CN1948340A

CN1948340A - Protein of interreaction with karyomitosis primary activator protein kinase p38 and its coding gene and application

Info

Publication number: CN1948340A
Application number: CN 200510108340
Authority: CN
Inventors: 顾军; 傅璟; 杨自强
Original assignee: Peking University
Current assignee: Peking University
Priority date: 2005-10-12
Filing date: 2005-10-12
Publication date: 2007-04-18
Anticipated expiration: 2025-10-12
Also published as: CN100455595C

Abstract

This invention discloses a protein interacting with mitogen activated protein kinase p38 and its coding gene and application. The purpose is to provide a protein interacting with mitogen activated protein kinase p38 and its coding gene, and the application in preparing therapeutic drug for the disease associated with mitogen activated protein kinase p38 signal iter. The protein is one of the following proteins with amino acid residue series. 1) SEQ ID NO1 in series table. 2) One to ten amino acid residue of SEQ ID NO:1 is displaced, absence or added to protein that interacting with mitogen activated protein kinase p38, and having regulation effect for itself.

Description

With the interactional albumen of mitogen activated protein kinase p 38 and encoding gene and application

Technical field

The present invention relates to albumen and encoding gene thereof and application, particularly relate to one with the interactional albumen of mitogen activated protein kinase p 38 and encoding gene thereof and its application in the curative drug of preparation and mitogen activated protein kinase p 38 signal path relative disease.

Background technology

Studies show that cell signal unusual and human diseases closely related (Mora-Garcia P, Sakamoto KM.Cell signaling defects and human disease.Mol Genet Metab 1999; 66 (3): 143-71.).The protein kinase that plays a crucial role in the signal transduction process is one of present most important disease treatment target (Cohen P.Protein kinases-the major drug targets of the twenty-firstcentury.Nature Reviews Drug Discovery.2002; 1 (4): 309-15.).Eukaryotic have a mitogen activated protein kinase (Mitogen-Activated Protein Kinase, MAPK) superfamily comprises ERK1/2, JNK1/2/3, p38 α/β/gamma/delta, ERK4/5 and ERK5/BMK1, they are responsible for various born of the same parents' external stimulus signals are passed to karyon, thereby regulate gene expression (Johnson G L and Lapadat R.Mitogen-ActivatedProtein Kinase Pathways Mediated by ERK, JNK, and p38 Protein Kinases.Science.2002; 298 (5600): 1911-2, Enslen H, Davi s RJ.Regulation of MAP kinases bydocking domains.Biol Cell.2001; 93 (1-2): 5-14.).Wherein, mitogen activated protein kinase p 38 (p38 NAPK) participates in inflammation, emergent, growth, cell growth and conducts (Herlaar E, Brown Z.p38MAPK signalling cascades in inflammatory disease.Mol Med Today.1999 with signal in physiology, the pathologic processes such as apoptosis, cell cycle regulating, ischemia damage and myocardial hypertrophy; 5 (10): 439-47, Ambrosino C, Nebreda AR.Cell cycle regulation by p38 MAP kinases.Biol Cell 2001; 93 (1-2): 47-51, Takeda K, Ichijo H.Neuronal p38 signalling:an emerging regulator of cell fate and function in the nervous system.GenesCells 2002; 7 (11): 1099-111, Bulavin DV, Higashimoto Y, Popoff IJ, et al.Initiation of a G2/M checkpoint after ultraviolet radiation requires p38kinase.Nature.2001; 411 (6833): 102-7).Suppress the p38 kinase activity, can effectively block the pathological signals transduction of its mediation, thereby alleviate even eliminate its pathological phenomenon (Lee JC, Kumar S, GriswoldDE.et al.Inhibition of p38 MAP kinase as a therapeutic strategy.Immunopharmacology.2000; 47 (2-3): 185-201, Lee JC, Kassis S, Kumar S, et al.p38 mitogen-activated protein kinase inhibitors--mechanisms and therapeuticpotentials.Pharmacol Ther.1999; 82 (2-3): 389-97).For example: the kinase whose inhibitor of p38 can alleviate ischemic brain injury (Barone FC, Irving EA, Ray AM, et al.SB 239063, asecond-generation p38 inhibitor.reduces brain injury and neurologicaldeficits in cerebral focal ischemia.J Pharmacol Exp Ther 2001; 296 (2): 312-21), formation (the Song C that stops the emergent fiber of beta amyloid inducing peptide Actin muscle, Perides G, Wang D, etal.beta-Amyloid peptide induces formation of actin stress fibers through p38mitogen-activated protein kinase.J Neurochem.2002; 83 (4): 828-36.).Therefore, the p38 kinases is drug targets (Lee JC, Kumar S, Griswold DE, the et al.Inhibition of p38 MAP kinase as a therapeutic strategy.Immunopharmacology.2000 that people comparatively pay close attention to; 47 (2-3): 185-201, Lee JC, Kassis S, Kumar S, et al.p38 mitogen-activatedprotein kinase inhibitors--mechanisms and therapeutic potentials.PharmacolTher.1999; 82 (2-3): 389-97, Adams JL, Badger AM, Kumar S, Lee JC.p38 MAPkinase:molecular target for the inhibition of pro-inflammatory cytokines.Prog Med Chem 2001; ).The conduction of interaction regulating cell signal (Souroujon MC, Mochly-Rosen D.Peptide modulators of protein-protein interactions inintracellular signaling.Nat Biotechnol.1999 between the protein; 16 (12): 919-24).Interaction between the interferencing protein, thereby suppress the transduction of pathological signals, become a kind of important disease treatment strategy (Huang Z.Structural chemistry and therapeutic intervention ofprotein-protein interactions in immune response, human immunodeficiency virusentry, and apoptosis.Pharmacol Ther.2000; 86 (3): 20l-15.).

The transcription product of normal gene can not form double-stranded RNA because of no complementary sequence in the organism, so has occurred double-stranded RNA in the organism and will excite RNAi (RNA interference, RNA interference) mechanism.The RNA perturbation technique is the biotechnology of specificity degraded target gene under the mediation of double-stranded RNA (ds RNA) molecule, can make the genetic expression silence, reaches the antagonism target gene and realizes biological (gene) therapeutic purpose.Double-stranded RNA is degraded into the siRNA fragment that length is 21-25nt (siRNA, small interfering RNA), and these small segments are mediation and its homologous single stranded RNA degraded further.

Summary of the invention

The purpose of this invention is to provide one and interactional albumen of mitogen activated protein kinase p 38 and encoding gene thereof.

Provided by the present invention and the interactional albumen of mitogen activated protein kinase p 38, name is called NP60, derives from Genus Homo people (Homo sapiens), is the protein with one of following amino acid residue sequences:

1) the SEQ ID № in the sequence table: 1;

2) with SEQ ID № in the sequence table: 1 amino acid residue sequence interacts through replacement, disappearance or the interpolation of one to ten amino-acid residue and with the mitogen activated protein kinase p 38, and it is had the protein of regulating and controlling effect.

SEQ ID № in the sequence table: 1 is made up of 553 amino-acid residues,

The gene (NP60) of coding and the interactional albumen NP60 of mitogen activated protein kinase p 38 is one of following nucleotide sequence:

1) SEQ ID № in the sequence table: 2 dna sequence dna;

2) SEQ ID № in the code sequence tabulation: 1 dna sequence dna;

3) under the rigorous condition of height can with SEQ ID № in the sequence table: the nucleotide sequence of the 2 dna sequence dnas hybridization that limit.

The rigorous condition of described height be 0.1 * SSPE (or 0.1 * SSC), in the solution of 0.1%SDS, hybridization and wash film under 65 ℃ of conditions.

SEQ ID № in the sequence table: 2 by 1662 based compositions, and its encoding sequence is that coding has SEQ ID № in the sequence table: the protein of 1 amino acid residue sequence from 5 ' end the 1st to 1662 bit base.

Contain expression carrier of the present invention, transgenic cell line and host bacterium and all belong to protection scope of the present invention.

Arbitrary segmental primer is to also within protection scope of the present invention among the amplification NP60.

The encoding gene of the RNA interfering of the gene NP60 of code book invention albumen NP60 is imported the host, make NP60 silence in the host cell, and then can suppress the activity of mitogen activated protein kinase p 38.Therefore encoding gene that can the NP60 RNA interfering is prepared into medicine as activeconstituents, is used for the treatment of and the relevant disease of mitogen activated protein kinase p 38 signal path, damages and myocardial hypertrophy etc. as inflammation, ischemia.

The encoding gene of described NP60 RNA interfering can import the host by the RNAi expression vector that contains described NP60 RNA interfering encoding gene; The carrier that sets out that is used for making up described RNAi expression vector can be any one can be at the carrier for expression of eukaryon of human body expression alien gene, as plasmid vectors such as pSuper, pSilencer 1.0-U6, pH1-siRNA siRNA, mU6pro or the carrier that on the virus vector basis, makes up, wherein, mu6pro is the carrier that preferably sets out.

Be the carrier that sets out with mU6pro, the RNAi expression vector of structure is NP60-SiRNA.

The invention provides one and interactional albumen NP60 of mitogen activated protein kinase p 38 and encoding gene thereof.Experimental results show that, this albumen can interact with the mitogen activated protein kinase p 38, regulate the activity of mitogen activated protein kinase p 38, thereby mediation mitogen activated protein kinase p 38 is pressed various kinds of cell emergency reactions such as change to inflammation, Premeabilisation of cells.Therefore, albumen NP60 of the present invention can be used as a new drug target molecule, by intervening the activity of mitogen activated protein kinase p 38 in the organism, treat the disease relevant thereby reach, as inflammation, ischemia damage and myocardial hypertrophy etc. with this signal path.The present invention has bigger practical significance and wide application prospect in medical science and field of biological pharmacy.

The present invention will be further described below in conjunction with specific embodiment.

Description of drawings

Fig. 1 detects NP60 and structure domain mutant and mitogen activated protein kinase p 38 results of interaction for the ELISA method

Fig. 2 detects the result of the upstream kinases of NP60 mediation to mitogen activated protein kinase p 38 activation for Western blotting

Fig. 3 detects the result of NP60 mediated cell osmotic pressure change to the active regulating effect of mitogen activated protein kinase p 38 for Western blotting

Embodiment

Method therefor is ordinary method if no special instructions among the following embodiment, and it is synthetic that the primer and probe are given birth to the worker by Shanghai.

The clone of embodiment 1, NP60

Adopt the method for yeast two-hybrid, with the mitogen activated protein kinase p 38 as bait protein, cDNA library (Ge, B., Gram in the stomach intestinal tissue in people source, H., Di Padova, F., Huang, B., New, L., Ulevitch, R.J., Luo, Y.and Han, J. (2002) .MAPKK-independent activation ofp38alpha mediated by TAB1-dependent autophosphorylation of p38alpha.Science295, screen 1291-4), concrete grammar is as follows:

Yeast two-hybrid is carried out in the cDNA library that personnel selection source stomach intestinal tissue obtains, the albumen that screening and p38 are harmonious.The result is 1.5 * 10 ⁷Screening has obtained clone (Ge, B., the Gram of four coding homologous geneses among the individual clone, H., DiPadova, F., Huang, B., New, L., Ulevitch, R.J., Luo, Y.and Han, J. (2002) .MAPKK-independent activation of p38alpha mediated by TAB1-dependentautophosphorylation of p38alpha.Science 295,1291-4).With this unnamed gene is NP60, sequencing result shows that this gene has SEQ ID № in the sequence table: 2 nucleotide sequence, SEQID № in the sequence table: 2 by 1662 based compositions, its encoding sequence is that coding has SEQ ID № in the sequence table: the protein of 1 amino acid residue sequence from 5 ' end the 1st to 1662 bit base.

Embodiment 2, ELISA method detect the interaction of NP60 and mitogen activated protein kinase p 38

One, the prokaryotic expression of NP60 and each structural domain deletion mutant thereof, mitogen activated protein kinase p 38

1, structure contains each structural domain deletion mutant gene of NP60, NP60, mitogen activated protein kinase p 38 Prokaryotic Expression carrier respectively

After the gene NP60 that embodiment 1 is obtained cut with restriction enzyme EcoR I and Xho I enzyme, (WI) connection obtained the prokaryotic expression carrier of NP60, called after GST-NP60-PGEX4T-1 for Promega, Madison with the carrier PGEX4T-1 that cuts through the same enzyme enzyme.The expression vector establishment of each structural domain deletion mutant of NP60: GST-NP60-PGEX4T-1 is a masterplate with NP60 full length sequence expression vector, PCR obtains three structural domain deletion mutants of NP60 sequence, with these three structural domain deletion mutants difference called after: Δ PWWP-NP60 (87-553aa), Δ AT-NP60 (185-553aa), Δ NAD-NP60 (1-353aa).Be cloned into respectively among the carrier PGEX4T-1 with above-mentioned three kinds of NP60 structural domain deletion mutant sequences of same procedure again, obtain the prokaryotic expression carrier of three kinds of NP60 structural domain deletion mutants pcr amplification.Mitogen activated protein kinase p 38 Prokaryotic Expression construction of carrier is referring to document (Jiang, Y., Chen.C, Li, Z.et al., Characterization of the structureand function of a new mitogen-activated protein kinase (p38beta), J.Biol.Chem., 1996,271 (30): 17920-17926.).

2, the prokaryotic expression of NP60 and each structural domain deletion mutant mitogen activated protein kinase p 38 thereof

The prokaryotic expression carrier GST-NP60-PGEX4T-1 of the NP60 that step 1 is made up and the prokaryotic expression carrier and the mitogen activated protein kinase p 38 Prokaryotic Expression carrier of each structural domain deletion mutant are used CaCl respectively ₂Method transformed into escherichia coli BL21 (DE3) bacterial strain through screening, obtains the positive colony of several reorganization bacterium; Several recons are inoculated in respectively in the 100mL liquid LB substratum, under 37 ℃, are cultured to OD _600nmAdd IPTG after=0.6 to final concentration 1mM, in 30 ℃ of abduction deliverings 3 hours.After cultivating end, expression product is carried out 12% SDS-PAGE electrophoresis detection, the result has obtained the albumen that molecular weight is respectively 86KD (GST-NP60 albumen), 77KD (GST-Δ PWWP-NP60 albumen), 66KD (GST-Δ AT-NP60 albumen), 64KD (GST-Δ NAD-NP60 albumen) and 40KD (his-p38 albumen) through expression, conform to expected results, then above-mentioned albumen is carried out purifying respectively.

Two, the ELISA method detects the interaction of NP60 and structural domain deletion mutant and mitogen activated protein kinase p 38

Method with ELISA detects the albumen NP60 purified in the step 1 and the interaction of structural domain deletion mutant and mitogen activated protein kinase p 38 thereof, concrete grammar is: the GST that gets the 1ug purifying, GST-NP60, GST-Δ PWWP-NP60, GST-Δ AT-NP60, GST-Δ NAD-NP60 and GST-ATF ₂(recombinant protein is through the Glutathione of AmershamBiosciences company Sepherose4B affinity column purifying, purification process sees operation instruction for details) wrap quilt respectively in 96 hole enzyme plates, (phosphate buffered saline buffer that contains 1 ‰ Tween20 is pH7.6) in 37 ℃ of sealing 1h to add the PBST that contains 3% skim-milk then.Other gets 0.1ug his-p38 and is dissolved in the PBST that contains 3% skim-milk, 37 ℃ in conjunction with 1h, with PBST liquid washing 3 times, add sheep anti-mouse antibody that mouse-anti-p38 monoclonal antibody and horseradish peroxidase be coupled then successively (all available from Santa Cruz Biotechnology, Santa Cruz, CA) (1: 1000) successively 37 ℃ in conjunction with 1h, add 100ul TMB room temperature reaction 15min after the PBST washing 3 times, add 50ul H then ₂SO ₄Termination reaction detects light absorption value in 450nm.Detected result is (GST, GST-NP60, Δ PWWP-NP60, Δ AT-NP60, Δ NAD-NP60, GST-ATF as shown in Figure 1 ₂The NP60 that represents GST albumen and merge with GST with and three mutant and ATF that structural domain lacks respectively ₂Albumen; "+" represents to participate in this sample this albumen of specified amount respectively, "-" expression does not participate in), the result compares with negative control GST albumen, and GST-NP60, GST-Δ PWWP-NP60, GST-Δ NAD-NP60 all can combine with p38, and GST-Δ AT-NP60 mutant does not then have the activity of combining with p38.Show that NP60 can interact with the mitogen activated protein kinase p 38, and bonded closes key sequence and may be arranged in AT-hook structural domain (sequence table SEQ ID №: 1 hold the 88th to 184 amino acids residue from N).

The upstream kinases of NP60 mediation is to the activation of mitogen activated protein kinase p 38 in embodiment three, detection 293T and the 293GP cell

One, the eukaryotic expression of NP60, mitogen activated protein kinase p 38

1, the Construction of eukaryotic of NP60, mitogen activated protein kinase p 38 gene and upstream kinases MKK6b gene thereof

Reverse transcription product with the 293T cell total rna is a masterplate, under the guiding of forward primer 5 '-CATATGGCGGCTGTGAGTCTG-3 ' and reverse primer 5 '-CTCGAGATGTATGTAGGCTCGGTA-3 ', pcr amplification NP60 gene, the PCR product of 1668bp is cut rear clone through restriction enzyme EcoR I and Xho I enzyme go into the carrier pcDNA6Myc/His A (Invitrogen that cuts through the same enzyme enzyme, Rockyille, MA) in.The construction process of the eukaryon expression plasmid of mitogen activated protein kinase p 38 gene and MKK6b gene is referring to document: (Han J, Lee JD, Jiang Y, Li Z, Feng L, Ulevitch RJ.Characterization of thestructure and function of a novel MAP kinase kinase (MKK6) J Biol Chem.1996Feb 9; 271 (6): 2886-91.).With the carrier for expression of eukaryon of the NP60, mitogen activated protein kinase p 38 gene and the upstream kinases MKK6b gene thereof that make up, called after myc-NP60, flag-p38 and HA-MKK6b respectively.

2, the eukaryotic expression of NP60, mitogen activated protein kinase p 38

The carrier for expression of eukaryon myc-NP60 of NP60, mitogen activated protein kinase p 38 gene and the upstream kinases MKK6b gene thereof that step 1 is made up, flag-p38 and HA-MKK6b use calcium phosphate method method transfecting eukaryotic cells 293T respectively, and instantaneous the mistake expressed.

Two, Western blotting detects the activation of the upstream kinases of NP60 mediation to the mitogen activated protein kinase p 38

The total protein of positive transfectional cell after cultivating of extraction step one, albumen with mitogen activated protein kinase p 38 phosphorylation is antibody (New England BioLabs, Beverley, MA), detect whether phosphorylation of mitogen activated protein kinase p 38 with Western blotting method.(p38a, myc-NP60, MKK6b represented respectively that expression NP60, mitogen activated protein kinase p 38 gene and MKK6b gene were in the 293T cell to detected result as shown in Figure 2, "+,-" implication represent transfection or untransfected respectively, * P-p38a represents the Western blot result that the antibody test with phosphorylation p38 obtains), showed and expressed the phosphorylation level that NP60 can strengthen p38, showed that NP60 can assist the activation of mitogen activated protein kinase p 38.

NP60 is to the active regulating effect of mitogen activated protein kinase p 38 when embodiment four, the change of detection Premeabilisation of cells pressure

One, the silence of the endogenous NP60 of cell

With the 293GP cell is example, and NP60 is to the active regulating effect of mitogen activated protein kinase p 38 when detecting the change of Premeabilisation of cells pressure.At first utilize the RNA perturbation technique, with the endogenous NP60 silence of 293GP cell, concrete grammar is as follows:

1, the structure of the RNAi interference carrier of NP60

The just sequence of interfering the target sequence encoding gene be 5 '-gctgtggatgctgttgaag-3 ' (19bp), antisense sequences be 5 '-cttcaacagcatccacagc-3 ' (19bp), with TTCG is Loop, be connected into interference vector mU6pro (Yu, J.Y., DeRuiter, S.L.and Turner, D.L. (2002) .RNA interference byexpression of short-interfering RNAs and hairpin RNAs in mammalian cells.ProcNatl Acad Sci USA 99,6047-52), with the RNAi interference carrier called after NP60-SiRNA of the NP60 that makes up.

2, the reticent and detection of the endogenous NP60 of cell

The RNAi interference carrier NP60-SiRNA of the NP60 that step 1 is made up is with calcium phosphate method method transfection 293GP cell, with the method for PCR positive transfectional cell is done further detection again, detection method is: extract cell total rna, with the reverse transcription product is masterplate, under the guiding of forward primer 5 '-gagtctagtaccgtgaaggg-3 ' and reverse primer 5 '-gatcccttgcagcacaccac-3 ', PCR detects the NP60 gene, with the actin gene is contrast, the primer of amplification actin gene fragment is 5 '-caccaactgggacgacat3 ' (forward primer) and 5 '-catactcctgcttgctgatc3 ' (reverse primer), and detected result shows the 293GP cell that has obtained the NP60 silence.

NP60 is to the active regulating effect of mitogen activated protein kinase p 38 when two, detecting the change of Premeabilisation of cells pressure

The 293GP cell of the NP60 silence that obtains with 0.3M Sorbitol Powder (sorbitol) stimulation step one 30 minutes.Then, collecting cell, the total protein of extraction cell.Detect whether phosphorylation of mitogen activated protein kinase p 38 with the method identical with embodiment 3, (mu6, NP60-SiRNA represent that respectively transfection has the 293GP cell of mU6pro and NP60-SiRNA interfere plasmid to detected result as shown in Figure 3,0.3M Sorbitol represents to give the stimulation of above-mentioned transfectional cell 0.3M sorbyl alcohol, "+,-" represents transfection or untransfected respectively and stimulates or do not stimulate; * P-p38, * P-JNK1/2, * P-ERK1/2, p38, represent to use phosphorylation p38 respectively, phosphorylation JNK1/2, the antibody of phosphorylation ERK1/2 is (all available from New England BioLabs, Beverley, MA) and the proteic antibody of p38 (Santa Cruz Biotechnology, Santa Cruz, CA) the western blot result of Jian Ceing), show in the 293GP cell of NP60 silence, Sorbitol stimulates the phosphorylation level of the p38 that causes to compare with control group and weakens, the phosphorylation level of two kinds of MAPKs:JNK1/2 and ERK1/2 no change then in addition shows that NP60 optionally regulates the phosphorylation level of the p38 that Sorbitol causes.In the 293GP of endogenous NP60 silence cell, Sorbitol Powder activates the loss of activity of mitogen activated protein kinase p 38, but shows that NP60 mediated cell osmotic pressure changes the active adjusting of mitogen activated protein kinase p 38.

Sequence table

<160>2

<210>1

<211>553

<212>PRT

＜213〉Genus Homo people (Homo sapiens)

<400>1

Met?Ala?Ala?Val?Ser?Leu?Arg?Leu?Gly?Asp?Leu?Val?Trp?Gly?Lys?Leu

1 5 10 15

Gly?Arg?Tyr?Pro?Pro?Trp?Pro?Gly?Lys?Ile?Val?Asn?Pro?Pro?Lys?Asp

20 25 30

Leu?Lys?Lys?Pro?Arg?Gly?Lys?Lys?Cys?Phe?Phe?Val?Lys?Phe?Phe?Gly

35 40 45

Thr?Glu?Asp?His?Ala?Trp?Ile?Lys?Val?Glu?Gln?Leu?Lys?Pro?Tyr?His

50 55 60

Ala?His?Lys?Glu?Glu?Met?Ile?Lys?Ile?Asn?Lys?Gly?Lys?Arg?Phe?Gln

65 70 75 80

Gln?Ala?Val?Asp?Ala?Val?Glu?Glu?Phe?Leu?Arg?Arg?Ala?Lys?Gly?Lys

85 90 95

Asp?Gln?Thr?Ser?Ser?His?Asn?Ser?Ser?Asp?Asp?Lys?Asn?Arg?Arg?Asn

100 105 110

Ser?Ser?Glu?Glu?Arg?Ser?Arg?Pro?Asn?Ser?Gly?Asp?Glu?Lys?Arg?Lys

115 120 125

Leu?Ser?Leu?Ser?Glu?Gly?Lys?Val?Lys?Lys?Asn?Met?Gly?Glu?Gly?Lys

130 135 140

Lys?Arg?Val?Ser?Ser?Gly?Ser?Ser?Glu?Arg?Gly?Ser?Lys?Ser?Pro?Leu

145 150 155 160

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

165 170 175

Asp?Glu?Lys?Asp?Leu?Thr?Ile?Pro?Glu?Ser?Ser?Thr?Val?Lys?Gly?Met

180 185 190

Met?Ala?Gly?Pro?Met?Ala?Ala?Phe?Lys?Trp?Gln?Pro?Thr?Ala?Ser?Glu

195 200 205

Pro?Val?Lys?Asp?Ala?Asp?Pro?His?Phe?His?His?Phe?Leu?Leu?Ser?Gln

210 215 220

Thr?Glu?Lys?Pro?Ala?Val?Cys?Tyr?Gln?Ala?Ile?Thr?Lys?Lys?Leu?Lys

225 230 235 240

Ile?Cys?Glu?Glu?Glu?Thr?Gly?Ser?Thr?Ser?Ile?Gln?Ala?Ala?Asp?Ser

245 250 255

Thr?Ala?Val?Asn?Gly?Ser?Ile?Thr?Pro?Thr?Asp?Lys?Lys?Ile?Gly?Phe

260 265 270

Leu?Gly?Leu?Gly?Leu?Met?Gly?Ser?Gly?Ile?Val?Ser?Asn?Leu?Leu?Lys

275 280 285

Met?Gly?His?Thr?Val?Thr?Val?Trp?Asn?Arg?Thr?Ala?Glu?Lys?Cys?Asp

290 295 300

Leu?Phe?Ile?Gln?Glu?Gly?Ala?Arg?Leu?Gly?Arg?Thr?Pro?Ala?Glu?Val

305 310 315 320

Val?Ser?Thr?Cys?Asp?Ile?Thr?Phe?Ala?Cys?Val?Ser?Asp?Pro?Lys?Ala

325 330 335

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

340 345 350

Pro?Gly?Lys?Cys?Tyr?Val?Asp?Met?Ser?Thr?Val?Asp?Ala?Asp?Thr?Val

355 360 365

Thr?Glu?Leu?Ala?Gln?Val?Ile?Val?Ser?Arg?Gly?Gly?Arg?Phe?Leu?Glu

370 375 380

Ala?Pro?Val?Ser?Gly?Asn?Gln?Gln?Leu?Ser?Asn?Asp?Gly?Met?Leu?Val

385 390 395 400

Ile?Leu?Ala?Ala?Gly?Asp?Arg?Gly?Leu?Tyr?Glu?Asp?Cys?Ser?Ser?Cys

405 410 415

Phe?Gln?Ala?Met?Gly?Lys?Thr?Ser?Phe?Phe?Leu?Gly?Glu?Val?Gly?Asn

420 425 430

Ala?Ala?Lys?Met?Met?Leu?Ile?Val?Asn?Met?Val?Gln?Gly?Ser?Phe?Met

435 440 445

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

450 455 460

Gln?Gln?Thr?Leu?Leu?Asp?Ile?Leu?Asn?Gln?Gly?Gln?Leu?Ala?Ser?Ile

465 470 475 480

Phe?Leu?Asp?Gln?Lys?Cys?Gln?Asn?Ile?Leu?Gln?Gly?Asn?Phe?Lys?Pro

485 490 495

Asp?Phe?Tyr?Leu?Lys?Tyr?Ile?Gln?Lys?Asp?Leu?Arg?Leu?Ala?Ile?Ala

500 505 510

Leu?Gly?Asp?Ala?Val?Asn?His?Pro?Thr?Pro?Met?Ala?Ala?Ala?Ala?Asn

515 520 525

Glu?Val?Tyr?Lys?Arg?Ala?Lys?Ala?Leu?Asp?Gln?Ser?Asp?Asn?Asp?Met

530 535 540

Ser?Ala?Val?Tyr?Arg?Ala?Tyr?Ile?His

545 550

<210>2

<211>1662

<212>DNA

＜213〉Genus Homo people (Homo sapiens)

<400>2

ATGGCGGCTG?TGAGTCTGCG?GCTCGGCGAC?TTGGTGTGGG?GGAAACTCGG?CCGATATCCT 60

CCTTGGCCAG?GAAAGATTGT?TAATCCACCA?AAGGACTTGA?AGAAACCTCG?CGGAAAGAAA 120

TGCTTCTTTG?TGAAATTTTT?TGGAACAGAA?GATCATGCCT?GGATCAAAGT?GGAACAGCTG 180

AAGCCATATC?ATGCTCATAA?AGAGGAAATG?ATAAAAATTA?ACAAGGGTAA?ACGATTCCAG 240

CAAGCGGTAG?ATGCTGTCGA?AGAGTTCCTC?AGGAGAGCCA?AAGGGAAAGA?CCAGACGTCA 300

TCCCACAATT?CTTCTGATGA?CAAGAATCGA?CGTAATTCCA?GTGAGGAGAG?AAGTAGGCCA 360

AACTCAGGTG?ATGAGAAGCG?CAAACTTAGC?CTGTCTGAAG?GGAAGGTGAA?GAAGAACATG 420

GGAGAAGGAA?AGAAGAGGGT?GTCTTCAGGC?TCTTCAGAGA?GAGGCTCCAA?ATCCCCTCTG 480

AAAAGAGCCC?AAGAGCAAAG?TCCCCGGAAG?CGGGGTCGGC?CCCCAAAGGA?TGAGAAGGAT 540

CTCACCATCC?CGGAGTCTAG?TACCGTGAAG?GGGATGATGG?CCGGACCGAT?GGCCGCGTTT 600

AAATGGCAGC?CAACCGCAAG?CGAGCCTGTT?AAAGATGCAG?ATCCTCATTT?CCATCATTTC 660

CTGCTAAGCC?AAACAGAGAA?GCCAGCTGTC?TGTTACCAGG?CAATCACGAA?GAAGTTGAAA 720

ATATGTGAAG?AGGAAACTGG?CTCCACCTCC?ATCCAGGCAG?CTGACAGCAC?AGCCGTGAAT 780

GGCAGCATCA?CACCCACAGA?CAAAAAGATA?GGATTTTTGG?GCCTTGGTCT?CATGGGAAGT 840

GGAATCGTCT?CCAACTTGCT?AAAAATGGGT?CACACAGTGA?CTGTCTGGAA?CCGCACTGCA 900

GAGAAATGTG?ATTTGTTCAT?CCAGGAGGGG?GCCCGTCTGG?GAAGAACCCC?CGCTGAAGTC 960

GTCTCAACCT?GCGACATCAC?TTTCGCCTGC?GTGTCGGATC?CCAAGGCGGC?CAAGGACCTG 1020

GTGCTGGGCC?CCAGTGGTGT?GCTGCAAGGG?ATCCGCCCTG?GGAAGTGCTA?CGTGGACATG 1080

TCAACAGTGG?ACGCTGACAC?CGTCACTGAG?CTGGCCCAGG?TGATTGTGTC?CAGGGGGGGG 1140

CGCTTTCTGG?AAGCCCCCGT?CTCAGGGAAT?CAGCAGCTGT?CTAATGACGG?GATGTTGGTG 1200

ATCTTAGCGG?CTGGAGACAG?GGGCTTATAT?GAGGACTGCA?GCAGCTGCTT?CCAGGCGATG 1260

GGGAAGACCT?CCTTCTTCCT?AGGTGAAGTG?GGCAATGCAG?CCAAGATGAT?GCTGATCGTG 1320

AACATGGTCC?AAGGGAGCTT?CATGGCCACT?ATTGCCGAGG?GGCTGACCCT?GGCCCAGGTG 1380

ACAGGCCAGT?CCCAGCAGAC?ACTCTTGGAC?ATCCTCAATC?AGGGACAGTT?GGCCAGCATC 1440

TTCCTGGACC?AGAAGTGCCA?AAATATCCTG?CAAGGAAACT?TTAAGCCTGA?TTTCTACCTG 1500

AAATACATTC?AGAAGGATCT?CCGCTTAGCC?ATTGCGCTGG?GTGATGCGGT?CAACCATCCG 1560

ACTCCCATGG?CAGCTGCAGC?AAATGAGGTG?TACAAAAGAG?CCAAGGCGCT?GGACCAGTCC 1620

GACAACGATA?TGTCCGCCGT?GTACCGAGCC?TACATACATT?AA 1662

Claims

1, with the interactional albumen of mitogen activated protein kinase p 38, be protein with one of following amino acid residue sequences:

1) the SEQ ID № in the sequence table: 1;

2, the according to claim 1 and interactional albumen of mitogen activated protein kinase p 38, it is characterized in that: described albumen has SEQ ID № in the sequence table: 1 amino acid residue sequence.

3, the described gene with mitogen activated protein kinase p 38 interaction protein of coding claim 1 is one of following nucleotide sequence:

1) SEQ ID № in the sequence table: 2 dna sequence dna;

2) SEQ ID № in the code sequence tabulation: 1 dna sequence dna;

4, the gene of coding according to claim 3 and mitogen activated protein kinase p 38 interaction protein, it is characterized in that: described gene has SEQ ID № in the sequence table: 2 dna sequence dna.

5, contain the described expression carrier of claim 3.

6, the transgenic cell line that contains the described gene of claim 3.

7, the host bacterium that contains the described gene of claim 3.

8, the application of the encoding gene of the RNA interfering of the described gene of claim 3 in the curative drug of preparation and mitogen activated protein kinase p 38 signal path relative disease.