CN1301870A - New Relatice protein kinase of human mitogen activated protein kinase and its code sequence - Google Patents

Abstract

The present invention discloses provides a kind of novel human MAP kinase-interacting kinase 2, shortly named hMnK2, protein expressed in adrenal gland tissue of normal human body and its coding sequence and well as the preparation and application of the protein and nucleic acid sequence and the method of detecting hMnK2 nucleic acid sequence and polypeptide in sample.

Description

New HUMAN MAP KINASE-INTERACTING KINASE 2 and encoding sequence thereof

The present invention relates to cytology, immunology, oncology and neurophysiology field, more specifically, the present invention relates to a kind of new HUMAN MAP KINASE-INTERACTING KINASE 2 hMnk2 (human MAP kinase-interacting kinase 2 abbreviates " hMnk2 " as) and nucleotide sequence thereof.The invention still further relates to the preparation method and the purposes of this albumen and nucleotide sequence.

Mitogen-activated protein kinase related protein kinase (Mnk) is a subfamily of more newfound serine/threonine kinase, and (MAP kinase) is closely related with mitogen-activated protein kinase.Studies show that Mnk albumen is that albumen (EMBO J 1997 Apr 15 are regulated in the downstream of multiple map kinase (kinase); 16 (8): 1909-20).

Map kinase is a kind of serine/threonine protein kitase, can be activated by the outer stimulation of various kinds of cell, as cytokine, somatomedin, neurohumor, hormone, cellular stress (cellular stress) and cell adhesion (cell adherence) (Physiol Rev 1999 Jan; 79 (1): 143-80).Map kinase all has expression in all eukaryotic cells, studies show that its all relevant (Acta Physiol Scand 1998 Dec with growth, signal conduction, differentiation and the apoptosis of cell; 164 (4): 611-21).

MAP regulation and control module is formed the phosphorylation cascade by three intracellular protein kinases, and they are map kinase (MAPK), map kinase protein kinase (MKK) and map kinase protein kinase protein kinase (MKKK).Their integrator cell membrane receptor activatory signals, the ability that enters nucleus phosphorylated nucleosides acid target sequence (as transcription factor) by the MAPK transposition will be converted into genomic response (Essay Biochem1997 from extracellular signal; 32:1-16).

Existing studies show that, map kinase family has the important physical function.The phosphorylation that map kinase family member born of the same parents regulate protein kinase (Extracellular Regulated Kinase) outward changes with nerve synapse and relevant (the Drugs Exp Clin Res 1999 of mnemonic learning process based on this; 25 (2-3): 99-103).Map kinase is the critical elements during mitogenesis stimulates, the mutual interchange between different map kinase signal transduction pathway determined cell to stress, reaction (the Postepy Hig Med Dosw1999 of apoptosis and generic physiological process; 53 (2): 291-303).Oestrogenic hormon and neurotrophic factor acceptor make the signal transduction path of the two assemble coupling (Front Neuroendocrinol 1999 Apr by the coexpression of MAP; 20 (2): 97-121).

The signal transduction pathway mediation oxidative stress of map kinase a kind of---SAPK2 (stress-activated protein kinase 2) and reorganization of vascular endothelial growth factor (VEGF) inductive Actin muscle and the cell movement of VEGF inductive, so this conduction path (Biochem Soc Symp 1999 that in inflammatory reaction and vasculogenesis reaction, plays an important role; 64:79-89).

Map kinase has multiple important effect.The conduction path of JNK in the map kinase family (c-Jun N-terminalkinase) is regulated the transcriptional activity of AP-1 (activator protein-1), so JNK is embryo's morphogenesis, the adjusting of cell proliferation apoptosis and necessary (the Biochem SocSymp 1999 of immunne response of cell; 64:1-12).MAP finally conducts to nucleus as the downstream pathway of growth factor receptor tyrosine kinase with signal, activates a plurality of transcription factor regulatory gene and expresses (Cell Signal 1999Jan; 11 (1): 1-14).Map kinase is all relevant with the growth and the apoptosis of vascular smooth muscle cell (VSMC), and this explanation map kinase is in cardiovascular disorder, as spontaneously hypertensive, atherosclerosis, in (Acta Physiol Scand 1998 Dec that play an important role; 164 (4): 611-21), this be the stimulation of somatomedin that Tyrosylprotein kinase is relied on make reply (Physiol Res 1998; 47 (4): 215-25).The transcription factor of map kinase and its regulation and control is with the damage of kidney and repair relevant (Curr Opin Nephrol Hypertens 1998Jul; 7 (4): 425-33).Two kinds of map kinases (p42/p44 and p38) mutually antagonism are regulated the protein kinase (CDKs) that cyclins (cyclin) rely on, so MAP (the Prog Cell Cycle Res 1996 that plays an important role in the reentering of cell cycle; 2:49-58).Map kinase also participates in physiological regulatory action (Mt Sinai J Med 1998 Mar of Angiotensin; 65 (2): 108-17).Multiple MAP plays an important role in the conduction of T lymphocytic signals, thereby can conducting from the TXi Baoshouti signal, they can regulate the expression that multiple transcription factor is controlled some important gene again, comprising interleukin-22 (IL-2) (Immunol Cell Biol1997 Dec; 75 (6): 528-45).(the Immunobiology 1997Dec thereby the activity of JNK and p38MAP kinases participation adjusting NF-kappa B transcription factor plays a significant role in inflammatory reaction and control necrocytosis; 198 (1-3): 35-49).Except somatomedin and some other signal of cytokine also can activate map kinase, as the component of ultraviolet ray or extracellular matrix, so map kinase may be played the part of important role (Baillieres Clin Endocrinol Metab 1996Jul in the regulation and control of people's epithelium proliferation and differentiation; 10 (3): 323-36).

Research hints, mitogen-activated protein kinase related protein kinase (Mnk) unusual relevant with some diseases, and therefore, to research and develop HUMAN MAP KINASE-INTERACTING KINASE 2 (Mnk) significant for therapeutic purpose.Yet before the application, still there is not to disclose or reported people hMnk2 albumen involved in the present invention.

The purpose of invention

First purpose of the present invention just provides a kind of new people's gene sequence-HUMAN MAP KINASE-INTERACTING KINASE 22 (human MAP kinase-interacting kinase 2, abbreviate " hMnk2 " as) gene order (Genbank Accession No.AF125532), a kind of new HUMAN MAP KINASE-INTERACTING KINASE 2 of this genes encoding.

Second purpose of the present invention provides a kind of new people's albumen hMnk2.

The 3rd purpose of the present invention provides a kind of recombinant technology that utilizes and produces the above-mentioned new people hMnk2 albumen and the method for nucleotide sequence.

The present invention also provides the application of this hMnk2 protein polypeptide and encoding sequence.

In one aspect of the invention, a kind of isolated dna molecular is provided, this molecule comprises: coding has the nucleotide sequence of the polypeptide of people hMnk2 protein active, shows at least 70% homology from the nucleotides sequence of Nucleotide 12-1253 position dna molecular among described nucleotide sequence and the SEQ ID NO.6; Perhaps described nucleotide sequence can be under the moderate stringent condition with SEQ ID NO.6 in from the nucleotide sequence hybridization of Nucleotide 12-1253 position.Preferably, described sequence encoding has the polypeptide of the aminoacid sequence shown in the SEQ ID NO.7.More preferably, described sequence has among the SEQ ID NO.6 nucleotide sequence from Nucleotide 12-1253 position.

In another aspect of this invention, provide a kind of isolated people hMnk2 protein and peptide, it comprises: have polypeptide or its conservative property variation polypeptide or its active fragments of SEQ ID NO.7 aminoacid sequence, or its reactive derivative.Preferably, this polypeptide is to have SEQ ID NO.7 polypeptide of sequence.

In another aspect of this invention, also provide a kind of carrier, it comprises above-mentioned dna molecular.

In another aspect of this invention, also provide a kind of usefulness above-mentioned carrier transformed host cells.This host cell is intestinal bacteria in an example; In another example, this host cell is an eukaryotic cell.

In another aspect of this invention, also provide a kind of generation to have the method for the polypeptide of people hMnk2 protein active, this method comprises:

(1) nucleotide sequence that coding is had a polypeptide of people hMnk2 protein-active operationally is connected in expression regulation sequence, form people hMnk2 protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 12-1253 position among described nucleotide sequence and the SEQ ID NO.6;

(2) change the expression vector in the step (1) over to host cell, form the proteic reconstitution cell of people hMnk2;

(3) under the condition that is fit to expressing human hMnk2 protein polypeptide, the reconstitution cell in the culturing step (2);

(4) isolate polypeptide with people hMnk2 protein-active.

Preferably, the nucleotide sequence that uses in the method has the sequence of 12-1253 position among the SEQ ID NO.6.

The present invention also provides and hMnk2 protein polypeptide specificity bonded antibody.

In the present invention, term " HUMAN MAP KINASE-INTERACTING KINASE 22 ", " hMnk2 albumen " and " hMnk2 polypeptide " are used interchangeably, all refer to HUMAN MAP KINASE-INTERACTING KINASE 22, especially the HUMAN MAP KINASE-INTERACTING KINASE 22 of total length form (SEQ ID NO:6).They comprise the HUMAN MAP KINASE-INTERACTING KINASE 22 that contains or do not contain initial methionine.

In the present invention, " isolating ", " purifying " or " pure substantially " DNA are meant, this DNA or fragment have been arranged in the sequence of its both sides and have separated under native state, refer to that also this DNA or fragment with under the native state follow the component of nucleic acid to separate, and separate with the protein of in cell, following it.

In the present invention, term " people hMnk2 albumen (or polypeptide) encoding sequence " refer to the encode nucleotide sequence of polypeptide with people hMnk2 protein-active is as 12-1253 position nucleotide sequence and degenerate sequence thereof among the SEQ ID NO.6.This degenerate sequence is meant, is arranged in the encoder block 12-1253 position Nucleotide of SEQ ID NO.6 sequence, and having one or more codons to be encoded, the degenerate codon of same amino acid replaces the back and the sequence that produces.Because the degeneracy of codon, thus with SEQ ID NO.6 in 12-1253 position nucleotide sequence homology be low to moderate about 70% the degenerate sequence described sequence of SEQ ID NO.7 of also encoding out.This term also comprises can be under the moderate stringent condition, better under the height stringent condition with SEQ ID NO.6 in from the nucleotide sequence of the nucleotide sequence hybridization of Nucleotide 12-1253 position.This term also comprise with SEQ ID NO.6 in from the homology of nucleotide sequence at least 70% of Nucleotide 12-1253 position, preferably at least 80%, more preferably at least 90%, at least 95% nucleotide sequence best.

This term also comprises encoding to have the variant form of open reading frame sequence among proteic, the SEQ ID NO.6 with people hMnk2 identical function.These variant forms comprise (but being not limited to): several (are generally 1-90, preferably 1-60, more preferably 1-20,1-10 best) disappearance, insertion and/or the replacement of Nucleotide, and several (are generally in 60 to hold interpolation 5 ' and/or 3 ', preferably being in 30, more preferably is in 10, is in 5 best) Nucleotide.

In the present invention, " pure substantially " protein or polypeptide are meant that it accounts at least 20% of the total material of sample at least, preferably at least 50%, more preferably at least 80%, and at least 90% (by dry weight or weight in wet base) best.Purity can be measured with any suitable method, as measure the purity of polypeptide with column chromatography, PAGE or HPLC method.Substantially pure polypeptide is substantially free of the component of following it under the native state.

In the present invention, term " people hMnk2 albumen or polypeptide " refers to have the SEQ ID NO.7 polypeptide of sequence of natural human hMnk2 protein-active.This term also comprises having and variant form people hMnk2 identical function, SEQ ID NO.7 sequence.These variant forms comprise (but being not limited to): several (are generally 1-50, preferably 1-30, more preferably 1-20,1-10 best) amino acid whose disappearance, insertion and/or replacement, and add one or several at C-terminal and/or N-terminal and (be generally in 20, preferably being in 10, more preferably is in 5) amino acid.For example, in the art, when replacing, can not change proteinic function usually with the close or similar amino acid of performance.Again such as, add one or several amino acid at C-terminal and/or N-terminal and also can not change proteinic function usually.This term also comprises proteic active fragments of hMnk2 and reactive derivative.

The variant form of inventor hMnk2 polypeptide comprises: homologous sequence, conservative property varient, allelic variant, natural mutation, induced mutation body, under high or low stringent condition can with the coded albumen of the DNA of people hMnk2 protein D NA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-people hMnk2 polypeptide to obtain.The present invention also provides other polypeptide, as comprises people hMnk2 polypeptide or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention also comprises the soluble fragments of people hMnk2 polypeptide.Usually, this fragment have people hMnk2 peptide sequence at least about 10 continuous amino acids, usually at least about 30 continuous amino acids, preferably at least about 50 continuous amino acids, more preferably at least about 80 continuous amino acids, best at least about 100 continuous amino acids.

In the present invention, " hMnk2 conservative property variation polypeptide " refers to compare with the aminoacid sequence of SEQ ID NO.7, has 10 at the most, and preferably at the most 8, more preferably 5 amino acid is replaced by similar performance or close amino acid and formed polypeptide at the most.These conservative property variation polypeptide are preferably replaced according to table 1 and are produced.

Table 1

Initial residue	Representational replacement	The preferred replacement
			Ala(A)	Val；Leu；Ile	Val
Arg(R)	Lys；Gln；Asn	Lys
			Asn(N)	Gln；His；Lys；Arg	Gln
Asp(D)	Glu	Glu
			Cys(C)	Ser	Ser
Gln(Q)	Asn	Asn
			Glu(E)	Asp	Asp
Gly(G)	Pro；Ala	Ala
			His(H)	Asn；Gln；Lys；Arg	Arg
Ile(I)	Leu；Val；Met；Ala；Phe	Leu
			Leu(L)	Ile；Val；Met；Ala；Phe	Ile
Lys(K)	Arg；Gln；Asn	Arg
			Met(M)	Leu；Phe；Ile	Leu
Phe(F)	Leu；Val；Ile；Ala；Tyr	Leu

Pro(P)	Ala	Ala
			Ser(S)	Thr	Thr
Thr(T)	Ser	Ser
			Trp(W)	Tyr；Phe	Tyr
Tyr(Y)	Trp；Phe；Thr；Ser	Phe
			Val(V)	Ile；Leu；Met；Phe；Ala	Leu

Invention also comprises the analogue of people hMnk2 albumen or polypeptide.The difference of these analogues and natural human hMnk2 polypeptide can be the difference on the aminoacid sequence, also can be the difference that does not influence on the modified forms of sequence, perhaps haves both at the same time.These polypeptide comprise natural or the inductive genetic variant.The induce variation body can obtain by various technology, as by radiation or be exposed to mutagenic compound and produce random mutagenesis, also can pass through site-directed mutagenesis method or the biological technology of other known moleculars.Analogue also comprises having the analogue that is different from the amino acid whose residue of natural L-(as D-amino acid), and has non-natural analogue that exist or synthetic amino acid (as β, gamma-amino acid).Should be understood that polypeptide of the present invention is not limited to the above-mentioned representational polypeptide that exemplifies.

(the not changing primary structure usually) form of modification comprises: the chemically derived form such as the acetylize or carboxylated of the polypeptide that body is interior or external.Modification also comprises glycosylation, carries out glycosylation modified and polypeptide that produce in the procedure of processing as those in the synthetic and processing of polypeptide or further.This modification can be carried out glycosylated enzyme (as mammiferous glycosylase or deglycosylating enzyme) and finishes by polypeptide is exposed to.Modified forms also comprises have the phosphorylated amino acid residue sequence of (as Tyrosine O-phosphate, phosphoserine, phosphothreonine).Thereby also comprise the polypeptide that has been improved its anti-proteolysis performance or optimized solubility property by modifying.

In the present invention, can select various carrier known in the art for use, the carrier as commercially available comprises plasmid, clay etc.When producing people hMnk2 polypeptide of the present invention, the hMnk2 encoding sequence operationally can be connected in expression regulation sequence, thereby form people hMnk2 protein expression vector.

As used herein, " operationally being connected in " refers to a kind of like this situation, and promptly some part of linear DNA sequence can influence the activity of same other parts of linear DNA sequence.For example, if signal peptide DNA as precursor expression and participate in the secretion of polypeptide, signal peptide (secretion leader sequence) DNA operationally is connected in polypeptid DNA so; If transcribing of promotor control sequence, it is operationally to be connected in encoding sequence so; When if ribosome bind site is placed in the position that can make its translation, it is operationally to be connected in encoding sequence so.Generally, " operationally being connected in " means adjacent, then means in reading frame adjacent for the secretion leader sequence.

In the present invention, term " host cell " comprises prokaryotic cell prokaryocyte and eukaryotic cell.The example of prokaryotic host cell commonly used comprises intestinal bacteria, Bacillus subtilus etc.Eukaryotic host cell commonly used comprises yeast cell, insect cell and mammalian cell.Preferably, this host cell is an eukaryotic cell, as Chinese hamster ovary celI, COS cell etc.

The present invention also provides the antibody special to people hMnk2, comprises polyclonal antibody and monoclonal antibody.

In the present invention, can use a series of methods known in the art to prepare special antibody at hMnk2.For example, the people hMnk2 gene product or its antigen fragment of purifying is injected in the animal body to produce polyclonal antibody.Equally, the cell of expressing human hMnk2 or its antigen fragment also can be used for animal is caused immunity and produces antibody.Antibody prepared in accordance with the present invention also can be monoclonal antibody, and these monoclonal antibodies can prepare (for example, Kohler et al., Nature 256:495,1975 with hybridoma technology; Kohler etal., Eur.J.Immunol.6:511,1976; Kohler et al., Eur.J.Immunol.6:292,1976).Antibody of the present invention comprises the antibody that can prevent the hMnk2 function, also can be the antibody that does not influence people hMnk2 function.Each antibody-like can produce by the fragment of people hMnk2 gene product or functional domain are caused immunity, and people hMnk2 gene product and fragment thereof can produce or synthesize with Peptide synthesizer with recombination method.With the hMnk2 gene product bonded antibody of non-modified forms, can come immune animal to obtain by being used in the gene product that prokaryotic cell prokaryocyte for example produces among the E.coli.With posttranslational modification form such as glycosylation or phosphorylated protein or polypeptide bonded antibody, can obtain by the immune animal that comes that is used in the gene product that produces in eukaryotic cell such as yeast or the insect cell.

People hMnk2 antibody of the present invention can be used for identifying the cell of expressing human hMnk2 albumen or polypeptide.For example, can with a kind of detectable molecule for example fluorescein isothiocyanic acid (FITC) come mark hMnk2 specific antibody, allow people hMnk2 specific antibody contact then, detect and hMnk2 specific antibody bonded cell with fluorescent microscope or flow cytometer again with cell sample.

Except cell surface detects people hMnk2, can also analyze this protein with the Western engram technology.Cell pyrolysis liquid can from culturing cell or take from patient's tissue sample such as suprarenal gland extract, and be dissolved in the lysis buffer that contains stain remover.Use sds polyacrylamide gel electrophoresis isolated cell extract (simultaneously with the people hMnk2 polypeptide of purifying as positive control) then, then it is transferred on the nitrocellulose by electrophoresis hybridization.In order to survey the hMnk2 polypeptide, can use typical antibodies detection method, for example radioautograph or alkaline phosphatase enzyme assay method with the immunity of Western trace.And can use the contrast of immunization serum or incoherent monoclonal antibody as non-specific responding.

Whether and quantity the expression of also available Nothern blotting technical Analysis hMnk2 gene product, the i.e. existence of rna transcription thing in cell of analyst hMnk2.

The Western engram analysis of the Nothern engram analysis of people hMnk2DNA and people hMnk2 specific antibody can also be united use, with the expression of confirmer hMnk2 in biological specimen.People hMnk2 DNA can also be used for Southern engram analysis or in situ hybridization analysis, with this assignment of genes gene mapping on karyomit(e), and can carry out genetic linkage analysis to find out other possible disease related gene.

In addition, the present invention also provides a kind of nucleic acid molecule that can be used as probe, and this molecule has the 8-100 of hMnk2 nucleotide coding sequence or more, preferably 15-50 continuous nucleotide usually.This probe can be used for whether existing in the test sample nucleic acid molecule of the hMnk2 that encodes.

The present invention also provides the method that whether has the hMnk2 nucleotide sequence in the test sample, and it comprises with above-mentioned probe and sample and hybridizing whether detection probes combination has taken place then.Preferably, this sample is the product behind the pcr amplification, and wherein the pcr amplification primer is corresponding to the hMnk2 nucleotide coding sequence, and can be positioned at the both sides or the centre of this encoding sequence.Primer length is generally 20-50 Nucleotide.

In addition, according to hMnk2 nucleotide sequence of the present invention and aminoacid sequence, can be on the homology basis of nucleic acid homology or marking protein, screening hMnk2 homologous gene or homologous protein.

In order to obtain and the people cDNAs of hMnk2 gene-correlation or the dot matrix of genomic dna s, can screen people cDNA or genome dna library with dna probe, these probes are under low stringent condition, with 32P hMnk2 all or part of cooked the radioactivity mark and.The cDNA library that most is suitable for screening is the library from the human adrenal gland tissue.Also can be used for screening purpose from the cDNA library that participates in endocrine other tissue or specific human body cell strain.Structure is that biology field is well-known from the method in the cDNA library of interested cell or tissue.In addition, many such cDNA libraries also can buy, for example available from Clontech, and Palo Alto, Cal..This screening method can be discerned the nucleotide sequence of the gene family relevant with hMnk2.

Can finish as follows according to Nucleotide similarity screening hMnk2 homologue.Human acth cDNA library, for example (Clontech, Palo Alto Cal.) can use one section all or part of random primer dna probe screening that comprises the hMnk2 gene order to Clontech Cat.#7430-l.Finish having clone's the evaluation of the DNA insertion sequence of 70% homology at least with the hMnk2 sequence, can use hybridization temperature is 55 ℃ hybridization solution, uses 0.5 * SSC and 0.1%SDS to clean then.Shi Bie clone's DNA insertion sequence can be further estimated the similarity of it and hMnk2 gene with DNA restriction endonuclease analysis and dna sequencing in this way.The distribution of tissue expression can be with above-mentioned Northern blotting technical Analysis.

The hMnk2 homologue also can be used at the antibody of hMnk2 albumen or polypeptide and discern.For example, can be with the method for standard to commercial or make up with currently known methods, from cell or organize for example adrenal expression library to screen.Pour the library into plate, on colony lift to a nitrocellulose membrane, the recombinant protein of expression is attached on the film.Just can carry out typical antibodies and detection then with specific people hMnk2 antibody.Identify the DNA insertion sequence among the clone in this way, can be further analyze to estimate the similarity of it and hMnk2 gene with DNA restriction endonuclease analysis and dna sequencing.The tissue expression of the gene of new identification distributes and can similarly analyze as stated above.

People hMnk2 Nucleotide full length sequence of the present invention or its fragment can obtain with the method for pcr amplification method, recombination method or synthetic usually.For the pcr amplification method, can be disclosed according to the present invention about nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the prepared cDNA storehouse of ordinary method well known by persons skilled in the art as template, amplification and must relevant sequence.When sequence is longer, usually needs to carry out twice or pcr amplification repeatedly, and then the fragment that each time amplifies is stitched together by proper order.

In case obtained relevant sequence, just can obtain relevant sequence in large quantity with recombination method.This normally is cloned into carrier with it, changes cell again over to, separates obtaining relevant sequence then from the host cell after the propagation by ordinary method.

In addition, also the method for available artificial chemosynthesis is synthesized relevant sequence.Before the application, prior art fully can be by first synthetic a plurality of polynucleotide small segments, and then connect and obtain the proteic nucleotide sequence of code book contriver hMnk2.Then, can be with in various existing dna moleculars (as carrier) and the cell in this nucleotide sequence introducing this area.In addition, also can will suddenly change and introduce in the protein sequence of the present invention by chemosynthesis.

Except producing with recombination method, the also available solid phase technique of the proteic fragment of the present invention is produced (people such as Stewart, (1969) Solid-Phase Peptide Synthesis, WHFreeman Co., San Francisco by direct peptide synthesis; Merrifield J. (1963) J.Am Chem.Soc 85:2149-2154).Can carry out by hand or automatically at external synthetic protein.For example, can (Foster City CA) synthesizes peptide automatically with the 431A type peptide synthesizer of AppliedBiosystems.Can distinguish proteic each fragment of chemosynthesis the present invention, be connected to produce the molecule of total length with chemical process then.

The proteic encoding sequence of the present invention can be used for the assignment of genes gene mapping.For example,, the cDNA clone is hybridized with the karyomit(e) of metaphase, can carry out chromosomal localization exactly by fluorescence in situ hybridization technique (FISH).This technology can be used the cDNA that is as short as about 500bp; Also can use and grow to about 2000bp or longer cDNA.For this technology, can be referring to people such as Verma, Human Chromosomes:A Manual ofBasic Techniques, Pergamon Press, New York (1988).

In case sequence is located in certain exact position on the karyomit(e), the physical location of sequence on karyomit(e) can be associated with the genetic map data.These genetic map data can obtain, for example by Mendelian (Mendelian) people genetic database (can obtain on the net by Johns Hopkins University Welch MedicalLibrary).Then, come identified gene by linkage analysis and be positioned dependency between the disease of same chromosomal region.

Then, be necessary to determine the cDNA between diseased individuals and the healthy individual or the difference of genome sequence aspect.Be not present in normal individual if a certain sudden change is present in part or all of diseased individuals, this sudden change may be exactly the paathogenic factor of this disease so.

Utilize people hMnk2 albumen of the present invention, by various conventional screening methods, can filter out with people hMnk2 take place interactional material or, as acceptor, inhibitor or antagonist etc.

Inventor hMnk2 albumen and antibody thereof, inhibitor, antagonist or acceptor etc. when using (administration) in treatment, can provide different effects.Usually, can these materials are formulated in nontoxic, inert and the pharmaceutically acceptable aqueous carrier medium, wherein pH is about 5-8 usually, and preferably pH is about 6-8, although the pH value can be with being changed to some extent by preparation Substance Properties and illness to be treated.The pharmaceutical composition for preparing can carry out administration by conventional route, comprising (but being not limited to): intramuscular, intraperitoneal, subcutaneous, intracutaneous or topical.

With people hMnk2 albumen of the present invention is example, can be with itself and suitable pharmaceutically acceptable carrier coupling.This class pharmaceutical composition contains protein and the pharmaceutically acceptable carrier or the vehicle for the treatment of significant quantity.This class carrier comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol and combination thereof.Pharmaceutical preparation should be complementary with administering mode.People hMnk2 albumen of the present invention can be made into the injection form, for example is prepared by ordinary method with the physiological saline or the aqueous solution that contains glucose and other assistant agents.Pharmaceutical composition such as tablet and capsule can be prepared by ordinary method.Pharmaceutical composition such as injection, solution, tablet and capsule should be made under aseptic condition.The dosage of activeconstituents is the treatment significant quantity, for example every day about 1 microgram/kg body weight-Yue 5 mg/kg body weight.In addition, polypeptide of the present invention also can use with the other treatment agent.

When people hMnk2 protein polypeptide of the present invention is used as medicine, this polypeptide of treatment effective dose can be applied to Mammals, wherein should treat effective dose usually at least about 10 micrograms/kg body weight, and in most of the cases be no more than about 8 mg/kg body weight, preferably this dosage is about 10 micrograms/kg body weight-Yue 1 mg/kg body weight.Certainly, concrete dosage also should be considered factors such as route of administration, patient health situation, and these all are within the skilled practitioners skill.

Find by the homology retrieval, new gene of the present invention has and delivers and be confirmed to be the proteic gene of mouse (Musmusculus) Mnk2 (GeneBank Accession Y11092) height homologous sequence, and new albumen of the present invention has the aminoacid sequence of mouse (Mus musculus) Mnk2 albumen (GenPept AccessionCAA71966) high conservative.So hMnk2 gene of the present invention is a homologous gene of mouse (Musmusculus) Mnk2 protein gene.

Find that by the homology retrieval new ZZ albumen of the present invention has the aminoacid sequence of Mnk albumen high conservative.Further studies show that, the present invention with deliver and be confirmed to be mouse Mnk2 gene and have high homology, homogeny is 96.4%, also has higher homology with other Mnk albumen (as Mnk1 albumen of mouse and people's Mnk1 albumen) in addition.So hMnk2 albumen of the present invention belongs to the Mnk protein family, be the proteic homologous protein of mouse Mnk2 and have same or analogous function.

Fig. 1 is that the homology of people hMnk2 of the present invention and mouse Mnk2 (mMnk2) gene nucleic acid sequence (GenBankAccession Y11092) compares (FASTA) figure.Wherein, identical Nucleotide marks with " | ".

Fig. 2 is that the homology of the aminoacid sequence of people hMnk2 albumen of the present invention and mouse Mnk2 (mMnk2) compares (FASTA) figure.MMnk2.pep: mouse Mnk2 Argine Monohydrochloride sequence (GenePept ACCESSION No.CAA71966); HMnk2: people Mnk2 Argine Monohydrochloride sequence.Wherein, identical amino acid marks with " | ", and different amino acid mark with "+".

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:ColdSpring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

Embodiment 1

The clone of hMnk2 gene

1. separate tissue (Tissue isolation)

Suprarenal gland derives from 5 normal adult male sex donors, takes out adrenal tissue in after death four hours, places the freezing preservation of liquid nitrogen immediately.

2.mRNA separation (mRNA isolation)

Take out tissue, grind, add the 50ml pipe that fills lysate, fully after the vibration, move in the glass homogenizer again with mortar.Move to 50ml after the homogenate and newly manage, and extracted total RNA (TRIzol Reagents, Gibco, NY, USA).Identify total RNA quality with the denaturing formaldehyde gel electrophoresis.Cellulose column with band Oligo d (T) separates mRNA among total RNA, quantitatively.

3.cDNA the structure in library (Construction of cDNA library)

With mRNA is template, and synthetic double chain cDNA, reverse transcription primer are seen SEQ ID NO.1.After mending flat end, add the joint that contains EcoR I point of contact, joint sequence is seen SEQ ID NO.2 and 3 respectively.Behind the phosphorylation EcoR I end, use Xho I digestion with restriction enzyme 1.5 hours, carry out fragment again and separate.Cross the fragment of post screening length＞500bp, use the phenol-chloroform extracting, ethanol sedimentation, the sterilized water dissolving is connected to Uni-ZAP XR carrier (Stratagene, CA9203, USA), with Zap-cDNA Gigapack III Gold Cloning Kit (Stratagene, CA9203, USA) pack, the host bacterium is used XL 1-Blue MRF ' bacterium.Coated plate is also measured titre.

4. order-checking and database are set up (Seqencing and Database Constructing)

Select the clone who has the external source fragment to insert in the library, amplification back extracting plasmid (Qiagen, Germany), with T3 and T7 universal primer as 3 ' end and 5 ' hold, adopt thing fluorescent mark (Big-Dye, Perkin-Elmer, method USA) of stopping, (Perkin-Elmer carries out the EST large scale sequencing on USA) at ABI 377 sequenators.Sequencing result is removed the carrier sequence with FACTURA software, is transferred to the processing of carrying out next step on SUN Ultra 450 Server.All sequence informations are used the GCG software package again, and (Wisconsin group, USA) BLAST in and the existing database of FASTA software search (Genebank+EMBL) are lower than 95% sequence with no homology or homology and are considered as new gene and set up database.

5. the full-length clone of gene (Cloning of Full-length cDNA)

On the new gene fragment order information basis that obtains, carry out the cDNA full-length clone, carry out in two stages:

(1) " electronic cloning " (Electronic Cloning)

Search the dbEST database with new gene fragment order as probe, with overlap＞50bp, homology is at (the Expressed Sequence Tag of the expressed sequence tag more than 98%, being called for short " EST ") sequence thinks same sequence (consensus sequence), take out and splice with AUTOASSEMBLER software, part EST can the extension probes sequence.Whether the sequence that is extended with the STRIDER software analysis has complete open reading frame (Open Reading Frame again, ORF), on Nucleotide and amino acid levels, whether homology is arranged with definite this sequence with BLAST search Genbank or SwissProt, to help how differentiate resulting full length gene integrity with other species.By the method for electronic cloning, can obtain the full length sequence of hMnk2 gene usually.

(2) the terminal rapid amplifying of cDNA (Rapid Amplification of cDNA Ends, RACE)

If do not obtain complete cDNA total length yet by " electronic cloning " method, then at 5 of existing sequence ' or 3 ' end design primer, (Clontech Lab, Inc carry out the long range PCR reaction in USA) in human suprarenal gland Marathon-Ready cDNA library.Then to PCR product cloning, order-checking.The sequence that is extended with AUTOASSEMBLER and STRIDER software analysis has or not complete ORF, as not having, repeats said process until obtaining total length.

(3)RT-PCR

For 5 ' and 3 ' end known sequences, the centre still has an intersegmental crack (gap) to obtain from existing public database or its data storehouse, can consider to adopt the method for RT-PCR.At sequence 5 ' end design primer, 3 ' end primer adopts Oligo-dT, increases in the total RNA of suprarenal gland storehouse.Then product is cloned, checked order.Splice at last and obtain total length.

By being used in combination above-mentioned 3 kinds of methods, obtained candidate's the proteic complete encoding sequence of people hMnk2.Obtain on the total length basis of (comprising complete open reading frame at least) in splicing, further design primer: oligonucleotide R1:5 '-CCGGACAGAAGATGGTGCAG-3 ' (SEQ ID NO.4) is a forward primer, oligonucleotide R2:5 '-CGGCATTGACAGTTGGTGTAAA-3 ' (SEQ ID NO.5) is a reverse primer, total RNA with adrenal tissue is a template, carry out the RT-PCR amplification, the PCR condition of R1/R2 be 94 ℃ 5 minutes, carried out 35 circulations in 1 minute with 94 ℃ 30 seconds, 59.5 ℃ 30 seconds and 72 ℃ thereupon, extended 5 minutes with 72 ℃ at last.The electrophoresis detection pcr amplification product, obtaining the purpose fragment length is 1473bp.Clone, check order with pcr amplification product according to a conventional method then, obtain the sequence shown in SEQ ID NO.6.

Embodiment 2

The sequence information of hMnk2 gene and homology analysis:

People hMnk2 full-length cDNA (the GenBank Accession No.AF125532 that the present invention is new.Because of applying for maintaining secrecy, so open before the application to the public) length be 1473bp, detailed sequence is seen SEQ ID NO.6, wherein open reading frame is positioned at 12-1253 position Nucleotide.Derive the aminoacid sequence of hMnk2 according to full-length cDNA, totally 414 amino-acid residues, molecular weight 46725.28 dalton, pI are 5.99.Detailed sequence is seen SEQ ID NO.7.

Full length cDNA sequence and coded protein thereof with hMnk2, in Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDStranslations+PDB+SwissProt+Superdate+PIR database, carry out the retrieval of Nucleotide and protein homology with blast program, found that all there is higher homology in the Mnk protein gene in it and people, mouse and the Africa xenopus.On nucleotide level, the 2-1106 bit base of the mRNA whole coding sequence (GenBank AccessionNo.Y11092) of it and mouse Mnk2 gene has 87.6% homogeny (Fig. 1).On amino acid levels, it and Mnk albumen all have higher homology, with mouse Mnk2 albumen 96.4% homogeny (Fig. 2) are arranged.Thereby can determine that hMnk2 albumen of the present invention is a kind of mitogen-activated protein(MAP) kinase enzyme related protein kinase.

People hMnk2 of the present invention is used for further functional study except can be used as this family's a member, also can be used for producing fusion rotein with other albumen, such as producing fusion rotein with immunoglobulin (Ig).In addition, inventor hMnk2 can also merge with other members of this family or exchange fragment, to produce new albumen.For example the N end of the N of inventor hMnk2 end with the Mnk2 of mouse exchanged, to produce the albumen that new activity is higher or have new features.

At the antibody of inventor hMnk2, be used to screen other members of this family, perhaps be used for affinity purification associated protein (as other members of this family).

In addition, inventor hMnk2 nucleic acid (encoding sequence or antisense sequences) can be introduced into cell, with expression level that improves people hMnk2 or the overexpression that suppresses people hMnk2.People hMnk2 albumen of the present invention or its active polypeptide fragment can be applied to patient, with treatment or alleviate because of people hMnk2 disappearance, no function or unusual cause related disorders arranged.In addition, can also be with carrying out relevant diagnosis or prognosis judgement based on nucleotide sequence of the present invention or antibody.

Embodiment 3

The proteic 26S Proteasome Structure and Function research of hMnk2:

1．hMnk2PROSITE ( ：http://www.motif.genome.ad.jp/motif-bin/ ) ( motif ) ,： 1 MVQKKPAELQ GFHRSFKGQN PFELAFSLDQ PDHGDSDFGL QCSARPDMPA 51 SQPIDIPDAK KRGKKKKRGR ATDSFSGRFE DVYQLQEDVL GEGAHARVQT101 CINLITSQEY AVKIIEKQPG HIRSRVFREV EMLYQCQGHR NVLELIEFFE151 EEDRFYLVFE KMRGGSILSH IHKRRHFNEL EASVVVQDVA SALDFLHNKG201 IAHRDLKPEN ILCEHPNQVS PVKICDFDLG SGIKLNGDCS PISTPELLTP251 CGSAEYMAPE LVEAFSEEAS IYDKRCDLWS LGVILYILLS GYPPFVGRCG301 SDCGWDRGEA CPACQNMLFE SIQEGKYEFP DKDWAHISCA AKDLISKLLV351 RDAKQRLSAA QVLQHPWVQG CAPENTLPTP MVLQRWDSHF LLPPHPCRIH401 VRPGGLVRTV TVNE

(1) in aminoacid sequence, there is following function motif:

(ⅰ) underscore district (39..44 165..170 232..237 300..305 405..410) is amidated site (amidation site);

(ⅱ) runic district (15..17 43..45 76..78) is protein kinase C phosphorylation site (Proteinkinase C phosphorylation site);

(ⅲ) italic district (106..109 191..194 243..246 270..273 321..324 411..414) is casein kinase phosphorylation site (Casein kinase II phosphorylation site);

(ⅳ) bold Italic district (128..134) is Tyrosylprotein kinase phosphorylation site (Tyrosine kinasephosphorylation site);

(ⅴ) wavy line district (90..113) is protein kinase A TP feature calmodulin binding domain CaM (Protein kinasesATP-binding region signature);

(ⅵ) two-wire district (201..213) is serine/threonine protein kitase feature activation site (Serine/Threonine protein kinases active-site signature).

(2) functional analysis:

Site (1)-(4) are relevant with posttranslational modification (post-translational modifications), are the sites that the hMnk2 activity is regulated and control, and can regulate its active its physiological function that realizes; Site (5) is the ATP-binding domain territory, and ATP is essential by catalyzed reaction provides energy to be that this enzymatic activity is achieved; Site (6) is serine/threonine protein kitase feature activation site, and promptly MAP is to its site that acts on.In sum, to the result of study explanation of motif, hMnk2 is the relevant modulin in the proteic downstream of MAP.

2. the hMnk2 amino acid sequence is retrieved in model (pattern) database (network address is www.isrec.isb-sib.ch) and obtained following result: 1 MVQKKPAELQ GFHRSFKGQN PFELAFSLDQ PDHGDSDFGL QCSARPDMPA, 51 SQPIDIPDAK KRGKKKKRGR ATDSFSGRFE DVYQLQEDVL GEGAHARVQT101 CINLITSQEY AVKIIEKQPG HIRSRVFREV EMLYQCQGHR NVLELIEFFE151 EEDRFYLVFE KMRGGSILSH IHKRRHFNEL EASVVVQDVA SALDFLHNKG201 IAHRDLKPEN ILCEHPNQVS PVKICDFDLG SGIKLNGDCS PISTPELLTP251 CGSAEYMAPE LVEAFSEEAS IYDKRCDLWS LGVILYILLS GYPPFVGRCG301 SDCGWDRGEA CPACQNMLFE SIQEGKYEFP DKDWAHISCA AKDLISKLLV351 RDAKQRLSAA QVLQHPWVQG CAPENTLPTP MVLQRWDSHF LLPPHPCRIH401 VRPGGLVRTV TVNE

(1) in aminoacid sequence, there is following functional domain:

(ⅰ) italic district (84..368): protein kinase structural domain (Protein kinase domain profile);

(ⅱ) runic district (83..368): eukaryotic protein kinase structural domain (Eukaryotic protein kinasedomain);

(ⅲ) wavy line district (181..236): the specific, activated site of tyrosine protein kinase (Tyrosine proteinkinases specific active-site signature);

(ⅳ) underscore district (90..113): protein kinase A TP feature calmodulin binding domain CaM (Protein kinases ATP-binding region signature);

(ⅴ) double underline district (201..213): the special activation of serine/threonine protein kitase site (Serine/Threonine protein kinases active-site signature).

(2) structural analysis:

Structural domain (ⅰ), (ⅱ) and (ⅳ) illustrated that people hMnk2 of the present invention has the physiological function as protein kinase; Structural domain (ⅲ), (ⅴ) are the action site of upstream protein kinase, have illustrated that hMnk2 has the structure of map kinase downstream modulin, thereby have had its function.

In sum, confirmed further that from proteic structure of hMnk2 and physicochemical property this gene is the member of Mnk gene family.Because protein structure has determined the specific biochemical theory of function, therefore people hMnk2 gene of the present invention has and the similar or identical functions of other Mnk protein gene.

Embodiment 4

The distribution expression pattern of hMnk2 gene

1. electronics Northern express spectra.Press people's such as Ton C. method (Ton C et al., BiochemBiophys Res Commun 1997 Dec 18; 241 (2): 589-594; Hwang DM, et al., Circulation 1997 Dec 16; 96 (12): 4146-4203), the BLAST retrieval will be done in the dbEST database of hMnk2 cDNA sequence in the GCG software package, in the human EST that obtains, the EST of probable value＜10e-10, homogeny＞95% has 21, can be considered the transcriptional expression of this gene in tissue originally, draw the tissue spectrum of expressing this gene thus, find that it all has expression in thymus gland, B cell germinal center, pineal gland, heart, lung, pancreas, embryo, neuro epithelium, uterus, testis, kidney, lymphoma, oophoroma, gland cancer and carcinoid tumor cell.This shows that this albumen all bringing into play important effect in the many tissues of human body.

Embodiment 5

The preparation of hMnk2 polypeptide and purification

In this embodiment, the hMnk2 encoding sequence of total length or fragment are built into commercial protein merge among the expression vector, to express and purification of recombinant proteins.

HMnk2 albumen or polypeptide carry out prokaryotic expression with the form of gst fusion protein in intestinal bacteria.

(a) construction of prokaryotic expression vector, and transformed into escherichia coli

Complete encoding sequence (SEQ ID NO.6) according to people hMnk2, design amplifies complete coding and reads the primer of frame (correspond respectively to encoding sequence 5 ' and about 20 above Nucleotide of 3 ' end), and on positive anti-primer, introduce restriction endonuclease sites (this is decided by the carrier of selecting for use) respectively, so that construction of expression vector.With the amplified production that obtains among the embodiment 1 is template, behind pcr amplification, with the hMnk2 gene guarantee to read be cloned under the correct prerequisite of frame the pGEX-2T carrier (Pharmacia, Piscataway, NJ).Identify that good expression vector utilizes CaCl ₂Method changes bacillus coli DH 5 alpha over to, and Screening and Identification obtains containing the engineering bacteria DH5 α-pGEX-2T-hMnk2 of pGEX-2T-hMnk2 expression vector.

(b) isolation identification of the engineering bacteria of expression GST-hMnk2 recombinant protein

DH5 α-pGEX-2T-hMnk2 the engineering bacteria of picking list bacterium colony contains jolting overnight incubation in the LB substratum of 100 μ g/ml penbritins in 3ml, draw nutrient solution by 1: 100 concentration and in new LB substratum (containing 100 μ g/ml penbritins), cultivated about 3 hours, to OD ₆₀₀After reaching 0.5, adding IPTG continues at 37 ℃ to final concentration 1mmol/L and cultivated respectively 0,1,2,3 hours.It is centrifugal to get the different 1ml bacterium liquid of incubation time, in the bacterial precipitation thing, add lysate (2 * SDS sample-loading buffer, 50 μ l, distilled water 45 μ l, 3-mercaptoethanol 5 μ l), the suspendible bacterial precipitation, boiled in the boiling water bath 5 minutes, 10000 left the heart 1 minute, and supernatant adds electrophoresis in the 12%SDS-PAGE glue.The bacterial strain that the protein content of dyeing back observation expection molecular weight size increases with the IPTG induction time is the engineering bacteria of expressing the GST-hMnk2 fusion rotein.

(c) the extraction purifying of GST-hMnk2 fusion rotein

The proteic engineering bacteria DH5 of abduction delivering GST-hMnk2 amalgamation and expression α-pGEX-2T-hMnk2 as stated above.Bacterium centrifugation after inducing adds the resuspended bacterium of 20ml PBS, ultrasonication bacterium by every 400ml bacterium.The ultrasonic completely liquid of broken bacterium adds 50% saturated Triptide Sepharose 4B of PBS by every milliliter of amount that adds 20 microlitres, 37 ℃ of joltings were in conjunction with 30 minutes, 10000rpm/min precipitated the Triptide Sepharose 4B that combines GST-hMnk2 in centrifugal 10 minutes, abandoned supernatant.Clean twice by the amount that every milliliter of ultrasonic liquid gained precipitation adds 100 μ l PBS, then add 10 μ l reduced glutathione elutriants by every milliliter of ultrasonic liquid gained precipitation, room temperature was put 10 minutes, centrifugal 10 minutes of 10000rpm/min, and supernatant is the fusion rotein of wash-out.Repeat twice of wash-out.The supernatant of wash-out is stored in-80 ℃, and carries out the SDS-PAGE electrophoresis, detects purification effect.Band at about 47kDa place is hMnk2 albumen.

Embodiment 6

HMnk2 albumen or polypeptide carry out eukaryotic cell expression in insect cell

1.hMnk2 the structure of rhabdovirus expression vector and transfection Sf 9 insect cell strain

According to the complete encoding sequence (SEQ ID NO.6) of people hMnk2, design amplifies the primer that complete coding is read frame, and introduces restriction endonuclease sites (this is decided by the carrier of selecting for use) respectively on positive anti-primer, so that construction of expression vector.With the amplified production that obtains among the embodiment 1 is template, behind pcr amplification, with hMnk2cDNA under the prerequisite that guarantees reading frame, be cloned into the pVL1392 carrier (Invitrogen, Carlsbad, CA).Identify good expression vector 3 μ g, wild-type linearized baculovirus dna (BaculoGold ^TMACMNPV DNA, Pharmingen, San Diego, CA) 1 μ g and Lipofection (Gibco-BRL, NY) 25 μ l add in the insect substratum of 1ml serum-free, the 15 seconds mixings that vibrate, incubated at room 15 minutes is standby.Get 1ml (2 * 10 ⁶) Sf9 insect cell suspension is in 60mm tissue culturing plate, change transfection media after adherent 1 hour, incubated at room was abandoned substratum after 15 minutes, add the dna vector transfection mixture for preparing previously, Parafilm seals culture plate, cultivated 4 hours in 27 ℃ of joltings of room temperature, then change perfect medium and cultivated 3 days, it is standby to collect supernatant.

(2) change the Screening and Identification of the insect cell line of recombinant expression vector over to

The insect cell of transfection after 3 days made cell suspension (2 * 10 with fresh culture ⁶/ 1ml), get the 1ml cell suspension and place 60mm tissue culture ware, add the 3ml substratum, the culture supernatant that 100 μ l collect, adherent 1 hour, abandon the 2ml substratum, continue incubated at room temperature 1 hour, and discarded all substratum, add the 3ml semisolid medium that contains 20 μ l 4%X-gal of preheating, cultivate after 5-7 days picking white cell clone and in 96 well culture plates, cultivated 3-5 days, then draw supernatant infection Sf9 insect cell.

Collect the cell that infects and carry out the Western evaluation.The SDS-PAGE electrophoresis will be carried out after the lysis, glue behind the electrophoresis prints to protein transduction on the nitrocellulose membrane in the half-dried electrotransfer instrument of the Multiphor of Pharmacia II, nitrocellulose membrane is placed confining liquid sealing 1 hour, then in the antibody-solutions of anti-hMnk2, sealed 1 hour, the jolting of TBS liquid is cleaned 5 minutes 2 times totally, then film is placed the anti-second antibody solution jolting of biotin labeled anti-hMnk2 one 1 hour, TBS cleans, adding avidin-alkaline phosphatase enzyme complex reacted 30 minutes, TBS cleans 2 times, adds freshly prepared colour developing liquid colour developing and observes protein band.

The Sf9 cell clone of picking high expression level hMnk2.

(3) the proteic extraction purifying of hMnk2

Supernatant with the Sf9 cell clone of high expression level hMnk2 infects the Sf9 cell in a large number, infects collecting cell after 48 hours, the PBS washing.Per 2 * 10 ⁸Cell adds 20ml cell pyrolysis liquid (0.5%Triton X-100,20 mM Na ₃PO ₄(sodium phosphate, pH7.8), 500mM NaCl, 1mM Na ₃VO ₄(vanadic acid sodium), 1mM Pefabloc, 1 μ g/ml pepstatin, leupeptin and aprotinin) broken cell, the centrifugal 20min of 12000 * g removes cell debris, and supernatant is by per 2 * 10 ⁸Cell add 2ml NTA-agarose (Qiagen, Germany), 4 ℃ of absorption 1 hour.Then with containing the His damping fluid washed twice of 100 nM imidazoles, with containing 20mM N, N '-two piperazine, 500mM NaCl, the buffer solution elution of 300mM imidazoles is to obtain the albumen of purifying.Elutriant is stored in 4 ℃, and carries out the proteic purity of hMnk2 that the SDS-PAGE electrophoresis detection is extracted.Band at about 47kDa place is hMnk2 albumen.

Embodiment 7

The preparation of anti-people hMnk2 antibody

1. the preparation of immune mouse and splenocyte: separate the people hMnk2 recombinant protein molecule that obtains among the embodiment 5 and 6 back standby with chromatography, also can separate with the SDS-PAGE gel electrophoresis, electrophoretic band is cut off from gel, and with isopyknic complete Freund ' s adjuvant emulsion.Get the female mouse of 6-8 week Balb/C in age, the albumen with 50-100 μ g/0.2ml emulsification carries out peritoneal injection to mouse.After 14 days, with the same antigen of non-complete Freund ' s adjuvant emulsion to mouse with the dosage of 50-100 μ g/0.2ml again booster immunization once be used for after 3-5 days merging.Wherein, E Zheng chief editor, " tissue culture and molecular cell learn a skill ", Beijing Publishing House, the 210th page are seen in the splenocyte preparation.

2. by " tissue culture and molecular cell learn a skill " (the same), the method in the 211st page, preparation feeder cell.

3. by " tissue culture and molecular cell learn a skill " (the same), the method in the 213rd page is carried out cytogamy.

4. detection of antibodies: after cytogamy 10-15 days, need to check by the hole, in case find vigorous hybrid cell colony growth, just use hMnk2 albumen and do the preliminary screening of antibody activity, method commonly used has: immunofluorescent test, emission immunity test (RIA), enzyme linked immunosorbent assay (ELISA).After checking out the hole of antibody activity, clone cultivation at once, and isolate antibody.

All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.

Sequence table (1) general information: (ⅰ) applicant: Nanfang Research Centre, State Human Gene Group (ⅱ) denomination of invention: new HUMAN MAP KINASE-INTERACTING KINASE 2 and encoding sequence thereof (ⅲ) sequence number: the information of 7 (2) SEQ ID NO.1

(ⅰ) sequence signature:

(A) length: 50bp

(B) type: Nucleotide

(C) chain: strand

(D) topological framework: linearity

(ⅱ) molecule type: oligonucleotide (ⅸ) sequence description: the information of SEQ ID NO.1GAGAGAGAGA GAGAGAGAGA ACTAGTCTCG AGTTTTTTTT TTTTTTTTTT 50 (2) SEQ ID NO.2

(ⅰ) sequence signature:

(A) length: 13bp

(B) type: Nucleotide

(C) chain: strand

(D) topological framework: linearity

(ⅱ) molecule type: oligonucleotide (ⅸ) sequence description: the information of SEQ ID NO.2AATTCGGCAC GAG 13 (2) SEQ ID NO.3

(ⅰ) sequence signature:

(A) length: 9bp

(B) type: Nucleotide

(C) chain: strand

(D) topological framework: linearity

(ⅱ) molecule type: oligonucleotide (ⅸ) sequence description: the information of SEQ ID NO.3GCCGTGCTC 9 (2) SEQ ID NO.4

(ⅰ) sequence signature:

(A) length: 22bp

(B) type: Nucleotide

(C) chain: strand

(D) topological framework: linearity

(ⅱ) molecule type: oligonucleotide (ⅸ) sequence description: the information of SEQ ID NO.4CCGGACAGAA GATGGTGCAG 20 (2) SEQ ID NO.5

(ⅰ) sequence signature:

(A) length: 19bp

(B) type: Nucleotide

(C) chain: strand

(D) topological framework: linearity

(ⅱ) molecule type: oligonucleotide (ⅸ) sequence description: the information of SEQ ID NO.5CGGCATTGAC AGTTGGTGTA AA 22 (2) SEQ ID NO.6

(ⅰ) sequence signature:

(A) length: 1473bp

(B) type: Nucleotide

(C) chain: two strands

( D ) ： ( ⅱ ) ： ( ⅸ ) ：SEQ ID NO.6CCGGACAGAA GATGGTGCAG AAGAAACCAG CCGAACTTCA GGGTTTCCAC CGTTCGTTCA 60AGGGGCAGAA CCCCTTCGAG CTGGCCTTCT CCCTAGACCA GCCCGACCAC GGAGACTCTG 120ACTTTGGCCT GCAGTGCTCA GCCCGCCCTG ACATGCCCGC CAGCCAGCCC ATTGACATCC 180CGGACGCCAA GAAGAGGGGC AAGAAGAAGA AGCGCGGCCG GGCCACCGAC AGCTTCTCGG 240GCAGGTTTGA AGACGTCTAC CAGCTGCAGG AAGATGTGCT GGGGGAGGGC GCTCATGCCC 300GAGTGCAGAC CTGCATCAAC CTGATCACCA GCCAGGAGTA CGCCGTCAAG ATCATTGAGA 360AGCAGCCAGG CCACATTCGG AGCAGGGTTT TCAGGGAGGT GGAGATGCTG TACCAGTGCC 420AGGGACACAG GAACGTCCTA GAGCTGATTG AGTTCTTCGA GGAGGAGGAC CGCTTCTACC 480TGGTGTTTGA GAAGATGCGG GGAGGCTCCA TCCTGAGCCA CATCCACAAG CGCCGGCACT 540TCAACGAGCT GGAGGCCAGC GTGGTGGTGC AGGACGTGGC CAGCGCCTTG GACTTTCTGC 600ATAACAAAGG CATCGCCCAC AGGGACCTAA AGCCGGAAAA CATCCTCTGT GAGCACCCCA 660ACCAGGTCTC CCCCGTGAAG ATCTGTGACT TCGACCTGGG CAGCGGCATC AAACTCAACG 720GGGACTGCTC CCCTATCTCC ACCCCGGAGC TGCTCACTCC GTGCGGCTCG GCGGAGTACA 780TGGCCCCGGA GTTAGTGGAG GCCTTCAGCG AGGAGGCTAG CATCTACGAC AAGCGCTGCG 840ACCTGTGGAG CCTGGGCGTC ATCTTGTATA TCCTACTCAG CGGCTACCCG CCCTTCGTGG 900GCCGCTGTGG CAGCGACTGC GGCTGGGACC GCGGCGAGGC CTGCCCTGCC TGCCAGAACA 960TGCTGTTTGA GAGCATCCAG GAGGGCAAGT ACGAGTTCCC CGACAAGGAC TGGGCCCACA 1020TCTCCTGCGC TGCCAAAGAC CTCATCTCCA AGCTGCTGGT CCGTGACGCC AAGCAGAGGC 1080TGAGTGCCGC CCAAGTCCTG CAACACCCCT GGGTTCAGGG GTGCGCCCCG GAGAACACCT 1140TGCCCACTCC CATGGTCCTG CAGAGGTGGG ACAGTCACTT CCTCCTCCCT CCCCACCCCT 1200GTCGCATCCA CGTGCGACCT GGAGGACTGG TCAGAACCGT TACTGTGAAT GAGTGAAGAT 1260CCTGGAGGAC CCTGGCCCCA GGCCAGCTCC CATCGCTGGG GGACGGTGAA CGGCCATGTG 1320TTAATGTTAC GATGTTTTTA AAAGACAAAA AAAAAAAAAA AACCTCAAAA GTTTTTTTAA 1380AGTGGGGGAA AAACATCCAA GCACTTTAAT TCCAATGTAC CAGGTGAACT GACGGAGCTC 1440AGAAGTTTTC CTTTACACCA ACTGTCAATG CCG 1473 ( 2 ) SEQ ID NO.7

(ⅰ) sequence signature:

(A) length: 414 amino acid

(B) type: amino acid

(C) chain: strand

(D) topological framework: linearity

(ⅱ) molecule type: polypeptide (ⅸ) sequence description: SEQ ID NO.7MVQKKPAELQ GFHRSPKGQN PFELAFSLDQ PDHGDSDFGL QCSARPDMPA SQPIDIPDAK 60KRGKKKKRGR ATDSFSGRFE DVYQLQEDVL GEGAHARVQT CINLITSQEY AVKIIEKQPG 120HIRSRVFREV EMLYQCQGHR NVLELIEFFE EEDRFYLVFE KMRGGSILSH IHKRRHFNEL 180EASVVVQDVA SALDFLHNKG IAHRDLKPEN ILCEHPNQVS PVKICDFDLG SGIKLNGDCS 240PISTPELLTP CGSAEYMAPE LVEAFSEEAS IYDKRCDLWS LGVILYILLS GYPPFVGRCG 300SDCGWDRGEA CPACQNMLFE SIQEGKYEFP DKDWAHISCA AKDLISKLLV RDAKQRLSAA 360QVLQHPWVQG CAPENTLPTP MYLQRWDSHF LLPPHPCRIH VRPGGLVRTV TVNE 414

Claims (10)

1. isolated dna molecular is characterized in that it comprises: coding has the nucleotide sequence of the polypeptide of people hMnk2 protein active,

And, show at least 70% homology from the nucleotides sequence of Nucleotide 12-1253 position dna molecular among described nucleotide sequence and the SEQ ID N0.6; Perhaps described nucleotide sequence can be under the moderate stringent condition with SEQ ID NO.6 in from the nucleotide sequence hybridization of Nucleotide 12-1253 position.

2. dna molecular as claimed in claim 1 is characterized in that described sequence encoding has polypeptide of sequence shown in the SEQ IDNO.7.

3. dna molecular as claimed in claim 1 is characterized in that, this sequence has among the SEQ ID NO.6 nucleotide sequence from Nucleotide 12-1253 position.

4. isolated people hMnk2 protein polypeptide is characterized in that it comprises: have polypeptide or its conservative property variation polypeptide or its active fragments of SEQ ID NO.7 aminoacid sequence, or its reactive derivative.

5. polypeptide as claimed in claim 4 is characterized in that, this polypeptide is to have SEQ ID NO.7 polypeptide of sequence.

6. a carrier is characterized in that, it comprises the described DNA of claim 1.

7. one kind with the described carrier transformed host cells of claim 6.

8. a generation has the method for the polypeptide of people hMnk2 protein active, it is characterized in that this method comprises:

(1) nucleotide sequence that coding is had a polypeptide of people hMnk2 protein-active operationally is connected in expression regulation sequence, form people hMnk2 protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 12-1253 position among described nucleotide sequence and the SEQ ID NO.6;

(2) change the expression vector in the step (1) over to host cell, form the proteic reconstitution cell of people hMnk2;

(3) under the condition that is fit to expressing human hMnk2 protein polypeptide, the reconstitution cell in the culturing step (2);

(4) isolate polypeptide with people hMnk2 protein-active.

9. energy and the described people hMnk2 of claim 7 protein polypeptide specificity bonded antibody.

10. a probe molecule is characterized in that, it comprises 8-100 continuous nucleotide in the described dna molecular of claim 1.

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