma of monoclonal antibody of the present invention with detection just in conjunction with the antibody aspect screening hybridoma culture supernatant of NIP45.
The method of replacing of the hybridoma of preparation secrete monoclonal antibody is, can be by (for example making up the immunoglobulin (Ig) library with NIP45 screening reorganization, the antibody phage display libraries) immunoglobulin library member of separation and combination NIP45 thus, and differentiate and separate monoclonal anti NIP45 antibody.Commercially available medicine box (for example, Pharmacia recombinant phages antibody system, the Directory Number 27-9400-01 that preparation and screening phage display library are arranged; SurfZAP with Stratagene TMPhage display medicine box t, Directory Number 240612).In addition, being specially adapted to produce and screen the example of the method for antibody display libraries and reagent can be referring to No. 5,223,409, United States Patent (USP) such as for example Ladener; International publication number WO92/18619 such as Kang; International publication number WO91/17271 such as Dower; International publication number WO92/20791 such as Winter; International publication WO92/15679 such as Markland; International publication number WO93/01288 such as Breitling; International publication number WO92/01047 such as McCafferty; International publication number WO92/09690 such as Garrard; International publication number WO90/02809 such as Ladner; Fuchs etc. (1991) Bio/Technology9:1370-1372; Hay etc. (1992) Hum Antibod Hybridomas3:81-85; Huses etc. (1989) Science246:1275-1281; Griffiths etc. (1993) EMBO J12:725-734; Hawkins etc. (1992) J Mol Biol226:889-896; C1arkson etc. (1991) Nature352:624-628; Gram etc. (1992) PNAS89:3576-3580; Garrad etc. (1991) Bio/Technology 9:1373-1377; Hoogenboom etc. (1991) Nuc Acid Res 19:4133-4137; Barbas etc. (1991) PNAS88:7978-7982; With Nature (1990) 348:552-554 such as McCafferty.
In addition, can with standard recombinant dna technology preparation, belong to scope of the present invention such as the anti-NIP45 antibody of reorganization chimeric and the peopleization monoclonal antibody that comprises people and non-human part.Can be by recombinant DNA technology known in the art, produce the monoclonal antibody of this chimeric and peopleization, for example use the technology that is described in following method: international monopolies such as Robinson are announced PCT/US86/02269; Akira waits european patent application 184,187; Taniguchi, M., european patent application 171,496; European patent applications such as Morrison 173,494; PCT such as Neuberger apply for WO86/01533; No. 4,816,567, United States Patent (USP)s such as Cabilly; European patent applications such as Cabilly 125,023; Better etc. (1988) Science240:1041-1043; Liu etc. (1987) PNAS84:3439-3443; Liu etc. (1987) J.Immunol.139:3521-3526; Sun etc. (1987) PNAS84:214-218; Nishimura etc. (1987) Canc.Res.47:999-1005; Wood etc. (1985) Nature314:446-449; With (1988) J.Natl.Cancer Inst.80:1553-1559 such as Shaw); Morrson, S.L. (1985) Science229:1202-1207; Oi etc. (1986) BioTechniques4:214; Winter United States Patent (USP) 5,225,539; Jones etc. (1986) Nature321:552-525; Verhoeyan etc. (1988) Science239:1534; With (1988) J.Immunol.141:4053-4060 such as Beidler.
Can use anti-NIP45 antibody (for example, monoclonal antibody) to separate NIP45 by standard technique such as affinity chromatography or immunoprecipitation.Anti-NIP45 antibody can be so that the NIP45 that the reorganization of expressing host cell from the natural NIP45 of cell purification and purifying produces.In addition, can use anti-NIP45 antibody test NIP45 albumen (for example, in cell pyrolysis liquid or cell conditioned medium liquid).By this antibody coupling (for example, physical connection) to detectable substance being convenient to detection.Therefore, in one embodiment, with detectable substance mark anti-NIP45 antibody of the present invention.The example of detectable substance comprises various enzymes, prothetic group, fluorescent substance, luminophore and radioactive substance.The example of suitable enzyme comprises horseradish peroxidase, alkaline phosphatase, beta galactosidase enzyme or acetylcholinesterase; The example of suitable prothetic group mixture comprises streptavidin/vitamin H and avidin/biotin; The example of suitable fluorescent substance comprises Umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazine amine fluorescein, dansyl chloride or phycoerythrin; The example of luminophore comprises luminol,3-aminophthalic acid cyclic hydrazide; The example of suitable radioactive substance comprises 125I, 131I, 35S or 3H.
D. medicinal compositions
NIP45 albumen of the present invention and anti-NIP45 antibody can be added the medicinal compositions that is suitable for giving.This composition comprises this albumen or antibody and pharmaceutically acceptable carrier usually.Term used herein " pharmaceutically acceptable carrier " comprises any He all and the medicinal solvent that adapts, dispersion medium, dressing, antiseptic-germicide and anti-mycotic agent, isotonic agent and absorption delay agent or the like.This medium and the medicine that are used for active medicinal matter are well-known in the art.Except with the situation of unmatched any conventional media of this active compound or medicine under, considered its application in said composition.Additional active compound can also be added in the said composition.
Prepare medicinal compositions of the present invention, its route of administration with plan is adapted.For example, the solution or the suspension that are used for non-enteron aisle, intracutaneous or subcutaneous use can comprise following component: sterile diluent, such as water for injection, salt brine solution, expressed oil, polyoxyethylene glycol, glycerine, propylene glycol or other synthetic; Antioxidant is such as the antibacterial agent of phenylcarbinol or methyl p-hydroxybenzoate (methyl parabens); Such as xitix or sodium bisulfite; Sequestrant is such as ethylenediamine tetraacetic acid (EDTA); Regulate the medicine of osmotic pressure, such as acetate, Citrate trianion or phosphatic damping fluid with such as sodium-chlor or glucose.Can regulate pH with the acid or the alkali of all example hydrochloric acids or sodium hydroxide.Disposable syringe or multiple doses cillin bottle that parenteral formulation can be packaged in peace bottle, make with glass or spear material.
The medicinal compositions that is suitable for injecting use comprises aseptic aqueous solution (if water-soluble) or dispersion and is used for preparing the sterilized powder of aseptic parenteral solution or dispersion temporarily.For intravenous administration, suitable carriers comprises physiological saline, bacteriostatic water, Cremophor EL TM(BASF, Parsippany, NJ) or phosphate-buffered saline (PBS).In all cases, said composition must be aseptic and should be that fluidic is to be easy to injection.It must be stablized under production and condition of storage and must carry out anticorrosion microbiological contamination effect with antagonism such as bacterium and fungi.This carrier can be to contain for example solvent or the dispersion medium of water, ethanol, polyvalent alcohol (for example glycerine, propylene glycol and liquid macrogol or the like) and their suitable mixture.Can be for example by use dressing such as Yelkin TTS, by the granular size that needing under the situation of dispersion, to keep with by using tensio-active agent to keep suitable fluidity.Can for example p-Hydroxybenzoate, trichloro-butyl alcohol, phenol, xitix, Thiomersalate or the like reach and prevent microbial process by various antiseptic-germicides and anti-mycotic agent.In many cases, be preferably in and comprise isotonic agent in the said composition, for example sucrose, polyvalent alcohol, sodium-chlor such as N.F,USP MANNITOL, sorbyl alcohol.Can be by the medicine that comprises that in said composition for example delay of aluminum monostearate and gelatin absorbs, the prolongation that reaches Injectable composition absorbs.
Can add in the appropriate solvent with a kind of above-mentioned composition of enumerating that needs or its combination by this active compound (for example NIP45 albumen or anti-NIP45 antibody), then carry out filtration sterilization and prepare aseptic parenteral solution requirement.Generally speaking, this active compound can be added in the aseptic solvent from above-mentioned other composition of enumerating that contain basic dispersion medium and needs and prepare dispersion.Under the situation of the sterile powder that is used to prepare aseptic parenteral solution, the preparation method of recommendation is vacuum-drying and lyophilize, produces this activeconstituents and any other needs the pulvis of composition from the solution of previous Sterile Filtration.
Oral compositions generally comprises inert diluent or edible carrier.They can be wrapped in gelatine capsule or be pressed into tablet.Be the purpose of oral therapeutic administration, this active compound can be added with vehicle, and use with tablet, lozenge or capsular form.Can also use fluid carrier to prepare oral compositions, to use as mouth-washes, wherein this compound in this fluid carrier can be oral, rinse and wash and cough and tell or swallow.The tackiness agent and/or the subsidiary material of pharmaceutically compatible can be included as the part of said composition.Tablet, pill, capsule, lozenge etc. can contain following composition or have compound any of similarity: tackiness agent, such as Microcrystalline Cellulose, tragakanta or gelatin; Such as the vehicle of starch or lactose, such as the disintegrating agent of alginic acid, Primogel or W-Gum; Wetting agent is such as the lubricant of Magnesium Stearate or Sterotes; Glidant is such as colloid silica; Sweeting agent is such as sucrose or asccharin; Seasonings is such as peppermint, wintergreen oil or orange seasonings.
In one embodiment, avoid this active compound of preparing carriers that body is removed fast with this compound of protection, described carrier for example comprises the controlled release preparation of the transfer system of implant and micro encapsulation.Can use biodegradable, biocompatible polymkeric substance, for example ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen protein, poe, poly(lactic acid).The method for preparing this preparation it will be apparent to those skilled in the art that.Also can be from AlzaCorporation and Nova Pharmaceuticals, commercially available this material that obtains of Inc..Can also use liposome suspension (comprising liposome) as pharmaceutically acceptable carrier with antiviral antigenic monoclonal antibody guiding cells infected.These can prepare according to method known to those skilled in the art, for example, and as United States Patent (USP) 4,522, No. 811 descriptions.
E. detect the active method of NIP45
The relevant use of another aspect of the present invention various NIP45 method for compositions of the present invention.For example, the invention provides and be used for detecting the method that NIP45 albumen or its mRNA exist at biological sample.This method relates to uses the medicine that can detect NIP45 albumen or its mRNA to contact this biological sample, makes and detects NIP45 albumen or the existence of its mRNA in this biological sample.The medicine of the detection NIP45mRNA that recommends be can with the labeling nucleic acid probe of NIP45mRNA hybridization.This nucleic acid probe can be NIP45cDNA or its part of for example SEQ ID NO:5, is at least 15,30,50,100,200,300,400,500,600,700,800,900 or 1000 Nucleotide and is enough under stringent condition oligonucleotide with NIP45mRNA hybridization such as length.Detect the proteic recommendation medicine of NIP45 and be can with the protein bound traget antibody of NIP45.Antibody can be polyclone or be more preferably monoclonal.Can use complete antibody or its part (for example, Fab or F (ab ') 2).The term " mark " relevant with this probe or antibody comprise by with detectable substance coupling (that is physical connection) to the direct mark of the probe of this probe or antibody or antibody and comprise by with the probe of the reaction of the reagent of another direct mark or the indirect labelling of antibody.The example of indirect labelling comprises the fluorescently-labeled secondary antibody of use and with vitamin H end mark dna probe it can be detected by fluorescently-labeled streptavidin, and the indirect labelling of detection one-level antibody.Term " biological sample " comprises tissue, cell and biological fluid.For example, the technology of detection NIP45 mRNA comprises Northern hybridization and in situ hybridization.Detect the proteic technology of NIP45 and comprise that enzyme linked immunological absorption detects (ELISA), Western trace, immunoprecipitation and immunofluorescence.VI. associating composition and medicine box
The present invention also provides the composition that comprises the modulability drug regimen.For example, the nucleotide sequence of the transcription factor of two or more coding and regulating Th2 relevant cell factor genetic expression can be added recombinant expression vector, and import host cell.For example, the invention provides recombinant vectors, import the host cell of this carrier, comprise cooperate with the NF-AT family protein second nucleotide sequence of second transcription factor that first nucleotide sequence and encoding of first transcription factor of regulating the genetic expression of Th2 relevant cell factor works to adjusting Th2 relevant cell factor gene of coding.Preferably, the first nucleotide sequence coded maf family protein (for example, c-Maf) or the NF-AT interaction protein (for example, NIP45).Preferably, the second nucleotide sequence coded transcription factor that is selected from NF-AT family protein, NF-AT interaction protein, maf family protein and AP-1 family protein.
The present invention also comprises the medicine box of regulating generation of Th2 relevant cell factor or the growth of Th1/Th2 subgroup.In one embodiment, medicine box of the present invention comprises at least a modulability medicine of packing with the specification sheets that uses generation of this modulability medicament adjusting Th2 relevant cell factor or Th1/Th2 subgroup to grow of the present invention.In one embodiment, this medicine box comprises at least a pungency medicine that is used to stimulate the Th2 relevant cell factor to produce or grows (or regulate the Th1 subgroup downwards and grow) to adjusted Th2 subgroup.In another embodiment, this medicine box comprises at least a suppressive drug that the Th2 relevant cell factor produced or regulated Th2 subgroup growth (or to the growth of adjusted Th1 subgroup) downwards that is used to suppress.The associating medicine box that comprises two kinds or multiple modulability of the present invention (for example, stimulate or suppress) medicine also is provided.VII. screening detects
Another aspect of the present invention is relevant differentiates that the screening of regulating compound detects, and described adjusting compound is regulated the transcription factor activity of Th2 relevant cell factor genetic expression.In various embodiments, these screenings detect and can differentiate the compound of for example regulating described transcription factor expression or functionally active, with the albumen of described transcription factor interaction and regulate the compound of these protein-protein interactions and in Th2 relevant cell factor gene, regulate described transcription factor and cis acting target site (for example, MARE) interactional compound.
In one embodiment, the invention provides the active compound of the transcription factor of differentiating adjusting and the genetic expression of activated T cells nf (NF-AT) family protein cooperation adjusting Th2 relevant cell factor, comprise
The indication composition is provided, and it has to cooperate with the NFAT family protein regulates the transcription factor activity of Th2 relevant cell factor genetic expression;
This indication composition is contacted with test compounds; With
Determine the influence of test compounds, with the compound of transcription factor activity that differentiate to regulate the adjusting Th2 relevant cell factor genetic expression of cooperating with the NFAT family protein thus to transcription factor activity in this indication composition.
This transcription factor can be a maf family protein for example, and for example c-Maf or little maf albumen are (for example, p18).Perhaps, this transcription factor can be and the interactional factor of NF-AT family protein, for example NIP45.
This indication composition can be a cell-free composite, or it can be a cell composition.For example, in one embodiment, this indication composition is a lymphoidocyte, for example the Th2 cell.In another embodiment, this indication composition is a yeast cell.
In recommended embodiment, this indication composition comprises indicator cells, and wherein said indicator cells comprises: (ⅰ) this transcription factor and the reporter gene of (ⅱ) this transcription factor being replied.Preferably, this indicator cells contains:
ⅰ) the encode recombinant expression vector of this transcription factor; With
ⅱ) comprise the carrier of adjusting sequence of the Th2 relevant cell factor gene of the reporter gene that is operably connected; And described method comprises:
A) contact this indicator cells with test compounds;
This report expression of gene level in this indicator cells when b) being determined at this test compounds of existence; With
The expression level of reporter gene and the expression level of reporter gene in the indicator cells when not having this test compounds in the indicator cells when c) relatively having this test compounds are to differentiate the compound of regulating this transcription factor activity thus.
In another recommended embodiment, the preparation that this indication composition comprises has: (ⅰ) this transcription factor and (ⅱ) this transcription factor bonded dna molecular, and
Described method comprises:
A) transfer test compounds to contact this indication composition
B) be determined at this transcription factor and the interactional degree of this dna molecular when having this test compounds; With
This transcription factor and the interactional degree of this dna molecular and this transcription factor and the interactional degree of this dna molecular when not having this test compounds when c) relatively having this test compounds are to differentiate the compound of regulating this transcription factor activity thus.
In an embodiment of preceding method, this transcription factor is that maf family protein and this dna molecular comprise maf response element (MARE).
In another recommended embodiment, this method is differentiated from the Th2 cell and albumen this transcription factor interaction.In this embodiment,
This indication composition is the indication cell, and this indicator cells comprises:
ⅰ) may be operably coupled to the reporter gene of transcriptional regulatory sequences; With
ⅱ) first mosaic gene of coding first fusion rotein, described first fusion rotein
Comprise and the collaborative Th2 of adjusting of NFAT family protein relevant cell factor gene table
The transcription factor that reaches;
This test compounds comprises the second mosaic gene library, this library second fusion rotein of encoding, and this second fusion rotein comprises the albumen that is derived from the Th2 cell;
This report expression of gene is to the interaction sensitivity between first fusion rotein, second fusion rotein and this transcriptional regulatory sequences; And
Wherein by measure this report expression of gene level in the indicator cells determine this test compounds to the influence of transcription factor in the indication composition to differentiate the proteic compound that is derived from the Th2 cell that comprises with this transcription factor interaction thus.
The present invention provides and differentiates the method for regulating interactional compound between NIP45 and the NF-AT family protein, comprises:
A) mix (ⅰ) and (ⅱ), wherein
(ⅰ) NIP45 or its NF-AT interaction part; With
(ⅱ) NF-AT family protein or its NIP45 interaction part;
Exist or do not exist under the situation of test compounds;
B) determine at (ⅰ) under the situation that has or do not exist test compounds and interactional degree (ⅱ); With
C) differentiate interactional compound between adjusting NIP45 and the NF-AT family protein.
The NIP45 of NF-AT family protein interacts and partly preferably comprises the Rel homeodomain of NF-AT family protein.In one embodiment, by with detectable substance mark (ⅰ) or (ⅱ), separate unmarked (ⅰ) or (ⅱ) and quantitatively with unmarked (ⅰ) or (ⅱ) (ⅰ) or (ⅱ) of bonded mark, and determine (ⅰ) and (ⅱ) between interactional degree.Can use this method discriminating or strengthen or weaken interactional compound between NIP45 and the NF-AT family protein.
Screen in the recommended embodiment of detection in the present invention,, just test of the influence of this test compounds then immunne response in case the differential test compound is regulated the purpose transcription factor activity.Therefore, screening method of the present invention can comprise again determine this compound to the influence of immunne response to differentiate the compound of regulating immunne response thus.In one embodiment, by measuring the influence that this compound is expressed Th2 relevant cell factor gene (for example, the interleukin-4 gene), determine of the influence of this compound to immunne response.In another embodiment, by measuring this compound, determine of the influence of purpose compound to immunne response to auxiliary 1 type (Th1) of T or the auxiliary cytocerastic influence of 2 types (Th2) of T.
The recombinant expression vector (referring to above-mentioned discussion, also can referring to embodiment) that can be used for expressing transcription factor known in the art at indicator cells.In one embodiment, in expression vector, this transcription factor encoding sequence be may be operably coupled to the adjusting sequence that allows this transcription factor constitutive expression in this indicator cells (for example, can use regulate sequence) such as the virus of cytomegalovirus promotor/enhanser.Preferably will allow the recombinant expression vector of this transcription factor of constitutive expression in indicator cells to be used to differentiate the compound that strengthens or suppress this transcription factor activity.In a displacement embodiment, in this expression vector, the transcription factor encoding sequence be may be operably coupled to the adjusting sequence (that is, being derived from the promotor regulatory region of this native gene) of endogenous corresponding transcription factor gene.Recommending wherein, transcription factor expression is subjected to the recombinant expression vector of endogenous adjusting sequence control to be used to differentiate the compound that strengthens or suppress this transcription factor transcriptional expression.
Used therein in the method for Th2 relevant cell factor gene, this Th2 relevant cell factor is interleukin-4 preferably.Before shown that Th2 specificity induction type IL-4 expression can be by the medium and small near-end IL-4 promotor guidance (Hodge, M. etc. (1995) J.Immunol.154:6397-6405) to 157bp of Th2 cell.Therefore, in one embodiment, method of the present invention use contain the district of this near-end IL-4 promotor, preferably the Nucleotide of IL-4 promotor-157 to+58 the reporter gene construction of (with respect to being positioned at+1 transcription initiation site).Perhaps, can use the longer part in IL-4 upstream regulation district of the upstream regulatory sequence of all 3kb according to appointment, reach stronger reporter gene and express.Suitable reporter gene construction is described in Todd, M. etc. (1993) J.Exp.Med.177:1663-1674.The description of relevant IL-4 reporter gene construction also can be referring to embodiment.
Various reporter genes are known in the art and are applicable to that screening of the present invention detects.The example of the reporter gene that is fit to comprises the reporter gene of those coding E.C. 2.3.1.28s, beta galactosidase enzyme, alkaline phosphatase or luciferase.The active standard method of these gene products of mensuration known in the art.
There are various cell types to be suitable for using as the indicator cells in the screening detection.Preferably use and do not express the clone of c-Maf, for example B cell (for example, M12B lymphoma cell line) or Th1 cell clone (for example, AE7 cell) usually.Can also use non-lymphoid cell line as indicator cells, for example HepG2 hepatocellular carcinoma cells system.
In one embodiment, when having test compounds in the indicator cells expression level of reporter gene be higher than when not having test compounds the expression level of reporter gene in the indicator cells, then this test compounds is differentiated to stimulating this transcription factor expression or active compound.In another embodiment, when having test compounds in the indicator cells expression level of reporter gene be lower than when not having test compounds the expression level of reporter gene in the indicator cells, then this test compounds is differentiated to suppressing this transcription factor expression or active compound.
Use the method for replacing of reporter gene construction to be, differentiate adjusting transcription factor expression or active compound by using other " to read ".For example, can use transcription factor expression carrier transfection indicator cells, cultivate existing or do not exist under the test compounds, can be (for example by cytokines mRNA in the detection indicator cells, IL-4mRNA) or be secreted in the culture supernatant cytokine (for example, the IL-4 secretion), estimating the Th2 relevant cell factor produces.The standard method that detects cytokines mRNA for example is a reverse transcriptase polymerase chain reaction (RT-PCR), is known in the art.The proteic standard method of cytokine for example detects (ELISA) for enzyme linked immunological adsorbs in the detection culture supernatant, and is also known in the art.Further describing of related detection cytokines mRNA and/or protein process also can be referring to embodiment.
As mentioned above, the invention provides the screening method of differentiating with purpose transcription factor (for example c-Maf or NF-AT) or the interactional albumen of NIP45 (for example, the albumen in the Th2 cell).Can based on known in the art pair of hybrid detection system (also be called interaction trap detect (interaction trap assay)) (referring to for example, No. 5,283,173, Field United States Patent (USP); Zervos etc. (1993) Cell72:223-232; Madura etc. (1993) J.Biol.Chem.268:12046-12054; Bartel etc. (1993) Biotechniques 14:920-924; With (1993) Oncogene8:1693-1696 such as Iwabuchi) design these detections.Two hybrids detect the albumen that generally is used to differentiate with the particular target protein-interacting.This detects and adopts gene fusion, can interact with the albumen of recombination function activating transcription factor to differentiate.This activating transcription factor is made up of in conjunction with territory and trans-activation domain DNA, and wherein these two territories all are essential for the genetic transcription that activates target sequence (for example upstream stimulating factor sequence (UAS) of GAL4) downstream.The proteic dna sequence dna of the target " bait " of will encoding merges to one of these territories, and the dna sequence dna library is merged to another territory.(for example can be bonded to this target-fusion rotein, target GAL4-merges " bait ") " fish " fusion rotein (producing) by this fusion library generally these two territories (DNA is in conjunction with territory and trans-activation domain) are brought to enough closely, be inserted in the transcribing of reporter gene in this target sequence downstream with activation.Therefore, can differentiate " fish " albumen by the ability of its recombination function trans-activator (for example, function GAL4 activating transcription factor).
By (for example making up target c-Maf fusion rotein, c-Maf/GAL4 in conjunction with the territory fusions as " bait ") and the cDNA library of " fish " fusion rotein (for example, cDNA/GAL4 activation domain library), and these constructions are imported also to contain (for example be connected to adjusting sequence that c-Maf is replied, the MARE sequence, the host cell of a reporter gene district of IL-4 promotor for example as discussed above), this common double hybrid system can be used for differentiating that Th2 cell and c-Maf are (perhaps, use similar approach and other purpose transcription factor) interactional albumen, wherein this cDNA library preparation is from the mRNA of Th2 cell.Trans-activation based on this report gene constructs can identifier number and the interactional proteic cDNA from the Th2 cell of c-Maf.
Perhaps, can use such as being described in Sieweke, " the single hybrid " of M.H. etc. (1996) Cell 85:49-60 detects, and differentiates and the interactional albumen from the Th2 cell of c-Maf.This detection is the modification of the two hybrid system of above-mentioned discussion.In this system, described " bait " is the transcription factor (for example, having removed the c-Maf of N-terminal trans-activation domain) of having removed trans-activation domain, and described " fish " is non-fusion cDNA library (for example, preparation is from the cDNA library of Th2 cell).These constructions are imported the host cell (for example, yeast cell) that also contains the reporter gene construction that is connected to adjusting sequence that this transcription factor is replied (for example, MARE sequence, for example district of this IL-4 promotor that c-Maf is replied).Trans-activation based on this report gene constructs can identifier number and the interactional proteic cDNA from the Th2 cell of c-Maf (or other purpose transcription factor).
As mentioned above, the invention provides screening and detect, regulate transcription factor of the present invention and the interactional compound of this transcription factor bonded dna molecular to differentiate, this transcription factor respectively in conjunction with in the IL-4 generegulation district such as c-Maf and MARE.Detection DNA known in the art is conjugated protein, and (for example, the electrophoretic mobility change detects, DNA enzyme I footprint detects or the like with the interactional detection of target DNA sequence; Relevant further description is referring to embodiment).By when existing or not having test compounds, carrying out this detection, can use these to detect to differentiate and regulate (in the example, suppress or strengthen) DNA conjugated protein with the interactional compound of its target DNA sequence.
In one embodiment, when having this test compounds and the amount of this dna fragmentation bonded transcription factor be higher than when not having this test compounds and the amount of this dna fragmentation bonded transcription factor, in this case, this test compounds is differentiated to strengthening this transcription factor bonded compound.In another embodiment, when having this test compounds and the amount of this dna fragmentation bonded transcription factor be lower than when not having this test compounds and the amount of this dna fragmentation bonded transcription factor, in this case, this test compounds is differentiated to suppressing this transcription factor bonded compound.
Regulate in the method for NIP45 and the interactional medicine of NF-AT family protein in discriminating of the present invention, can use isolating NIP45 and/or NF-AT family protein in the method, perhaps can only use the part of NIP45 and/or NF-AT family protein.For example, isolating NF-AT Rel Si Yuan territory (or comprise bigger NF-AT subprovince of the RHD) can be used as the NIP45 of the NF-AT part that interacts.Equally, can use can be in conjunction with the NIP45 part of this NF-AT RHD.In a recommended embodiment, one or two in (ⅰ) and (ⅱ) all is fusion roteins, for example gst fusion protein (for example, GST-NF-AT RHD can interact as the NIP45 of NF-AT part use).By with one of them albumen of detectable substance (for example, radio-labeled) mark, separate unmarked albumen and quantitatively and the amount of unmarked protein bound detectable substance and determine (ⅰ) and (ⅱ) between interactional degree.Can use this method discriminating or stimulation or suppress interactional medicine between NIP45 and the NF-AT family protein.Based on its interaction degree when not having this medicine compare enhancing (ⅰ) and (ⅱ) between the ability of interaction degree, differentiating stimulates NIP45 and the interactional medicine of NF-AT family protein, and based on its interaction degree when not having this medicine compare reduction (ⅰ) and (ⅱ) between the ability of interaction degree, differentiate and suppress NIP45 and the interactional medicine of NF-AT family protein.The detection system that can adopt the discriminating of No. 5,352,660, the United States Patent (USP) that is described in Pawson to regulate SH2 territory-ligand interaction is differentiated and is regulated the interactional medicine of NIP45/NF-AT RHD.
Further describe the present invention by the following embodiment that should not be considered as limiting.The content of all reference, patent and the disclosed patent application that please quote in this is attached to herein by reference.The nucleotide sequence and the aminoacid sequence that are stored in the public database that this paper quotes also are attached to herein by reference.Embodiment 1: the cytokine specificity is because pros-and-cons type incitant rather than because aporepressor
Can reach tissue specificity by aporepressor or reticent proteic effect.Therefore possibility IL-2 and IL-4 gene are initiatively checked in Th2 and Th1 cell respectively.For detecting the existence of aporepressor, between Th1 of different mhc class i haplotypes (D1.1) and Th2 (D10) clone, carry out somatocyte and merge.Merge Th1 clone D1.1 (Kd) and Th2 clone D10 (Kk) according to " suspension cell fusion " program (Lane, R.D. etc. (1986) MethodsEnzymol.121:183-192).After the fusion, make cellular-restoring 8 hours, put together anti-Kk antibody with PE then and FITC puts together anti-Kd antibody (Phamingen, La Jolla, CA) two dyeing.Based on big young pathbreaker's cell sorting, distinguishing not fused cell from heterokaryon and homokaryons, and by the positive and two positive cells of fluorescence identification list.As be shown in the diagram of this method of Figure 1A, sorting three kind of groups: big PE positive cell (D1.1 * D1.1), big FITC positive cell (D10 * D10) and big PE and FITC positive cell (D1.1 * D10).The cell of expressing mhc class i Kb and Kk mark is a heterokaryon, and only the cell of expressing K b or Kk is represented homokaryons and as contrast.
Cultivating this three kind of groups of moderate stimulation with anti-cd 3 antibodies then, with the expression of activating cells factor gene, and preparation is used for the RNA of RT-PCR and Northem engram analysis.Obtain about 5 * 105 cells from each colony.Usually, the cell of 5-10% merges.Then these three colonies respectively are divided into 2 half, general-semicell is transferred to prewashed anti-CD3 bag by plate, remaining half is transferred to does not wrap by plate.After 4 hours, harvested cell, and (Statagene, La Jolla CA) separate polyadenylation RNA to use Micro-Fast TrackTM medicine box.Use SuperScript medicine box (Gibco/BRL, Bethesda, MD) preparation cDNA, and be used for using commercially available to mouse IL-2, IL-4 and the specific primer of β Actin muscle (Stratagene, La Jolla, pcr analysis CA) according to manufacturer specification.PCR reaction comprise 0.5 μ Ci α-32P-dCTP (3000Ci/mmol, NENDupont).With ethanol sedimentation PCR product, and by non-sex change PAGE separate, dry and pass through autoradiography observation.
The RT-PCR analytical results of mRNA expression of cytokines is shown among Figure 1B.Th1 and Th2 clone and Th homokaryons are only transcribed IL-2 (Th1) or IL-4 (Th2) respectively, and the Th1/Th2 heterokaryon produces this two kinds of cytokines.In contrast, the existence of aporepressor should cause eliminating these two kinds of cytokines in heterokaryon.Reach a conclusion from these experiments and to be: is by the mediation of Th specificity pros-and-cons type incitant at Th1 to the cytokine specificity the Th2 cell, rather than protein mediated by selective silence.Embodiment 2: it is special to separate Th2 from preparation from anti-CD3 activated Th2 clone's cDNA library
Property c-maf gene
In that (relevant this system description is referring to No. 5,283,173, Field United States Patent (USP) for example by the two hybrid systems of yeast; Zervos etc. (1993) Cell72:223-232; Madura etc. (1993) J.Biol.Chem.268:12046-12054; Bartel etc. (1993) Biotechniques 14:920-924; With (1993) Oncogene8:1693-1696 such as Iwabuchi) with regard to the screening preparation of NF-AT interaction protein aspect in the process in the cDNA library of anti-CD3 activated Th2 clone D10, separated a plurality of cDNA, these cDNA are extremely weak interaction factors.By using one group of Th1 and Th2 clone's Northern engram analysis, the Th that estimates all cDNA that obtain in this screening is specific expressed then.(in 140 60) this cDNA of a repeated isolation only detects transcript at the RNA from Th2 clone (D10, CDC35) preparation, rather than from or Th1 clone (AR5, OS6, D1) or detect transcript from the RNA of B cell lymphoma M12 preparation, as be depicted in that Northern engram analysis among Fig. 2 A describes.In addition, when being activated with being connected of anti-cd 3 antibodies by this TXi Baoshouti, detected transcript level significantly increases in the D10Th2 cell.When anti-CD3 handles, transcribe not induce by detected this of this cDNA clone and in the Th1 clone, take place.Contrast probe GAPDH confirms that the RNA applied sample amount is basic identical in all swimming lanes.Therefore, this cDNA is cloned in expression in this lymph sample pedigree, and to look like Th2 specific, and the signal sensitivity to transmitting by this TXi Baoshouti.For these Northern traces, use Trizol (GIBCO/BRL) to prepare total RNA according to manufacturer specification.With the total RNA of 10 μ g of each sample fractional separation and be transferred to nylon membrane on the formaldehyde agarose gel.(Boehringer Mannheim, Indianapolis IN), derive from the 300bp Dra I fragment of 3 ' non-translational region of this separating clone with α-32P-dCTP mark to use random primer dna marker medicine box.(Stratagene, La Jolla CA) is hybridized to use QuikHyb according to manufacturer specification.
For determine that whether this expression of gene is tissue specificity and is regulated, and carries out following experiment in normal Th cell development process.By existing cytokine and anti-cytokine antibodies (for Th1 IFN γWith anti-IL-4, for Th2 with IL-4 and anti-IFNg) situation under, drive splenocyte (Th precursor (Thp) cell) originally with anti-CD3 processing along Th1 or Th2 approach.Prepare splenocyte suspension from 6-8 Balb/c mouse in age in week, cultivate in the RPMI 1640 that has added 10%FCS with the density of 106 cells/ml, and have anti-IL4 antibody of 5 μ g/ml (11B11) (for the Th1 pedigree) or the anti-IFN of 5 μ g/ml γStimulate in conjunction with anti-cd 3 antibodies with dull and stereotyped under the situation of antibody (XMG-1) (for the Th2 pedigree).Stimulated 24 hours afterwards, and 50 U/ml IL2 were added all cultures, and 500 U/ml IL4 (Genzyme) are added the Th2 culture.Behind primary stimulus 7 days, gather in the crops all cells, clean cell and stimulate again with dull and stereotyped bonded anti-cd 3 antibodies.Use the method for foregoing description, cell in the differentiation of first (0-8 days) and the different time points results during reply (0-20 hour) once more after stimulation is carried out the Northern engram analysis, and pass through elisa assay culture supernatant IL-10 and IFN γ, determine to differentiate the Thp cell that is divided into Th1 or Th2.As the following quantitative ELISA of cytokine that carries out.All anti-cytokine antibodies are all available from Pharmingen.ELISA is carried out in explanation according to Pharmingen, except using 1: 500 dilution avidin-alkaline phosphatase (Sigrna) replacement avidin-peroxidase among the PBS/BSA.Use substrate buffer solution (10% diethanolamine, 0.5mM MgCl 2, 0.02% sodiumazide, pH9.8) in the phosphoric acid p-nitrophenyl (GIBCO BRL) of 4mg/ml as substrate.
Be shown in two independent experiments of Fig. 2 B at its representative result, this analysis is arranged in when being shown in the 0th day that the expression level of this cDNA of splenocyte originally of baseline is low maybe can't to be detected.In the culture of Th2 approach differentiation, a large amount of transcript of generation is induced when 8 days of primary stimulus and stimulate once more 20 hours.In contrast, along not inducing in the cell of Th1 approach driving.Contrast probe (GAPDH) shows that the RNA applied sample amount is basic identical in all swimming lanes.The low-level transcript that exists in the cell that drives along the Th1 approach may reflect and has residual Th2 cell, because in this vitro differentiation system complete deflection does not take place.
In general, these experiments disclose this and separate optionally expression in the Th2 clone of cDNA, and it is induced when t cell activation, and it is not present in Th1 clone and B lymphoma.In addition, when this gene when the Th2 pedigree drives is being induced in normal Thp, and in growing, Th1 do not induced.
The cDNA that use obtains from the two hybrid screenings of yeast separates full-length cDNA from the D10Th2 cell cdna library by the standard hybridizing method as probe.Check order from this Th2 cell library separation 4.3kb cDNA and with standard method.Sequential analysis discloses, this Th2 specific gene aspect sequence corresponding to the c-maf proto-oncogene.The ectopic expression of embodiment 3:c-Maf in Th1 and B cell causes the IL-4 promotor to activate
Be described in separation cDNA among the embodiment 2 differentiate for the member of AP-1/CREB/ATF gene family with and in the Th2 cell selective expression, improved the possibility of the IL-4 gene organization specific transcriptional of c-Maf control.In addition, in the Th2 cell and among four three of transforming in the mast cell line that detect, the existence of the transcript of coding c-maf is very relevant with the expression of IL4.Can trans-activation IL-4 promotor for whether testing c-Maf, carried out cotransfection experiments.
Thl clone and B lymphoma M12.4.C3 (M12) neither express c-maf and do not transcribe the IL-4 gene again.If c-Maf is the transcription factor to control IL-4 genetic expression key, forced expression should make IL-4 genetic expression in these cells so.For detecting this point, with the Sal I site that total length (4.3kb) c-maf cDNA clone inserts the pMex-NeoI mammalian expression vector, this carrier utilizes cmv enhancer to drive the expression of insertion sequence.Then this c-Maf expression vector and IL-4 promotor report construction cotransfection are gone into Th1 clone AE7 and B lymphoma M12.At Hodge, the wild-type IL4CAT report construction that preparation contains from-157 to+68 the IL4 promoter fragment that may be operably coupled to chloramphenicol acetyl transferasegene has been described among M. etc. (1995) J.hnmunol.154:6397-6405.This Th1 clone is incubated among the RPMI1640 of the rat spleen cells supernatant liquor of having added 10%FCS and 10%Con-A stimulation, and by keeping with stimulating once in suitable antigen and per two weeks of APC.With the M12 cell cultures in the RPMI1640 that has added 10%FCS.
Clone AE7 or M12B lymphoma cell by the various plasmids with 20 μ g (AE7) or 5 μ g (M12) in 10 minutes transient transfection Thl of room temperature preincubation 0.4ml cell, this 0.4ml cell contains 2 * 107 cells/ml AE7 or 3 * 106 cells/mlM12 cell in serum-free RPMI 1640.(BIO-RAD, Richmond CA) carry out electroporation to sample, and place immediately 10 minutes on ice with the BIO-RAD gene pulse producer that is set in 975 μ F, 280V then.Make cells transfected in perfect medium, recover to spend the night, and with dull and stereotyped in conjunction with anti-cd 3 antibodies ({ Pharmningen, San Diego, 1 μ/the ml of CA} in 1XPBS spends the night in 4 ℃) or with 50ng/ml PMA (Sigma, St.Louis, MO) and 1 μ M ionomycin (CalbiochemCorp., La Jolla Califomia) stimulate 24 hours.By in 0.25M Tris-Cl, the freeze-thaw cracking prepares cell pyrolysis liquid among the pH7.8.The albumen (5-20 μ g) of equivalent is used for the CAT detection.As Todd, the described CAT that carries out of M etc. (1993) J.Exp.Med.177:1663-1674 detects.
Before shown that Th2 specificity induction type IL-4 expression can be by the medium and small near-end IL-4 promotor guidance (Hodge, M. etc. (1995) J.Irmmunol.154:6397-6405) to 157bp of Th2 cell.In the result is summarized in cotransfection experiments among Fig. 3 A, confirm c-Maf ectopic expression in Th1 clone AE7 cause stimulating by TXi Baoshouti after the remarkable activity of this IL-4 promotor reporter.It is only to use observed 5 times of empty carrier approximately that observed multiple is induced.Do not express in AE7 cell subclone used herein although before observed the report construction that contains near-end (157 to+58) IL-4 promoter sequence, and confirmed and can in other clone of AE7, detect a spot of IL-4mRNA by RT-PCR.For more strictly testing c-Maf, in B lymphoma cell line M12, carry out same experiment in the ability that non-IL-4 produces trans-activation IL-4 promotor in the cell.Normal B cell and B lymphoma cell do not produce IL-4.The representative result of cotransfection experiments is depicted in Fig. 3 B, and the summary of three independent experiments is shown in the following table 1.
Table 1
CAT activity (multiple is induced)
Plasmid The PMA/ ionomycin The experiment I * The experiment II The experiment III
pMEX-NeoⅠ/pREP4 - 1 ?1 ?1
+ 7.6 ?1 ?1.4
pMEX-Maf/pREP4 - 95 ?5 ?18.6
+ 186 ?7 ?37
pMEX-c-Fos/pREP4 - 2.7 ?1 ?0.8
+ 7.6 ?1.2 ?1
pMEX-JunD/pREP4 - ND ** ?0.9 ?0.5
+ ND ?1.4 ?1.9
pMEX-NeoⅠ/pREP4-NF-ATp - 14.2 ?1.6 ?0.3
+ 41.2 ?3.5 ?0.3
pMEX-Maf/pREP4-NF-ATp - 136 ?54 ?26.3
+ 138 ?100 ?54.7
pMEX-c-Fos/pREP4-NF-ATp - 7.4 ?1.6 ?3
+ 15.4 ?1.9 ?6.1
* in the experiment I, with 20mg cell pyrolysis liquid incubation 2 hours.In experiment II and III, only, do not do with the synergy * * ND=that discloses between c-Maf and the NF-ATp with 5mg cell pyrolysis liquid incubation 1 hour
Result in the M12B lymphoma cell has confirmed the discovery in the Th1 clone.The ectopic expression of c-Maf cause or with or the M12 cell that stimulates without the PMA/Ca++ ionophore in the remarkable activity of IL-4 promotor.Compare with the control vector transfection, observed multiple is not induced and on average is about 50 in stimulating the M12 cell.Stimulate the M12 cell with the sub-carrier of PMA/Ca++, this stimulation should cause the NF-AT transposition to examining and induce other AP-1 family member (Flanagan, W.M. (1991) Nature352:803-807; Jain, J. etc. (1993) Nature365:352-355), the basis that increases the IL-4 promotor is active, but still has significantly inducing (on average being about 25 times) of the promoter activity that caused by c-Maf.The not trans record of C-Maf activates the contrast reporter that is driven by the many bodies of NF-AT, confirms the specificity of trans-activation.
As the c-Maf specific contrast opposite, utilize in the M12 cell of the mouse full-length cDNA of coding c-Fos and JunD in mammalian expression vector pMEX-Neo I and IL-4 report plasmid also overexpression c-Fos and c-Jun albumen with other AP-1 family member.By one of these two AP-1 family members of overexpression in the M12 cell, can not reach the IL-4 promoter activity.Therefore, c-Maf has IL-4 gene transcription in unique ability driving M12B cell.In addition, be that forced expression c-Maf also causes IL-4 promotor trans-activation among the HepG2 at hepatocellular carcinoma cells.These experiment confirms, supply c-Maf are given negative Th1 of c-Maf or B cell or are made this cell trans-activation IL-4 promotor for non-lymphoidocyte (for example, hepatocellular carcinoma cells system).
Shown that NF-AT albumen is all of crucial importance in regulating IL-4 and IL-2 cytokine.NF-ATp is first separated member (McCaffrey, P.G. etc. (1993) Science262:750-754) of this family.AE7 and M12 cell all have endogenous NF-ATp albumen, but do not transcribe IL-4.Therefore although NF-ATp is not the reason of selectivity IL-4 genetic transcription, test meaningfully whether in the M12 cell that does not stimulate or stimulate overexpression NF-ATp can further strengthen the trans-activation of the IL-4 promotor that causes by c-maf.With IL-4 report construction and or NFAPp expression vector (pREP4-NF-ATp, it also carries hygromycin gene) self or NFAPp expression vector and c-Maf expression vector cotransfection M12 cell.Only overexpression NF-ATp causes the restraining that has of IL-4 promotor to be expressed in the M12 cell.This trans-activation is strengthened significantly by the ectopic expression of c-Maf, this enhancing be not superimposed type but collaborative (referring to Fig. 3 B and table 1).Contrast with it, c-Fos overexpression can further not strengthen by what NF-ATp reached a restraining trans-activation.These results show that c-maf and NF-ATp interact, and express with the maximum that reaches this IL-4 promotor, and this tissue specificity is provided by c-Maf.The ectopic expression of embodiment 4:c-Maf activates endogenous IL-4 gene transcribing in the B lymphoma
As in embodiment 3, confirming trans-activation IL-4 promotor during the transient transfection of c-Maf in Th1, B and non-lymphoidocyte detects.Whether can activate endogenous IL-4 and transcribe for test c-maf produces to express in the cell at non-IL-4, with the expression vector of coding c-maf, NF-ATp or encode the two or coding junD and with or without NF-ATp as contrasting stable transfection B lymphoma M12.Be stable transfection, as transfection M12 cell among the above-mentioned embodiment 3.Add concentration be 400 μ g/ml Xin Meisu (GIBCO/BRL, Gaithersburg, MD) and Totomycin (Calbiochem Corp.) makes cells transfected recover in perfect medium 48 hours before.Every other day add cells transfected with fresh culture.
The M12 cell of stable transfection is paved plate with equal densities, and after 24 hours, gather in the crops supernatant liquor, measure cytokine by ELISA.As the ELISA that carries out that describes among the embodiment 2.The result who is shown among Fig. 4 confirms that the M12 cell with c-maf, junD or NF-ATp self transfection in these experiments does not produce the IL-4 that ELISA can measure.Yet it is detectable but be low-level IL-4 to produce ELISA with the M12 cell of c-maf and NF-ATp stable transfection together.These results confirm the RT-PCR from the RNA of these transfectional cells.On the contrary, these cells do not produce detectable IL-2.Need these factors of noticing in the transient transfection experiment in c-maf and NF-ATp and the M12 cell consistent simultaneously in the synergistic effect of trans-activation IL-4 promotor.Contrast with it, transfection can strengthen the AP-1 family member's that IL-4 expresses junD self or with the NF-ATp transfection, not cause IL-4 to produce in the Th2 cell.These results confirm, c-Maf is instructing the aborning essential and selectively acting of the endogenous IL-4 of tissue specificity.A site in the embodiment 5:IL-4 promotor is cloned by Th2 rather than Th1 clone's extract footprint covers
Embodiment 3 and the 4 experiment confirm c-maf that describe the clearly effect in control tissue specificity IL-4 expresses.In addition, the c-maf transcript is expressed in the Th2 cell rather than in the Th1 cell.Yet, when using preparation, detect (EMSA) by the electrophoretic mobility change and do not detect the DNA-albumen composition from the nuclear extract of Th2 cell.For the albumen that further detects in the Th2 nuclear extract whether can use more responsive DNA enzyme I footprint technique in conjunction with MARE or near sequence.By TXi Baoshouti is connected in conjunction with anti-cd 3 antibodies with dull and stereotyped, activates two Th2 clones (D10, CDC35) and two Th1 clone (AE7, S53), and in 0 o'clock (stimulation), prepare nuclear extract after 2 hours and 6 hours.According to standard method, use the end-labelled IL-4 promoter fragment of Ke Lienuo (157 to+68) to carry out the analysis of DNA enzyme I footprint then.The results are shown in Fig. 5 A.Cover the AP-1 site (Rooney of two NF-AT sites and upstream, NF-AT site, previously described distally from the equal footprint of the stimulation nuclear extract of Th1 and Th2 cell, (1995) Immunity2:545-553 such as J.), regulate function consistent (Rooney, J. etc. (1995) Immunity2:545-553 of IL-2 and IL-4 promotor with NF-AT that confirms and AP-1 albumen; Rooney, J. etc. (1995) Mol.Cell.Biol.15:6299-6310).In addition, check that this radioautograph discloses, when the extract that uses from the Th1 cell of Th2 that stimulates rather than stimulation, super quick district is arranged at the residue-28 and-29 places of noncoding strand.Not the Th cell extract of Ci Jiing not footprint cover this district.Observed Th2 footprint is not dense, but can repeat in two experiments, and to be arranged in that previous confirmation activates at the Th2 cell the IL-4 promotor be crucial site (Hodge, M. etc. (1995) J.Immunol.154:6397-6405).Diagram summary by the site that occupies in the IL-4 promotor of footprint analyzing and testing is shown among Fig. 5 B.These results show that before being shown as the important site in near-end IL-4 promotor of function is arranged in activated Th2 cell, rather than are arranged in activated Th1 cell.Embodiment 6: reorganization c-maf is bonded to the MARE site in the IL-4 promotor
This Th2 specificity footprint does not contain c-maf response element (MARE).But, check that this near-end IL-4 promotor discloses the half c-maf binding site (MARE) (residue-42 is to-37) (being shown in Fig. 5 B) near downstream, this near-end NF-AT site (residue-56 is to-51).Previous this site mutation that confirmed has been eliminated the activity of this IL-4 promotor in the Th2 cell (Hodge, M. etc. (1995) J.Immunol.154:6397-6405).For whether detecting c-Maf in conjunction with this site, the c-Maf recombinant protein of brachymemma that will contain b-slide fastener territory (amino acid/11 71-371) is at expression in escherichia coli, and on S-mark agarose column purifying, and be used for using the electrophoretic mobility change of radiolabeled MARE oligonucleotide to detect.
CDNA fragment by the c-Maf amino-acid residue 171-317 that will encode (is disclosed in Kurschner C. and Morgan, J.I. (1995) Mol.Cell.Biol.15:246-254) insert pET29 (Novagen, Inc.Madison, Not I site WI), the expression vector of structure reorganization c-Maf.The c-Maf albumen that uses the T7 polysaccharase in BL21 (DE3) strain, to express this brachymemma.By adding 1mM IPTG and in 3 hours inducing cells of 37 ℃ of incubations.The inductive cell is also then passed through the supersound process cracking at lX combination/cleaning buffer solution (20mM Tris-HCl pH7.5,150mM NaCl, 0.1%TritonX-100).Use the specification sheets of S-mark purifying medicine box (Novagen), from soluble fractions purifying c-Maf albumen according to manufacturers.Two other albumen NF-ATp and c-Jun also are used for EMSA and detect.Use contains the segmental in-vitro transcription of the cDNA/translation carrier TP7-NF-ATp in the Rel territory of coding mouse NF-ATp, expresses the reorganization NF-ATp in the Rel territory of containing mouse NF-ATp.By the Pst I site that total length mouse c-Jun cDNA is inserted pGEM4, make up c-Jun expression vector pGEM-c-Jun.From this T7 promoter transcription every kind of plasmid DNA of 1 μ g, and by using the TnT coupling to transcribe/translate medicine box (Promega, Madison WI) translate in rabbit reticulocyte lysate.
Change detection (EMSA) as the following electrophoretic mobility of carrying out.Double chain oligonucleotide usefulness γ-32P-dATP (DuPont NEN Research Product, wilmington, DE) usefulness T4 polynucleotide kinase (Pharmacia LKB Biotechnology, Inc., Piscataway, NJ) end mark with 100ng.With double chain oligonucleotide fractional separation on the 15-20% polyacrylamide gel of this mark, in 1X TE, spend the night and in ethanol, precipitate in 37 ℃ of wash-outs.Carry out combination in room temperature and detect 20 minutes, 0.5 μ g recombinant protein in 20mM HEPES (pH7.9), 100mMKCl, 5% glycerine, 1mM EDTA, 5mM DTT, 0.1%NP-40 and the 0.5mg/ml BSA of 15 μ l volumes or the external translation product of 0.4 μ l, 500ng poly (dI-dC) and 20,000cpm probe have been used in this detection.In containing 4% non-denaturing polyacrylamide gel of 0.5X TBE, this sample classification is separated then in room temperature.
The oligonucleotide that is derived from mouse IL4 promotor that is used for EMSA is :-59 to-27:5 '-CTCATTTTCCCTTGGTTTCAGCAACTTTAACTC-3 ' (SEQ ID NO:1);-79 to-60:5 '-ATAAAATTTTCCAATGTAAA-3 ' (SEQ ID NO:2); With-88 to-61:5 '-TGGTGTAATAAAATTTTCCAATGTAAA-3 ' (SEQ ID NO:3).The sequence that is used for the MARE oligonucleotide of EMSA is: 5 '-GGAATTGCTGACTCAGCATTACT-3 ' (SEQ ID NO:4). with its corresponding reverse complemental chain annealing of all oligonucleotide, to form double chain oligonucleotide.
Use reorganization c-Maf EMSA the results are shown in Fig. 6.This reorganization c-Maf albumen and total MARE oligonucleotide and be present in the 33bp oligonucleotide that contains this NF-AT site and MARE among this IL-4 and all combine well.In conjunction with being competed by unmarked homologue rather than contrast probe specificity ground.In addition, c-Maf5 does not combine with the oligonucleotide of the NF-AT target sequence that only contains reorganization NF-ATp good combination.C-Maf and this IL-4 promoter probe bonded ability are specific, because in vitro translated c-Jun albumen does not combine with this oligonucleotide.This c-Jun albumen is functional, because it can combine with the total MARE that contains core TRE site.These results show that c-Maf rather than another AP-1 family member (c-Jun) can combine with the MARE site in the near-end IL-4 promotor.
NF-AT and AP-1 family member albumen interact collaboratively, are being formed on higher mixture (Jain, J. (1993) Nature365:353-355 of mobility on IL-2 and the IL-4 promoter DNA on the EMSA; Rooney, J. etc. (1995) Immunity2:545-553).The functional study prompting NF-AT albumen of describing among the previous embodiment may interact with c-maf.For determining whether c-Maf interacts with NF-AT when having DNA, contain among the EMSA of this NF-AT site and the 33bp oligonucleotide in contiguous MARE site in use, independently or together use reorganization NF-ATp and c-Maf.The results are shown in Fig. 6.Every kind of independent protein binding is to the IL-4 promoter DNA.Reorganization c-Maf adds reorganization NF-ATp albumen and produces these mixtures, and forms the higher mixture of mobility in addition.When using c-Jun and NF-ATp, do not observe the mixture of high mobility, with c-Jun can not be consistent in conjunction with this site.These results show, c-Maf can externally specifically be bonded to the sequence that is positioned at near-end IL-4 promotor, it is the function key that this sequence before had been presented in the Th2 cell, and as other AP-1 albumen, c-Maf can external and NF-AT protein-interacting.The ability of embodiment 7:c-Maf transcriptional activation IL-4 promotor is reflected to MARE and Th-2 spy
On the opposite sex footprint
By high resolving power mutagenesis characterized be positioned at the required area (Hodge, M. etc. (1995) J.Immunol.154:6397-6405) of the IL-4 promotor of TATA element upstream just.Mutagenesis to this 33bp district (59 to-28) confirms induction type IL-4 in the Th2 cell is transcribed necessary a plurality of site.These sites comprise district that NF-AT target sequence, Th2 extract footprint cover and the site that is identified as MARE now.Used cross over the IL-4 reporter gene constructions that four base pair joints that this district produces scan mutant a series of comprising, the target sequence that is utilized in the body in the M12 cell by c-Maf with mark.With this c-maf expression vector and these cells of this series mutation IL-4 promotor construction cotransfection.These the results are shown in Fig. 7 A.MARE (mut3 and 4) or (mut3) c-maf of transfection by the sudden change cancellation (mut2 and 4) in the site (mut2) of Th2 footprint definition or partial cancellation and drive the ability that IL-4 transcribes.Also in destroying the mutant 8 of this NF-AT sequence, observe the gentle effect that reduces the c-maf trans-activation, and have endogenous NF-ATp consistent and and the NF-ATp that formerly confirms among the embodiment and the synergy unanimity between the c-maf in the M12 cell.Mutant 6 and 7 does not have significant effect, and mutant 5 has enhanced trans-activation ability and before in the Th2 cell observed consistent (Hodge, M. etc. (1995) J.Immunol.154:6397-6405).These trans-activation data are with consistent with reorganization c-Maf albumen and the EMSA that carries out as the oligonucleotide that contains this 33bp district of probe with as the same series mutation oligonucleotide of cold competition thing.These EMSA result of experiment are shown in Fig. 7 B.These experiments show the MARE that c-Maf is bonded to specifically and trans-activation near-end IL-4 promotor is interior, and this vicinity Th2 specificity element participates in combination and the function of c-Maf closely.Embodiment 8: use the two hybrid interaction traps of yeast to detect and separate NIP45cDNA
Use the two hybrid interaction traps of yeast to detect to separate can be directly in conjunction with the albumen of the RHD of NF-ATp.By 900bp fragment (McCaffrey with the leap amino acid 228-520 of mouse NF-ATp, (1993) Science262:750-754 such as P.G.) is cloned into carrier pEG202 (Gyuris, (1993) Cell75:791-803 such as J.) " bait " during BamH I site, preparation NF-ATp (RHD)-Gal4 fusion rotein are detected as the two hybrids of yeast.Confirmed the framework endomixis of this NF-AT (p) peptide sequence and this Gal4 sequence by dna sequence analysis.Use methods known in the art (referring to Gyuris, (1993) Cell75:791-803 such as J.), with this bait be used for screening preparation from the cDNA library of mouse T clone D10, be implemented in plasmid pJG4-5, to select the clone of coding and the interactional polypeptide of this bait.
Separated class coding and had interaction factor and called after NIP45 (NF-AT interaction protein 45) the fusion rotein of NF-ATp (RHD)-obvious high-affinity of Gal4 bait (by high-level betagalactosidase activity and give leucine anauxotrophic ability performance).Fig. 8 shows the photo (each plasmid combination Three Represents) of yeast colony, this yeast with the NIP45 plasmid and or this NF-ATp-RHD bait or contrast bait (Max-Gla4, CDK2-Gal4 and control vector pEG202 only express epi-position mark Gal4 albumen) and LacZ report plasmid pSH18 cotransformation.Described yeast colony is selected on suitable substratum, and selected and contain on Xgal and the non-flat board that checks the carbon source semi-lactosi.Yeast colony with NIP45 plasmid and NF-ATp-RHD bait cotransformation is blue, confirm this LacZ report plasmid expression (indication NIP-45/NF-ATp-RHD interacts), and be white with the yeast colony that this NIP45 plasmid and this contrast bait transform, show NIP45 and contrast bait and do not have interaction.Also lack tested transformant on the leucic substratum containing semi-lactosi, only those contain the yeast growth of this NIP45 plasmid and this NF-ATp-RHD bait, further show the specificity interaction of NIP45 and NF-ATp-RHD.Detecting isolating NIP45cDNA by this pair hybrid is the 1.9kb dna fragmentation.Embodiment 9:NIP45 and NF-ATp interact in the mammalian cell endosome
In mammalian cell, detected interactional ability in NIP45 polypeptid specificity ground and the NF-ATp body.The 1.9kb NIP45cDNA insertion fragment subclone that to select in the two hybrid systems of yeast (described in the embodiment 8) is gone into mammalian expression vector, this carrier merges the coding region to epi-position mark, carrier pCEP4-HA (Herrshcer from influenza blood lectin (HA) peptide, (1995) Genes Dev.9:3067-3082 such as R.F.), to produce expression vector NIP45-HA.Then the construction and the NF-ATp expression plasmid cotransfection of this mark are gone into HepG2 cell (the low-level NF-ATp of this cell expressing).As contrast, also use the parental generation expression vector (that is, this NF-ATp does not insert segmental expression vector) of NIP45-HA and NF-ATp construction or with this NF-ATp expression vector and the outer fusions cotransfection HepG2 cell of the NIP45 framework with this epi-position mark.Prepare lysate from cells transfected, and precipitate with anti-NF-ATp antibody mediated immunity.Use or anti-NF-ATp antibody or anti-HA antibody carry out the Westem engram analysis to this immunoprecipitation material then.
This result of experiment is shown in Fig. 9.Use HA monoclonal antibody specific (mAb) that the western engram analysis that these samples carry out is confirmed, be used for the anti-NF-ATp antibody of immunoprecipitation and the NIP45 polypeptide coimmunoprecipitation of this HA mark.Show that the swimming lane (middle swimming lane) of only using the NIP45-HA transfection is disclosed in the NF-ATp that has low endogenous levels in these cells.By using NF-ATp expression plasmid cotransfection, further increased the proteic amount of NIP45 of the HA mark of immunoprecipitation, confirm this interactional specificity (the right swimming lane).The Western engram analysis of lysate of being untreated confirms to have expressed the NIP45-HA polypeptide of par in the sample of the anti-NF-ATp antibody of test NIP45-HA coimmunoprecipitation.In addition, when using normal rabbit serum to carry out immunoprecipitation, do not detect to or the immunoreactivity material of NF-ATp or this HA labelled protein.These experiment confirms, NF-AT and NIP45 physical bond in the body in mammalian cell.Embodiment 10:NIP45cDNA structural analysis
The 1.9kb NIP45cDNA that uses two hybrids to detect isolating clone is inserted fragment (being described in embodiment 1) be used to screen D10.G4 T cell λ zap II cDNA library (Stratagene), to differentiate full-length clone.The library that screening contains 8 * 105 clones that have an appointment has produced 7 hybridization clones, and they mostly are no more than 5 ' end of original isolate.Yet the sequential analysis of Changke grand (2.8kb) confirms and original 5 ' the terminal identity that is cloned in.The structure that in Figure 10, has compared original 1.9kbcDNA isolate and the longest 2.8kb cDNA isolate.This 2.8kb cDNA isolate contains another fragment of the 180bp that is positioned at original clone 5 ' terminal downstream 868bp.Show that at the catenation sequence of this 180 nucleotide fragments end it is that the conceptual translation (conceptual translation) of not montage intron and the nucleotide sequence in this district discloses terminator codon in the framework.The major part of this another sequence in this clone all is positioned at the extensibility 3 ' non-translational region (referring to Figure 10) that connects the polyadenylation tail after 3 ' end is also represented.In many genes, all observe this extensibility 3 ' non-translational region.If this 2.8kb cDNA clones coding and the identical polypeptide of original 1.9kb isolate are expected by this little intron of montage and this single open reading-frame (ORF) district of translation.
The nucleotide sequence of this 1.9kb cDNA isolate and the aminoacid sequence of expection are shown in Figure 11 (and being shown in SEQ ID NO:5 and 6 respectively).First terminator codon in from first initiator codon to framework shows this coding region.Nucleotide and amino acid position are indicated the right in original series.The conceptual translation of this 1.9kb nucleotide sequence has disclosed 412 amino acid whose polypeptide with 45Kd molecular weight, and therefore with this albumen called after NF-AT interaction protein 45 (NIP45).Check that the aminoacid sequence of NIP45 is disclosed in a highly alkaline territory of N-terminal, wherein 13 in 32 amino acid are alkaline.In Figure 11, should distinguish the mark underscore.The wetting ability that this basic region looks like in the hydrophobicity profile that shows among Figure 12 is extended (stretch).The tissue expression of embodiment 11:NIP45mRNA
Carry out from the Northem engram analysis of the RNA of different rat tissues tissue expression with investigation NIP45mRNA.Will from the total RNA of 10 μ g separation on the sex change sepharose of various tissues, trace and with radiolabeled 1.4kb MP45cDNA fragment hybridization.Go up sample by the equivalent RNA that compares ethidium bromide fluorescence control sample.This Northern engram analysis the results are shown in Figure 13.This hybridization discloses the transcript of about 3.1kb, and its size is suitable with the longest cDNA clone.RNA from testis suspects other 1.4Kb hybridization kind.In spleen, thymus gland and testis, observe the highest NIP45 transcript level.Preferential expression in lymphoid organ may show the specific function of NIP45 in immunity system.NIP45cDNA clone's low strength hybridization signal is relative rare information with few NIP45RNA that shows in this T cell cdna library.The Subcellular Localization of embodiment 12:NIP45
The proteic Subcellular Localization of NIP45 by indirect immunofluorescence assay epi-position mark.Use methods known in the art (referring to Heald, R. etc. (1993) Cell74:463-474), with the encode expression constructs transfection bhk cell of NIP45 (pCEP4-HA) of HA epi-position mark of 1 μ g.As described with cells transfected incubated overnight, fixing, saturatingization (Heald, R. etc. (1993) are seen above-mentioned) also survey with the donkey anti-mouse antibody (Jackson ImmunoResearch) of anti-HA mAb 12CA5 (Boehringer Mannheim) and indocarbocyanine mark, redye with dyestuff Hoechst33258 then.The results are shown in Figure 14 A-B.By comparing, observed the nuclear staining (referring to Figure 14 A) of NIP45 with the second class grade chemical of indocarbocyanine mark with the same cell of redying with DNA dyestuff Hoechst33258 (referring to Figure 14 B).The fluorescence pattern shows that NIP45 distributes equably in nuclear.In addition, this pattern meets in usefulness NF-AT4 transfection and observed situation (Shibasaki, F. etc. (1996) Nature382:370-373 in the ionomycin stimulated cells; Also referring to following).Stimulate the Subcellular Localization that does not influence this NIP45 with PMA and/or ionomycin.
Also the bhk cell with the NF-AT4 transfection has been carried out control experiment.With cell overnight incubation in substratum, and or directly fixing or before fixing, at first stimulated 10 minutes with the 1mM ionomycin, and then as above-mentioned carrying out.The results are shown in Figure 14 C-F.With anti-NF-AT4 specific antibody, then second class grade chemical and the Hoechst33258 with the indocarbocyanine mark surveys (Figure 14 C and 14D) and (Figure 14 E and 14F) the NF-AT4 transfectant of ionomycin processing that does not stimulate.Indocarbocyanine fluorescence confirms, the localized NF-AT4 of kytoplasm and in the dyeing pattern of the localized NF-AT4 of stimulated cells center (Figure 14 E) in the transfectant (Figure 14 C) that does not stimulate.Contiguous plate (being respectively Figure 14 D and 14F) has shown that exposure is to detect by the same area with the painted nuclear of Hoechst33258.
Also investigated the influence of NIP45 to the transposition of NF-AT4 nuclear.With or NF-AT4 or NF-AT4 add the NIP45 transfection HepG 2 cell, and stimulated 0,2,4,8 or 15 minute in inferior daily 1 μ M ionomycin.For a sample,, clean with fresh culture then and make it to leave standstill again 15 minutes (in table 1, indicating) with " leaving standstill in 15 minutes+15 minutes " with ionomycin irritation cell 15 minutes.Design this analysis to detect the function of NIP45 as the nuclear retention factors.Shown 15 minutes exporting tenuigenin to for NF-AT4 is competent time (Shibasaki, F. etc. (1996) Nature382:370-373).Fix all samples then, and as it is above-mentioned by immunofluorescence analysis NF-AT4 transposition.The result is summarized in following table 2.The Subcellular Localization of NF-AT4 in tenuigenin indicates that with (-) the nuclear transposition of NF-AT4 is indicated with (+).
The nuclear transposition of table 2:NF-AT4
0 minute 2 minutes 4 minutes 8 minutes 15 minutes 15 minutes+15 minutes time left standstill Ionomycin-+/-+/-++- Ionomycin+NIP45-+/-+/-++-
Do not observe the difference that NF-AT4 nuclear inputs or outputs speed when having NIP45, show the cellular calcium level is changed the influence that the NF-AT4 nuclear transportation of replying is not subjected to external source NIP45 overexpression.The functionally active of embodiment 13:NIP45 in regulatory gene is expressed
For detect NIP45 NF-AT drive transcribe in function, with high level expression NIP45 in the HepG2 cell.Selecting the HepG2 cell is because they have low-level endogenous NF-AT, and the proteic ectopic expression of NF-AT family member has been presented at transcribe (Hoey, T. etc. (1995) Immunity2:461-472) that trans-activation NF-AT drives in this clone when lacking exogenous stimulation.Use 3X NF-AT-CAT reporter gene (Venkataraman, L. etc. (1994) Immunity1:189-196) from the IL-2 gene and NIP45 and NF-AT family member's (NF-ATp, NF-ATc, NF-AT3, NF-AT4) control plasmid or expression vector transfection HepG 2 cell.By being described in Hoey, T. etc. (1995) see above-mentioned deae dextran method transfection HepG 2 cell, and carry out CAT according to standard method and detect.The results are shown in Figure 15.A representativeness of each combination is detected the active histogram vicinity of the relative CAT that is shown in each group of representative.By the CAT activity of normalization method, and each parental generation expression vector is normalized to 1, calculates multiple and induce with this CAT reporter gene cells transfected.The numerical value representative exceeds the level relatively of the CAT expression of this contrast transfection.At least three transfections have all been carried out in each the representative radioautograph that shows.
Only NIP45 is transfected into the phenomenal growth that the HepG2 cell with 3X NF-AT-CAT reporter gene does not cause CAT to express, confirms that NIP45 can not only depend on self formula activation NF-AT target sequence.Independent NF-ATp overexpression causes a large amount of trans-activations (exceeding 6 times of control vector) of this NF-AT-CAT reporter gene, with previous report (Hoey, T. etc. (1995) see above-mentioned) unanimity.The cotransfection that NIP45 adds NF-ATp causes with respect to the active 4-5 of increasing of the CAT that only uses the NF-ATp transfection doubly, and surpasses 25-30 times that only uses the carrier transfection.When using sudden change 3X NF-AT-CAT reporter gene or contrast MHC II class promotor reporter gene, do not observe this increase, therefore prove the specificity of its target site.For this polypeptide product of confirming this NIP45cDNA coding is responsible for this enhanced trans-activation, phase shift mutation is imported this coding region by making two base deletions in Nucleotide 50.This variation results in amino acid/11 3 importing missense mutation and this polypeptide termination behind additional 22 residues.Use the detection of this NIP45 Δ construction confirm it can be when existing or not have NF-ATp this NF-AT reporter gene of trans-activation, confirm that therefore observed enhanced trans-activation is because from the NIP45cDNA polypeptide expressed.Also carrying out the trans-activation experiment in B clone M12 and T cell clone D10, obtain result similar but that significance is lower, may be because of high-caliber endogenous NIP45 or NF-ATp in these later clones.These experiment confirms, NIP45 strengthen significantly and specifically by the NF-ATp inductive transcribes, and this activity need interact with NF-ATp.
NF-AT albumen is shared about 70% identity in RHD, improved NIP45 can also with the interactional possibility of other NF-AT family member.For testing this possibility, the expression constructs of NIP45 and coding or NF-ATc, NF-AT3 or NF-AT4 is added 3X NF-AT-CAT report plasmid such as above-mentioned cotransfection.These result of experiment also are shown in Fig. 8.Confirmed before that when overexpression in the HepG2 cell, all NF-AT family members can trans-activation be contained the reporter gene in the NF-AT/AP1 site of three parts of copies, although level difference (Hoey, T. etc. (1995) see above-mentioned).When lacking NIP45, NF-ATp is the trans-activator of the most effective NF-AT-CAT reporter gene, secondly is NF-ATc and NF-AT3, and NFAT4 has only faint trans-activation, with previous data (McCaffrey, P.G. etc. (1993) Science262:750-754) unanimity.When NF-ATc, NF-AT3 or NF-AT4 and NIP45 cotransfection, NIP45 significantly strengthens the trans-activation of NF-ATc and NF-AT3 driving, and faintly strengthens the trans-activation (Figure 15) of NF-AT4 mediation.In the HepG2 cell, cooperate consistent with NF-ATc with NIP45 and the interactional observation in yeast cell of NF-ATcRHD bait.Generally, the trans-activation that the NIP45 overexpression causes NF-ATc to cause increases by 4 times, and the trans-activation that NF-AT3 drives increases by 3 times, and transcribing that NF-AT4 drives increases by 2 times.The active ability that the height sequence conservation of known NF-AT family member's RHD, NIP45 strengthen all NF-AT family members is N/R.The sequence in NF-AT RHD territory relatively discloses, and compares the sequence identity higher (Hoey, T. etc. (1995) see above-mentioned) of N-terminal part with C-terminal.Therefore, the 5 ' part that might this NIP45/NF-AT interaction sites be positioned at this RHD.
Provide the responsive method of measuring the trans-activation that is caused by NF-AT and NIP45 although contain the report construction of multiple copied NF-AT binding site, we seek to determine whether NIP45 has function under the situation of natural NF-AT dependency promotor.The IL-4 expression is highly tissue-specific and is confined to the Th2 subgroup and the mastocyte of T cell.This IL-4 promotor contains and shows a plurality of NF-AT binding sites (Rooney, J.w. etc. (1995) Immunity2:473-483) of IL-4 being expressed key.In addition, shown that proto-oncogene c-Maf instructs the tissue specific expression (embodiment 3 and 4) of IL-4.Therefore, this IL-4 promotor is inactive but can be activated by importing NF-ATp and c-Maf in HepG2 clone.As the above-mentioned cotransfection experiments that carries out in, with IL-4-CAT report construction (extend to this IL-4 promotor-732bp) and the expression vector of NIP45, NF-ATp and c-Maf or contrast transfection HepG 2 cell.The contrast of NIP45 is in the phase shift mutant of amino acid/11 3.The contrast of NF-ATp and c-Maf is respectively empty expression vector pREP4 and pMEX (Ho, I.C. etc. (1996) Cell85:973-983).These result of experiment are shown in Figure 16 (detecting and histogram as describe representative CAT in Figure 15).These data show, together import NIP45 with NF-ATp and c-Maf and cause with respect to only increasing by 9 times again with NF-ATp and the observed IL-4 promoter activity of c-Maf.IL-4 promoter activity when NIP45 also increases the NF-ATp that lacks transfection, this effect might be because interact with endogenous NF-ATp.Embodiment 14: instantaneous overexpression NIP45 and NF-ATp and c-Maf cause endogenous IL-4 to produce
For whether the combination of determining NIP45, NF-ATp and c-Maf is enough to induce the endogenous IL-4 of the cell that does not normally produce IL-4 express, with expression vector and NIP45 or the pCI vehicle Control transient cotransfection M12B lymphoma cell of NF-ATp and c-Maf.As described previously by electroporation transient transfection M12 cell (Ho, I.C. etc. (1996) Cell85:973-983), promptly before 975 μ F, 280V electroporation in room temperature with every kind of plasmid incubation of 3x106 cell among the 0.4ml PBS and 5 μ g 10 minutes.Measure the IL-4 level of the cell conditioned medium liquid of results after 72 hours except described modification (Ho, I.C. etc. (1996) see above-mentioned) according to manufacturer specification by commercially available IL-4ELISA (Pharmingen).Carry out independently four groups of transient transfections, and detected the secretion of IL-4 in this culture supernatant.The representative experimental result of one of these four independent experiments is shown in Figure 17.For every group of transfection, comprise that NIP45 causes significantly increasing IL-4 and produces.Give birth to 50-200 endogenous IL-4 doubly with the NIP45 cells transfected than the cell fecund of not accepting NIP45, wherein IL-4 produces near the limit that detects.Embodiment 15: external t helper cell between the differentiation phase p18mRNA express and to be regulated downwards
Maf family protein p18 is one of aminoterminal " little " maf albumen member who lacks the c-Maf 2/3rds that contains trans-activation domain.For detecting the expression of p18 transcript during normal t helper cell differentiation becomes the Th2 phenotype, described in above-mentioned embodiment 2, carry out the vitro differentiation experiment.By existing cytokine and anti-cytokine antibodies (for Th1 IFN γWith anti-IL-4, for Th2 IL-4 and anti-IFN γ) situation under handle with anti-CD3, drive splenocyte (Th precursor (Thp) cell) originally along Th1 or Th2 approach.Noble cells in post-stimulatory each time point (the 0th, 1,3,5 or 7 day) results is carried out the Northem engram analysis, to analyze the expression of p18 and c-maf transcript.Vitro differentiation Th2 cell c-maf and p18 express the results are shown in Figure 18.Consistent with The above results, to express be low-levelly maybe can't detect to the c-maf transcript in the time of the 0th day, but in that it is expressed and increases along Th2 approach differentiation phase.In contrast, the p18 transcript is detectable when being expressed in the 0th day (promptly not breaking up in the T cell), but reduces to undetectable substantially level at described this cell along Th2 approach differentiation phase.These results show that during normal t helper cell was divided into the Th2 phenotype, p18 expressed and regulated downwards.Embodiment 16:p18 checks the IL-4 promoter activity
Express whether influence the IL-4 promoter activity for detecting p18, in the M12B lymphoma cell, carry out cotransfection experiments.The method that these experiments are used is to describe in the foregoing description 3.With IL-4 promotor/CAT reporter gene construction with or c-Maf expression vector, p18 expression vector or c-Maf and p18 expression vector be transfected into the M12 cell.The representative result that CAT detects is shown in Figure 19.Only express c-Maf (5 μ g plasmid) and cause this IL-4 promotor construction to activate (referring to the swimming lane 2 of Figure 19), confirm by detectable CAT in this M12 cell is active.Cotransfection p18 expression vector (2.5,5 or 10 μ g) causes active reduce (referring to the swimming lane 3,4 and 5 of Figure 19) of CAT with c-Maf, and the increase of p18 amount causes observed CAT activity to reduce more.In the M12 cell, only express p18 and do not cause detectable CAT activity in cell (referring to the swimming lane 6 of Figure 19).These results confirm that p18 can check the IL-4 promoter activity that is stimulated by c-Maf.Embodiment 17: the transgenic mice of overexpression c-Maf
Present embodiment is described in overexpression c-Maf albumen in the T cell of transgenic mice.Illustrating of this c-maf transgenosis construct in Figure 20.This construction comprises first intron of 4kb mouse c-mafcDNA and mouse c-maf gene.The expression of this c-maf cDNA is controlled by CD4 promotor/enhanser regulatory region, and this district may be operably coupled to 5 ' end of first intron of c-maf, gives this construction T cell specific expression thus.A SV40 polyadenylation site is connected to the 3 ' end of this c-mafcDNA.This c-maf transgenosis construct microinjection is gone into the oocyte of mouse of fertilization, and prepare transgenic mice according to standard program.
Use several different detections that phenotype of these c-maf transgenic animal and the phenotype of wild-type animal are compared.At first, the total cell count in the quantitative assay lymphoid organ (lymphoglandula, spleen and thymus gland), it the results are shown in Figure 21.This experiment confirm, c-maf transgenic mice show to be compared cell count with wild-type mice and reduces in all three lymphoid organs that detect.Secondly, use flow cytometer, relatively the abundance of specific thymocyte colony in c-maf transgenic mice and the wild-type mice.Quantitative assay two positives (CD4+CD8+), jack to jack adapter (CD4-CD8-) and single positive thymocyte (CD4+ or CD8+).Also measured the ratio (CD4/CD8) of CD4 and cd8 cell.The result is summarized in the following table 3.
The abundance of specific thymocyte colony body in the table 3:c-Maf transgenic mice
CD4 -CD8 - CD4 +CD8 + CD4 + CD8 + CD4/CD8 *
Wild-type 4.2±0.6 71.6±9.5 8.5±1.6 2.8±0.6 3.2±0.8
Transgenosis 2.8±0.6 11.8±8.7 3.8±0.8 2.0±0.6 2.0±0.2
These results confirm to compare with wild-type mice, and the two positive thymocyte digital display work of c-maf transgenic mice reduces.In addition, the single positive thymocyte digital display work of the CD4+ of c-maf transgenic mice reduces.At last, quantitative assay the basal level of IgE in c-maf transgenic mice and the wild-type mice serum.The results are shown in Figure 22.This experiment confirm is compared with wild-type mice, and the c-maf transgenic mice shows SERUM IgE basal level to be increased.
The phenotype that phenotype of above-mentioned c-maf transgenic mice (being that little, the two positive thymocyte of spleen and thymus gland and single positive CD4+ thymocyte number reduction, SERUM IgE basal level increase) and IL-4 overexpression transgenic mice are described is (referring to (1990) Cell62:457 such as Tepper; With (1991) J.Exp.Med.173:89 such as Lewis) very similar.Equivalent
Those skilled in the art will discern many equivalence of the particular of the present invention described herein that maybe can determine to use normal experiment.This equivalence is included in following claims.
Sequence table (1) general information
(ⅰ) applicant
(A) title: President and Fellows of Harvard College
(B) street: 124Mount Auburn Street
(C) city: Cambrige
(D) state: Massachusetts
(E) country: the U.S.
(F) postcode: 02138
(ⅱ) denomination of invention: regulate the method and composition of T cell subsets by regulating transcription factor activity
(ⅲ) sequence quantity: 6
(ⅳ) contact address:
(A) contact person: LAHIVE﹠amp; COCKFIELD
(B) street: 60State Street, suite510
(C) city: Boston
(D) state: Massachusetts
(E) country: the U.S.
(F) postcode: 02109-1875
(ⅴ) computer-reader form:
(A) media types: floppy disk
(B) computer: IBMPC compatible
(C) operating system: PC-DOS/MS-DOS
(D) software: PatentIn Release#1.0, the current request for data of version #1.25 (ⅵ):
(A) application number: US
(B) submission date:
(C) classification: (ⅵ) request for data formerly:
(A) application number: US08/636,602
(B) submission date: on April 23rd, 1996
(C) classification: (ⅵ) request for data formerly:
(A) application number: US08/755,592
(B) submission date: on November 25th, 1996
(C) classification: (ⅵ) request for data formerly:
(A) application number: US08/755,584
(B) submission date: on November 25th, 1996
(C) classification: (ⅷ) attorney/proxy's information:
(A) name: Kara, Catherine J.
(B) number of registration: P41,106
(C) reference/file number: HUI-021CPPC (ⅸ) telecom information:
(A) phone: (617) 227-7400
(B) fax: the information of (617) 227-5941 (2) SEQ IDNO:1:
(ⅰ) sequence signature:
(A) length: 33 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topology: linearity
(ⅱ) molecule type: oligonucleotide
(ⅹ ⅰ) sequence description: the information of SEQ ID NO:1CTCATTTTCC CTTGGTTTCA GCAACTTTAA CTC 33 (2) SEQ ID NO:2:
(ⅰ) sequence signature:
(A) length: 20 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topology: linearity
(ⅱ) molecule type: oligonucleotide
(ⅹ ⅰ) sequence description: the information of SEQ ID NO:2ATAAAATTTT CCAATGTAAA 20 (2) SEQ ID NO:3:
(ⅰ) sequence signature:
(A) length: 27 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topology: linearity
(ⅱ) molecule type: oligonucleotide
(ⅹ ⅰ) sequence description: the information of SEQ ID NO:3TGGTGTAATA AAATTTTCCA ATGTAAA 27 (2) SEQ ID NO:4:
(ⅰ) sequence signature:
(A) length: 23 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topology: linearity
(ⅱ) molecule type: oligonucleotide
(ⅹ ⅰ) sequence description: the information of SEQ ID NO:4GGAATTGCTG ACTCAGCATT ACT 23 (2) SEQ ID NO:5:
(ⅰ) sequence signature:
(A) length: 1946 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topology: linearity
(ⅱ) molecule type: cDNA
(ⅸ) feature:
(A) title/key: CDS
(B) position: 13...1248
(ⅹ ⅰ) sequence description: SEQ ID NO:5 ACAGTGTGGG AG ATG GCG GAA CCA CTG AGG GGA CGT GGT CCG AGG TCC 48
Met?Ala?Glu?Pro?Leu?Arg?Gly?Arg?Gly?Pro?Arg?Ser
1???????????????5??????????????????10CGC?GGT?GGC?CGA?GGC?GCT?CGG?AGA?GCC?CGA?GGC?GCC?CGT?GGC?CGG?TGT???????96Arg?Gly?Gly?Arg?Gly?Ala?Arg?Arg?Ala?Arg?Gly?Ala?Arg?Gly?Arg?Cys
15??????????????????20??????????????????25CCT?CGC?GCC?CGG?CAG?TCT?CCG?GCT?AGG?CTC?ATT?CCA?GAC?ACC?GTG?CTT???????144Pro?Arg?Ala?Arg?Gln?Ser?Pro?Ala?Arg?Leu?Ile?Pro?Asp?Thr?Val?Leu
30??????????????????35??????????????????40GTG?GAC?TTG?GTC?AGT?GAC?AGC?GAC?GAA?GAG?GTC?TTG?GAA?GTC?GCA?GAC???????192Val?Asp?Leu?Val?Ser?Asp?Ser?Asp?Glu?Glu?Val?Leu?Glu?Val?Ala?Asp?45??????????????????50??????????????????55??????????????????60CCA?GTA?GAG?GTG?CCG?GTC?GCC?CGC?CTC?CCC?GCG?CCG?GCT?AAA?CCT?GAG???????240Pro?Val?Glu?Val?Pro?Val?Ala?Arg?Leu?Pro?Ala?Pro?Ala?Lys?Pro?Glu
65??????????????????70??????????????????75CAG?GAC?AGC?GAC?AGT?GAC?AGT?GAA?GGG?GCG?GCC?GAG?GGG?CCT?GCG?GGA???????288Gln?Asp?Ser?Asp?Ser?Asp?Ser?Glu?Gly?Ala?Ala?Glu?Gly?Pro?Ala?Gly
80??????????????????85??????????????????90GCC?CCG?CGT?ACA?TTG?GTG?CGA?CGG?CGG?CGG?CGG?CGG?CTG?CTG?GAT?CCC???????336Ala?Pro?Arg?Thr?Leu?Val?Arg?Arg?Arg?Arg?Arg?Arg?Leu?Leu?Asp?Pro
95?????????????????100?????????????????105GGA?GAG?GCG?CCG?GTG?GTC?CCA?GTG?TAC?TCC?GGG?AAG?GTA?CAG?AGC?AGC???????384Gly?Glu?Ala?Pro?Val?Val?Pro?Val?Tyr?Ser?GlY?Lys?Val?Gln?Ser?Ser
110?????????????????115?????????????????120CTC?AAC?CTC?ATT?CCA?GAT?AAT?TCA?TCC?CTC?TTG?AAA?CTG?TGC?CCT?TCA???????432Leu?Asn?Leu?Ile?Pro?Asp?Asn?Ser?Ser?Leu?Leu?Lys?Leu?Cys?Pro?Ser125?????????????????130?????????????????135?????????????????140GAG?CCT?GAA?GAT?GAG?GCA?GAT?CTG?ACA?AAT?TCT?GGC?AGT?TCT?CCC?TCT???????480Glu?Pro?Glu?Asp?Glu?Ala?Asp?Leu?Thr?Asn?Ser?Gly?Ser?Ser?Pro?Ser
145?????????????????150?????????????????155GAG?GAT?GAT?GCC?CTG?CCT?TCA?GGT?TCT?CCC?TGG?AGA?AAG?AAG?CTC?AGA???????528Glu?Asp?Asp?Ala?Leu?Pro?Ser?Gly?Ser?Pro?Trp?Arg?Lys?Lys?Leu?Arg
160?????????????????165?????????????????170AAG?AAG?TGT?GAG?AAA?GAA?GAA?AAG?AAA?ATG?GAA?GAG?TTT?CCG?GAC?CAG???????576Lys?Lys?Cys?Glu?Lys?Glu?Glu?Lys?Lys?Met?Glu?Glu?Phe?Pro?Asp?Gln
175?????????????????????180?????????????185GAC?ATC?TCT?CCT?TTG?CCC?CAA?CCT?TCG?TCA?AGG?AAC?AAA?AGC?AGA?AAG???????624Asp?Ile?Ser?Pro?Leu?Pro?Gln?Pro?Ser?Ser?Arg?Asn?Lys?Ser?Arg?Lys
190?????????????????195?????????????????200CAT?ACG?GAG?GCG?CTC?CAG?AAG?CTA?AGG?GAA?GTG?AAC?AAG?CGT?CTC?CAA???????672His?Thr?Glu?Ala?Leu?Gln?Lys?Leu?Arg?Glu?Val?Asn?Lys?Arg?Leu?Gln205?????????????????210?????????????????215?????????????????220GAT?CTC?CGC?TCC?TGC?CTG?AGC?CCC?AAG?CAG?CAC?CAG?AGT?CCA?GCC?CTT???????720Asp?Leu?Arg?Ser?Cys?Leu?Ser?Pro?Lys?Gln?His?Gln?Ser?Pro?Ala?Leu
225?????????????????230?????????????????235CAG?AGC?ACA?GAT?GAT?GAG?GTG?GTC?CTA?GTG?GAA?GGG?CCT?GTC?TTG?CCA???????768Gln?Ser?Thr?Asp?Asp?Glu?Val?Val?Leu?Val?Glu?Gly?Pro?Val?Leu?Pro
240?????????????????245?????????????????250CAG?AGC?TCT?CGA?CTC?TTT?ACA?CTC?AAG?ATC?CGG?TGC?CGG?GCT?GAC?CTA???????????????????816Gln?Ser?Ser?Arg?Leu?Phe?Thr?Leu?Lys?Ile?Arg?Cys?Arg?Ala?Asp?Leu
255?????????????????260????????????????265GTG?AGA?CTG?CCT?GTC?AGG?ATG?TCG?GAG?CCC?CTT?CAG?AAT?GTG?GTG?GAT???????????????????864Val?Arg?Leu?Pro?Val?Arg?Met?Ser?Glu?Pro?Leu?Gln?Asn?Val?Val?Asp
270?????????????????275?????????????????280CAC?ATG?GCC?AAT?CAT?CTT?GGG?GTG?TCT?CCA?AAC?AGG?ATT?CTT?TTG?CTT???????????????????912His?Met?Ala?Asn?His?Leu?Gly?Val?Ser?Pro?Asn?Arg?Ile?Leu?Leu?Leu285?????????????????290?????????????????295?????????????????300TTT?GGA?GAG?AGT?GAA?CTG?TCT?CCT?ACT?GCC?ACC?CCT?AGT?ACC?CTA?AAG???????????????????960Phe?Gly?Glu?Ser?Glu?Leu?Ser?Pro?Thr?Ala?Thr?Pro?Ser?Thr?Leu?Lys
305?????????????????310?????????????????315CTT?GGA?GTG?GCT?GAC?ATC?ATT?GAT?TGT?GTG?GTG?CTA?GCA?AGC?TCT?TCA??????????????????1008Leu?Gly?Val?Ala?Asp?Ile?Ile?Asp?Cys?Val?Val?Leu?Ala?Ser?Ser?Ser
320?????????????????325?????????????????330GAG?GCC?ACA?GAG?ACA?TCC?CAG?GAG?CTC?CGG?CTC?CGG?GTG?CAG?GGG?AAG??????????????????1056Glu?Ala?Thr?Glu?Thr?Ser?Gln?Glu?Leu?Arg?Leu?Arg?Val?Gln?Gly?Lys
335?????????????????340?????????????????345GAG?AAA?CAC?CAG?ATG?TTG?GAG?ATC?TCA?CTG?TCT?CCT?GAT?TCT?CCT?CTT??????????????????1104Glu?Lys?His?Gln?Met?Leu?Glu?Ile?Ser?Leu?Ser?Pro?Asp?Ser?Pro?Leu
350?????????????????355?????????????????360AAG?GTT?CTC?ATG?TCA?CAC?TAT?GAG?GAA?GCC?ATG?GGA?CTC?TCT?GGA?CAC??????????????????1152Lys?Val?Leu?Met?Sar?His?Tyr?Glu?Glu?Ala?Met?Gly?Leu?Ser?Gly?His365?????????????????370?????????????????375?????????????????380AAG?CTC?TCC?TTC?TTC?TTT?GAT?GGG?ACA?AAG?CTT?TCA?GGC?AAG?GAG?CTG??????????????????1200Lys?Leu?Ser?Phe?Phe?Phe?Asp?Gly?Thr?Lys?Leu?Ser?Gly?Lys?Glu?Leu
385?????????????????390?????????????????395CCA?GCT?GAT?CTG?GGC?CTG?GAA?TCC?GGA?GAT?CTC?ATC?GAA?GTC?TGG?GGC??????????????????1248Pro?Ala?Asp?Leu?Gly?Leu?Glu?Ser?Gly?Asp?Leu?Ile?Glu?Val?Trp?Gly
400 405 410TGAAGCTCTC ACCCTGTTCG GACGCAAAGC CAAGACATGG AGACAATAGC TCCCAATTTT 1308ATTATTGTGA TTTTTCGCCC CATAAGGGCT AACAGAAACT GAATTAGAAC TTGTTTACTT 1368ATTTATTTCT GGTGCTGGGG ATTGAACCCC AGACTATGCA CATGCTAAGG ATGTATGAAG 1428TGGAGGCAAA ACCAAGGCAT TACCTTTAGC CAGCCTCTAG TAGACTGTAG TGTCAAGCAA 1488GTGGCTACTT GGTAGTTGTG TGGCTCTGTG TATGTTTGTG CTGTATTTGG CAGCCCCTGG 1548GGCACATAGA AGGGACCTTG GCTTCCCTAC CATTTCACGT TCGCTGGTGC CCTTTCCTTC 1608ATCAGATGAC TTCTGTGAAG CTGCCTATGT TGAGTGTGTT GAACTAAATG AGCTCTGCTT 1668TGGGTGTCCA GGCCTGGGGT TTGTGCCGCA GTTGGAGCCA GCAGTGACTT CACTCTGACT 1728TGGGACTGAG AATGCATTTC CTGGTGGAGA CACTCGGGTG CAGAAATATA ACAGAAGGTG 1788ACATACATGC TGAAGCTGAG GACTAGGTCG AAAGTTAACG ACGTTGCATT TTCAGCCTTG 1848GGTATCCTCT CTGCCTGCCA GGACTCTAGC CAGTGTCTGG TACACACTTC TTGGCATGGA 1908CACCTAGGTC GACGCGGGCG CGATTCGGCC GACTCGAG 1946 ( 2 ) SEQ ID NO:6：
(ⅰ) sequence signature:
(A) length: 412 amino acid
(B) type: amino acid
(D) topology: linearity
(ⅱ) molecule type: albumen
(ⅹ ⅰ) sequence description: SEQ ID NO:6Met Ala Glu Pro Leu Arg Gly Arg Gly Pro Arg Ser Arg Gly Gly Arg 15 10 15Gly Ala Arg Arg Ala Arg Gly Ala Arg Gly Arg Cys Pro Arg Ala Arg
20??????????????????25??????????????????30Gln?Ser?Pro?Ala?Arg?Leu?Ile?Pro?Asp?Thr?Val?Leu?Val?Asp?Leu?Val
35??????????????????40??????????????????45Ser?Asp?Ser?Asp?Glu?Glu?Val?Leu?Glu?Val?Ala?Asp?Pro?Val?Glu?Val
50??????????????????55??????????????????60Pro?Val?Ala?Arg?Leu?Pro?Ala?Pro?Ala?Lys?Pro?Glu?Gln?Asp?Ser?Asp?65??????????????????70??????????????????75??????????????????80Ser?Asp?Ser?Glu?Gly?Ala?Ala?Glu?Gly?Pro?Ala?Gly?Ala?Pro?Arg?Thr
85??????????????????90??????????????????95Leu?Val?Arg?Arg?Arg?Arg?Arg?Arg?Leu?Leu?Asp?Pro?Gly?Glu?Ala?Pro
100?????????????????105?????????????????110Val?Val?Pro?val?Tyr?Ser?Gly?Lys?val?Gln?Ser?Ser?Leu?Asn?Leu?Ile
115?????????????????120?????????????????125Pro?Asp?Asn?Ser?Ser?Leu?Leu?Lys?Leu?Cys?Pro?Ser?Glu?Pro?Glu?Asp
130?????????????????135?????????????????140Glu?Ala?Asp?Leu?Thr?Asn?Ser?Gly?Ser?Ser?Pro?Ser?Glu?Asp?Asp?Ala145?????????????????150?????????????????155?????????????????160Leu?Pro?Ser?Gly?Ser?Pro?Trp?Arg?Lys?Lys?Leu?Arg?Lys?Lys?Cys?Glu
165?????????????????170?????????????????175Lys?Glu?Glu?Lys?Lys?Met?Glu?Glu?Phe?Pro?Asp?Gln?Asp?Ile?Ser?Pro
180?????????????????185?????????????????190Leu?Pro?Gln?Pro?Ser?Ser?Arg?Asn?Lys?Ser?Arg?Lys?His?Thr?Glu?Ala
195?????????????????200?????????????????205Leu?Gln?Lys?Leu?Arg?Glu?Val?Asn?Lys?Arg?Leu?Gln?Asp?Leu?Arg?Ser
210?????????????????215?????????????????220Cys?Leu?Ser?Pro?Lys?Gln?His?Gln?Ser?Pro?Ala?Leu?Gln?Ser?Thr?Asp225?????????????????230?????????????????235?????????????????240Asp?Glu?Val?Val?Leu?Val?Glu?Gly?Pro?Val?Leu?Pro?Gln?Ser?Ser?Arg
245?????????????????250?????????????????255Leu?Phe?Thr?Leu?Lys?Ile?Arg?Cys?Arg?Ala?Asp?Leu?Val?Arg?Leu?Pro
260?????????????????265?????????????????270Val?Arg?Met?Ser?Glu?Pro?Leu?Gln?Asn?Val?Val?Asp?His?Met?Ala?Asn
275?????????????????280?????????????????285His?Leu?Gly?Val?Ser?Pro?Asn?Arg?Ile?Leu?Leu?Leu?Phe?Gly?Glu?Ser
290?????????????????295?????????????????300Glu?Leu?Ser?Pro?Thr?Ala?Thr?Pro?Ser?Thr?Leu?Lys?Leu?Gly?Val?Ala305?????????????????310?????????????????315?????????????????320Asp?Ile?Ile?Asp?Cys?Val?Val?Leu?Ala?Ser?Sar?Ser?Glu?Ala?Thr?Glu
325?????????????????330?????????????????335Thr?Ser?Gln?Glu?Leu?Arg?Leu?Arg?Val?Gln?Gly?Lys?Glu?Lys?His?Gln
340?????????????????345?????????????????350Met?Leu?Glu?Ile?Ser?Leu?Ser?Pro?Asp?Ser?Pro?Leu?Lys?Val?Leu?Met
355?????????????????360?????????????????365Ser?His?Tyr?Glu?Glu?Ala?Met?Gly?Leu?Ser?Gly?His?Lys?Leu?Set?Phe
370?????????????????375?????????????????380Phe?Phe?Asp?Gly?Thr?Lys?Leu?Ser?Gly?Lys?Glu?Leu?Pro?Ala?Asp?Leu385?????????????????390?????????????????395?????????????????400Gly?Leu?Glu?Ser?Gly?Asp?Leu?Ile?Glu?Val?Trp?Gly
405?????????????????410

Claims (87)

1. regulate the method for auxiliary 2 types (Th2) the relevant cell factor generation of T of cell, comprise with regulating transcription factor expression or active medicine and contact this cell, the feasible generation of regulating the Th2 relevant cell factor of cell, wherein the expression of Th2 relevant cell factor is regulated in this transcription factor and the cooperation of activated T cells nf (NF-AT) family protein.
2. the process of claim 1 wherein that this transcription factor is the Th2 idiosyncratic transcription factor.
3. the process of claim 1 wherein that this transcription factor is the maf family protein.
4. the method for claim 3, wherein this maf family protein is c-Maf.
5. the method for claim 3, wherein this maf family protein is little maf albumen.
6. the method for claim 5, wherein this little maf albumen is p18.
7. the process of claim 1 wherein that this transcription factor and NF-AT family protein interact.
8. the method for claim 7, wherein the Rel homeodomain of this transcription factor and NF-AT family protein interacts.
9. the method for claim 8, wherein this transcription factor is NIP45.
10. the method for each of claim 1-9, wherein this medicine works in born of the same parents and regulates the expression or the activity of this transcription factor.
11. the method for each of claim 1-9, wherein this medicine is the nucleic acid molecule of this transcription factor of coding, and wherein this nucleic acid molecule is imported into this cell with the form that is suitable for this transcription factor of expression in this cell.
12. the method for each of claim 1-9, wherein this medicine is a binding molecule in the born of the same parents.
13. the method for each of claim 1-9 wherein contacts this cell with at least a other medicines, described other medicines are regulated at least a to regulating the activity of other transcription factor that Th2 relevant cell factor gene works.
14. the method for claim 13, wherein at least a other transcription factor is selected from NF-AT family protein, NF-AT interaction protein, maf family protein and AP-1 family protein.
15. the method for claim 11 wherein imports this cell with at least a other nucleic acid molecule, described at least a other nucleic acid molecule encoding is at least a to regulating other transcription factor that Th2 relevant cell factor gene works.
16. the method for claim 15, wherein this at least a other transcription factor is selected from NF-AT family protein, NF-AT interaction protein, maf family protein and AP-1 family protein.
17. the method for each of claim 1-9, wherein this cell is auxiliary 1 type (Th1) cell, B cell or a non-lymphoidocyte of T.
18. the method for each of claim 1-9, the generation of this cell of its moderate stimulation Th2 relevant cell factor.
19. the method for each of claim 1-9 wherein suppresses the generation of this cell Th2 relevant cell factor.
20. the method for each of claim 1-9, wherein this Th2 relevant cell factor is an interleukin-4.
21. the method for each of claim 1-9 comprises and gives the curee with this cell, to regulate auxiliary 1 type (Th1) of T or auxiliary 2 types (Th2) cells whose development of T among the curee thus.
22. recombinant expression vector, it comprises the nucleotide sequence of coding maf family protein, and described nucleotide sequence functionally is connected to and instructs this maf family protein specific expressed adjusting sequence in lymphoidocyte.
23. the recombinant expression vector of claim 22, wherein sequence-directed this maf family protein of this adjusting is expressed in the T cell specifically.
24. the recombinant expression vector of claim 22, wherein sequence-directed this maf family protein of this adjusting is expressed in the B cell specifically.
25. recombinant expression vector, it comprises the nucleotide sequence of coding maf family protein, and described nucleotide sequence functionally is connected to and instructs this maf family protein specific expressed adjusting sequence in hemopoietic stem cell.
26. imported the host cell of the recombinant expression vector of coding maf family protein, wherein this host cell is a lymphoidocyte.
27. the host cell of claim 26, it is the T cell.
28. the host cell of claim 26, it is the B cell.
29. imported the host cell of the recombinant expression vector of coding maf family protein, wherein this host cell is a hemopoietic stem cell.
30. isolated nucleic acid molecule, it comprises the nucleotide sequence of coding NIP45 or its biologic activity part.
31. isolated nucleic acid molecule, it comprises the nucleotide sequence of proteins encoded, and wherein this albumen comprises the aminoacid sequence at least 60% homologous aminoacid sequence with SEQ ID NO:6, and interacts with the Rel homeodomain of NF-AT family protein.
32. the isolated nucleic acid molecule of claim 31, wherein this albumen comprises the aminoacid sequence at least 70% homologous aminoacid sequence with SEQ IDNO:6.
33. the isolated nucleic acid molecule of claim 31, wherein this albumen comprises the aminoacid sequence at least 80% homologous aminoacid sequence with SEQ IDNO:6.
34. the isolated nucleic acid molecule of claim 31, wherein this albumen comprises the aminoacid sequence at least 90% homologous aminoacid sequence with SEQ IDNO:6.
35. length is at least the isolated nucleic acid molecule of 15 Nucleotide, it under stringent condition with comprise the making nucleic acid molecular hybridization of the nucleotide sequence of SEQ ID NO:5.
36. the isolated nucleic acid molecule of claim 35, it comprises naturally occurring nucleotide sequence.
37. the isolated nucleic acid molecule of claim 36, its encoding murine NIP45.
38. the isolated nucleic acid molecule of claim 36, its human NIP45 that encodes.
39. comprise the isolated nucleic acid molecule in the nucleotide sequence coded district of SEQ ID NO:5.
40. the isolated nucleic acid molecule of claim 39 comprises the nucleotide sequence of SEQ ID NO:5.
41. the isolated nucleic acid molecule of the aminoacid sequence of coding SEQ ID NO:6.
42. the isolated nucleic acid molecule of coding NIP45 fusion rotein.
43. isolated nucleic acid molecule with the nucleic acid molecule antisense of claim 30.
44. isolated nucleic acid molecule, it and the coding strand antisense of nucleic acid molecule of the nucleotide sequence that comprises SEQ ID NO:5.
45. the isolated nucleic acid molecule of claim 44, the coding region antisense of the nucleotide sequence coded chain of it and SEQ ID NO:5.
46. the isolated nucleic acid molecule of claim 44, the non-coding region antisense of the nucleotide sequence coded chain of it and SEQ ID NO:5.
47. comprise the carrier of each nucleic acid molecule among the claim 30-46.
48. the carrier of claim 47, it is a recombinant expression vector.
49. contain the host cell of the carrier of claim 47 or 48.
50. produce the proteic method of NIP45, the host cell that is included in cultivation claim 49 in the suitable substratum is until producing NIP45 albumen.
51. the method for claim 50 comprises from this substratum or this host cell again and separates NIP45 albumen.
52. isolating NIP45 albumen or its biologic activity part.
53. isolating albumen, it comprises the aminoacid sequence at least 60% homologous aminoacid sequence with SEQ ID NO:6, and interacts with the Rel homeodomain of NF-AT family protein.
54. the isolating albumen of claim 53, aminoacid sequence at least 70% homology of it and SEQ ID NO:6.
55. the isolating albumen of claim 53, aminoacid sequence at least 80% homology of it and SEQ ID NO:6.
56. the isolating albumen of claim 53, aminoacid sequence at least 90% homology of it and SEQ ID NO:6.
57. comprise the fusion rotein of the NIP45 polypeptide that functionally is connected to non-NIP45 polypeptide.
58.NIP45 antigen peptide, it comprises at least 8 amino-acid residues of the aminoacid sequence that is shown in SEQ ID NO:6, this peptide comprises the NIP45 epi-position, the antibody and NIP45 formation specific immunity complex body of anti-this peptide that makes preparation.
59. specificity is in conjunction with the proteic antibody of NIP45.
60. the antibody of claim 59, it is a monoclonal antibody.
61. the antibody of claim 59, it is coupled on the detectable substance.
62. medicinal compositions, it comprises among the claim 59-61 each antibody and pharmaceutically acceptable carrier.
63. non-human transgenic animal, it contains and carries coding NIP45 proteic genetically modified cell.
64. the non-human transgenic animal of claim 63, it comprises the cell of the endogenous NIP45 gene with change.
65. non-human transgenic animal contains the genetically modified cell that carries coding maf family protein.
66. the non-human transgenic animal of claim 65, wherein this maf family protein is preferentially expressed in the T of this animal cell.
67. differentiate the method for the compound of regulating transcription factor activity, Th2 relevant cell factor expression of gene is regulated in wherein said transcription factor and the cooperation of activated T cells nf (NF-AT) family protein, this method comprises
The indication composition is provided, and it has the activity of regulating the transcription factor of Th2 relevant cell factor genetic expression with the cooperation of NF-AT family protein;
Contact this indication composition with test compounds; With
Determine that this test compounds regulates the compound of regulating the transcription factor activity of Th2 relevant cell factor genetic expression with the cooperation of NFAT family protein to the influence of this transcription factor activity in this indication composition to differentiate thus.
68. the method for claim 67, wherein this transcription factor is the maf family protein.
69. the method for claim 68, wherein this maf family protein is c-Maf.
70. the method for claim 68, wherein this maf family protein is little maf albumen.
71. the method for claim 67, wherein this transcription factor and NF-AT family protein interact.
72. the method for claim 71, wherein this transcription factor is NIP45.
73. the method for claim 67, wherein this indication composition is a lymphoidocyte.
74. the method for claim 73, wherein this lymphoidocyte is the Th2 cell.
75. the method for claim 67, wherein this indication composition is a yeast cell.
76. each method among the claim 67-75, wherein this indication composition comprises indicator cells, and wherein said indicator cells comprises: (ⅰ) this transcription factor and the reporter gene of (ⅱ) this transcription factor being replied.
77. the method for claim 76, wherein said indicator cells contains:
ⅰ) the encode recombinant expression vector of this transcription factor; With
ⅱ) comprise the carrier of the Th2 relevant cell factor sequential gene regulating that functionally connects reporter gene; And described method comprises:
A) contact this indicator cells with test compounds;
B) determine when having this test compounds this report expression of gene level in this indicator cells; With
This report expression of gene level and this report expression of gene level in the indicator cells when not having this test compounds in the indicator cells when c) relatively having this test compounds are to differentiate the compound of regulating this transcription factor activity thus.
78. arbitrary method of claim 67-72, wherein this indication composition comprises following preparation: (ⅰ) this transcription factor and (ⅱ) this transcription factor bonded dna molecular, and
Described method comprises:
A) contact this indication composition with test compounds;
This transcription factor and the interactional degree of this dna molecular when b) determining to have this test compounds; With
This transcription factor and the interactional degree of this dna molecular and this transcription factor and the interactional degree of this dna molecular when not having this test compounds when c) relatively having this test compounds are to differentiate the compound of regulating this transcription factor activity thus.
79. the method for claim 78, wherein this transcription factor is that maf family protein and this dna molecular comprise maf response element (MARE).
80. each method among the claim 67-75, its is differentiated from the Th2 cell and albumen this transcription factor interaction, wherein:
This indication composition is the indication cell, and this indicator cells comprises:
ⅰ) functionally be connected to the reporter gene of transcriptional regulatory sequences; With
ⅱ) first mosaic gene of coding first fusion rotein, described first fusion rotein comprises the transcription factor of regulating the genetic expression of Th2 relevant cell factor with the cooperation of NFAT family protein;
This test compounds comprises the library of second mosaic gene, this library second fusion rotein of encoding, and this second fusion rotein comprises the albumen that is derived from the Th2 cell.
Expression is to the reporter gene of the interaction sensitivity between first fusion rotein, second fusion rotein and this transcriptional regulatory sequences; With
Wherein, determine of the effect of this test compounds, comprise from the Th2 cell and compound this transcription factor interaction with discriminating thus to this transcription factor in this indication composition by determining this report expression of gene level in this indicator cells.
81. differentiate the method for regulating interactional compound between NIP45 and the NF-AT family protein, comprise:
A) exist or mix when not having test compounds:
(ⅰ) NIP45 or its NF-AT interaction part; With
(ⅱ) NF-AT family protein or its NIP45 interaction part;
B) when existing and not having this test compounds, determine (ⅰ) and (ⅱ) between interactional degree; With
C) differentiate adjusting NIP45 and the interactional compound of NF-AT family protein.
82. the method for claim 81, wherein the NIP45 of this NF-AT family protein interacts and partly comprises the Rel homeodomain of this NF-AT family protein.
83. the method for claim 81, wherein by with detectable substance mark (ⅰ) or (ⅱ), separate unmarked (ⅰ) or (ⅱ) and quantitative assay and unmarked (ⅰ) or (ⅱ) bonded mark (ⅰ) or amount (ⅱ), definite (ⅰ) and (ⅱ) between interactional degree.
84. arbitrary method of claim 67-75 or 81-83 comprises and determines the influence of this compound to immunne response, to differentiate the compound of regulating immunne response thus.
85. the method for claim 84 is wherein determined the influence of this compound to immunne response by measuring this compound to the influence of Th2 relevant cell factor genetic expression.
86. the method for claim 85, wherein interleukin-4 gene during this Th2 relevant cell factor gene.
87. the method for claim 84 is wherein determined the influence of purpose compound to immunne response by measuring this compound to auxiliary Class1 (Th1) of T or the auxiliary cytocerastic influence of type 2 (Th2) of T.
CN97195740A 1996-04-23 1997-04-23 Method and compositions for regulating T cell subsets by modulating transcription facter activity Pending CN1225682A (en)

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US08/636,602 1996-04-23
US08/636,602 US5958671A (en) 1996-04-23 1996-04-23 Methods and compositions for regulating T cell subsets by modulating transcription factor activity
US08/755,584 1996-11-25
US08/755,592 1996-11-25
US08/755,592 US6537810B1 (en) 1996-04-23 1996-11-25 Methods for regulating T cell subsets by modulating transcription factor activity
US08/755,584 US5858711A (en) 1996-11-25 1996-11-25 NF-AT-interacting protein NIP45 and methods of use therefor

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GB9722388D0 (en) * 1997-10-24 1997-12-24 Zeneca Ltd NIP45 human homolog
US6274338B1 (en) 1998-02-24 2001-08-14 President And Fellows Of Harvard College Human c-Maf compositions and methods of use thereof
WO1999061908A1 (en) * 1998-05-28 1999-12-02 President And Fellows Of Harvard College METHODS AND COMPOSITIONS RELATING TO MODULATION OF CARTILAGE CELL GROWTH AND/OR DIFFERENTIATION BY MODULATION OF NFATp ACTIVITY
MXPA02006433A (en) * 1999-12-30 2002-11-29 Harvard College Methods and compositions relating to modulation of hepatocyte growth, plasma cell differentiation or t cell subset activity by modulation of xbp1 activity.
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WO2012109238A2 (en) 2011-02-07 2012-08-16 President And Fellows Of Harvard College Methods for increasing immune responses using agents that directly bind to and activate ire-1
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WO2015019125A1 (en) 2013-08-05 2015-02-12 Societe De Developpement Et De Recherche Industrielle Molecular targets for the treatment of wounds, in particular chronic wounds
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