CN1255547A - Human protein and its coding sequence, preparing process and application - Google Patents

Abstract

The present invention discloses a new human gene nucleotide sequence, that is, the CDNA sequence of human nek4. The protein coded with it is the honolog of murinus nek/protein and human nek protein. The polypeptide coded with said nucleotide sequence, the application of said polynucleotide and polypeptide, and the process for preparing said polynucleotide and polypeptide are disclosed.

Description

A kind of new people's albumen and encoding sequence thereof, and method for making and purposes

The present invention relates to the genetically engineered field, particularly, the present invention relates to a kind of new human gene nucleotide sequence.More particularly, the present invention relates to the cDNA sequence of people nek4, this albumen is mouse nek1 and the proteic homologue of people nek.The invention still further relates to by this nucleotide sequence coded polypeptide the application of these polynucleotide and polypeptide, and the production method of described polynucleotide and described polypeptide.

NIMA, nek1 are two genes finding from fungi and mouse respectively, belong to two members in the serine/threonine protein kitase family of cell cycle regulation, and they are considered to regulate the cell division cycle activity relevant.Cell division cycle is the ubiquitous biological phenomena of organic sphere, is the significant process of higher organism cell proliferation and growth.Cell division cycle is finished by common adjusting of various kinds of cell cyclin, and these cyclins are normal regulatory functions of performance under the regulating effect of the protein kinase of cyclin dependent.

The serine/threonine protein kitase of cell cycle regulation all extensively exists in various cell type and population, and has the conservative property of height, from the fungi to the mouse and the mankind all contain this albumen.People find and clone to have obtained NIMA gene (J Cell.Biol 96,1155-1158,1983) from Aspergillus nidulans (Aspergillus.nidulans); Subsequently, people such as Kenneth Letwin separated from mouse in 1992 obtained the nek1 gene (EMBOJ.1992,11 (10), 3521-3531).People such as Sharo J. have obtained people nek1, nek2 and nek3 gene (Cell Growth in separation in 1994; Differ.1994,5,625-635).Discover, these genes on structure and function all be high conservative (Proc.Natl.Acad.Sci.USA 1998,95:114-119).

Yet, before the present invention, this new albumen of the present invention or its encoding sequence were not disclosed.

An object of the present invention is to provide a kind of new polynucleotide sequence, this polynucleotide sequence coding people's a kind of and mouse nek1 homologous albumen, people of the present invention is named as people nek4 with mouse nek1 homologous gene.

Another object of the present invention provides a kind of new albumen, and this albumen is named as people nek4 albumen.

A further object of the present invention provides a kind of recombinant technology that utilizes and produces the described new proteic method of people nek4.

The present invention also provides the application of this people nek4 nucleotide sequence and protein polypeptide.

In one aspect of the invention, a kind of isolated dna molecular is provided, it comprises: coding has the nucleotide sequence of the polypeptide of people nek4 protein-active, shows at least 70% homology from the nucleotides sequence of Nucleotide 78-1019 position among described nucleotide sequence and the SEQ ID NO.5; Perhaps described nucleotide sequence can be hybridized from the Nucleotide of Nucleotide 78-1019 position under the moderate stringent condition with among the SEQ ID NO.5.Preferably, described sequence encoding one polypeptide, this polypeptide has the sequence shown in the SEQ ID NO.6.More preferably, this sequence has the nucleotide sequence of 78-1019 position among the SEQ ID NO.5.

In another aspect of this invention, provide a kind of isolating people nek4 protein polypeptide, it comprises: have polypeptide or its active fragments of SEQ ID NO.6 aminoacid sequence, or its reactive derivative.Preferably, this polypeptide is to have SEQ ID NO.6 polypeptide of sequence.

In another aspect of this invention, provide a kind of carrier, it contains above-mentioned isolated DNA.

In another aspect of this invention, provide a kind of described carrier transformed host cells.

In another aspect of this invention, provide a kind of generation to have the method for the polypeptide of people nek4 protein-active, this method comprises:

(a) nucleotide sequence that coding is had a polypeptide of people nek4 protein-active operationally is connected in expression regulation sequence, form people nek4 protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 78-1019 position among described nucleotide sequence and the SEQ ID NO.5;

(b) change the expression vector in the step (a) over to host cell, form the proteic reconstitution cell of people nek4;

(c) under the condition that is fit to expressing human nek4 protein polypeptide, the reconstitution cell in the culturing step (b);

(d) isolate polypeptide with people nek4 protein-active.

In a specific embodiments of the present invention, isolating polynucleotide total length of the present invention is 1189 Nucleotide, and its detailed sequence is seen SEQ ID NO.5, and wherein open reading frame is positioned at 78-1019 position Nucleotide.

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 nek4 albumen (or polypeptide) encoding sequence " refer to the encode nucleotide sequence of polypeptide with people NEK4 protein-active is as 78-1019 position nucleotide sequence and degenerate sequence thereof among the SEQ ID NO.5.This degenerate sequence is meant, is arranged in the encoder block 78-1019 position Nucleotide of SEQ ID NO.5 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.5 in 78-1019 position nucleotide sequence homology be low to moderate about 70% degenerate sequence described sequence of SEQ ID NO.6 of also encoding out.This term also comprises can be under the moderate stringent condition, more preferably under the height stringent condition with SEQ ID NO.5 in from the nucleotide sequence of the nucleotide sequence hybridization of Nucleotide 78-1019 position.Also term also comprise with SEQ ID NO.5 in from Nucleotide 78-1019? homology of nucleotide sequence at least 70%, preferably at least 80%, at least 90% nucleotide sequence more preferably.

This term also comprises encoding to have variant form with proteic, the SEQ ID NO.6 sequence of people Nek4 identical function.These variant forms comprise (but being not limited to): the disappearance of several Nucleotide, insertion and/or replacement, and at 5 ' and/or 3 ' several Nucleotide of end interpolation.

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 " nek4 protein polypeptide " refers to have the SEQ ID NO.6 polypeptide of sequence of people nek4 protein-active.This term also comprises having and variant form people nek4 identical function, SEQ ID NO.6 sequence.These variant forms comprise (but being not limited to): several amino acid whose disappearances, insertion and/or replacement, and at C-terminal and/or N-terminal interpolation one or several 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 people nek4 and reactive derivative.

The variant form of this polypeptide comprises: homologous sequence, allelic variant, natural mutation, induced mutation body, under high or low rigorous degree condition can with the coded albumen of the DNA of people nek4 DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-people nek4 polypeptide to obtain.The present invention also provides other polypeptide, as comprises people nek4 polypeptide or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention also comprises the soluble fragments of people nek4 polypeptide.Usually, this fragment have people nek4 polypeptid coding 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.

Invention also provides the analogue of people nek4 albumen or polypeptide.The difference of these analogues and natural human nek4 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 glycosylation 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, " people nek4 conservative property variation polypeptide " refers to compare with the aminoacid sequence of SEQ ID No.6, has 10 at the most, preferably at the most 8, more preferably at the most 5,3 amino acid is replaced by similar performance or close amino acid and is formed polypeptide at the most best.These conservative property variation polypeptide preferably carry out the amino acid replacement according to table 1 and produce.

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; Nor-leucine	????Leu
			Leu(L)	Nor-leucine; 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; Nor-leucine	????Leu

The present invention also comprises people nek4 polypeptid coding sequence and segmental antisense sequences thereof.This antisense sequences can be used for suppressing the expression of people nek4 in the cell.

The present invention also comprises a kind of nucleic acid molecule that can be used as probe or primer, and this molecule has 8-100 of people nek4 polypeptid coding sequence, preferably 15-50 continuous nucleotide usually.This probe can be used for whether existing in the test sample nucleic acid molecule of coding people nek4.

The present invention also comprises the method that detects people nek4 nucleotide sequence, 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 encoding sequence of people nek4 polypeptide, and can be positioned at the both sides or the centre of this encoding sequence.Primer length is generally 20-50 Nucleotide.

In the present invention, can select various carrier known in the art for use, as commercially available carrier.

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.

On the other hand, the present invention also comprises people nek4 DNA or the polypeptide of its fragment coding has specific polyclonal antibody and monoclonal antibody, especially monoclonal antibody.Here, " specificity " is meant that antibody capable is incorporated into people nek4 gene product or fragment.Preferably, refer to that those can combine with people nek4 gene product or fragment but nonrecognition and be incorporated into the antibody of other irrelevant antigen molecule.Among the present invention antibody comprise those can in conjunction with and suppress the proteic molecule of people nek4, comprise that also those do not influence the antibody of people nek4 protein function.The present invention also comprise those can with modify or without the people nek4 gene product bonded antibody of modified forms.

The present invention not only comprises complete mono-clonal or polyclonal antibody, but also comprises having immunocompetent antibody fragment, as Fab ' or (Fab) ₂Fragment; Heavy chain of antibody; Light chain of antibody; Genetically engineered strand Fv molecule (people such as Ladner, U.S. Patent No. 4,946,778); Or chimeric antibody, as have the murine antibody binding specificity but still keep antibody from people's antibody moiety.

Antibody of the present invention can be prepared by the known various technology of those skilled in that art.For example, the people nek4 gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing human nek4 or its have antigenic segmental cell and can be used to immune animal and produce antibody.Antibody of the present invention also can be monoclonal antibody.This type of monoclonal antibody can utilize hybridoma technology to prepare that (see people such as Kohler, Nature 256; 495,1975; People such as Kohler, Eur.J.Immunol.6:511,1976; People such as Kohler, Eur.J.Immunol.6:292,1976; People such as Hammerling, In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981).Antibody of the present invention comprises the antibody that can block people nek4 function and the antibody that does not influence people nek4 function.Each antibody-like of the present invention can utilize the fragment or the functional zone of people nek4 gene product, obtains by the routine immunization technology.These fragments or functional zone can utilize recombinant methods or utilize Peptide synthesizer synthetic.Can come immune animal and produce with the gene product of producing in the prokaryotic cell prokaryocyte (for example E.Coli) with the unmodified form bonded antibody of people nek4 gene product; With posttranslational modification form bonded antibody (as the albumen or the polypeptide of glycosylation or phosphorylation), can come immune animal and obtain with the gene product that produces in the eukaryotic cell (for example yeast or insect cell).

People nek4 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 relevant nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or the cDNA storehouse of press the currently known methods preparation as template, increase and must be about 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 synthetic is synthesized relevant sequence, especially fragment length more in short-term.Usually, by first synthetic a plurality of small segments, and then connect and to obtain the very long fragment of sequence.

NIMA, nek1 are the serine/threonine protein kitase genes of two cell cycle regulations finding in fungi and the mouse respectively.The nek1 height homology of people nek4 gene of the present invention and mouse, simultaneously also higher with the same cell cycle regulating serine/threonine protein kitase source property of fungi NIMA, a member in the serine/threonine protein kitase family of this announcement nek4 genus cell cycle regulation, relevant with the regulating effect of cell division cycle, and nek4 is as the homologous gene of mouse nek1 in the people, may have similar in appearance to the biological function of mouse nek1, as regulating the propagation and the process of growth of cell.

In the accompanying drawings, Fig. 1 is the homology comparison diagram of people nek4 albumen of the present invention (HNEK4) with the aminoacid sequence of people nek1, nek2, nek3 albumen (being designated as HNEK1, HNEK2 and HNEK3 respectively).Wherein, identical amino acid marks with " * ".Conservative amino acid marks with " ".

Fig. 2 is the homology comparison diagram of the aminoacid sequence of people nek4 albumen of the present invention (HNEK4) and mouse nek1 albumen (NEK1P).Wherein, identical amino acid marks with " | " between two sequences, and similar amino acid marks with " ".Similar amino acid is: A, S, T; D, E; N, Q; R, K; I, L, M, V; F, Y, W.

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, such as people such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring HarborLaboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

Embodiment 1:

The clone and the mensuration of the cDNA sequence of people nek4

1. material

8 kinds of people cDNA library of moleculess (deriving from human liver, kidney, brain, cardiac muscle, marrow, placenta, muscle and peripheral blood leucocyte respectively) and contain MTN (the Multiple Tissue Northern) film (deriving from human liver, brain, kidney, placenta, lung, skeletal muscle, pancreas, spleen, thymus gland, prostate gland, testis, ovary, small intestine, colon and peripheral blood leucocyte respectively) of 16 kinds of tissue mRNA are all available from U.S. Clontech company; Two primers on the λ gt11 carrier: S1 primer 5 '-GTT CAA CAT CAG CCG CTA CA-3 ' (SEQ IDNO.1) and S2 primer 5 '-CAC CAG ACC AAC TGG TAA TG-3 ' (SEQ ID NO.2) and two special primers: A2 primer 5 '-CTG TGC ATC CTC CTG ACC CAC AG-3 ' (forward) (SEQ IDNO.3), B primer 5 '-CCT CCC TCA GCT GCT CTG CCT TC-3 ' (oppositely) (SEQ IDNO.4).Above primer is synthetic by Shanghai Sangon company entirely.

2.cDNA segmental pcr amplification and clone

Special primer and the collocation of carrier primer in 8 kinds of cDNA library of moleculess, use PCR instrument (PT-200 type U.S. PE company) to increase.Amplified production utilizes special primer collocation amplification again.The PCR reaction conditions :-72 ℃ of-62 ℃ of annealing of 93 ℃ of pre-sex change-93 ℃ of sex change in 3 minutes 1 minute 1 minute extend 1 minute-72 ℃ extended totally 35 circulations again 5 minutes.Amplified production carries out sequence verification at sequenator (377 type U.S. PE company) after with PCR purification kit (German QIAGEN company) amplified production purifying.The amplified production of purifying is further packed on the carrier pGEMT (U.S. Promage company), is cloned in the JM103 intestinal bacteria (U.S. Promage company).

3.cDNA the screening of full-length clone and evaluation

The fragment that adopts the PCR method mark to be increased.Mark system: each 1 μ l of 25pmol/ μ lA2 primer and B primer, 2mmol/ μ ldGTP, each 1.5 μ l of dCTP, dTTP, α 32p-dATP5 μ l (μ UCi), 10* damping fluid 5 μ l, TaqDNA polysaccharase 2U (U.S. Promage), adding water to final volume is 50 μ l.Flag condition is with above-mentioned pcr amplification condition.The probe that mark is good is hybridized with the film for preparing after Sephadex G-50 (U.S. Promage company) crosses post, by relevant document (J. Sa nurse Brooker; E.F. Ritchie not; T. Manny A Disi work (Jin Dongyan translates), molecular cloning experiment guide, 1995) described in method carry out membrane prepare and probe hybridization.After hybridization finished, positive colony and sequence verification were selected in radioautograph.Through the pcr amplification of twice different primer collocation, in people's placenta cDNA library of molecules, obtained the cDNA fragment of a segment length for 1.1kb.Further utilize this fragment to make probe screening by hybridization in people's placenta cDNA library of molecules, obtained 70 positive colonies, wherein clone 4 and confirm that through primer B and S1 PCR order-checking its 5 ' end has extended about 100bp forward.With computer software splicing order, obtain full length cDNA sequence at last, be total to 1189bp, detailed sequence is seen SEQ ID NO.5, and wherein open reading frame is positioned at 78-1019 position Nucleotide.

Derive the aminoacid sequence of people nek4 according to the full length cDNA sequence that obtains, totally 313 amino-acid residues, its aminoacid sequence sees SEQ ID NO.6 for details.

Embodiment 2

Northern hybridization

PCR method label probe (system and condition are the same), the purifying probe is hybridized with the MTN Northern film that contains 16 kinds of tissue mRNA.Concrete operations are referring to relevant document.

With the standard beta-actin positive is contrast, and the Northern engram analysis result of nek4 shows: people nek4 has three kinds of transcripts (transcript), is respectively 1.8kb, 3.0kb and 9.5kb.1.8kb has higher expression in prostate gland and ovary, and ovary is the highest.Three kinds of transcripts all do not detect the obvious expression signal in cerebral tissue, and the expression of its hetero-organization is between three's centre.

Embodiment 3 homologies relatively

Full-length cDNA encoding sequence and proteins encoded thereof with people nek4 of the present invention, in Non-redundantGenBank+EMBL+DDBJ+PDB database and Non-redundant GenBank CDStranslations+PDB+SwissProt+Spupdate+PIR database, carry out nucleic acid and albumen homology retrieval with BLAST software, found that itself and mouse nek1 gene and encoded protein thereof have higher homology, the identity on protein level is 32%, similarity is 46% (Fig. 2).So people nek4 of the present invention is the homologue of mouse nek1 in the people.

Research finds between the amino acid of the protein kinase structural domain of nek1 and the NIMA composition higher similarity is arranged also, and similarity is about 42%, and particularly their functional zone are high conservatives (EMBO J.1992,11 (10): 3521-3531).N-terminal at mouse nek1 and NIMA all has a catalyst structure domain, this structural domain is the critical function district that albumen plays a role, this structural domain can be divided into 11 subdomains again, nek4 albumen of the present invention also has most feature of this catalyst structure domain, and itself and nek1 catalyst structure domain have higher homology degree.Studies show that, the characteristic aminoacid sequence that three high conservatives are arranged in the catalyst structure domain of nek1, NIMA: promptly the Gly-Thr-Pro-(Tyr/Phe) among the subdomain VIII-Tyr-X-(Ser/Pro)-Pro-Glu (wherein, X is expressed as arbitrary amino acid, any one in the bracket in these two amino acid of expression.); Asp-X-Trp-X-X-Gly among Asn-among the subdomain VIB (Iso/Val)-Phe-Leu and the subdomain IX.Correspondingly aminoacid sequence is respectively Gly Thr Pro Tyr TyrMet Ser Pro Glu (209-217 position among the SEQ ID NO.4), Asn Val Phe Ile (177-180 position among the SEQ ID NO.4) and Asp Ile Trp Ser Leu (229-233 position among the SEQ ID NO.4) (Cell Growth ﹠amp in albumen nek4 of the present invention; Differ.1993,4:821-830).Shown further that thus people nek4 of the present invention and nek1, NIMA belong to the member in the cyclin kinase family, and had similar function.

Also separate having obtained nek1, nek2 and three kinds of albumen of nek3 in the people, their catalytic domain is compared, the identity of nek2 and NIMA is 47%, and the identity of itself and mouse nek1 is 43%.The identity of nek3 and NIMA is 43%, and with the identity of mouse nek1 be 58%.This shows that the similarity of people nek1, nek2 and nek3 three's catalytic domain is not high yet, only is 24%-26%, but some special site height homology of catalytic domain.People nek4 of the present invention and other three proteic catalytic domains of people nek are carried out homology relatively with PCGENE software, and the similarity of finding them only is 22.8%, but also homology (Fig. 1) highly of some special site of its catalytic domain and other three kinds of albumen.This shows that further nek4 albumen of the present invention and other three people nek albumen belong to the member in the cyclin kinase family together, and have similar function.

Nek1, NIMA belong to the member in the protein kinase family that depends on cyclin.Cell division cycle is the significant process of biomass cells propagation and growth.Cell division cycle is finished by common adjusting of various kinds of cell cyclin, and these cyclins are normal functions of performance under the regulating effect of the protein kinase of cyclin dependent.The protein kinase that depends on cyclin generally is to finish the proteic adjusting of cell cycle by the phosphorylation in some special Serine of cell cycle albumen and Threonine site and dephosphorylation, these albumen are generally at fissional G1 phase to S phase and G2 phase to the M phase (Cell1990 that plays a role, 64,1111-1122).

As everyone knows, cell division cycle is biomass cells propagation and the significant process of growing, and it also plays important effect for growing of living organism, so this hint nek4 plays an important role in vivo.

The multicellular organism individuality is by diversified cellularity, biont all is through many times cell fission and be grown to serve as complete, as to have a vitality individuality at first by zygote, therefore, cell fission is biomass cells propagation and the significant process that produces offspring.To the RNA analysis revealed that nek1 expresses, nek1 is equal high expression level in the sexual cell of male and female mice, and therefore, it may directly affect the mitotic division of sexual cell.Nek1 is as the protein kinase of cyclin dependent, mainly come the activating cells cyclin by some special amino acid sites of phosphorylation cyclin, thereby regulate the time length of cell division cycle and the process of carrying out (EMBO J.1992,11 (10): 3521-3531).The catalysis district of nek4 and some serine/threonine protein kitases, particularly there is extremely similar place in the catalysis district to man bunch of NIMA albumen.Therefore can conclude tentatively that nek4 itself is a kind of phosphorylating kinase, it has important regulation in the cell cycle.From nek4 expression pattern analysis result, although the expression of people nek4 does not have tangible tissue specificity, its expression dosage in different tissues has huge difference.The expression amount of people nek4 in prostate gland and ovary tissue is the highest, and expression amount is minimum in cerebral tissue, illustrates that people nek4 does not express in well differentiated tissue.Therefore, can conclude that people nek4 not only also has important function in mitotic division but also in reduction division.Discover that the protein kinase that depends on cyclin not only pair cell division is absolutely necessary, its pair cell differentiation also plays a part very important.The sudden change of the protein kinase that some are relevant will cause some cytodifferentiation variation (Nature, 1990,344,503-508).Therefore, the variation of people nek4 can cause cell fission normally to carry out usually.

People nek4 of the present invention and nek1 and NIMA have height homologous protein kinase catalysis district, therefore think that it also has above these functions.Promptly regulating the activity of cell division cycle in vivo.

People nek4 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 nek4 can also merge with other members of this family or exchange fragment, to produce new albumen.For example the N end of inventor nek4 and the N end of mouse nek1 are exchanged, to produce the albumen that new activity is higher or have new features.

At the antibody of inventor nek4, 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 nek4 nucleic acid (encoding sequence or antisense sequences) can be introduced into cell, with expression level that improves people nek4 or the overexpression that suppresses people nek4.People nek4 albumen of the present invention or its active polypeptide fragment can be applied to patient, with treatment or alleviate because of people nek4 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 4

The expression of people nek4 in intestinal bacteria

In this embodiment, will be template with people's placenta cDNA, use corresponding to 5 of this dna sequence dna ' and the PCR Oligonucleolide primers of 3 ' end increase, obtain people nek4 cDNA as inserting fragment.

5 ' the Oligonucleolide primers sequence of using in the PCR reaction is:

5’-CACACTGCAGATGGCAGGACAGCCCGGCC-3’(SEQ?ID?NO.7)，

This primer contains the restriction enzyme site of PstI restriction enzyme, is 19 Nucleotide of the nek4 encoding sequence that begun by initiator codon after this restriction enzyme site;

3 ' end primer sequence is:

5-CCAAAAGCTTTCAGGTGCTGGACATCCAG-3i(SEQ?ID?NO.8)，

This primer contains the part encoding sequence of restriction enzyme site, translation termination and the nek4 of HindIII restriction enzyme.

The restriction enzyme site of the restriction enzyme on the primer is corresponding to bacterial expression vector pQE-9 (Qiagen Inc., Chatsworth, the CA) restriction enzyme digestion sites on, this plasmid vector coding antibiotics resistance (Amp ^r), a bacterium replication orgin (ori), an adjustable promotor/operon of IPTG-(P/O), a ribosome bind site (RBS), a 6-histidine mark thing (6-His) and restriction enzyme cloning site.

With PstI, HindIII digestion pQE-9 carrier and insertion fragment, will insert fragment subsequently and be connected to the pQE-9 carrier and keep open reading frame initial at bacterium RBS.Transform available from Qiagen with connecting mixture subsequently, the E.coli bacterial strain of commodity M15/rep4 by name, M15/rep4 contains the plasmid pREP4 of multiple copied, and it is expressed the lacI repressor and carries kalamycin resistance (Kan ^r).Screen transformant containing on the LB culture dish of Amp and Kan, the extracting plasmid cuts with the BamHI enzyme and to identify and insert clip size and direction, and the cDNA fragment of sequence verification nek4 has correctly been inserted carrier.

Incubated overnight (O/N) contains the positive transformant clone of required construction in the LB liquid nutrient medium of adding Amp (100 μ g/ml) and Kan (25 μ g/ml).Spend the night (O/N) culture with 1: 100-1: 250 thinning ratio dilution is inoculated in the large volume substratum culturing cell to 600 optical density(OD) (OD then ₆₀₀) when being 0.4-0.6, add IPTG (" isopropylthio-") to final concentration be 1mM.By making lacI repressor inactivation, IPTG induces startup P/O to cause gene expression dose to improve.Continued culturing cell 3-4 hour, centrifugal subsequently (6000 * g, 20 minutes).The ultrasonic degradation inclusion body, collecting cell also is dissolved in cell precipitation in the Guanidinium hydrochloride of 6M.After the clarification, by containing under the condition that 6-His marker albumen combines closely making, with nickel-chelate column chromatography purifying dissolved nek4 from solution.With 6M Guanidinium hydrochloride (PH5.0) wash-out nek4 from post.Available several method is the sex change protein precipitation from Guanidinium hydrochloride.Perhaps, use the dialysis step to remove Guanidinium hydrochloride, perhaps from nickel-chelate column, isolate purifying protein.Protein behind the purifying is incorporated in second post, has the linear Guanidinium hydrochloride gradient of successively decreasing in this post.Protein denaturation when being attached to this post is used Guanidinium hydrochloride (PH5.0) wash-out subsequently.At last, soluble protein is dialysed with PBS, then protein is kept in the stock solution that final concentration is 10% (w/v) glycerine.

SDS-PAGE glue with 12% carries out electrophoresis, identifies that the molecular weight size of expressing protein is about 36KDa.

In addition, the amino acid that reaches proteic N end and each 10 amino acid length of C end is checked order, find consistent with the sequence of SEQ ID NO.6 with ordinary method.

Embodiment 5

The expression of people nek4 in eukaryotic cell (Chinese hamster ovary celI strain)

In this embodiment, will be template with people's placenta cDNA, use 5 ' and 3 ' the PCR Oligonucleolide primers of holding to increase corresponding to this dna sequence dna, obtain people nek4 cDNA as inserting fragment.

5 ' the Oligonucleolide primers sequence of using in the PCR reaction is:

5’-CACAAAGCTTATGGCAGGACAGCCCGGCC-3’(SEQ?ID?NO.9)，

This primer contains the restriction enzyme site of HindIII restriction enzyme, is 19 Nucleotide of the nek4 encoding sequence that begun by initiator codon after this restriction enzyme site;

3 ' end primer sequence is:

5i-CCAATCTAGATCAGGTGCTGGACATCCAG-3 _i(SEQ?ID?NO.10)

This primer contains the part encoding sequence of the restriction enzyme site of XbaI restriction enzyme, translation termination and nek4.

The restriction enzyme site of the restriction enzyme on the primer is corresponding to the restriction enzyme digestion sites on the expressing cho cell carrier pcDNA3, this plasmid vector coding antibiotics resistance (Amp ^rAnd Neo ^r), a phage replication starting point (f1 ori), a virus replication starting point (SV40 ori), T7 promotor, a viral promotors (P-CMV), a Sp6 promotor, a SV40 promotor, a SV40 tailing signal and corresponding polyA order, a BGH tailing signal and a corresponding polyA order.

With HindIII, XbaI digestion pcDNA3 carrier and insertion fragment, will insert fragment subsequently and be connected to the pcDNA3 carrier.Subsequently with connecting mixture Transformed E .coli DH5 α bacterial strain.Screen transformant containing on the LB culture dish of Amp, incubated overnight (O/N) contains the clone of required construction in the LB liquid nutrient medium of adding Amp (100 μ g/ml).The extracting plasmid cuts evaluation with the BamHI enzyme and insert clip size and direction, and the cDNA fragment of sequence verification nek4 has correctly been inserted carrier.

The plasmid transfection Chinese hamster ovary celI is to adopt lipofection, carries out with Lipofectin test kit (GiBco Life).After the transfection 48 hours, through the lasting G418 pressurization screening in 2-3 week, collecting cell and cell conditioned medium are measured the expressing protein enzyme activity.Remove G418, continuous passage is cultivated; To mixing the clone cell Method of Limited Dilution, select to have the cell subclone of higher protein-active.The above-mentioned positive subclone of a large amount of according to a conventional method cultivations.After 48 hours, beginning collecting cell and supernatant are with ultrasonic degradation method smudge cells.With 50mMTris á Hcl (PH7.6) solution that contains 0.05%Triton is balance liquid and elutriant, uses through the Superdex of pre-equilibration G-75 post and collects above-mentioned proteic active peak.Using 50mMTris á Hcl (PH8.0) equilibrated DEAE-Sepharose post again, is that elutriant carries out gradient elution with 50mMTris á Hcl (PH8.0) solution that contains 0-1M NaCl, collects above-mentioned proteic active peak.Be that dialyzate is dialysed to expressing protein solution with PBS (PH7.4) then.Last freeze-drying is preserved.

SDS-PAGE glue with 12% carries out electrophoresis, identifies that the molecular weight size of expressing protein is 36KDa.

In addition, the amino acid that reaches proteic N end and each 10 amino acid length of C end is checked order, find consistent with the sequence of SEQ ID NO.6 with ordinary method.

Embodiment 6 preparation antibody

The recombinant protein that obtains in embodiment 4 and 5 is used for immune animal to produce antibody, specific as follows.Recombinant molecule is standby after separating with chromatography.Also available SDS-PAGE gel electrophoresis separates, electrophoretic band downcut from gel, and with isopyknic complete Freund ' s adjuvant emulsion.Albumen with 50-100 μ g/0.2ml emulsification carries out peritoneal injection to mouse.After 14 days,, mouse is carried out peritoneal injection with booster immunization with the dosage of 50-100 μ g/0.2ml with the same antigen of non-complete Freund ' s adjuvant emulsion.Carried out booster immunization one time every 14 days, carry out at least three times.The sero-fast specific reaction that obtains is active to be assessed in the ability of external precipitation people nek4 gene translation product with it.Found that antibody can precipitate with albumen of the present invention specifically.

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: Fudan University is denomination of invention (ii): a kind of new people's albumen and encoding sequence thereof reach (iii) sequence number of method for making and purposes: information (i) sequence signature of 10 (2) SEQ ID NO:1

(A) length: 20 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: information (i) sequence signature of SEQ ID NO:1:GTTCAACATC AGCCGCTACA 20 (2) SEQ ID NO:2

(A) length: 20 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: information (i) sequence signature of SEQ ID NO:2:CACCAGACCA ACTGGTAATG 20 (2) SEQ ID NO:3

(A) length: 23 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: information (i) sequence signature of SEQ ID NO:3:CTGTGCATCC TCCTGACCCACAG 23 (2) SEQ ID NO:4

(A) length: 23 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: the information of SEQ ID NO:4:CCTCCCTCAG CTGCTCTGCC TTC 23 (2) SEQ ID NO.5: (i) sequence signature:

(A) length: 1189bp

(B) type: nucleic acid

(C) chain: strand

( D ) ： ( ii ) ：cDNA ( xi ) ：SEQ ID NO.5：CGTCGACAGC AGACCTTCCC TGGAGCTTCT CAGGAGCCAC TTCAAAAGTT CGTGCCCTCG 60TGAGGCTGGC ATGCAGGATG GCAGGACAGC CCGGCCACAT GCCCCATGGA GGGAGTTCCA 120ACAACCTCTG CCACACCCTG GGGCCTGTGC ATCCTCCTGA CCCACAGAGG CATCCCAACA 180CGCTGTCTTT TCGCTGCTCG CTGGCGGACT TCCAGATCGA AAAGAAGATA GGCCGAGGAC 240AGTTCAGCGA GGTGTACAAG GCCACCTGCC TGCTGGACAG GAAGACAGTG GCTCTGAAGA 300AGGTGCAGAT CTTTGAGATG ATGGACGCCA AGGCGAGGCA GGACTGTGTC AAGGAGATCG 360GCCTCTTGAA GCAACTGAAC CACCCAAATA CCATCAAGTA TTTGGACTCG TTTATCGAAG 420ACAACGAGCT GAACATTGTG CTGGAGTTGG CTGACGCAGG GGACCTCTCG CAGATGATCA 480AGTACTTTAA GAAGCAGAAG CGGCTCATCC CGGAGAGGAC AGTATGGAAG TACTTTGTGC 540AGCTGTGCAG CGCCGTGGAG CACATGCATT CACGCCGGGT GATGCACCGA GACATCAAGC 600CTGCCAACGT GTTCATCACA GCCACGGGCG TCGTGAAGCT CGGTGACCTT GGTCTGGGCC 660GCTTCTTCAG CTCTGAGACC ACCGCAGCCC ACTCCCTAGT GGGGACGCCC TACTACATGT 720CACCGGAGAG GATCCATGAG AACGGCTACA ACTTCAAGTC CGACATCTGG TCCTTGGGCT 780GTCTGCTGTA CGAGATGGCA GCCCTCCAGA GCCCCTTCTA TGGAGATAAG ATGAATCTCT 840TCTCCCTGTG CCAGAAGATC GAGCGGTGTG ACTACCCCCC ACTCCCCGGG GAGCATTACT 900CCGAGAAGTT ACGAGAACTG GTCAGCATGT GCATCTGCCC TGACCCCCAC CAGAGACCTG 960ACATCGGATA CGTGCACCAG GTGGCCAAGC AGATGCACAT CTGGATGTCC AGCACCTGAG 1020CGTGGATGCA CCGTGCCTTA TCAAAGCCAG CACCACTTTG CCTTACTTGA GTCGTCTTCT 1080CTTCGAGTGG CCACCTGGTA GCCTAGAACA GCTAAGACCA CAGGGTTCAG CAGGTTCCCC 1140AAAAGGCTGC CCAGCCTTAC AGCAGATGCT GAAGGCAGAG CAGCTGAGG 1189 ( 2 ) SEQ ID NO.6： ( i ) ：

(A) length: 313 amino acid

(B) type: amino acid

( D ) ： ( ii ) ： ( xi ) ：SEQ ID NO.6：Met Ala Gly Gln Pro Gly His Met Pro His Gly Gly Ser Ser Asn 15Asn Leu Cys His Thr Leu Gly Pro Val His Pro Pro Asp Pro Gln 30Arg His Pro Asn Thr Leu Ser Phe Arg Cys Ser Leu Ala Asp Phe 45Gln Ile Glu Lys Lys Ile Gly Arg Gly Gln Phe Ser Glu Val Tyr 60Lys Ala Thr Cys Leu Leu Asp Arg Lys Thr Val Ala Leu Lys Lys 75Val Gln Ile Phe Glu Met Met Asp Ala Lys Ala Arg Gln Asp Cys 90Val Lys Glu Ile Gly Leu Leu Lys Gln Leu Asn His Pro Asn Thr 105Ile Lys Tyr Leu Asp Ser Phe Ile Glu Asp Asn Glu Leu Asn Ile 120Val Leu Glu Leu Ala Asp Ala Gly Asp Leu Ser Gln Met Ile Lys 135Tyr Phe Lys Lys Gln Lys Arg Leu Ile Pro Glu Arg Thr Val Trp 150Lys Tyr Phe Val Gln Leu Cys Ser Ala Val Glu His Met His Ser 165Arg Arg Val Met His Arg Asp Ile Lys Pro Ala Asn Val Phe Ile 180Thr Ala Thr Gly Val Val Lys Leu Gly Asp Leu Gly Leu Gly Arg 195Phe Phe Ser Ser Glu Thr Thr Ala Ala His Ser Leu Val Gly Thr 210Pro Tyr Tyr Met Ser Pro Glu Arg Ile His Glu Asn Gly Tyr Asn 225Phe Lys Ser Asp Ile Trp Ser Leu Gly Cys Leu Leu Tyr Glu Met 240Ala Ala Leu Gln Ser Pro Phe Tyr Gly Asp Lys Met Asn Leu Phe 255Ser Leu Cys Gln Lys Ile Glu Arg Cys Asp Tyr Pro Pro Leu Pro 270Gly Glu His Tyr Ser Glu Lys Leu Arg Glu Leu Val Ser Met Cys 285Ile Cys Pro Asp Pro His Gln Arg Pro Asp Ile Gly Tyr Val His 300Gln Val Ala Lys Gln Met His Ile Trp Met Ser Ser Thr 313 ( 2 ) SEQ ID NO：7 ( i ) ( A ) ：29 ( B ) ： ( C ) ： ( D ) ： ( ii ) ： ( xi ) ：SEQ ID NO：7：CACACTGCAG ATGGCAGGAC AGCCCGGCC 29 ( 2 ) SEQ ID NO：8 ( i )

(A) length: 29 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: information (i) sequence signature of SEQ ID NO:8:CCAAAAGCTT TCAGGTGCTG GACATCCAG 29 (2) SEQ ID NO:9

(A) length: 29 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: information (i) sequence signature of SEQ ID NO:9:CACAAAGCTT ATGGCAGGAC AGCCCGGCC 29 (2) SEQ ID NO:10

(A) length: 29 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: SEQ ID NO:10:CCAATCTAGA TCAGGTGCTG GACATCCAG 29

Claims (14)

1. isolated dna molecular, it is characterized in that, it comprises: coding has the nucleotide sequence of the polypeptide of people nek4 protein-active, shows at least 70% homology from the nucleotides sequence of Nucleotide 78-1019 position among described nucleotide sequence and the SEQ ID NO.5; Perhaps described nucleotide sequence can be hybridized from the Nucleotide of Nucleotide 78-1019 position under the moderate stringent condition with among the SEQ ID NO.5.

2. dna molecular as claimed in claim 1 is characterized in that, described sequence encoding one polypeptide, and this polypeptide has the sequence shown in the SEQ ID NO.6.

3. dna molecular as claimed in claim 1 is characterized in that, this sequence has the nucleotide sequence of 78-1019 position among the SEQ ID NO.5.

4. isolating nek4 protein polypeptide is characterized in that it comprises: have polypeptide or its active fragments of SEQ ID NO.6 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.6 polypeptide of sequence.

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

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

8. host cell as claimed in claim 7 is characterized in that this cell is intestinal bacteria.

9. host cell as claimed in claim 7 is characterized in that this cell is an eukaryotic cell.

10. a generation has the method for the polypeptide of people nek4 protein-active, it is characterized in that this method comprises:

(a) nucleotide sequence that coding is had a polypeptide of people nek4 protein-active operationally is connected in expression regulation sequence, form people nek4 protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 78-1019 position among described nucleotide sequence and the SEQ ID NO.5;

(b) change the expression vector in the step (a) over to host cell, form the proteic reconstitution cell of nek4;

(c) under the condition that is fit to expressing human nek4 protein polypeptide, the reconstitution cell in the culturing step (b);

(d) isolate polypeptide with people nek4 protein-active.

11. method as claimed in claim 10 is characterized in that, this sequence is from Nucleotide 78-1019 position among the SEQ ID NO.5.

12. energy and the described people nek4 of claim 6 protein polypeptide specificity bonded antibody.

13. a nucleic acid molecule is characterized in that, it is the antisense sequences of the described dna molecular of claim 1.

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

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