CN1696155A - Gene of restraining activation NF-kB and NFAT, and coded polypeptide - Google Patents

Abstract

A human gene NFIF1 for suppressing the activation of NF-kB and NFAT, its cDNA sequence, the polypeptide coded by it, the process for testing its functions by dual-leuciferinase report gene method, and its application in preparing the medicines for preventing and treating inflammation, anaphylactia, autoimmunopathy, tumor etc are disclosed.

Description

Suppress NF-κ B and NFAT activatory gene and encoded polypeptides thereof

Technical field

The present invention relates to the gene expression regulation field, specifically relate to suppress human body cell nf NF-κ B and NFAT activatory gene, the preparation method of this gene, contain this expression carrier, its encoded polypeptides, the antibody of polypeptide, and described polypeptide, antibody be in preparation prevention and treatment and the human body cell nf NF-κ B disease relevant with NFAT, especially the medicine of inflammation, anaphylactic disease, autoimmune disease and tumour and in the purposes of developing on adjusting lymphocytic activation, propagation and the apoptosis medicine.

Background technology

NFAT and NF-kB protein are the members of transcription factor superfamily, though they all have expression in a variety of cells, they bring into play crucial effect in the activation of regulation and control lymphocyte series in propagation and the necrocytosis of activation inductive.NFAT and NF-κ B have many general character, comprise after similar DNA land, the antigenic stimulation nuclear translocation etc. fast.Mutual again antagonism of overlapped while between them, they have constituted meticulous gene expression regulation network system in lymphocyte.The NF-κ B regulation and control natural immunity is replied with acquired immunity and is replied, and NFAT regulation and control acquired immunity is replied.On NFAT and the NF-κ B function significant difference is arranged, the necrocytosis of NFAT regulation and control T/B cell activation inductive, and the NF-kB protein usually plays very strong anti-apoptotic effect in lymphocyte and other cell.The anti-apoptosis activity of NF-κ B is that they have oncogene potential, and the pro-apoptosis bioactivity of NFAT makes it to become the tumor inhibitor in the lymphocyte.

NFAT has similar DNA combined function territory (about 300Aa) with the NF-kB protein.NF-κ B is the Rel functional domain, and NFAT is the RSD functional domain.Though RSD and Rel functional domain have only 20% sequence homology, they all have 10 B lamellas and 2 annular structures in same position, in order to transcribe molecule such as AP-1 interaction (Chen in conjunction with similar dna sequence dna with other, Glover, Hogan, Rao ， ﹠amp; Harrison, 1998).

Nucleus factor NF-κ B is one of most important transcription factor of human body, and it participates in inducing diversified cell and virusization expression of gene, plays pleiotropic effects in the broad variety gene expression of cells is regulated.These gene pairs inflammation, immunity and apoptotic signal are replied and are activated, comprising many cytokines, chemokine, acceptor, adhesion molecule etc.It is initial that NF-κ B participates in also that viral promotors transcribes, for example I type human immunodeficiency virus (HIV-1).

Mammiferous NF-κ B comprises 5 kinds of albumen: p65 (RelA), p50 (NF-κ B-1), RelB, c-Rel, p52 (NF-κ B-2, p50B), and they have 300 amino acid similarities in the N-terminal district, be called the Rel homologous region.5 kinds of NF-kB protein matter molecules can form homology or heterodimer, combine with specific DNA recognition site with different binding specificities, have different transactivation activities.

Because the activation of NF-κ B can promote the transcriptional expression of many proinflammatory factors, so, generation, anaphylactic disease and autoimmune disease that the overactivity of NF-κ B can cause inflammation; Because the activation of NF-κ B can promote cell growth-inhibiting apoptosis, so it plays an important role in the generation evolution of tumour; In addition, the activation of NF-κ B can be blocked the apoptosis of some cells, so it also has important function aspect Growth and Differentiation of regulating cell.

TNF α (tumor necrosis factor alpha), PMA (TETRADECONIC ACID ripple boil ester), IL-1 (interleukin 1), LPS (lipopolysaccharides) all can promote the NF-kB activation; The expression of MEKK (the kinase whose kinases of mitogen activated protein kinase), TRAF NF-κ B signal path positive regulator genes such as (TNF (tumor necrosis factor) receptor associated factor) also can promote the NF-kB activation; IL-10 (interleukin 10), FK506 (fujimycin 506), glucocorticosteroid, acetylsalicylic acid etc. then can suppress the activation of NF-κ B significantly.

In view of NF-κ B is regulating human body physiological function, and vital role (the Burke J.R.Curr Opin Drug Discov Devel.20036 (5) that in the generation of multiple disease, development, is risen, 720-8), the inhibitor of NF-κ B and suppressor gene will have important application in the treatment of inflammatory diseases and neoplastic disease.The NF-kB activation is regulated the focus that the field has become preclinical medicine, clinic study and drug development, for example the adjusting albumen (United States Patent (USP) 5,597,898) relevant with the NF-kB activation such as I-KappaB-α, I-KappaB-β at present.In addition, the many negative regulation things of NF-κ B such as the treatment of anti-IL-8-1, the treatment of anti-tumor necrosis factor, hormonotherapy, Asprin, non-hormone anti-inflammatory drug etc. (with reference to survey article Nature ReviewImmunology, 2002,2:725; J Clin Invest, January 2001, Volume 107, Number 2,135-142) be widely used in clinical, but these medicines have specificity strong, suppress inadequately obviously, stable shortcoming inadequately, and side effect is often arranged.Therefore, be necessary further to explore the new regulatory gene that suppresses the NF-kB activation, and study it, thereby provide the foundation of necessity for the medicine of exploitation treatment inflammation, anaphylactic disease, autoimmune disease and tumour in the regulatory function of intracellular expression to the NF-kB activation.

NFAT is a kind of multi-functional molecule of transcribing, it is expressed in most of immunocytes, and in the expression of a series of cytokines of regulation and control and cell surface part, play the role of a nucleus, and these cytokines and cell surface part play a crucial role to regulating immunne response.The antigen receptor of immune stimulatory cell, cause flow of calcium ions, produce activatory calcineurin (CaN), its catalyzing N FAT albumen dephosphorylation, also promote them to change in the nucleus thereby activate NFAT, and combine with AP-1 genes involved that interleukin-22 starts on the factor.So NFAT and Ca ²⁺The signal path that-CaN relies on is closely related.

There are 5 members in NFAT family, NFAT1/p, NFAT2/c, NFAT3, NFAT4/x and NFAT5, and wherein preceding four kinds regulated by calcineurin.Multiple lymphoma comprises R-S cell and the disperse large B cell lymphoid tumor and the Burkitt lymphoma of He Jiejin lymphomas, the decline that all shows the NFATc1 expression level.Retrovirus inserts the label experiment and shows that NFATc1 and NFAT5 gene are nonrandom insertion sites, thinks that these genes have participated in the tumour generating process.Virus will cause quickening the generation of T cell tumour if the dna rat of disappearance NFATc2 and/or NFATc3 infects lymphoma SL3-3, and compares NFATc3 absence type mouse death rate height with wild-type.These explanations NFAT transcription factor is vital for lymphocytic activation of balance and elimination, and can avoid because the lymphoma that the variation of lymphocytic elimination defective or activation levels causes takes place.In addition, NF-AT3 is the most important downstream procedures of signal transduction path that CaN relies on, nuclear is gone in NF-AT3 transposition behind the CaN dephosphorylation, performance signal transfer function, NF-AT3 can produce in examining after by phosphorylation, thereby stop the effect of loose signal, therefore the phosphorylation of NF-AT3 also is one of means that suppress the CaN signal pathway, also may become the effect target that stops myocardial hypertrophy, thereby specific heart inhibition medicine can be provided, avoid complication such as immunosuppression.

On the one hand, NFAT plays critical effect in the genetic transcription process that immunne response causes, its regulation and control acquired immunity answering system.On the one hand, NFAT is in lymphocytic activation, propagation, with play a key role in the apoptotic process, it promotes activation inductive T/B necrocytosis (AICD), this pro-apoptosis bioactivity makes it can be used as the tumor inhibitor of lymphoid lineage cell, and lymphocytic activation of balance and elimination are avoided because the lymphoma that the variation of lymphocytic elimination defective or activation threshold level causes takes place.On the other hand, in the T cell, NFAT can mediate Ca as the downstream substrate of CaN ²⁺Signal is to the adjusting of genetic expression.In cardiac muscle, also may exist a kind of like this with cell Ca ²⁺Signal and cardiac gene transcription activating link coupled potential mechanism.In the vitro culture myocardial cell, find that CaN, GATA4 and NT-AT are under the condition of activated form existence, can make the activation of sharp sodium polypeptide (BNP) promotor of Type B increase by 100 times (Molkentin JD, Lu JR, Antos CL, et al.Cell, 1998).And this is by Ca ²⁺The signal path that activatory, CaN rely on may play important effect in the developing of myocardial hypertrophy.And CaN participates in the adjusting of various kinds of cell function as a kind of multifunctional signal enzyme, and .1997 such as () Guerini except the effect of immunity system and cardiac muscle, growing of neurocyte, plays an important role in the adjusting of synaptic plasticity; In skeletal muscle, can participate in the adjusting that the myofiber type transforms .1998 such as () Chin.There are some researches show that CaN may participate in Ca in the vascular smooth muscle cell (VSMC) ²⁺Adjusting.Therefore, the regulation and control that the signal path that relies on for the generation development of immunity system and myocardial hypertrophy etc. and CaN of the transcriptional activity of NFAT is relevant are significant.

NFAT is the main target of immunosuppressor such as cyclosporin A and FK-506.They are mainly by the neural calcineurin of inhibition, and then the NFAT in the suppressor T cell nuclear, and its gene can not be transcribed and translate, thereby suppressed IL-2,3,4,8, the expression of cytokine such as IFN γ and IL-2 acceptor and Transferrins,iron complexes, and suppress the transformation reactions of IgE mediation.These biological immunosuppressor are one of important trend of future drugs development, have broad application prospects.Some is gone on the market by drugs approved by FDA, but majority is in experimental stage.Anti-IL-8 monoclonal antibody has been used for psoriatic, and certain curative effect is arranged.Use IL-10/11, can suppress the Th1 cell and produce IL-2 and IFN-γ, help the balance of Th1/Th2, to psoriatic effectively (Ellis CN, Barker NWN.Psoriasis.CurrProb Dermatol, 2000,12:45-50).And most of such medicine can suppress CaN in non-immunocyte, poor selectivity, toxic side effect are big, so need exploitation not have toxic action, the direct reagent of target NFAT, thereby help to develop selectivity by force, novel immunomodulator safely and effectively.

Summary of the invention

Deficiency at prior art and existing medicine or reagent, the purpose of this invention is to provide a kind of inhibition human body cell nf NF-κ B and NFAT activatory human gene NFIF1, and determined that its expression product has and suppressed NF-κ B and NFAT activatory function, and suppress NF-κ B and the effect of NFAT activatory very obviously, stable.In addition, external source high expression level NFIF can more obviously cause the apoptosis of some cell in cell.Thereby provide this gene in the pharmaceutical composition of preparation prevention and treatment inflammation, anaphylactic disease, autoimmune disease and tumour and the purposes of regulating immunoregulation druges such as lymphocytic activation, propagation and apoptosis.

Technical scheme of the present invention comprises following content:

1, the polypeptide (327 amino acid) of its aminoacid sequence shown in sequence in the sequence table 2, or its conservative property variation polypeptide, or its active fragments.

2, the gene of the polypeptide of the above item 1 of coding.

3, above 2 gene is characterised in that to have the nucleotide sequence shown in the sequence 1 (NFIF1) in the sequence table, or its fragment, or homology is more than 95%, especially the sequence more than 99%.

4, contain above 2 or 3 gene or its segmental expression vector.

5, the method for the polypeptide of the above item 1 of preparation comprises gene NFIF1 or its segmental expression vector transformed host cell with above item 4, cultivates, and isolates described polypeptide from culture.

6, a kind of genetically engineered host cell is characterized in that it is a host cell with above 2 described gene transformation or transduction, or the host cell that transforms or transduce with above 3 described carriers.

7, above 1 polypeptide or 2 gene prevent and/or treat purposes in the medicine with human body cell nf NF-κ B diseases associated in preparation, or regulate lymphocytic activation and/or the medicine of propagation and/or apoptosis or the purposes in the reagent in preparation.

8, according to above 7 purposes, wherein said disease is selected from inflammation, anaphylactic disease, autoimmune disease or tumour.

9, contain above 1 the polypeptide and the pharmaceutical composition of pharmaceutically acceptable carrier.

10, a kind of can with the antibody of above 1 polypeptide specific combination.

11, the external detection method of a kind of diseases associated with inflammation or tumour is characterised in that existence or the level of the antibody test of item 10 more than utilizing from the polypeptide in host's sample.

Polypeptide of the present invention can be a recombinant polypeptide, synthetic polypeptide etc., preferably recombinant polypeptide.Polypeptide of the present invention comprises conservative property variation polypeptide, or its active fragments, and they are meant and keep biological function or the active polypeptide that polypeptide is identical therewith.

Nucleotide sequence of the present invention or fragment comprise its complementary strand.The invention still further relates to the carrier that comprises polynucleotide of the present invention, with the genetically engineered host cell of carrier manufacturing of the present invention and the polypeptide products of producing with recombinant technology of the present invention.Polynucleotide sequence of the present invention can be included in any one of numerous expression vectors of being used for express polypeptide.Suitable carrier for example comprises bacterial plasmid, adenovirus, adeno-associated virus etc.Expression vector can be protokaryon or carrier for expression of eukaryon.The carrier that contains gene order of the present invention can be used for transforming suitable host cell so that this polypeptide of host expresses.

Host cell can be a higher eucaryotic cells, as mammalian cell, or prokaryotic cell prokaryocyte, for example intestinal bacteria.

Utilize gene recombination technology, thus can mass production polypeptide of the present invention.

Polypeptide of the present invention has the human body cell nf NF-κ B of inhibition and the effect of NFAT activatory, human body cell nf NF-kB activation and multiple disease-relateds such as inflammation, anaphylactic disease, autoimmune disease and tumour, and the NFAT activation is relevant with lymphocytic activation, propagation and apoptosis.Therefore polypeptide of the present invention can be effective to prevention or treat these diseases.

Polypeptide of the present invention can be separately or is used in combination with suitable pharmaceutical carrier.This composition can comprise polypeptide and the pharmaceutically acceptable carrier or the excipient for the treatment of significant quantity, and such carrier includes but not limited to: salt solution, and buffer saline, glucose, water, glycol, ethanol, or mixture, these preparations should be fit to administering mode.

Pharmaceutical composition of the present invention administration by rights is as by part, intravenously, intraperitoneal, intramuscular, administration such as subcutaneous.The consumption that delivers medicine to the patient depends on many factors, as administering mode, and curer's physical qualification and doctor's experience.

Polypeptide of the present invention also can use by express these polypeptide at live body.For example patient's cell can carry out the genetically engineered operation by the gene NFIF1 at external use code book invention polypeptide, then engineering cell is offered the patient, makes engineering cell this peptide species of high expression level in vivo, thereby reaches the purpose of treatment.

Polypeptide of the present invention, or conservative variations polypeptide or fragment, or the cell of expressing them can be used as antigen and is used for producing antibody, be used for the clinical diagnosis, treatment, therapeutic evaluation of diseases associated with inflammation, anaphylactic disease, autoimmune disease and tumour etc. clinically, also can be used for NFIF1 and suppress NF-κ B and NFAT activated molecule Study on Mechanism.Described antibody can be monoclonal antibody, or polyclonal antibody, also comprise embedding and, strand and humanized antibody and Fab fragment, or the product of Fab expression library.The antibody of polypeptide of the present invention can be by obtaining this polypeptide direct injection to animal.The method for preparing mono-clonal and polyclonal antibody is known in the art, and is described in numerous documents.This antibody can be used to detect the existence or the level of polypeptide of the present invention, or is used to detect gene.In inflammation or tumor disease,, can be used to diagnose these diseases by level or the existence that detects this polypeptide.

The NFIF1 gene healthy tissues that all are used in experiment, fetal tissue, tumor tissues all have expression, illustrate that it is important NFKB of human body self and NFAT regulatory molecule; Expression amount just has difference in different tissues, and the degree difference of its performance function in different tissues is described.

Description of drawings

Fig. 1 pGEM-T Easy carrier cloning NFIF1 gene coding region cDNA synoptic diagram.Triangle icon wherein is for inserting NFIF1 gene coding region cDNA fragment place.

The agarose gel electrophoresis figure of Fig. 2 NFIF1 gene pcr amplification product in tissue.Among the figure: A is 14 kinds of human normal tissues, wherein is followed successively by: A0 blank, the A1 heart, A2 pancreas, A3 testis, A4 ovary, A5 prostate gland, A6 colon, A7 small intestine, A8 skeletal muscle, A9 thymus gland, A10 lymphoglandula, A11 tonsilla, A12 white corpuscle; B is 6 kinds of people's tumor tissues, wherein is followed successively by: B0 blank, B1 lung cancer, B2 carcinoma of the pancreas, B3 ovarian cancer, B4 prostate cancer, B5 colorectal carcinoma, B6 mammary cancer; C is 8 kinds of fetal tissues, wherein is followed successively by: C0 blank, C1 tire lung, C2 fetal rhythm, C3 tire liver, C4 tire spleen, C5 tire kidney, C6 tire brain, C7 tire skeletal muscle, C8 tire thymus gland; M is a molecular weight marker; 2-1 figure and 2-2 figure among Fig. 2 are the pcr amplification product agarose gel electrophoretogram of NFIF1 gene, and 2-3 figure is the pcr amplification product agarose gel electrophoretogram of house-keeping gene G3PDH.

The structure synoptic diagram of Fig. 3 carrier for expression of eukaryon pcDNA3.1B-NFIF1.

The structure iron of Fig. 4 pNF-KB-luc and pNFAT-luc luciferase gene reporter plasmid.Wherein last figure is the structure iron of pNF-κ B-luc luciferase gene reporter plasmid, and figure below is the structure iron of pNFAT-luc luciferase gene reporter plasmid.

Fig. 5 NFIF1 suppresses NF-kB activation and TNF inductive NF-kB activation.Be set at 1 with pcDNA3.1B and pNF-κ B-1uc cotransfection 293T cell, uciferase activity ratio (Photinus pyralis LUC activity/jellyfish luciferases activity) when non-stimulated, wherein the left side square column be a blank, and the right side square column is TNF α.

Fig. 6 NFIF1 suppresses NF-kB activation and PMA+ ionomycin inductive NF-kB activation.

Among the figure: be set at 1 with pcDNA3.1B and pNF-κ B-luc cotransfection 293T cell, uciferase activity ratio when non-stimulated, wherein the left side square column is P+I, and the right side square column be a blank.

Fig. 7 NFIF1 suppresses MEKK inductive NF-kB activation.Uciferase activity ratio when pcDNA3.1B and pNF-κ B-luc cotransfection 293T cell is set at 1.Wherein the left side square column is TNFc, and middle square column is P+I, and the right side square column is blank.

Fig. 8 NFIF1 suppresses NFAT activation and PMA+ ionomycin inductive NFAT activation.Among the figure: be set at 1 with pcDNA3.1B and pNFAT-luc cotransfection 293T cell, uciferase activity ratio when non-stimulated.Wherein the left side square column is P+I, and the right side square column is blank.Data among Fig. 5-Fig. 8 are the result of three revision test gained.

Below embodiment of the present invention are further described.

1, isolate the cDNA of NFIF1 gene from tissue, measure its nucleotide sequence, and Further infer the amino acid sequence that obtains its coding.

The present invention carries out the retrieval of people's Unknown Function predicted gene by the nr database to NCBI, obtains People's unknown function gene order (Ref:XM_114657; Locus ID:203245; UniGene: Hs.373606), this nucleotide sequence is shown in sequence in the sequence table 3.

Further utilize the Human_est database to carry out sequence by the BLASTn method and proofread and correct, the site of correction is as follows:

1) 37 bit bases are A by the G correction;

2) 562 bit bases are C by the T correction;

3) insert 45 bases between 1283 bit bases and 1284 bit bases, predict that this insertion does not change ORF (ORFs);

4) after last base of this sequence, extend 405 bases, predict that this extension does not change ORF; The sequence that obtains after the correction is shown in sequence in the sequence table 4.

In addition, analyze to have a variable spliced body, predict that this spliced body does not change ORF, itself and former Sequence be not located on 3 ' non-translational region, sequence is shown in sequence in the sequence table 5.

This gene is named as NFIF1. NFIF1 gene chromosome mapping 9q34.13 is outside 3 Aobvious son and 2 intrones form (its spliced body is made up of 2 extrons and 1 introne). Prediction ORF total length 984bp, 327 amino acid of encoding.

According to the NFIF1 gene order, design NFIF1 gene specific primer, (5 ' primer: 5 '-gggggtgggtgaggggc-3 '; 3 ' primer: 5 '-acccctgccctcactggatg-3 ') from mixing people's tissue In the cDNA library (brain, lung, colon, testis, Clometech, K14201-1, K1241-1) Obtain NFIF1 gene coding region cDNA fragment by pcr amplification. Adopt Tag in the pcr amplification Archaeal dna polymerase obtains containing the amplified production of 3 ' outstanding base A, after electrophoresis is identified with its time Receive, insert behind the purifying the linear pGEM-T Easy carrier that contains 3 ' outstanding base T (Promega, A1360), transformed into escherichia coli DH50 α extracts plasmid, with ABI PRISM 3700 DNA Analyzer (Perkin-Elmer/applied Biosystem) order-checking obtains the NFIF1 gene cDNA Fragment 1091bp (wherein 984bp is the open code district of reading). The sequencing result confirms center of sequence It is that C is correct that 562 bit bases are proofreaied and correct by T, and the correction in other site is not because at the pcr amplification model Enclose interior and be not confirmed. Sequencing result is shown in sequence 1.

The pcr amplified fragment of pGEM-T Easy carrier cloning NFIF1 gene coding region cDNA Schematic diagram as shown in Figure 1.

Analyze 2 of the amino acid sequence of NFIF1 dna encoding the protein such as sequences according to sequencing result Show.

2, the structure of carrier for expression of eukaryon

In order to detect NFIF1 to the inhibit feature of NF-κ B and NFAT activation, at first make up and contain The carrier for expression of eukaryon pcDNA3.1B-NFIF1 of NFIF1 cDNA (below be abbreviated as PcDB-NFIF1). The present invention adopts carrier for expression of eukaryon pcDNA3.1/MycHis (-) B (Invitrogen, V85520 below are abbreviated as pcDB) make up eucaryon with the NFIF1 genetic recombination Expression vector. PcDB is designed for recombinant protein the carrying of high expressed in the mammalian cell yarn Body, it contains human cytomegalovirus CMV promoter, can realize high in the mammalian cell yarn Express. With EcoRI the NFIF1 cDNA of above-mentioned insertion pGEM-T Easy carrier is downcut, with The time EcoRI enzyme cut pcDB, both connect rear transformed into escherichia coli DH5 α, select transformant and extract Plasmid is selected correct forward by order-checking and is inserted the clone, namely obtains containing the true of NFIF1 cDNA Nuclear expression carrier pcDB-NFIF1, Fig. 3 is for making up the schematic diagram of this carrier for expression of eukaryon.

3, detect the method that the NFIF1 gene suppresses the function of NF-κ B and NFAT activation

With pcDB-NFIF1 and pNF-κ B-luc (Stratagane, 219077), pRL-SV40 (Promega, E2231) cotransfection human embryo kidney 293T cell (ATCC Number: CRL-11268), adopt the luciferase reporter gene method, measure NFIF1 cDNA at 293T Expression product in the cell is to TNF α, PMA (Sigma)+ionomycin (Calbiochem) The NF-kB activation of inducing with MEKK and to PMA (Sigma)+ionomycin The inhibitory action of the NFAT activation of (Calbiochem) inducing.

The present invention adopts luciferase reporter gene method detection NFIF1 gene pairs NF-kB activation Inhibit feature, what the method adopted is signal transduction pathway cis report system in the body, wherein used PNF-κ B-luc reporter plasmid is loaded with firefly luciferase gene, and the expression of this gene is by synthetic Promoter is regulated and control, and wherein comprises basic promoter element (TATA box), and transcription factor The binding site of NF-κ B, the structure chart of pNF-κ B-luc reporter plasmid as shown in Figure 4. When carefully When the NF-κ B in the born of the same parents was activated by certain signal transduction pathway, NF-κ B just can be incorporated into newspaper Accuse the enhancer element of plasmid pNF-κ B-luc, thereby start reporter gene firefly luciferase base Transcribing of cause further translated into luciferase in the born of the same parents, causes luciferase quantity increase in the cell, Increased activity; On the contrary, when intracellular NF-kB activation is suppressed, the table of luciferase The amount of reaching and active just low. So, by detecting the activity of luciferase, just can reflect different stimulated Whether the genes of interest of thing and cotransfection has activation to NF-κ B in the cell or suppresses activation Function. The use principle of pNFAT-luc reporter plasmid is similar to pNF-κ B-luc reporter plasmid. The pRL-SV40 reporter plasmid is loaded with the jellyfish luciferases gene, and the expression of this gene is by simian virus 40 (SV40) enhancers/promoters is regulated and control, and can produce stronger base behind the transfection mammalian cell The expression of the jellyfish luciferases of this level, and the activity of expressing be not subjected to NF-κ B and NFAT activation with No adjusting is so be used as the internal reference reporter gene in experiment.

Plasmid pcDB-NFIF1 and pNF-κ B-luc, pRL-SV40 cotransfection 293T cell, After the transfection 24 hours, with TNF α or PMA+ ionomycin activation 293T cell, after 8 hours, Cell lysis, the activity of detection luciferase. Record the result and show that the NFIF1 gene is at the 293T cell Interior expression product has inhibitory action to the activation of NF-κ B. Illustrate that NFIF1 has negative the accent to NF-κ B The joint effect.

The method of the function of detection NFIF1 gene inhibition NFAT activation is the same, only alone PMA+ Ionomycin activation 293T cell. Record the result and show that the NFIF1 gene is at the intracellular table of 293T Reach product the activation of NFAT is had inhibitory action. Illustrate that NFIF1 has down regulation to NFAT.

Advantage of the present invention:

1, provides cDNA sequence and the coded polypeptide thereof of human gene NFIF1, and sent out first Now this gene transfection has the function that suppresses the NF-kB activation;

2, NFIF1 has the effect that suppresses simultaneously NF-κ B and NFAT activation, and efficient, Stable, show that NFIF1 has higher affinity and effectiveness in cell;

3, NFIF1 expresses at the most normal cells of body, illustrate its be self important NF κ B and The NFAT regulatory molecule.

4, based on 3 above-mentioned advantages, the present invention is the regulation relationship of further studying between NFIF1 and NF-κ B and the NFAT, and the novel drugs of developing treatment inflammation, anaphylactic disease, autoimmune disease and tumour and regulating lymphocytic activation, propagation and apoptosis, establish necessary base for starting new clinical diagnosis, therapeutic evaluation and prognostic indicator.

Specific embodiments

The clone of embodiment 1, NFIF1 gene cDNA

Nr database to NCBI carries out the retrieval of people's Unknown Function predicted gene, obtains people's unknown function gene order (Ref:XM_114657; Locus ID:203245; UniGene:Hs.373606), this sequence is set at sequence 3, and utilizes the Human_est database to carry out sequence by the BLASTn method and proofread and correct, and the sequence that finally obtains is set at sequence 4.Design the NFIF1 gene specific primers according to sequence nucleotide sequence 4:

5 ' primer: 5 '-GGGGGTGGGTGAGGGG-3 '

3 ' primer: 3 '-ACCCCTGCCCTCACTGGATG-3 '

Use above-mentioned primer, with mix people's tissue cDNA library (brain, lung, pancreas, testis, ClonetechK1420-1 K1241-1) carry out pcr amplification reaction for template, and reaction conditions is as follows:

Reaction volume 50 μ l, wherein contain:

Mix people's tissue cDNA template 2 μ l (its midbrain, lung, colon, each 0.5 μ l of testis)

Primer 5 ' primer, each 0.2 μ M of 3 ' primer final concentration

Each 200 μ M of dNTP final concentration

Taq archaeal dna polymerase 2.5U

10 * Taq dna polymerase buffer liquid, 5 μ l

Complement to 50 μ l volumes with distilled water.

Temperature of reaction, time: 94 ℃, after the sex change in 5 minutes, 94 ℃ of sex change 30 seconds, 63 ℃ of annealing 30 seconds, 72 ℃ were extended 80 seconds, and 30 circulations of increase are at last 72 ℃ of extensions 7 minutes down.

Amplified production is 3 ' 3 ' the outstanding cohesive end fragment of base A to be arranged, reclaim test kit (Qiagen with the quick glue of QIA, 28704) carry out purifying by product description, then with 3 ' the linear pGEM-T EASY carrier (Promega that base T arranged, A1360) connection is spent the night under 16 ℃, use the 2mm pole cup, 2500V transformed into escherichia coli DH5 α, conversion product is grown containing on the LB plate culture medium of penbritin, and selected clone extracts plasmid, use AbI PRISM 3700DNA analyser (Perkin-Elmer/Applied Biosystem) order-checking, obtain the cDNA1091bp of NFIF1 gene, ORF total length 984bp wherein, 327 amino acid of encoding.

Embodiment 2, RT-PCR method detect the special primer of the transcription product of NFIF1 gene in tissue with the NFIF1 gene:

5 ' primer: 5 '-TGTGTCCGTCGCCATGACAG-3 '

3 ' primer: 5 '-TGGTTGAGTGGCAGGTGAGG-3 '

Respectively with 12 kinds of human normal tissues (heart, pancreas, testis, ovary, prostate gland, colon, small intestine, skeletal muscle, thymus gland, lymphoglandula, tonsilla, white corpuscle); 6 kinds of people's tumor tissues (lung cancer, carcinoma of the pancreas, ovarian cancer, prostate cancer, colorectal carcinoma, mammary cancer); (Clonetch, K1420-1 1241-1) carry out pcr amplification for template to organize the cDNA library of amassing (tire lung, fetal rhythm, tire liver, tire spleen, tire kidney, tire brain, tire skeletal muscle, tire thymus gland) with 8 kinds of fetuses.

The pcr amplification condition is as follows:

Reaction volume 50 μ l, wherein contain:

Mix people's tissue cDNA template 2 μ l (its midbrain, lung, colon, each 0.5 μ l of testis)

Primer 5 ' primer, each 0.2 μ M of 3 ' primer final concentration

Each 200 μ M of dNTP final concentration

Taq archaeal dna polymerase 2.5U

10 * Taq dna polymerase buffer liquid, 5 μ l

Complement to 50 μ l volumes with distilled water.

Reaction conditions:

94 ℃, after the sex change in 5 minutes, 94 ℃ of sex change 30 seconds, 58 ℃ of annealing 30 seconds, 72 ℃ were extended 30 seconds, and 30 circulations of increase are at last 72 ℃ of extensions 7 minutes down.In addition, use the house-keeping gene special primer: 5 ' primer: 5 '-ACCACAGTCCATGCCATCAC-3 ', 3 ' primer: 5 '-TCCACCACCCTGTTGCTGTA-3 ' carry out pcr amplification with above-mentioned same template respectively, as internal reference.The PCR reaction system is the same, and reaction conditions is as follows: 94 ℃, and after the sex change in 5 minutes, 94 ℃ of sex change 30 seconds, 58 ℃ of annealing 30 seconds, 72 ℃ were extended 30 seconds, and 25 circulations of increase are at last 72 ℃ of extensions 7 minutes down.The PCR reaction result as shown in Figure 2, the result of amplification shows, the cDNA (with reference to Fig. 2) that all has the NFIF1 gene in the cell of these tissues, illustrate that NFIF1 has produced NFIF1 gene transcription product in the cell of these tissues, wider expression Tu Spectrum is arranged, in multiple tissue, participate in the adjusting of transcription factor.

The structure of embodiment 3, NFIF1 gene eukaryotic expression vector

With EcoRI (Promega) with the cDNA fragment of NFIF1 gene from pGEM-T Easy carrier (Promega, A1360) go up cutting-out, cut carrier for expression of eukaryon pcDNA3.1/mycHis (-) B (Invitrogen with the EcoRI enzyme simultaneously, V85520), according to J.Sambrook etc., the described method of molecular cloning experiment guide second edition, NFIF1 after enzyme cut is connected under 16 ℃ with carrier and spends the night, transformed into escherichia coli DH5 α, conversion product grow containing on the LB plate culture medium of penbritin, select the bacterium colony of growth, extract plasmid, cut with the EcoRI enzyme, enzyme is cut product and is identified with agarose gel electrophoresis, has selected and has inserted segmental positive colony, (use ABI PRISM3700DNA analyser by order-checking, the same), select correct forward and insert clone, called after pcDNA3.1B-NFIF1.

Collect nutrient solution simultaneously, analyze protein precipitation, obtain the NFIF1 polypeptide with SDS-PAGE.

NFIF1 analysis of protein result shows: the NFIF1 protein sequence shown in sequence table sequence 2, molecular weight 35.1kD, iso-electric point 6.82.Striding film distinguishes and to analyse (TMHMM analyzes, http://www.cbs.dtu.dk/services/TMHMM) and show not have and stride the film district.The nuclear localization signal analysis does not have tangible nuclear localization signal.Subcellular Localization analysis prompting is positioned at nuclear possibility maximum (nucleus: 39.1%; Plastosome: 30.4%; Cytoplasm: 17.4%; Excretory system vesicle: 8.7%; Cytoskeleton: 4.3).

Embodiment 4, two luciferase reporter gene method are measured NFIF1 induces the NF-kB activation to TNF α, PMA+ ionomycin restraining effect.

With goal gene pcDB-NFIF1 and reporter gene pNF-κ B-luc, pRL-SV40 cotransfection human embryo kidney 293T cell is measured the activity of NF-κ B by detecting two kinds of uciferase activities respectively.The method of cotransfection is the liposome transfection method, adopts LipfectanineTM2000 (Invitrogen, 11668027), is undertaken by product description is described.

The transfection operation steps is as follows:

(1) cell cultures: 293T cell (ATCC Number:CRL-11268) (2.0 * 104) is used DMEM (Dulbecco ' s modified Eagle ' s medium) substratum (Hyclone that contains 10% foetal calf serum, SH0022.02) be layered on 96 porocyte culture plate (Costar, 3599) on, at 5%CO2, cultivated 16 hours in 37 ℃ the incubator.

(2) preparation DNA-LipfectamineTM2000 mixture: the DMEM substratum that does not contain serum with 25 μ l dilutes 20ng pNF-KB-luc, and 2ngpRL-SV40 and 0-80ngpcDB-NFIF1 slowly mix; Same LipfectaminTM2000 with 25 μ l DMEM substratum dilution appropriate amount slowly mixes, at room temperature be incubated 5 minutes after, slowly mix with the DNA of dilution, room temperature was placed 20 minutes, with formation DNA-Lipofectamine 2000 mixtures.

(3) transfection: DNA-Lipofectamine 2000 mixtures are slowly splashed into Tissue Culture Plate (50 μ l/ hole), slightly shake up.5%CO2 cultivated 24 hours in 37 ℃ the incubator.

Use TNF α (tumor necrosis factor alpha, IYX300-01A2, final concentration 20ng/ml) or PMA (TETRADECONIC ACID Buddhist ripple ester after 24 hours, Calbiochem, 524400, final concentration 50ng/ml)+ionomycin (Calbiochem, 407950, final concentration 1nM) irritation cell, or do not add stimulator; Discard substratum after 8 hours, add Reporter Lysis Buffer (Promega, E4030), placed 30 minutes down, take out the back and melt naturally in room temperature at-80 ℃, make lysis, then cell pyrolysis liquid is moved into fluorescent plate (Gronier, 655075), with Dual-Luciferase ReporterAssay System 10-Pack (Promaga, E1960), detect uciferase activity by Fluostar OPTIMA (BMGLabtechnologies).

Set transfection pcDB empty carrier, intracellular uciferase activity ratio (Photinus pyralis LUC fluorescence intensity/jellyfish luciferases fluorescence intensity is represented) is 1 when not adding stimulator, intracellular uciferase activity ratio is represented with relative value as standard under other conditions.

The result:

(1) NFIF1 induces the restraining effect of NF-kB activation to TNF α

With reference to Fig. 5, use 0,10,20,40 respectively, 80ng pcDNA3.1B-NFIF1 and 20ngpNF-B-luc cotransfection 293T cell, increase along with pcDNA3.1B-NFIF1 consumption in the cotransfection, activity with luciferase in the 293T cell of TNF α stimulation descends rapidly significantly, and the activity of luciferase descends comparatively not slowly in the 293T cell that stimulates with TNF α, shows that the expression product of NFIF1 in the 293T cell induce the NF-kB activation that strong restraining effect is arranged to TNF α.

(2) NFIF1 induces the restraining effect of NF-kB activation to the PMA+ ionomycin

With reference to Fig. 6, use 0,10,20,40 respectively, 80ng pcDNA3.1B-NFIF1 and 20ng pNF-κ B-luc cotransfection 293T cell, induce similar with TNF α, increase along with the consumption of pcDNA3.1B-NFIF1 in the cotransfection, induce the activity of luciferase in the 293T cell of stimulation to descend rapidly significantly with the PMA+ ionomycin, and the activity of luciferase descends gently in the 293T cell that stimulates with the PMA+ ionomycin, shows that equally the expression product of NFIF1 in the 293T cell induce the NF-kB activation that strong restraining effect is arranged to the PMA+ ionomycin.

Embodiment 5, two luciferase reporter gene method are measured the restraining effect of NFIF1 to MEKK inductive NF-kB activation.Except with 20ng pNF-κ B-luc, 2ngpRL-SV40,0-80ngpcDNA3.1B-NFIF1 (supplying with the pcDNA3.1B carrier during not enough 80ng) and 20ngpFC-MEKK (Stratageme, 219077) cotransfection 293T cell, and transfection is after 24 hours directly beyond the lysing cell, and all the other are identical with embodiment 4.

The result:

With reference to Fig. 7, use 0,10,20,40 respectively, 80ng pcDNA3.1B-NFIF1 and 20ngpNF-κ B-luc, 2ngpRL-SV40 and 20ng pFC-MEKK cotransformation 293T cell, NFIF1 to similar to TNF, PMA+ ionomycin inductive, illustrates that NFIF1 has strong restraining effect to MEKK inductive NF-kB activation to the restraining effect of MEKK inductive NF-kB activation thus.

According to above experimental result, can determine that when the activation of NF-κ B caused transcriptional expression activation with some disease related gene, NFIF1 can stop the generation and the development of this disease to the restraining effect of NF-kB activation.Therefore, the present invention can be used to prepare the medicine of prevention and treatment inflammation, anaphylactic disease, autoimmune disease and tumour.

Embodiment 6, two luciferase reporter gene method are measured NFIF1 to PMA+ ionomycin inductive NFAT activatory restraining effect.

Except replace pNF-κ B-luc transfection with 20ng pNFAT-luc, all the other with embodiment 4 in NFIF1 induce the restraining effect of NF-kB activation to test identical to the PMA+ ionomycin.

The result:

With reference to Fig. 8, use 0,10,20,40 respectively, 80ng pcDNA3.1B-NFIF1 and 20ngPnfat-luc cotransfection 293T cell, increase along with the consumption of pcDNA3.1B-NFIF1 in the cotransfection, induce the activity of luciferase in the 293T cell of stimulation to descend rapidly significantly with the PMA+ ionomycin, and the activity of luciferase descends gently in the 293T cell that stimulates with the PMA+ ionomycin, shows that equally the expression product of NFIF1 in the 293T cell induce the NFAT activation that strong restraining effect is arranged to the PMA+ ionomycin.

Therefore, the present invention can be used to regulate the exploitation of the novel drugs of lymphocytic activation, propagation and apoptosis.

Embodiment 7, Antibody Preparation

Antigen is selected the NFIF1 albumen total length or the partial peptide section of prokaryotic cell prokaryocyte or eukaryotic cell expression for use, also can synthesize polypeptide as antigen.

Polyclonal Antibody Preparation: immune animal is selected bull new zealand rabbit or BALb/c mouse for use, after initial immunity uses 200ug (new zealand rabbit) or 20ug (BALb/c mouse) antigen and equal-volume Freund's complete adjuvant (FCA) fully emulsified, the subcutaneous multi-point injection in the back.Behind the initial immunity 21,42,63 days, with Freund's incomplete adjuvant (FIA) emulsive antigen protein fully, each booster immunization 1 time, consumption was the same.Each immunity back 7～10 days, the ELISA method detects serum titer, and when reaching 1 * 10-4, bloodletting separation of serum .Western blot identifies antibodies specific.

Monoclonal Antibody: immune BALb/c mouse is the same, gets spleen and makes the B cell suspension, with the myeloma cell SP2/0 fusion of logarithmic phase, by HAT (H, xanthoglobulin; A, aminopterin-induced syndrome; T, thymidine) the selectivity cultivation, obtain hybrid cell line, detect antibody titer by the ELISA method again, filter out specific hybridoma cell line, and obtain monoclonal antibody.

NFIF1 antibody can be used for the clinical diagnosis, treatment, therapeutic evaluation of diseases associated with inflammation, anaphylactic disease, autoimmune disease and tumour etc. clinically, also can be used for NFIF1 and suppresses NF-κ B and NFAT activated molecule Study on Mechanism.

SEQUENCE?LISTING

＜110〉Sinogenomax Co., Ltd. of Peking University

＜120〉a kind of inhibition human body cell nf NF-κ B and NFAT activatory gene and encoded polypeptides and purposes

<130>

<160>5

<170>PatentIn?version?3.1

<210>1

<211>1091

<212>DNA

＜213〉people

<220>

<221>CDS

<222>(98)..(1078)

<223>

<400>1

gggggtgggt?gaggggcgag?gctgaggaag?aagagaaggg?gaattggggc?gcttgagggg 60

attataattt?ctttaaaaag?aggggagggg?agaggcc?atg?gcc?gtc?cca?gcc?aag 115

Met?Ala?Val?Pro?Ala?Lys

1 5

aaa?agg?aag?atg?aac?ttc?tca?gag?cgg?gag?gtg?gag?atc?atc?gtg?gag 163

Lys?Arg?Lys?Met?Asn?Phe?Ser?Glu?Arg?Glu?Val?Glu?Ile?Ile?Val?Glu

10 15 20

gag?ctg?gag?ctg?aag?aag?cac?ctg?ctg?gtg?aac?cac?ttc?aac?gcc?ggg 211

Glu?Leu?Glu?Leu?Lys?Lys?His?Leu?Leu?Val?Asn?His?Phe?Asn?Ala?Gly

25 30 35

gta?ccc?ctg?gcc?gcc?aag?agt?gcg?gcc?tgg?cac?ggc?atc?ctg?aga?agg 259

Val?Pro?Leu?Ala?Ala?Lys?Ser?Ala?Ala?Trp?His?Gly?Ile?Leu?Arg?Arg

40 45 50

gtc?aac?gcc?gtg?gcc?acc?tgc?cgc?aga?gag?ctg?cct?gag?gtc?aag?aag 307

Val?Asn?Ala?Val?Ala?Thr?Cys?Arg?Arg?Glu?Leu?Pro?Glu?Val?Lys?Lys

55 60 65 70

aag?tgg?tct?gac?ctc?aag?acc?gag?gtc?cgt?cgc?aag?gtt?gcc?cag?gtc 355

Lys?Trp?Ser?Asp?Leu?Lys?Thr?Glu?Val?Arg?Arg?Lys?Val?Ala?Gln?Val

75 80 85

cgg?gcc?gcc?gtg?gag?ggt?ggt?gag?gcg?ccg?ggg?ccc?act?gag?gag?gac 403

Arg?Ala?Ala?Val?Glu?Gly?Gly?Glu?Ala?Pro?Gly?Pro?Thr?Glu?Glu?Asp

90 95 100

gga?gct?ggg?ggg?cct?ggg?aca?ggc?ggt?ggc?agt?ggc?ggc?ggt?ggc?cca 451

Gly?Ala?Gly?Gly?Pro?Gly?Thr?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Pro

105 110 115

gct?gta?gcc?cca?gtg?ctg?ctg?acc?ccc?atg?caa?caa?cgt?atc?tgc?aac 499

Ala?Val?Ala?Pro?Val?Leu?Leu?Thr?Pro?Met?Gln?Gln?Arg?Ile?Cys?Asn

120 125 130

ctg?ctg?ggc?gag?gcc?acc?atc?atc?agc?ctg?ccc?agc?acc?aca?gag?atc 547

Leu?Leu?Gly?Glu?Ala?Thr?Ile?Ile?Ser?Leu?Pro?Ser?Thr?Thr?Glu?Ile

135 140 145 150

cac?cct?gtg?gcc?ctc?gga?ccc?tcg?gcc?acc?gca?gcc?gca?gcc?acg?gtc 595

His?Pro?Val?Ala?Leu?Gly?Pro?Ser?Ala?Thr?Ala?Ala?Ala?Ala?Thr?Val

155 160 165

acc?ctg?aca?cag?atc?ccc?aca?gag?acc?acc?tat?cac?acg?ctg?gag?gag 643

Thr?Leu?Thr?Gln?Ile?Pro?Thr?Glu?Thr?Thr?Tyr?His?Thr?Leu?Glu?Glu

170 175 180

ggc?gtg?gtg?gag?tac?tgc?acg?gct?gag?gcg?ccc?cca?cct?ctg?cca?cca 691

Gly?Val?Val?Glu?Tyr?Cys?Thr?Ala?Glu?Ala?Pro?Pro?Pro?Leu?Pro?Pro

185 190 195

gag?acc?cct?gtg?gac?atg?atg?gcc?cag?cat?gca?gac?acg?tcg?gtc?aag 739

Glu?Thr?Pro?Val?Asp?Met?Met?Ala?Gln?His?Ala?Asp?Thr?Ser?Val?Lys

200 205 210

ccg?caa?gcg?ctc?aag?agc?cgc?att?gct?ctc?aac?tcc?gcc?aag?ctg?ata 787

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

215 220 225 230

cag?gag?cag?cgg?gtc?acc?aac?ctg?cat?gtg?aag?gag?atc?gca?cag?cac 835

Gln?Glu?Gln?Arg?Val?Thr?Asn?Leu?His?Val?Lys?Glu?Ile?Ala?Gln?His

235 240 245

ctg?gaa?cag?cag?aac?gac?cta?ctg?cag?atg?atc?cgc?cgc?tcc?cag?gaa 883

Leu?Glu?Gln?Gln?Asn?Asp?Leu?Leu?Gln?Met?Ile?Arg?Arg?Ser?Gln?Glu

250 255 260

gtg?cag?gcc?tgt?gcc?cag?gag?cgc?cag?gcc?cag?gcc?atg?gag?ggc?aca 931

Val?Gln?Ala?Cys?Ala?Gln?Glu?Arg?Gln?Ala?Gln?Ala?Met?Glu?Gly?Thr

265 270 275

cag?gct?gcc?ctg?agc?gtc?ctc?atc?cag?gtc?ctc?cgg?cct?atg?atc?aaa 979

Gln?Ala?Ala?Leu?Ser?Val?Leu?Ile?Gln?Val?Leu?Arg?Pro?Met?Ile?Lys

280 285 290

gat?ttc?cgc?cgc?tac?ctg?cag?agc?aac?aca?gct?aac?ccg?gcc?ccc?gcc 1027

Asp?Phe?Arg?Arg?Tyr?Let?Gln?Ser?Asn?Thr?Ala?Asn?Pro?Ala?Pro?Ala

295 300 305 310

tct?gac?cct?ggg?cag?gtg?gcc?cag?aat?ggg?cag?cca?gac?agc?atc?atc 1075

Ser?Asp?Pro?Gly?Gln?Val?Ala?Gln?Asn?Gly?Gln?Pro?Asp?Ser?Ile?Ile

315 320 325

cag?tgagggcagg?ggt 1091

Gln

<210>2

<211>327

<212>PRT

＜213〉people

<400>2

Met?Ala?Val?Pro?Ala?Lys?Lys?Arg?Lys?Met?Asn?Phe?Ser?Glu?Arg?Glu

1 5 10 15

Val?Glu?Ile?Ile?Val?Glu?Glu?Leu?Glu?Leu?Lys?Lys?His?Leu?Leu?Val

20 25 30

Asn?His?Phe?Asn?Ala?Gly?Val?Pro?Leu?Ala?Ala?Lys?Ser?Ala?Ala?Trp

35 40 45

His?Gly?Ile?Leu?Arg?Arg?Val?Asn?Ala?Val?Ala?Thr?Cys?Arg?Arg?Glu

50 55 60

Leu?Pro?Glu?Val?Lys?Lys?Lys?Trp?Ser?Asp?Leu?Lys?Thr?Glu?Val?Arg

65 70 75 80

Arg?Lys?Val?Ala?Gln?Val?Arg?Ala?Ala?Val?Glu?Gly?Gly?Glu?Ala?Pro

85 90 95

Gly?Pro?Thr?Glu?Glu?Asp?Gly?Ala?Gly?Gly?Pro?Gly?Thr?Gly?Gly?Gly

100 105 110

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

115 120 125

Gln?Gln?Arg?Ile?Cys?Asn?Leu?Leu?Gly?Glu?Ala?Thr?Ile?Ile?Ser?Leu

130 135 140

Pro?Ser?Thr?Thr?Glu?Ile?His?Pro?Val?Ala?Leu?Gly?Pro?Ser?Ala?Thr

145 150 155 160

Ala?Ala?Ala?Ala?Thr?Val?Thr?Leu?Thr?Gln?Ile?Pro?Thr?Glu?Thr?Thr

165 170 175

Tyr?His?Thr?Leu?Glu?Glu?Gly?Val?Val?Glu?Tyr?Cys?Thr?Ala?Glu?Ala

180 185 190

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

195 200 205

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

210 215 220

Asn?Ser?Ala?Lys?Leu?Ile?Gln?Glu?Gln?Arg?Val?Thr?Asn?Leu?His?Val

225 230 235 240

Lys?Glu?Ile?Ala?Gln?His?Leu?Glu?Gln?Gln?Asn?Asp?Leu?Leu?Gln?Met

245 250 255

Ile?Arg?Arg?Ser?Gln?Glu?Val?Gln?Ala?Cys?Ala?Gln?Glu?Arg?Gln?Ala

260 265 270

Gln?Ala?Met?Glu?Gly?Thr?Gln?Ala?Ala?Leu?Ser?Val?Leu?Ile?Gln?Val

275 280 285

Leu?Arg?Pro?Met?Ile?Lys?Asp?Phe?Arg?Arg?Tyr?Leu?Gln?Ser?Asn?Thr

290 295 300

Ala?Asn?Pro?Ala?Pro?Ala?Ser?Asp?Pro?Gly?Gln?Val?Ala?Gln?Asr?Gly

305 310 315 320

Gln?Pro?Asp?Ser?Ile?Ile?Gln

325

<210>3

<211>1416

<212>DNA

＜213〉people

<400>1

cattacgctg?ctggctggca?gcggccgggc?cggtcggggc?tgggccctac?gcactttgcg 60

tagcgagggg?ggttaccaaa?ggcctagtgc?ttggcctcga?gcaagcctgg?cctatcccct 120

gtagggggtg?ggtgaggggc?gaggctgagg?aagaagagaa?ggggaattgg?ggcgcttgag 180

gggattataa?tttctttaaa?aagaggggag?gggagaggcc?atggccgtcc?cagccaagaa 240

aaggaagatg?aacttctcag?agcgggaggt?ggagatcatc?gtggaggagc?tggagctgaa 300

gaagcacctg?ctggtgaacc?acttcaacgc?cggggtaccc?ctggccgcca?agagtgcggc 360

ctggcacggc?atcctgagaa?gggtcaacgc?cgtggccacc?tgccgcagag?agctgcctga 420

ggtcaagaag?aagtggtctg?acctcaagac?cgaggtccgt?cgcaaggttg?cccaggtccg 480

ggccgccgtg?gagggtggtg?aggcgccggg?gcccactgag?gaggacggag?ctggggggcc 540

tgggacaggc?ggtggcagtg?gtggcggtgg?cccagctgta?gccccagtgc?tgctgacccc 600

catgcaacaa?cgtatctgca?acctgctggg?cgaggccacc?atcatcagcc?tgcccagcac 660

cacagagatc?caccctgtgg?ccctcggacc?ctcggccacc?gcagccgcag?ccacggtcac 720

cctgacacag?atccccacag?agaccaccta?tcacacgctg?gaggagggcg?tggtggagta 780

ctgcacggct?gaggcgcccc?cacctctgcc?accagagacc?cctgtggaca?tgatggccca 840

gcatgcagac?acgtcggtca?agccgcaagc?gctcaagagc?cgcattgctc?tcaactccgc 900

caagctgata?caggagcagc?gggtcaccaa?cctgcatgtg?aaggagatcg?cacagcacct 960

ggaacagcag?aacgacctac?tgcagatgat?ccgccgctcc?caggaagtgc?aggcctgtgc 1020

ccaggagcgc?caggcccagg?ccatggaggg?cacacaggct?gccctgagcg?tcctcatcca 1080

ggtcctccgg?cctatgatca?aagatttccg?ccgctacctg?cagagcaaca?cagctaaccc 1140

ggcccccgcc?tctgaccctg?ggcaggtggc?ccagaatggg?cagccagaca?gcatcatcca 1200

gtgagggcag?gggtcaggcc?agccttctgc?catgatggga?tgaaaactcc?atggacttac 1260

tcaccatcaa?ttaccaaggc?ccttgcctca?gccacatatg?accaatggtt?acaactcagg 1320

gctccagacc?tcagctaaaa?agagaagacg?ctgccctcct?gggcacgaac?gtttagaatg 1380

ctcaactcct?ctattgtgac?cacaggaagg?tggccc 1416

<210>4

<211>1866

<212>DNA

＜213〉people

<400>1

cattacgctg?ctggctggca?gcggccgggc?cggtcgaggc?tgggccctac?gcactttgcg 60

tagcgagggg?ggttaccaaa?ggcctagtgc?ttggcctcga?gcaagcctgg?cctatcccct 120

gtagggggtg?ggtgaggggc?gaggctgagg?aagaagagaa?ggggaattgg?ggcgcttgag 180

gggattataa?tttctttaaa?aagaggggag?gggagaggcc?atggccgtcc?cagccaagaa 240

aaggaagatg?aacttctcag?agcgggaggt?ggagatcatc?gtggaggagc?tggagctgaa 300

gaagcacctg?ctggtgaacc?acttcaacgc?cggggtaccc?ctggccgcca?agagtgcggc 360

ctggcacggc?atcctgagaa?gggtcaacgc?cgtggccacc?tgccgcagag?agctgcctga 420

ggtcaagaag?aagtggtctg?acctcaagac?cgaggtccgt?cgcaaggttg?cccaggtccg 480

ggccgccgtg?gagggtggtg?aggcgccggg?gcccactgag?gaggacggag?ctggggggcc 540

tgggacaggc?ggtggcagtg?gcggcggtgg?cccagctgta?gccccagtgc?tgctgacccc 600

catgcaacaa?cgtatctgca?acctgctggg?cgaggccacc?atcatcagcc?tgcccagcac 660

cacagagatc?caccctgtgg?ccctcggacc?ctcggccacc?gcagccgcag?ccacggtcac 720

cctgacacag?atccccacag?agaccaccta?tcacacgctg?gaggagggcg?tggtggagta 780

ctgcacggct?gaggcgcccc?cacctctgcc?accagagacc?cctgtggaca?tgatggccca 840

gcatgcagac?acgtcggtca?agccgcaagc?gctcaagagc?cgcattgctc?tcaactccgc 900

caagctgata?caggagcagc?gggtcaccaa?cctgcatgtg?aaggagatcg?cacagcacct 960

ggaacagcag?aacgacctac?tgcagatgat?ccgccgctcc?caggaagtgc?aggcctgtgc 1020

ccaggagcgc?caggcccagg?ccatggaggg?cacacaggct?gccctgagcg?tcctcatcca 1080

ggtcctccgg?cctatgatca?aagatttccg?ccgctacctg?cagagcaaca?cagctaaccc 1140

ggcccccgcc?tctgaccctg?ggcaggtggc?ccagaatggg?cagccagaca?gcatcatcca 1200

gtgagggcag?gggtcaggcc?agccttctgc?catgatggga?tgaaaactcc?atggacttac 1260

tcaccatcaa?ttaccaaggc?ccttgcatct?ccccagatcg?tttcccaagt?ttggcgattt 1320

gctgtcggtg?cctcagccac?atatgaccaa?tggttacaac?tcagggctcc?agacctcagc 1380

taaaaagaga?agacgctgcc?ctcctgggca?cgaacgttta?gaatgctcaa?ctcctctatt 1440

gtgaccacag?gaaggtggcc?ctgaagatgc?accgaagaca?gctgggaggt?gactgctgta 1500

ctgtcagcct?ctctgtggag?gcattcgtgc?agtgccagct?aaaagggagg?tgaaggggga 1560

tatcggaccc?agcgagggag?ttgctggtag?aaggaaagct?cttctcagtg?tggctggatt 1620

aagagcagcc?tagcagcttg?ggcacctcca?ctctgtgcgg?tctgatggcc?ccagcaaggt 1680

cgctgcaggg?acttcctgag?gacttggttt?ggtttttttc?tggggttgga?aatctgagcc 1740

aatattgtgt?ctgttccatt?tgggtatgaa?gaggaagtct?ggatcactta?aactgactag 1800

ttatttccgg?gtcataattt?taaattaaag?acatatcact?tttttataaa?aaaaaaaaaa 1860

aaaaaa 1866

<210>5

<211>1362

<212>DNA

＜213〉people

<400>1

cattacgctg?ctggctggca?gcggccgggc?cggtcgaggc?tgggccctac?gcactttgcg 60

tagcgagggg?ggttaccaaa?ggcctagtgc?ttggcctcga?gcaagcctgg?cctatcccct 120

gtagggggtg?ggtgaggggc?gaggctgagg?aagaagagaa?ggggaattgg?ggcgcttgag 180

gggattataa?tttctttaaa?aagaggggag?gggagaggcc?atggccgtcc?cagccaagaa 240

aaggaagatg?aacttctcag?agcgggaggt?ggagatcatc?gtggaggagc?tggagctgaa 300

gaagcacctg?ctggtgaacc?acttcaacgc?cggggtaccc?ctggccgcca?agagtgcggc 360

ctggcacggc?atcctgagaa?gggtcaacgc?cgtggccacc?tgccgcagag?agctgcctga 420

ggtcaagaag?aagtggtctg?acctcaagac?cgaggtccgt?cgcaaggttg?cccaggtccg 480

ggccgccgtg?gagggtggtg?aggcgccggg?gcccactgag?gaggacggag?ctggggggcc 540

tgggacaggc?ggtggcagtg?gcggcggtgg?cccagctgta?gccccagtgc?tgctgacccc 600

catgcaacaa?cgtatctgca?acctgctggg?cgaggccacc?atcatcagcc?tgcccagcac 660

cacagagatc?caccctgtgg?ccctcggacc?ctcggccacc?gcagccgcag?ccacggtcac 720

cctgacacag?atccccacag?agaccaccta?tcacacgctg?gaggagggcg?tggtggagta 780

ctgcacggct?gaggcgcccc?cacctctgcc?accagagacc?cctgtggaca?tgatggccca 840

gcatgcagac?acgtcggtca?agccgcaagc?gctcaagagc?cgcattgctc?tcaactccgc 900

caagctgata?caggagcagc?gggtcaccaa?cctgcatgtg?aaggagatcg?cacagcacct 960

ggaacagcag?aacgacctac?tgcagatgat?ccgccgctcc?caggaagtgc?aggcctgtgc 1020

ccaggagcgc?caggcccagg?ccatggaggg?cacacaggct?gccctgagcg?tcctcatcca 1080

ggtcctccgg?cctatgatca?aagatttccg?ccgctacctg?cagagcaaca?cagctaaccc 1140

ggcccccgcc?tctgaccctg?ggcaggtggc?ccagaatggg?cagccagaca?gcatcatcca 1200

gtgagggcag?gggtcaggcc?agccttctgc?catgatggga?tgaaaactcc?atggacttgt 1260

aagtgggtcc?tgtgattggc?cttggcctta?gaccggccac?gtgcacagct?ccctctttaa 1320

taaacgctta?ggggttgcac?tgttaaaaaa?aaaaaaaaaa?aa 1362

Claims (11)

1, the polypeptide of its aminoacid sequence shown in sequence in the sequence table 2, or its conservative property variation polypeptide, or its active fragments.

2, the gene of the polypeptide of coding claim 1.

3, the gene of claim 2 is characterised in that to have the nucleotide sequence shown in the sequence 1 (NFIF1) in the sequence table, or its fragment, or the sequence of homology more than 95%.

4, the gene or its segmental expression vector that contain claim 2 or 3.

5, the method for the polypeptide of preparation claim 1 comprises gene or its segmental expression vector transformed host cell with claim 4, cultivates, and isolates described polypeptide from culture.

6, a kind of genetically engineered host cell is characterized in that it is a host cell with the described gene transformation of claim 2 or transduction, or the host cell that transforms or transduce with the described carrier of claim 3.

7, the gene of the polypeptide of claim 1 or claim 2 prevents and/or treats purposes in the medicine with human body cell nf NF-κ B diseases associated in preparation, or regulates lymphocytic activation and/or the medicine of propagation and/or apoptosis or the purposes in the reagent in preparation.

8, according to the purposes of claim 7, wherein said disease is selected from inflammation, anaphylactic disease, autoimmune disease or tumour.

9, contain the polypeptide of claim 1 and the pharmaceutical composition of pharmaceutically acceptable carrier.

10, a kind of can with the antibody of the polypeptide specific combination of claim 1.

11, the external detection method of a kind of diseases associated with inflammation or tumour is characterised in that the antibody test that utilizes claim 10 existence or the level from the polypeptide in host's sample.

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