CN1600789A - Promotive factor for activating NF-kappa B and usage - Google Patents

Abstract

In the invention, a human's idiogene promoting activity of NF-kappa B (nuclear factor-kappa B) is found. It is confirmed by experiment that said gene and coded protein are adjusting factor promoting activity of NF-kappa B, which can be screened to provide design target and treating sign of inflammation for medicine intervening NF-kappa B activity and used for preparing medicine treating diseases related to NF-kappa B activity imbalance.

Description

The NF-kappaB activatory promotes factor and uses thereof

Technical field:

The present invention relates to promote transcription factor NF-kappaB (nuclear factor-kappaB) activatory nf, be specifically related to a kind of promotion NF-kappaB (nuclear factor-kappaB) activatory people's specific gene, the protein sequence of this genes encoding and they in the lack of proper care purposes of aspects such as medicine of relative disease of preparation control and NF-kappaB activation.

Background technology:

NF-kappaB (nuclear factor-kappaB) is a kind of human body that is present in, can be in all kinds cell transcription factor of wide expression, usually combine with supressor IkappaBs (inhibit kappaB), be present in the tenuigenin with inactive form.NF-kappaB regulates a large amount of gene transcription relevant with the cell emergency rating, particularly relate to the gene transcription of immunity and inflammatory reaction, comprise cytokine (somatomedin), acceptor, adhesion molecule, acute phase protein, virogene, transcription factor and regulatory factor etc.

NF-kappaB plays a significant role in immune response and inflammatory reaction.Human many illnesss are lacked of proper care directly related (Barnes et al., 1997) with the NF-kappaB activatory, as cancer, nerve degenerative diseases, telangiectasis dystaxia, rheumatoid arthritis, asthma, enteritis and other inflammation in a large number; NF-kappaB plays a significant role in epithelial cell and the lymphoid organ structural development process at some hematopoietic cell simultaneously.Recent studies have shown that the NF-kappaB family member has also participated in the formation of nerve synapse and the migration of tumour (Karin and Ben-Neriah, 2000).

Therefore the key factor in the NF-kappaB activated channel all can be used as the target of medicinal design screening, and the research of the active factor of adjusting NF-kappaB provides new approach for the prevention and the treatment of immunity and inflammation.Effective inhibition that for example the most effective anti-asthmatic medicament adrenocortical hormone is NF-kappaB vigor in the lymphocyte (Auphan et al., 1995); The research and development of USA I MUNEX company can be blocked TNF and induce NF-kappaB activated medicine Enbrel to be used for the treatment of rheumatoid arthritis.

Summary of the invention:

The purpose of this invention is to provide a kind of promotion NF-kappaB (nuclear factor-kappaB) activatory cDNA sequence, and the albumen of this genes encoding, for the activation of intervening NF-kappaB provides the medicinal design target.

The present invention utilizes yeast two-hybrid screening to obtain to promote the new regulatory factor of NF-kappaB activatory.

The present invention has found to have the cDNA of sequence shown in the SEQ ID NO:1, called after NKAP (NuclearNF-kappaB Activating Protein).Experimental result proves that the NKAP albumen of NKAP gene and coding thereof (having aminoacid sequence shown in the SEQ ID NO:2) is to promote NF-kappaB (nuclear factor-kappaB) activatory regulatory factor.

Above-mentioned NKAP gene and albumen be with the known kinases RIP that in TNF-R1 inductive NF-kappaB reactivation process, plays a significant role as bait, obtain with the interactional with it albumen of yeast two-hybrid method screening.

Sequential analysis shows, NKAP and known do not have the obvious sequence homology and between each species sequence conservative.Overexpression NKAP can dose-dependently activatable NF-kappaB in 293 cells.The sense-rna experiment is proof further, and the decline of NKAP expression amount can suppress the activation of NF-kappaB, and makes TNF and IL-1 induce NF-kappaB activated ability obviously to reduce.The experimental result of immunofluorescence dyeing shows that NKAP is positioned in the nucleus.Above experimental result proves that NKAP is that TNF and IL-1 activate a kind of new regulatory factor in the NF-kappaB signal pathway.

Because can be used as the activity that the potential protein drug is directly regulated NF-kappaB to the active albumen that plays regulating effect of NF-kappaB, perhaps as the target of medicinal design, the activation of the active indirect regulation NF-kappaB of medicine by regulating it.

NKAP gene provided by the invention and albumen can be regulated and control the activation of NF-kappaB, and therefore the diagnostic flag of design target and inflammation can be provided for the molecule of intervening the drug screening of NF-kappaB activatory.Behind various pharmaceutically-acceptable excipients, pharmaceutical adjuvant compatibility, NKAP gene and albumen full-length molecule or fragment directly can be made various types of medicines as protein drug, increase the activity of NF-kappaB; Perhaps design suppresses medicine at NKAP, by suppressing the activity that NKAP reduces NF-kappaB.Be used to prevent or treatment and the relevant disease of NF-kappaB activatory imbalance.

Description of drawings:

Fig. 1 is NKAP to NF-kappaB and the NKAP fluorescence report gene test comparing result to the effect of transcription factor AP-1.Can find out among the figure that NKAP can activate NF-kappaB, and the activity of AP-1 is not influenced.

Fig. 2 is the fluorescence report genetic test result of NKAP sense-rna stable expression cell line and control cells system.Show among the figure that the decline of NKAP expression amount can suppress the activation of NF-kappaB.

Embodiment:

One. material

Human embryo kidney (HEK) 293 cells are available from ATCC;

Cell cultures DMEM, foetal calf serum, 0.05% pancreatin solution, Sodium.alpha.-ketopropionate solution, green grass or young crops, chain enzyme solution are available from GIBCO and Hyclone;

Yeast culture uses Peptone available from Difco, addition of C SM-Trp-, and CSM-Trp-Leu-, CSM-His-Leu-Trp-is available from Q-BIO Gene; Salmon sperm DNA is available from Roche;

Mammalian cell expression vector pRK-HA is made up by this laboratory;

NF-kappaB fluorescence report plasmid is so kind as to give by Dr.Gary Johnson (University of Colorado Health ScienceCenter);

PRL-SV40 Renilla fluorescence report plasmid is purchased in Promega;

Yeast two-hybrid " bait " carrier pGBT ₉Available from CLONTECH; Human B cell cDNA library is available from ATCC;

Coli expression carrier PET-15b, e. coli bl21 (DE3) bacterial strain and nickel ion column purification test kit are available from Novagen;

The EST clone BG391661 that contains NKAP part encoding sequence is used to make up the carrier pIRESneo3 of NKAP sense-rna available from CLONTECH available from ATCC;

The required restriction enzyme of vector construction is available from Promega; DNA reclaims test kit Geneclean III Kit available from Q-BIO Gene;

Determined dna sequence is given birth to worker bio-engineering corporation by Chinese Shanghai and is finished; The PCR primer is given birth to worker bio-engineering corporation by Chinese Shanghai and is synthesized;

Two fluorescence report gene detecting kits are available from Promega;

The used magnetic bead Protein of co-immunoprecipitation G-Sepharose is available from Amersham Biosciences;

Western blot usefulness nitrocellulose filter Hybond ECL and HRP link coupled sheep anti-mouse igg, sheep anti mouse rabbit igg are available from Amersham pharmacia biotech.; The chemical luminous substrate test kit is available from PIERCE;

The people TNF of reorganization, IL-1 is available from R﹠amp; D Systems Inc.;

The HA monoclonal antibody is available from Sigma;

The proteic polyclonal antibody of the anti-people NKAP of rabbit is by the epi chamber preparation of heredity institute of the Chinese Academy of Sciences;

TXRD link coupled sheep anti-mouse igg, FITC link coupled goat anti-rabbit igg, DAPI is available from Southern BiotechnologyAssociates Inc.; Anti-cancellation mountant Gel/mountTM is available from Biomeda Corp.;

CaCl2, PEG4000, LiAc, DMSO is available from Sigma; CsCl is available from Fisher Biotech.; β-gal is available from Promega; Other chemical reagent is available from chemical reagent shop, Beijing.

Two. method:

1. plasmid construction and DNA purifying

PCR obtains the RIP full-length molecule from 293 cell cdna libraries, is building up to yeast two-hybrid usefulness, the carrier pGBT of encoding transcription factor Gal4 binding domains ₉, obtain the plasmid that in yeast cell, to express: pGBT ₉-RIP;

GeneBank retrieval and EST (expression sequece tag) sequence assembly obtain the complete encoding sequence of NKAP molecule, design 5 ' end and 3 ' end primer respectively according to this sequence, from the B cell cdna library, be cloned into the NKAP full-length molecule by PCR, insert respectively after SalI/NotI or XhoI/BamHI enzyme are cut among mammalian cell expression vector pRK-HA and the coli expression carrier PET-15b, obtain mammalian cell expression plasmid pRK-HA-NKAP and colibacillus expression plasmid PET-15b-NKAP.

The mammalian cell expression plasmid that makes up is all with reference to " molecular cloning " (J. Sa nurse Brooker etc., 1999) a large amount of extraction methods of plasmid, through CsCl density gradient ultracentrifugation purifying.

2. yeast two-hybrid screening

Reference literature method (Chen et al., 2002).

3. cell cultures and cell transfecting

Human embryo kidney (HEK) 293 cell cultures 37,5% CO2gas incubator in the high sugared DMEM substratum that contains 10% foetal calf serum is cultivated.

293 cells (1 * 10 ⁵) be inoculated in 12 orifice plates, carry out the transfection of calcium phosphate mediation after 18 hours with reference to " molecular cloning experiment guide " (J. Sa nurse Brooker etc., 1999).

4. fluorescence report gene test

293 cells (～1 * 105) are inoculated in 12 orifice plates, carry out the transfection of calcium phosphate mediation after 18 hours.Each sample is established three parallel laboratory test groups, adds empty carrier in right amount and equates with the DNA total amount that keeps every group of transfection.Every hole adds 0.1 μ gNF-kappaB fluorescence report plasmid; For the balance transfection efficiency, add 0.1 μ g pRL-SV40-Renilla fluorescence report plasmid simultaneously.Detected in 14-18 hour after the transfection, with the fluorescence detection reagent kit detection NF-kappaB fluorescence report gene of Promega company and the activity of pRL-SV40-Renilla fluorescence report gene, method is respectively with reference to the description of product.

5. co-immunoprecipitation and Western blot

293 cells (～2 * 106) are inoculated in the 10cm culture dish, use 1ml cell pyrolysis liquid (20mM Tris after 20 hours, 150mM NaCl, 1%Triton, 1mM EDTA, 10 μ g/ml aprotinin, 10 μ g/ml leupeptin, 1mMphenylmethylsulfonyl fluoride pH7.5) lysing cell, antiserum(antisera) or the preimmune serum of adding 2 μ gNKAP in the 0.5ml cell pyrolysis liquid, add 30 μ l protein A link coupled Sepherose again, 4 rotation mixed 4 hours.After the cleaved liquid of magnetic bead cleans three times, add 2 * SDS polyacrylamide gel electrophoresis sample buffer, 20 μ l, carry out the 10%SDS polyacrylamide gel electrophoresis behind the boiling water bath, electrophoresis and commentaries on classics membrane operations are with reference to " molecular cloning experiment guide " (J. Sa nurse Brooker etc., 1999).Nitrocellulose filter confining liquid 5% skimmed milk-TBST (150mM NaCl, 25mM Tris, 0.05%Tween-20, pH7.5) room temperature incubation 20mins, the antiserum(antisera) or the preimmune serum that add 0.5 μ g/mlNKAP, room temperature incubation 2hrs adds HRP link coupled sheep anti-mouse igg or sheep anti mouse rabbit igg (dilution in 1: 1000) after TBST washs three times, room temperature incubation 1hrs detects with the fluorogenic substrate detection kit after TBST washs three times.

6. Recombinant Protein Expression and purifying

The PET-15b-NKAP plasmid is transformed into e. coli bl21-DE3, amplification cultivation is to 500ml, induced 5 hours with 1mM IPTG, with reference to the purifying of previous method (Li Lianyun etc., 2001) and Novagen nickel ion column purification test kit explanation carrying out NKAP recombinant protein.

7. the screening of the structure of antisense RNA expression vector and stable expression cell line

Expression plasmid (carrier is pCMV-sport6) among the EST clone BG391661 contains the about 700bp encoding sequence of NKAP molecule 5 ' end, and the 145bp non-coding sequence before the NKAP promotor.To insert in the corresponding site of carrier pIRESneo3 behind this plasmid usefulness NheI and the EcoRI double digestion, because NheI and the position opposite of EcoRI on these two carrier multiple clone site, we have obtained the antisense RNA expression vector pIRESneo3-AS-NKAP of NKAP.

PIRESneo3-AS-NKAP is transfected in 293 cells with the calcium phosphate mediated method, changes nutrient solution after cultivating 24hr, and add G418 (1 μ g/ml) and screen, two week back picking mono-clonals clone cultivation.

8. immunofluorescence dyeing

293 cell cultures on slide glass, are carried out transfection and cultivated 20hrs with expression vector or corresponding empty carrier; With the ice-cold methyl alcohol of cell and acetone fixing 10mins respectively.After the PBS rinsing, soak 3mins with 0.1%Trition X-100 PBS, use the monoclonal antibody (Sigma) of HA respectively, rabbit anti-people NKAP protein antiserum and contrast mouse IgG or rabbit preimmune serum be in 37 incubation 2hrs, after the PBS rinsing, more respectively with TXRD link coupled sheep anti-mouse igg, FITC link coupled goat anti-rabbit igg covers light incubation 1hr in 37, after the PBS rinsing, add DAPI room temperature effect 5mins, the Gel/Mount mounting.Lecai DMR/XA (DMRB) fluorescent microscope 100 * object lens are observed.

The clone of three .NKAP and Function Identification

1.NKAP the clone of cDNA

We are bait with the RIP full-length molecule, and yeast two-hybrid screening is carried out in human B cell cDNA library.We have screened 5 * 10 altogether ⁶Individual yeast clone, beta galactose coloration result show 26 yeast clones and are positive.We so respectively the B cell cdna library plasmid among these 26 clones has been carried out sequencing, the gene of finding the insertion fragment of 9 clone library plasmids wherein and encoding transcription factor GAL4 activation structure territory is not in same single open reading frame; In 17 correct clones of single open reading frame, all the encode partial sequence of FADD of two clones' insertion fragment, FADD be a kind of reported have death domain and can with RIP bonded joint albumen; The unknown function albumen that has 3 clones' insertion fragment to encode different respectively in addition, one of them our called after NKAP.

We have carried out EST (expressed sequece tag) sequence retrieval and splicing to the NKAP molecular moiety encoding sequence that order-checking obtains, obtained the full length sequence (shown in the SEQ ID NO:1) of NKAP, (initiator codon is positioned at 256-258, ends codon and is positioned at 1501-1503).5 ' end in NKAP molecule initiator codon has the terminator codon (202-204) that is positioned at same single open reading frame, and 3 ' end has poly (A).We have designed 5 ' primer and 3 ' primer according to this sequence, have obtained the NKAP full-length cDNA by PCR from the B cell cdna library.The full-length cDNA of NKAP is 1248bp, 416 amino acid (shown in the SEQ ID NO:2) of encoding, and sequential analysis shows that NKAP is the protein molecular of an alkalescence, and very conservative during evolution.The amino acid identity of people and mouse NKAP is up to 90%.

2.NKAP protein expression

We with the NKAP protein Preparation of purifying the proteic antiserum(antisera) of the anti-people NKAP of rabbit.Western blot result show the antiserum(antisera) of preparation can specific recognition 293 cells in the NKAP albumen of endogenous expression.Molecular weight is about 52kD, and is consistent with the NKAP molecular weight of albumen of purifying, is slightly less than the HA-NKAP of overexpression, illustrates that we have been cloned into the full-length molecule of NKAP.

3.NKAP cellular localization

We have studied the distribution of NKAP in 293 cells with immunofluorescence dyeing.293 cells of overexpression pRK-HA-NKAP dye with the monoclonal antibody of anti-HA, and the result shows that the NKAP of overexpression is gathered in the nuclear.With 293 cells of NKAP antiserum(antisera) mark untransfected, the NKAP albumen that background is expressed in the same showed cell of result is positioned in the nuclear.Above presentation of results NKAP is a nf.

4.NKAP Function Identification

(1) NKAP promotes the activation of NF-kappaB

293 transit cells are gone into 0.1 μ g NF-kappaB or 0.1 μ g AP-1 fluorescence report plasmid, and 0.1 μ g pRL-SV40 Renilla fluorescence report plasmid is with the balance transfection efficiency.Change the get the bid NKAP expression plasmid of fluence of figure simultaneously over to.The activity of about 16 hours detection fluorescence report genes after the transfection.Each sample is provided with three parallel laboratory tests, and Fig. 1 result is the average of three independent experiment gained results.

NF-kappaB fluorescence report gene test result shows, NKAP can dose-dependently activatable NF-kappaB (Fig. 1) in 293 cells during overexpression.Whether have specificity in order to detect this activation, we have detected the effect of NKAP to another kind of transcription factor AP-1 simultaneously, and the result shows that NKAP does not significantly activate or restraining effect (Fig. 1) AP-1.Illustrate that NKAP is special relatively to the activation of NF-kappaB.

(2) NKAP suppresses the activation of TNF and IL-1 inductive NF-kappaB

In order to detect the function of NKAP under physiological condition, we have detected the activation that can disappearance that NKAP expresses influence NF and IL-1 inductive NF-kappaB.We have made up the NKAP antisense RNA expression plasmid and it have been changed in 293 cells, obtain the cell strain AS-NKAP-293 of stably express NKAP sense-rna through screening, and Western blot result shows that the sense-rna of NKAP can suppress the proteic expression of NKAP.

TNF and IL-1 induce NF-kappaB activatory ability drop in the AS-NKAP-293 cell.Change RIP expression plasmid or control plasmid and 0.5 μ g NF-kappaB fluorescence report plasmid in 293 cells and the AS-NKAP-293 cell respectively over to, change 0.5 μ g pRL-SV40 Renilla fluorescence report plasmid simultaneously over to the balance transfection efficiency.Handle cell with TNF (20ng/ml) and IL-1 (20ng/ml) in 14 hours after the transfection, detect the activity of fluorescence report gene after 6 hours.

Fluorescence report gene experimental result shows, compares active obviously reduce (Fig. 2) of the NF-kappaB of background level in the AS-NKAP-293 cell with 293 cells that change empty carrier over to.And TNF and IL-1 induce the activated ability of NF-kappaB to be subjected to obvious inhibition in the AS-NKAP-293 cell.

Above presentation of results NKAP is a new NF-kappaB activatory regulatory factor.

Sequence table

＜110〉Peking University

＜120〉the NF-kappaB activatory promotes factor and uses thereof

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

1 5 10 15

Ser?Gly?Gly?Arg?Arg?Arg?Ser?Ser?Ser?Lys?Ser?Pro?Lys?Pro?Ser?Lys

20 25 30

Ser?Ala?Arg?Ser?Pro?Arg?Gly?Arg?Arg?Ser?Arg?Ser?His?Ser?Cys?Ser

35 40 45

Arg?Ser?Gly?Asp?Arg?Asn?Gly?Leu?Thr?His?Gln?Leu?Gly?Gly?Leu?Ser

50 55 60

Gln?Gly?Ser?Arg?Asn?Gln?Ser?Tyr?Arg?Ser?Arg?Ser?Arg?Ser?Arg?Ser

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Arg?Glu?Arg?Pro?Ser?Ala?Pro?Arg?Gly?Ile?Pro?Phe?Ala?Ser?Ala?Ser

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Ser?Ser?Val?Tyr?Tyr?Gly?Ser?Tyr?Ser?Arg?Pro?Tyr?Gly?Ser?Asp?Lys

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Pro?Trp?Pro?Ser?Leu?Leu?Asp?Lys?Glu?Arg?Glu?Glu?Ser?Leu?Arg?Gln

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Lys?Arg?Leu?Ser?Glu?Arg?Glu?Arg?Ile?Gly?Glu?Leu?Gly?Ala?Pro?Glu

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Val?Trp?Gly?Leu?Ser?Pro?Lys?Asn?Pro?Glu?Pro?Asp?Ser?Asp?Glu?His

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Thr?Pro?Val?Glu?Asp?Glu?Glu?Pro?Lys?Lys?Ser?Thr?Thr?Ser?Ala?Ser

165 170 175

Thr?Ser?Glu?Glu?Glu?Lys?Lys?Lys?Lys?Ser?Ser?Arg?Ser?Lys?Glu?Arg

180 185 190

Ser?Lys?Lys?Arg?Arg?Lys?Lys?Lys?Ser?Ser?Lys?Arg?Lys?His?Lys?Lys

195 200 205

Tyr?Ser?Glu?Asp?Ser?Asp?Ser?Asp?Ser?Asp?Ser?Glu?Thr?Asp?Ser?Ser

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Asp?Glu?Asp?Asn?Lys?Arg?Arg?Ala?Lys?Lys?Ala?Lys?Lys?Lys?Glu?Lys

225 230 235 240

Lys?Lys?Lys?His?Arg?Ser?Lys?Lys?Tyr?Lys?Lys?Lys?Arg?Ser?Lys?Lys

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Ser?Arg?Lys?Glu?Ser?Ser?Asp?Ser?Ser?Ser?Lys?Glu?Ser?Gln?Glu?Glu

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Phe?Leu?Glu?Asn?Pro?Trp?Lys?Asp?Arg?Thr?Lys?Ala?Glu?Glu?Pro?Ser

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Lys?Pro?Leu?Asn?Tyr?Gly?His?Ala?Leu?Leu?Pro?Gly?Glu?Gly?Ala?Ala

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Met?Ala?Glu?Tyr?Val?Lys?Ala?Gly?Lys?Arg?Ile?Pro?Arg?Arg?Gly?Glu

325 330 335

Ile?Gly?Leu?Thr?Ser?Glu?Glu?Ile?Ala?Ser?Phe?Glu?Cys?Ser?Gly?Tyr

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

355 360 365

Lys?Glu?Asn?Gln?Ile?Tyr?Ser?Ala?Asp?Glu?Lys?Arg?Ala?Leu?Ala?Ser

370 375 380

Phe?Asn?Gln?Glu?Glu?Arg?Arg?Lys?Arg?Glu?Asn?Lys?Ile?Leu?Ala?Ser

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Phe?Arg?Glu?Met?Val?Tyr?Arg?Lys?Thr?Lys?Gly?Lys?Asp?Asp?Lys

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Claims (10)

1. the DNA that has sequence shown in the SEQ ID NO:1.

2. the described DNA of claim 1 regulates NF-kappaB activatory purposes.

3. with the purposes of the described DNA of claim 1 at the medicine for preparing prevention or the treatment disease relevant with the imbalance of NF-kappaB activatory.

4. the described DNA of claim 1 intervenes the purposes of NF-kappaB activatory medicine in screening.

5. with the purposes of the described DNA of claim 1 as the diagnostic flag of inflammation.

6. the polypeptide that has aminoacid sequence shown in the SEQ ID NO:2.

7. the described aminoacid sequence of claim 6 is regulated NF-kappaB activatory purposes.

8. with the purposes of the described aminoacid sequence of claim 6 at the medicine for preparing prevention or the treatment disease relevant with the imbalance of NF-kappaB activatory.

9. the described aminoacid sequence of claim 6 is intervened the purposes of NF-kappaB activatory medicine in screening.

10. with the purposes of the described aminoacid sequence of claim 6 as the diagnostic flag of inflammation.

Images