CN1325874A - Human C-type lectin-like receptor and its coding sequence and application - Google Patents

Abstract

A novel C-type lectin-like receptor, the polynucleotide for coding it, the process for preparing said protein molecule by recombination, the application of said polynucleotide, the characteristics of said protein molecule in naturally killing human cell surface expression, and the antagonist of said protein molecule and its medical action, especially treating tumor, are disclosed.

Description

New C LECTIN-LIKE MOLECULE, its encoding sequence and purposes

The invention belongs to biotechnology and medical field, specifically, the present invention relates to the polynucleotide of new coding C LECTIN-LIKE MOLECULE (C lectin-like molecule abbreviates " CLL-1 " as), and the polypeptide of this polynucleotide encoding.This acceptor is worn membrane glycoprotein for the II type, goes up at natural killer cell (natural killer cell, NK cell) and expresses.The invention still further relates to the purposes and the preparation of these polynucleotide and polypeptide.Specifically, polypeptide of the present invention is a kind of new relevant cell surface molecule of the treatment with tumour and virus infection.The NK cell is lymphocytic a kind of, mainly is present in blood and the lymphoid tissue, can the cell of tumour cell and virus infection be killed and wounded.Because the NK cell does not have the specific antigens identification receptor, this killing and wounding is nonspecific.Studies show that in recent years, killing and wounding of NK cell is subjected to (the majorhistocompatibility complex of main histocompatibility complex, MHC) class effect, the NK cell need not sensitization can kill and wound tumour cell (the Yokoyama and Seaman that does not express or hang down the expression major histocompatibility antigen, 1993), for the virus infection that can reduce the mhc class i developed by molecule or tumour cell, the effect of NK cell is the important supplement to T cellular immunization regulating effect.

The NK cell receptor is a series of surface moleculars of expressing on the NK cell, in the identification of NK cell and target cell, plays a significant role in the process of target lethal effect that NK is cell-mediated and signal conduction.The NK cell receptor can be classified according to different angles (Yokoyama et al, 1995): 1. whether the part by the NK cell receptor is the mhc class i molecule, the NK cell receptor can be divided into mhc class i molecular receptor and non-mhc class i molecular receptor; 2. can be divided into immunoglobulin superfamily (IgSF) and C type Sugar receptors superfamily (C-lectin SF) by the receptor structure feature; 3. can be divided into KIR family, CD94/NKG2 family, Ly family and NKR-P1 family etc. with NK kdr transfected cell location; 4. can be divided into inhibition acceptor and activation receptor by function.C type Sugar receptors (C-type lectin) receptor family is an integral part of NK cell receptor family, at present the C type Sugar receptors class NK cell receptor of finding comprises Ly49 family, NKR-P1 family and the people's of mouse NKG2 family etc., encode by the NK gene composite, the encoding gene of mouse is positioned at karyomit(e) No. 6, and people's encoding gene is positioned at karyomit(e) No. 12.Also have some NK cell C type Sugar receptors acceptors, comprise CD94, CD69, AICL (activation-induced C-type lectin) etc. by the single copy gene coding.These gene products are the II type membrane-spanning protein that is expressed on the NK cell, and Ca is arranged ²⁺Rely on the glycosyl cog region, at Ca ²⁺Can combine with part under the condition that exists, in activation or inhibition NK cell killing mechanism, play a role (Lanier et al, 1998).

The NK cell can be expressed one or more C type lectin-likes NK cell receptor, can combine with corresponding M HC class part.The inhibition acceptor combines the back and transmit to suppress signal with part, can suppress the NK cell to the killing and wounding of target cell, and activation receptor then can stimulate the NK cell that target cell is killed and wounded with after part combines.The NK cell receptor has popularity with combining of part, and for example, Ly49C can combine with multiple H-2 molecule, and has cross reaction (Brennan et al, 1 995) with other Ly49 molecules.Therefore, the newcomer who finds NK cell receptor family combines for NK cell receptor and part, and Mechanism Study such as signal conduction are significant.

Ly49 family and CD94/NKG2 family product mostly are NK cell inhibition acceptor, and some are also arranged is activation receptor; And NKR-P1 family product mostly is NK cell-stimulating acceptor.So far, Ly49 family has 9 members at least, and NKG2 family has 5 members at least, plays a role with the CD94 covalent attachment.Found a plurality of NKR-P1 genes up to now, each all has allelotrope.Along with the development of Protocols in Molecular Biology, constantly there is new C type lectin-like NK cell receptor to be found.This is for further understanding NK nonspecific immunity mechanism and use the NK cell and tumour and virus infection are treated have very great help.

Research shows that unusual the or disappearance of C type lectin-like receptor is relevant with numerous disease, and therefore, the C LECTIN-LIKE MOLECULE is significant for the therapeutic purpose research and development.

The purpose of this invention is to provide a kind of new C LECTIN-LIKE MOLECULE (C lectin-like molecule abbreviates " CLL-1 " as) albumen with and fragment, analogue and derivative.

Another object of the present invention provides the polynucleotide of these polypeptide of coding.

Another object of the present invention provides the method for these polypeptide of production and the purposes of this polypeptide and encoding sequence.

In a first aspect of the present invention, novel isolated CLL-1 polypeptide is provided, this polypeptide is the people source, it comprises: polypeptide or its conservative property variation polypeptide or its active fragments or its reactive derivative with SEQ ID NO:2 aminoacid sequence.Preferably, this polypeptide is the polypeptide with SEQ ID NO:2 aminoacid sequence.

In a second aspect of the present invention, the polynucleotide of isolating these polypeptide of coding are provided, these polynucleotide comprise a nucleotide sequence, and this nucleotide sequence is shown at least 70% homogeny with a kind of nucleotides sequence that is selected from down group: (a) polynucleotide of the above-mentioned people CLL-1 polypeptide of coding; (b) with polynucleotide (a) complementary polynucleotide.Preferably, this polynucleotide encoding has the polypeptide of aminoacid sequence shown in the SEQ ID NO:2.More preferably, the sequence of these polynucleotide is be selected from down group a kind of: the sequence that (a) has 170-967 position among the SEQ ID NO:1; (b) has the sequence of 1-1566 position among the SEQ ID NO:1.

In a third aspect of the present invention, the carrier that contains above-mentioned polynucleotide is provided, and has been transformed or host cell of transduceing or the host cell that is directly transformed or transduce by above-mentioned polynucleotide by this carrier.

In a fourth aspect of the present invention, provide preparation to have the method for the polypeptide of people CLL-1 protein-active, this method comprises: (a) under the proteic condition of suitable expressing human CLL-1, cultivate the above-mentioned host cell that is transformed or transduce; (b) from culture, isolate polypeptide with people CLL-1 protein-active.

In a fifth aspect of the present invention, provide and above-mentioned people CLL-1 polypeptid specificity bonded antibody.The nucleic acid molecule that can be used for detecting also is provided, and it contains a successive 10-3018 Nucleotide in the above-mentioned polynucleotide.

In a sixth aspect of the present invention, the compound of simulation, promotion, antagonism people CLL-1 polypeptide active is provided, and the compound that suppresses people CLL-1 polypeptide expression.The method of screening and/or prepare these compounds also is provided.Preferably, this compound is encoding sequence or its segmental antisense sequences of people CLL-1 polypeptide.

In a seventh aspect of the present invention, provide and whether had the proteic method of CLL-1 in the test sample, it comprises: sample is contacted with the proteic specific antibody of CLL-1, observe whether form antibody complex, formed antibody complex and just represented to exist in the sample CLL-1 albumen.

In a eighth aspect of the present invention, a kind of disease relevant with people CLL-1 polypeptide unconventionality expression or method of disease susceptibility of detecting is provided, this method comprises: whether have sudden change in the nucleotide sequence of detection coding said polypeptide.

In a ninth aspect of the present invention, provide the purposes of polypeptide of the present invention and encoding sequence.Polypeptide for example of the present invention can be used to screen the agonist that promotes people CLL-1 polypeptide active, and perhaps screening suppresses the antagonist of people CLL-1 polypeptide active or is used to the peptide finger print identification.The proteic encoding sequence of people CLL-1 of the present invention or its fragment can be used as primer and be used for pcr amplification reaction, perhaps are used for hybridization as probe, perhaps are used to make gene chip or microarray.

In a tenth aspect of the present invention, a kind of pharmaceutical composition is provided, it contains people CLL-1 polypeptide of the present invention or its agonist, antagonist and the pharmaceutically acceptable carrier of safe and effective amount.These pharmaceutical compositions can be treated illnesss such as tumour.

Others of the present invention are because disclosing of the technology of this paper is conspicuous to those skilled in the art.

Following accompanying drawing is used to illustrate specific embodiments of the present invention, and is not used in qualification by the scope of the invention that claims defined.

Fig. 1 is the cDNA sequence of people CLL-1 of the present invention, and wherein non-coding sequence is represented with lowercase, and encoding sequence is represented with capitalization.

Fig. 2 is the proteic full length amino acid sequence of people CLL-1 of the present invention.

Fig. 3 is the amino acid sequence homology comparison diagram of people CLL-1 albumen of the present invention and mouse KLR2 (aaf36830) and people CD94 albumen (q60660).The top sequence is people CLL-1, and the below sequence is mouse KLR2 and people CD94 albumen.Identical amino acid marks with black between two sequences, and similar amino acid marks with shade.

Fig. 4 is the hydrophobicity graphic representation of the proteic Kyte-doolitte hydrophobicity analysis of people CLL-1 of the present invention.

In the present invention, term " CLL-1 albumen ", " CLL-1 polypeptide " or " C type lectin-like is subjected to Body CLL-1 " be used interchangeably, all refer to have C LECTIN-LIKE MOLECULE CLL-1 amino acid sequence (SEQ ID NO:2) albumen or polypeptide. They comprise the C type lectin-like that contains or do not contain initial methionine Acceptor CLL-1.

As used herein, " separation " refers to that material separates (if natural thing from its primal environment Matter, primal environment namely are natural surroundingses). Such as the polynucleotide under the native state in the active somatic cell and polypeptide be Do not have separation and purification, but same polynucleotide or polypeptide as from native state with in other materials that exist Separately, then for separation and purification.

As used herein, it is natural that " CLL-1 albumen or the polypeptide of separation " refers to that the CLL-1 polypeptide is substantially free of Relative other albumen, lipid, carbohydrate or other material. Those skilled in the art can use the albumen of standard Matter purification technique purifying CLL-1 albumen. Basically pure polypeptide can produce on non-reduced polyacrylamide gel Single master tape. The purity of CLL-1 polypeptide can be used amino acid sequence analysis.

Polypeptide of the present invention can be recombinant polypeptide, natural polypeptides, synthetic polypeptide, preferred recombinant polypeptide. This Bright polypeptide can be the product of natural purifying, or the product of chemical synthesis, or uses recombinant technique from protokaryon Or produce in the eucaryon host (for example, bacterium, yeast, higher plant, insect and mammalian cell). According to The host that the recombinant production scheme is used, polypeptide of the present invention can be glycosylated, maybe can be nonglycosylated. Polypeptide of the present invention also can comprise or not comprise initial methionine residues.

The present invention also comprises fragment, derivative and the analog of people CLL-1 albumen. As used herein, term " sheet Section ", " derivative " refer to basically keep natural human CLL-1 albumen of the present invention identical with " analog " Biological function or active polypeptide. Polypeptide fragment of the present invention, derivative or analog can be that (ⅰ) has one Or a plurality of conservative or substituted polypeptide of non-conservation amino acid residue (preferred conservative amino acid residue), and like this The amino acid residue of replacement can be also can be by the genetic code coding, or (ⅱ) at one or more ammonia Base has the polypeptide of substituted radical in the sour residue, or (ⅲ) mature polypeptide and another compound (such as prolonging polypeptide partly The compound of phase, for example polyethylene glycol decline) merge formed polypeptide, or (ⅳ) additional amino acid sequence is fused to This peptide sequence and the polypeptide that forms are (such as targeting sequencing or secretion sequence or be used for sequence or the albumen of this polypeptide of purifying Former sequence, or with the fusion of the formation of antigen I gG fragment). According to the instruction of this paper, these fragments, spread out Biological and analog belongs to the known scope of those skilled in the art.

In the present invention, term " people CLL-1 polypeptide " refers to have the SEQ ID NO. of people CLL-1 protein active The polypeptide of 2 sequences. This term also comprise have with people CLL-1 albumen identical function, SEQ ID NO.2 order The variant form of row. 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 (being generally in 20, preferably is in 10, better for one of the terminal interpolation of C end and/or N or several Ground is in 5) amino acid. For example, in the art, the amino acid close or similar with performance replaces The time, the common function that can not change protein. Again such as, add one or several in that C end and/or N are terminal Amino acid also can not change the function of protein usually. This term also comprise people CLL-1 albumen active fragment and Reactive derivative.

The variant form of this polypeptide comprises: homologous sequence, conservative variant, allelic variant, natural sudden change Body, induced mutation body, under high or low stringency condition, can compile with the DNA of people CLL-1 DNA hybridization The albumen of code and the polypeptide or the albumen that utilize the antiserum acquisition of anti-people CLL-1 polypeptide. The present invention also provides Other polypeptide, as comprise the fusion of people CLL-1 polypeptide or its fragment. Except the polypeptide of total length almost, The present invention has also comprised the soluble fragments of people CLL-1 polypeptide. Usually, this fragment has people CLL-1 polypeptide order Row at least about 10 continuous amino acids, usually at least about 30 continuous amino acids, preferably at least about 50 Continuous amino acid is more preferably at least about 80 continuous amino acids, best at least about 100 continuous amino acids.

Invention also provides the analog of people CLL-1 albumen or polypeptide. These analogs and natural human CLL-1 polypeptide Difference can be difference on the amino acid sequence, also can be the difference that does not affect on the modified forms of sequence, or The person haves both at the same time. These polypeptide comprise genetic variant natural or that induce. The induce variation body can be by various skills Art obtains, as by radiation or be exposed to mutagens and produce random mutagenesis, and also can be by direct mutagenesis method or other The biological technology of known molecular. Analog also comprises having and is different from the amino acid whose residue of natural L-(such as D-amino Acid) analog, and the analog with that non-natural exists or synthetic amino acid (such 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.

(usually the not changing primary structure) form of modification comprises: chemically derived form such as the second of the polypeptide that body is interior or external Acidylate or carboxylated. Modify and also to comprise glycosylation, such as those in the synthetic and processing of polypeptide or further add work step Carry out glycosylation modified in rapid and polypeptide that produce. This modification can by polypeptide is exposed to carry out glycosylated Enzyme (such as mammiferous glycosylase or deglycosylating enzyme) and finishing. Modified forms also comprises having phosphorylation amino The sequence of acid residue (such as phosphotyrosine, phosphoserine, phosphothreonine). Thereby also comprising being modified improves Its anti-proteolysis performance or optimized the polypeptide of solubility property.

In the present invention, " people CLL-1 albumen conservative variation polypeptide " refers to the amino acid order with SEQ ID NO:2 Row are compared, and have 10 at the most, and preferably at the most 8, more preferably at the most 5,3 amino acid quilts at the most best Similar performance or close amino acid are replaced and are formed polypeptide. These conservative variation polypeptide preferably advance according to table 1 Row amino acid substitution and producing.

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

Polynucleotides of the present invention can be dna form or rna form. Dna form comprises cDNA, base Because of group DNA or artificial synthetic DNA. DNA can be strand or double-stranded. DNA can be coding Chain or noncoding strand. The coding region sequence of encoding mature polypeptide can with the code area order shown in the SEQ ID NO:1 Be listed as the variant of identical or degeneracy. As used herein, " variant of degeneracy " refers to coding in the present invention Protein with SEQ ID NO:2, but with the differentiated nucleic acid of coding region sequence shown in the SEQ ID NO:1 Sequence.

The polynucleotides of the mature polypeptide of coding SEQ ID NO:2 comprise: the coded sequence of an encoding mature polypeptide; The coded sequence of mature polypeptide and various additional code sequence; The coded sequence of mature polypeptide is (with optional additional volume The code sequence) and non-coding sequence.

Term " polynucleotides of coded polypeptide " can be the polynucleotides that comprise this polypeptide of encoding, and also can be The polynucleotides that also comprise additional code and/or non-coding sequence.

The invention still further relates to the variant of above-mentioned polynucleotides, its coding has identical amino acid sequence with the present invention Polypeptide or fragment, analog and the derivative of polypeptide. The variant of these polynucleotides can be natural generation etc. The variant that position variant or non-natural take place. These nucleotide diversity bodies comprise replacement variant, deletion mutation body With the insertion variant. As known in the art, allelic variant is the replacement form of polynucleotides, and it may Replacement, disappearance or the insertion of one or more nucleotides, but can be from the merit of the polypeptide that changes in fact its coding Energy.

The invention still further relates to and above-mentioned sequence hybridization and two sequences between have at least 50%, preferably at least 70%, the polynucleotide of at least 80% homogeny more preferably.The present invention be more particularly directed under stringent condition and the interfertile polynucleotide of polynucleotide of the present invention.In the present invention, " stringent condition " is meant: (1) than hybridization under low ionic strength and the comparatively high temps and wash-out, as 0.2 * SSC, and 0.1%SDS, 60 ℃; Or (2) hybridization the time is added with denaturing agent, as 50% (v/v) methane amide, 0.1% calf serum/0.1%Ficoll, 42 ℃ etc.; Or (3) only at the homogeny between the two sequences at least more than 90%, be more preferably 95% and just hybridize when above.And the polypeptide of interfertile polynucleotide encoding has identical biological function and activity with the mature polypeptide shown in the SEQ ID NO:2.

The invention still further relates to nucleic acid fragment with above-mentioned sequence hybridization.As used herein, the length of " nucleic acid fragment " contains 15 Nucleotide at least, better is at least 30 Nucleotide, is more preferably at least 50 Nucleotide, preferably more than at least 100 Nucleotide.Nucleic acid fragment can be used for the amplification technique (as PCR) of nucleic acid to determine and/or the proteic polynucleotide of separation coding CLL-1.

Polypeptide among the present invention and polynucleotide preferably provide with isolating form, more preferably are purified to homogeneous.

People CLL-1 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 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.

At present, can be fully obtain the dna sequence dna of code book invention albumen (or its fragment, or derivatives thereof) by chemosynthesis.This dna sequence dna can be introduced in various existing dna moleculars as known in the art (or as carrier) and the cell then.In addition, also can will suddenly change and introduce in the protein sequence of the present invention by chemosynthesis.

Use method (Saiki, the et al.Science 1985 of round pcr DNA amplification/RNA; 230:1350-1354) be optimized for acquisition gene of the present invention.When particularly being difficult to obtain the cDNA of total length from the library, can preferably use RACE method (the terminal rapid amplifying method of RACE-cDNA), the primer that is used for PCR can suitably be selected according to sequence information of the present invention disclosed herein, and available ordinary method is synthetic.Available ordinary method is as the DNA/RNA fragment by gel electrophoresis separation and purifying amplification.

The present invention also relates to comprise the carrier of polynucleotide of the present invention, and the host cell that produces through genetically engineered with carrier of the present invention or CLL-1 albumen coded sequence, and the method that produces polypeptide of the present invention through recombinant technology.

Recombinant DNA technology (Science, 1984 by routine; 224:1431), can utilize polymerized nucleoside acid sequence of the present invention to can be used to express or produce the CLL-1 polypeptide of reorganization.In general following steps are arranged:

(1). with the polynucleotide (or varient) of coding of the present invention people CLL-1 polypeptide, or with the recombinant expression vector that contains these polynucleotide proper host cell that transforms or transduce;

(2). the host cell of in suitable medium, cultivating;

(3). separation, protein purification from substratum or cell.

Among the present invention, people CLL-1 polynucleotide sequence can be inserted in the recombinant expression vector.Term " recombinant expression vector " refers to that bacterial plasmid well known in the art, phage, yeast plasmid, vegetable cell virus, mammalian cell virus are as adenovirus, retrovirus or other carriers.The carrier of Shi Yonging includes but not limited in the present invention: and the expression vector based on T7 of in bacterium, expressing (Rosenberg, et al.Gene, 1987,56:125); The pMSXND expression vector of in mammalian cell, expressing (Lee and Nathans, J Bio Chem.263:3521,1988) and at the carrier that derives from baculovirus of expressed in insect cells.In a word, as long as can duplicate in host and stablize, any plasmid and carrier can be used.A key character of expression vector is to contain replication orgin, promotor, marker gene and translation controlling elements usually.

Method well-known to those having ordinary skill in the art can be used to make up and contains people CLL-1 DNA sequences encoding and suitable transcribing/the translate expression vector of control signal.These methods comprise (Sambroook, et al.Molecular Cloning, a Laboratory Manual, coldSpring Harbor Laboratory.New York, 1989) such as extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technologys of body.Described dna sequence dna can effectively be connected on the suitable promotor in the expression vector, and is synthetic to instruct mRNA.The representative example of these promotors has: colibacillary lac or trp promotor; Lambda particles phage P _LPromotor; Eukaryotic promoter comprises LTRs and some other known may command gene expression promoter in protokaryon or eukaryotic cell or its virus of CMV immediate early promoter, HSV thymidine kinase promoter, early stage and late period SV40 promotor, retrovirus.Expression vector also comprises ribosome bind site and the transcription terminator that translation initiation is used.

In addition, expression vector preferably comprises one or more selected markers, to be provided for selecting the phenotypic character of transformed host cells, cultivate Tetrahydrofolate dehydrogenase, neomycin resistance and the green fluorescent protein (GFP) of usefulness as eukaryotic cell, or be used for colibacillary tsiklomitsin or amicillin resistance.

Comprise the carrier of above-mentioned suitable dna sequence dna and suitable promotor or control sequence, can be used to transform appropriate host cell, so that it can marking protein.

Host cell can be a prokaryotic cell prokaryocyte, as bacterial cell; Or eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as mammalian cell.Representative example has: intestinal bacteria, streptomyces; The bacterial cell of Salmonella typhimurium; Fungal cell such as yeast; Vegetable cell; The insect cell of fruit bat S2 or Sf9; The zooblast of CHO, COS, 293 cells or Bowes melanoma cells etc.

When polynucleotide of the present invention are expressed in higher eucaryotic cells, be enhanced if will make to transcribe when in carrier, inserting enhancer sequence.Enhanser is the cis acting factor of DNA, and nearly 10 to 300 base pairs act on promotor transcribing with enhancing gene usually.Can for example be included in the SV40 enhanser of 100 to 270 base pairs of replication origin side in late period one, at the polyoma enhanser of replication origin side in late period one and adenovirus enhanser etc.

Persons skilled in the art all know how to select appropriate carriers, promotor, enhanser and host cell.

Can carry out with routine techniques well known to those skilled in the art with the recombinant DNA transformed host cell.When the host was prokaryotic organism such as intestinal bacteria, the competent cell that can absorb DNA can be used CaCl in exponential growth after date results ₂Method is handled, and used step is well-known in this area.Another kind method is to use MgCl ₂If desired, transforming also the method for available electroporation carries out.When the host is an eukaryote, can select following DNA transfection method for use: coprecipitation of calcium phosphate method, conventional mechanical method such as microinjection, electroporation, liposome packing etc.

The transformant that obtains can be cultivated with ordinary method, expresses the polypeptide of coded by said gene of the present invention.According to used host cell, used substratum can be selected from various conventional substratum in the cultivation.Under the condition that is suitable for the host cell growth, cultivate.After host cell grows into suitable cell density, induce the promotor of selection with suitable method (as temperature transition or chemical induction), cell is cultivated for some time again.

The extracellular can be expressed or be secreted into to recombinant polypeptide in the above methods in cell or on cytolemma.If desired, can utilize its physics, the separating by various separation methods with other characteristic and the albumen of purification of Recombinant of chemistry.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation handles, with protein precipitant handle (salt analysis method), centrifugal, the broken bacterium of infiltration, superly handle, the combination of super centrifugal, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography (LC) technology and these methods.

The people CLL-1 albumen or the polypeptide of reorganization are of use in many ways.These purposes include, but is not limited to: the direct disease due to the low or forfeiture and be used to screen and promote or antibody, polypeptide or other part of antagonism CLL-1 protein function as pharmacological agent CLL-1 protein function.The peptide molecule that can suppress or stimulate people CLL-1 protein function that can be used for seeking therapeutic value with the recombinant human CLL-1 protein screening peptide library of expressing.

On the other hand, the present invention also comprises people CLL-1 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 CLL-1 gene product or fragment.Preferably, refer to that those can combine with people CLL-1 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 CLL-1, comprise that also those do not influence the antibody of people CLL-1 protein function.The present invention also comprise those can with modify or without the people CLL-1 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 CLL-1 gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing human CLL-1 albumen 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 CLL-1 protein function and the antibody that does not influence people CLL-1 protein function.Each antibody-like of the present invention can utilize the fragment or the functional zone of people CLL-1 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 CLL-1 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).

The proteic antibody of anti-people CLL-1 can be used in the immunohistochemistry technology, detects the people CLL-1 albumen in the biopsy specimen.In addition, with the also available labelled with radioisotope of the protein bound monoclonal antibody of people CLL-1, inject in the body and can follow the tracks of its position and distribution.This radiolabeled antibody can be used as a kind of atraumatic diagnostic method and is used for the location of tumour cell and has judged whether transfer.

Antibody among the present invention can be used for treating or prevention and the relevant disease of people CLL-1 albumen.The antibody that gives suitable dosage can stimulate or block proteic generation of people CLL-1 or activity.

Antibody also can be used for being designed to the immunotoxin at a certain privileged sites in the body.As the monoclonal antibody of people CLL-1 albumen high-affinity can with bacterium or plant poison (as diphtheria toxin, ricin, abrine etc.) covalent attachment.A kind of usual method is with sulfydryl linking agent such as SPDP, attacks the amino of antibody, by the exchange of disulfide linkage, toxin is incorporated on the antibody, and this hybrid antibody can be used for killing the cell (for example lymph-node cell etc.) of people CLL-1 protein positive.

The production of polyclonal antibody can choose CLL-1 albumen or polypeptide immune animal, as rabbit, mouse, rat etc.Multiple adjuvant can be used for the enhancing immunity reaction, includes but not limited to freund's adjuvant etc.

Utilize albumen of the present invention,, can filter out with CLL-1 albumen interactional material takes place, as acceptor, inhibitor, agonist or antagonist etc. by various conventional screening methods.

Albumen of the present invention and antibody thereof, inhibitor, agonist, 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, intravenously, subcutaneous, intracutaneous or topical.

Polypeptide of the present invention can be directly used in disease treatment, for example, is used for the treatment of tumour aspect.When using CLL-1 albumen of the present invention, also can use the other treatment agent simultaneously, as IFN-α, IFN-β, TNF-α, TNF-β etc.

The present invention also provides a kind of pharmaceutical composition, and it contains CLL-1 polypeptide of the present invention or its agonist, antagonist and the pharmaceutically acceptable carrier or the vehicle of safe and effective amount.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.Pharmaceutical composition 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 making pharmaceutical composition, be that the CLL-1 albumen of safe and effective amount or its antagonist, agonist are applied to Mammals, wherein this safe and effective amount is 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.

The proteic polynucleotide of people CLL-1 also can be used for multiple therapeutic purpose.Gene therapy technology can be used for treating because cell proliferation, growth or the metabolic disturbance due to the proteic expression of CLL-1 of the proteic nothing expression of CLL-1 or unusual/non-activity.The CLL-1 albumen that the gene therapy vector (as virus vector) of reorganization can be designed to express variation is to suppress endogenic CLL-1 protein-active.Deriving from viral expression vector such as retrovirus, adenovirus, adeno-associated virus (AAV), hsv, parvovirus etc. can be used for the CLL-1 transgenosis to cell.The method that structure carries the recombinant viral vector of CLL-1 gene be found in existing document (Sambrook, etal.).Recombinant human CLL-1 gene can be packaged in the liposome in addition, and then is transferred in the cell.

Suppress the oligonucleotide (comprising sense-rna and DNA) of people CLL-1 mRNA and ribozyme also within the scope of the invention.Ribozyme is the enzyme sample RNA molecule that a kind of energy specificity is decomposed specific RNA, and its mechanism of action is to carry out the endonuclease effect after ribozyme molecule and the hybridization of complementary target RNA-specific.The RNA of antisense and DNA and ribozyme can obtain with existing any RNA or DNA synthetic technology, as the technology widespread use of solid phase phosphoamide chemical synthesis synthetic oligonucleotide.Antisense rna molecule can be transcribed acquisition by the dna sequence dna of this RNA that encodes in external or body.This dna sequence dna has been incorporated into the downstream of rna polymerase promoter of carrier.In order to increase the stability of nucleic acid molecule, available several different methods is modified it, and as increasing the sequence length of both sides, the connection between the ribonucleoside is used phosphoric acid thioester bond or peptide bond but not phosphodiester bond.

Polynucleotide imports tissue or intracellular method comprises: directly be injected into polynucleotide in the in-vivo tissue; Or external by carrier (as virus, phage or plasmid etc.) earlier with the polynucleotide transfered cell in, again cell is transplanted in the body etc.

Can be incorporated into the rondom polypeptide storehouse that solid formation forms by the various amino acid that may make up by screening with the protein bound peptide molecule of people CLL-1 obtains.During screening, must carry out mark to people CLL-1 protein molecular.

The invention still further relates to the diagnostic testing process of quantitative and detection and localization people CLL-1 protein level.These tests are known in the art, and comprise that FISH measures and radioimmunoassay.The people CLL-1 protein level that is detected in the test can be with laying down a definition the importance of people CLL-1 albumen in various diseases and be used to the disease of diagnosing CLL-1 albumen to work.

Whether having the proteic method of CLL-1 in a kind of detection test sample is to utilize the proteic specific antibody of CLL-1 to detect, and it comprises: sample is contacted with the CLL-1 protein specific antibody; Observe whether form antibody complex, formed antibody complex and just represented to exist in the sample CLL-1 albumen.

The proteic polynucleotide of CLL-1 can be used for the diagnosis and the treatment of CLL-1 protein related diseases.Aspect diagnosis, the proteic polynucleotide of CLL-1 can be used for detecting the proteic expression of CLL-1 CLL-1 abnormal exprssion whether or under morbid state.Can be used for the hybridization of biopsy specimen to judge the proteic abnormal expression of CLL-1 as the CLL-1 dna sequence dna.Hybridization technique comprises the Southern blotting, Northern blotting, in situ hybridization etc.These technological methods all are disclosed mature technologies, and relevant test kit all can obtain from commercial channels.Part or all of polynucleotide of the present invention can be used as probe stationary on microarray (microarray) or DNA chip (being called " gene chip " again), is used for analyzing the differential expression analysis and the gene diagnosis of tissue gene.Carry out RNA-polymerase chain reaction (RT-PCR) amplification in vitro with the special primer of CLL-1 albumen and also can detect the proteic transcription product of CLL-1.

The sudden change that detects the CLL-1 gene also can be used for the disease of diagnosing CLL-1 albumen relevant.The form of CLL-1 protein mutation comprises that the point mutation compared with normal wild type CLL-1 dna sequence dna, transposition, disappearance, reorganization and other are any unusual etc.Available existing technology such as Southern blotting, dna sequence analysis, PCR and in situ hybridization detect sudden change.In addition, sudden change might influence proteic expression, therefore can judge indirectly that with Northern blotting, Western blotting gene has or not sudden change.

Sequence of the present invention identifies it also is valuable to karyomit(e).In brief, the proteic cDNA of CLL-1 prepares PCR primer (preferred 15-35bp) according to the present invention, sequence can be positioned on the karyomit(e).Then, these primers are used for the somatocyte hybrid cell that the PGR screening contains each bar human chromosome.Have only those hybrid cells that contain corresponding to the people's gene of primer can produce the fragment of amplification.

In case sequence is positioned to chromosome position accurately, the physical location of this sequence on karyomit(e) just can be associated with the gene map data.These data for example are found in, V.Mckusick, Mendelian Inheritance in Man (can by with the online acquisition of Johns Hopkins University Welch Medical Library).Can pass through linkage analysis then, determine gene and navigated to relation between the disease on the chromosomal region already.

In an example of the present invention, a kind of isolating polynucleotide are provided, its coding has the polypeptide of aminoacid sequence shown in the SEQ ID NO:2.Polynucleotide of the present invention are isolated from human dendritic cell cDNA library.Its sequence is shown in SEQ ID NO:1, and the polynucleotide sequence total length that it comprises is 1566 bases, and its open reading frame is positioned at the 170-967 position, and the coding total length is 265 amino acid whose people CLL-1 albumen (SEQ IDNO:2).This CLL-1 albumen belongs to C type lectin-like receptor family molecule.The Northern engram analysis shows high expression level in liver, spleen and peripheral blood cells.

The research prompting of having carried out, CLL-1 is a kind of candidate's a NK cell receptor, its tool potential antineoplastic action.Therefore, CLL-1 albumen or its relevant antagonist, agonist etc. can be diseases such as treating tumour provides new treatment approach, thereby has great application prospect.

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 people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York:Cold SpringHarbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.Embodiment 1: the clone of people CLL-1 cDNA

Extract the total RNA of human dendritic cell with Trizol (Gibco company).Then, from total RNA, separate poly (A) mRNA.Poly (A) mRNA after reverse transcription forms cDNA, is inserted into cDNA fragment orientation on the multiple clone site of carrier with SuperScript II clone's test kit (available from Gibco), transforms DH5 α bacterium and form the cDNA plasmid library.Measure random choose clone's 5 ' terminal sequence with dideoxy method.CDNA sequence and the existing public dna sequence data storehouse measured are compared, found that a cDNA clone's dna sequence dna is new full-length cDNA.By synthetic a series of primers the contained dna sequence dna of new clone is carried out two-way mensuration.Computer Analysis shows that cloning contained full-length cDNA is a new cDNA sequence (shown in SEQ ID NO:1), the new protein (shown in SEQ ID NO:2) of encoding.This protein is named as C LECTIN-LIKE MOLECULE CLL-1, its encoding gene called after C LECTIN-LIKE MOLECULE CLL-1 gene.

Sequence SEQ ID NO:1 total length is 1566bp, comprises 3 ' end non-coding region of 5 of 169bp ' end non-coding region and 599bp, and coding contains 265 amino acid whose polypeptide.Calculating not in theory, the molecular weight of glycosylated ripe molecule is 31.8kD.

They are different with known for the BLAST analysis revealed, protein level and people C type Sugar receptors homology, and supposition may be a new C type lectin-like receptor, called after CLL-1 albumen.

CLL-1 albumen is carried out Kyte-doolitte hydrophobicity analysis (Fig. 4), show that it is that an II type is worn membrane glycoprotein.Embodiment 2: with the proteic encoding sequence of RT-PCR method human cloning CLL-1

Be in the total RNA of logarithmic phase human dendritic cell with Trizol (Gibco company) extraction, get 6 μ g cell total rnas and 0.5 μ g Oligo-dT _12-18Mix, carry out reverse transcription.The reverse transcription system is 20 μ l, and reaction adds 80 μ l ddH after finishing ₂O dilutes.The used primer of pcr amplification is as follows: adopted primer 5 ' GGGTGATTGGTACAGTAGGTTTATAAACAG (SEQ ID NO:3) is arranged, antisense primer 5 ' GCCTTTTCGTTTTTGTTTATTGGCTTTTAC (SEQ ID NO:4).The PCR reaction volume is 50 μ l, wherein contain reverse transcription template 10 μ l, 0.4 μ M primer, 0.2 μ M dNTP and 1U ExTaq archaeal dna polymerase (Takara Inc.), the amplification parameter be 94 ℃ 30 seconds, 60 ℃ 30 seconds, 72 ℃ 45 seconds, capable 2% agarose gel electrophoresis of PCR product is tentatively confirmed after 25 circulations.Dna sequence analysis is the result show, the coding region (170-967) shown in the dna encoding sequence of this PCR product and the SEQ ID NO:1 is identical.The proteic Northern engram analysis of embodiment 3 CLL-1

Carry out the Northern trace by following ordinary method: filter membrane to be checked places the hybridization solution of 10ml through 68 ℃ of preheatings, in hybrid heater (Bellco) in 68 ℃ of prehybridizations 30 minutes; The cDNA probe that mark is good was in 95～100 ℃ of sex change 2～5 minutes, and (cDNA probe final concentration is 2～10ng/ml or 1～2 * 10 to put rapid cooling back adding hybridization solution on ice ⁶Cpm/ml), fully mixing was hybridized 2 hours in 68 ℃.After hybridization finishes, filter membrane with 2 * SSC, the drip washing of 0.05%SDS room temperature for several times, the washing lotion several is changed in continuing vibration flushing 30～40 minutes therebetween.Use 0.1 * SSC, 0.1%SDS in 50 ℃ of vibration flushings 20～40 minutes subsequently.Last filter membrane wrapped up with plastic fresh-keeping membrane, in-70 ℃ of exposure X line films 24～48 hours.

The Northern blot hybridization shows: CLL-1 is high expression level in liver, spleen and peripheral blood cells.Embodiment 4 people CLL-1 albumen are recombinant expressed

In this embodiment, be template with the pcr amplification product among the embodiment 2, increase with the PCR Oligonucleolide primers of 5 ' and 3 following ' end of sequence, obtain people CLL-1DNA as inserting fragment.

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

5 ' end primer: 5 '-cg gga tcc ATC AGT GAA GAG CTC CAG AGA A-3 ' (SEQ IDNO:5),

3 ' end primer: 5 '-cG gaa ttc TCA TGC CTC CCT AAA ATA TGT A-3 ＇ (SEQ IDNO:6).

The PCR product purification after BamH I/EcoR I enzyme cut and recombinate according to a conventional method with plasmid pUC18 again and be converted into competence DH5 α, the picking positive colony is identified back purifying and order-checking (377 type sequenators of ABI company, BigDye Terminator test kit, PE company).The CLL-1 albumen cDNA endonuclease bamhi of correct sequence is cloned into expression vector pGEX-2T and is converted into DH5 α.Enzyme is cut the capable 2% agarose gel electrophoresis analysis of product.Confirm through order-checking, inserted complete CLL-1 encoding sequence.

Choosing the positive DH5a clone who expresses CLL-1 is inoculated in 100ml 2 * YTA substratum, 37 ℃ of 300rpm shaking culture 12-15hr, 2 * YTA the substratum that is diluted in preheating at 1: 10 continues shaking culture 1.5hr, add behind the 100mM IPTG to 0.1mM 30 ℃ and induce 2-6hr, 5,4 ℃ of centrifugal 10min of 000g remove supernatant, put and use 50ml 1 * PBS (0.14M NaCl on ice, 2.7mM KCl, 10.1mM Na ₂HPO ₄, 1.8mMKH ₂PO ₄PH7.3) resuspended, add 20%Triton X-100 to 1% jog 30min again after ultrasonic (B.Braun Labsonic U) fragmentation, then 12,4 ℃ of centrifugal 10min of 000g, supernatant with 0.8 μ m membrane filtration after, cross 1ml 50% glutathione S epharose 4B chromatography column, behind 1 * PBS thorough washing, (pH8.0) room temperature leaves standstill after 30 minutes and collects elutriant for 10mM gsh, 50mM Tris-HCl to add 500ul gsh elution buffer, repeat wash-out 2-3 time, obtain people CLL-1 albumen.

Obtain CLL-1 albumen after zymoplasm (thrombin) (Sigma company) enzyme cuts except that GST, molecular weight is about 31.8kD.

Carry out the n terminal amino acid sequential analysis with the Edams hydrolysis method, confirmed that its N terminal sequence conforms to the N terminal sequence shown in the SEQ ID NO:2.Embodiment 5: the generation of anti-people CLL-1 protein antibodies

The recombinant protein c LL-1 albumen that obtains among the embodiment 4 is used for immune animal to produce antibody, and concrete grammar is 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 CLL-1 protein 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:

(ⅱ) denomination of invention: new C LECTIN-LIKE MOLECULE, its encoding sequence and purposes

(ⅲ) sequence number: the information of 6 (2) SEQ ID NO:1:

(ⅰ) sequence signature:

(A) length: 1566bp

(B) type: nucleic acid

(C) chain: two strands

(D) topological framework: linearity

(ⅱ) molecule type: cDNA

( ⅹⅰ ) ：SEQ ID NO:1:gggtgattgg tacagtaggt ttataaacag aagtttaaac ttgtaagctt aagcttccgt 60ttataaacag aagtttaaaa ttataggtcc tgtttaacat tcagctctgt taactcactc 120atctttttgt gtttttacac tttgtcaaga tttctttaca tattcatcaA TGTCTGAAGA 180AGTTACTTAT GCAGATCTTC AATTCCAGAA CTCCAGTGAG ATGGAAAAAA TCCCAGAAAT 240TGGCAAATTT GGGGAAAAAG CACCTCCAGC TCCCTCTCAT GTATGGCGTC CAGCAGCCTT 300GTTTCTGACT CTTCTGTGCC TTCTGTTGCT CATTGGATTG GGAGTCTTGG CAAGCATGTT 360TCATGTAACT TTGAAGATAG AAATGAAAAA AATGAACAAA CTACAAAACA TCAGTGAAGA 420GCTCCAGAGA AATATTTCTC TACAACTGAT GAGTAACATG AATATCTCCA ACAAGATCAG 480GAACCTCTCC ACCACACTGC AAACAATAGC CACCAAATTA TGTCGTGAGC TATATAGCAA 540AGAACAAGAG CACAAATGTA AGCCTTGTCC AAGGAGATGG ATTTGGCATA AGGACAGCTG 600TTATTTCCTA AGTGATGATG TCCAAACATG GCAGGAGAGT AAAATGGCCT GTGCTGCTCA 660GAATGCCAGC CTGTTGAAGA TAAACAACAA AAATGCATTG GAATTTATAA AATCCCAGAG 720TAGATCATAT GACTATTGGC TGGGATTATC TCCTGAAGAA GATTCCACTC GTGGTATGAG 780AGTGGATAAT ATAATCAACT CCTCTGCCTG GGTTATAAGA AACGCACCTG ACTTAAATAA 840CATGTATTGT GGATATATAA ATAGACTATA TGTTCAATAT TATCACTGCA CTTATAAACA 900AAGAATGATA TGTGAGAAGA TGGCCAATCC AGTGCAGCTT GGTTCTACAT ATTTTAGGGA 960GGCATGAggc atcaatcaaa tacattgaag gagtgtaggg ggtgggggtt ctaggctata 1020ggtaaattta aatattttct ggttgacaat tagttgagtt tgtctgaaga cctgggattt 1080tatcatgcag atgaaacatc caggtagcaa gcttcagaga gaatagactg tgaatgttaa 1140tgccagagag gtataatgaa gcatgtccca cctcccactt tccatcatgg cctgaaccct 1200ggaggaagag gaagtccatt cagatagttg tggggggcct tcgaattttc attttcattt 1260acgttcttcc ccttctggcc aagatttgcc agaggcaaca tcaaaaacca gcaaatttta 1320attttgtccc acagcgttgc tagggtggca tggctcccca tctcgggtcc atcctatact 1380tccatgggac tccctatggc tgaaggcctt atgagtcaaa ggacttatag ccaattgatt 1440gttctaggcc aggtaagaat ggatatggac atgcatttat tacctcttaa aattattatt 1500ttaagtaaaa gccaataaac aaaaacgaaa aggcaaaaaa aaaaaaaaaa aaaaaaaaaa 1560aaaaaa 1566 ( 2 ) SEQ ID NO:2：

(ⅰ) sequence signature:

(A) length: 265 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: polypeptide

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:2:MSEEVTYADL QFQNSSEMEK IPEIGKFGEK APPAPSHVWR PAALFLTLLC 50LLLLIGLGVL ASMFHVTLKI EMKKMNKLQN ISEELQRNIS LQLMSNMNIS 100NKIRNLSTTL QTIATKLCRE LYSKEQEHKC KPCPRRWIWH KDSCYFLSDD 150VQTWQESKMA CAAQNASLLK INNKNALEFI KSQSRSYDYW LGLSPEEDST 200RGMRVDNIIN SSAWVIRNAP DLNNMYCGYI NRLYVQYYHC TYKQRMICEK 250MANPVQLGST YFREA 265 (2) SEQ ID NO:3

(ⅰ) sequence signature

(A) length: 30 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ⅱ) molecule type: oligonucleotide

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:3GGGTGATTGG TACAGTAGGT TTATAAACAG 30 (2) SEQ ID NO:4

(ⅰ) sequence signature

(A) length: 30 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ⅱ) molecule type: oligonucleotide

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:4GCCTTTTCGT TTTTGTTTAT TGGCTTTTAC 30 (2) SEQ ID NO:5

(ⅰ) sequence signature

(A) length: 30 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ⅱ) molecule type: oligonucleotide

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:5cgggatccAT CAGTGAAGAG CTCCAGAGAA 30 (2) SEQ ID NO:6

(ⅰ) sequence signature

(A) length: 30 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ⅱ) molecule type: oligonucleotide

(ⅹ ⅰ) sequence description: SEQ ID NO:6cggaattcTC ATGCCTCCCT AAAATATGTA 30

Claims (14)

1. an isolating people CLL-1 polypeptide is characterized in that it comprises: polypeptide or its conservative property variation polypeptide or its active fragments or its reactive derivative with SEQ ID NO:2 aminoacid sequence.

2. polypeptide as claimed in claim 1 is characterized in that, this polypeptide is the polypeptide with SEQ ID NO:2 aminoacid sequence.

3. isolating polynucleotide is characterized in that, it comprises a nucleotide sequence, and this nucleotide sequence is shown at least 70% homogeny with a kind of nucleotides sequence that is selected from down group:

(a) coding is as the polynucleotide of polypeptide as described in claim 1 and 2;

(b) with polynucleotide (a) complementary polynucleotide.

4. polynucleotide as claimed in claim 3 is characterized in that, this polynucleotide encoding has the polypeptide of aminoacid sequence shown in the SEQ ID NO:2.

5. polynucleotide as claimed in claim 3 is characterized in that, the sequence of these polynucleotide is selected from down a kind of of group:

(a) has the sequence of 170-967 position among the SEQ ID NO:1;

(b) has the sequence of 1-1566 position among the SEQ ID NO:1.

6. a carrier is characterized in that, it contains the described polynucleotide of claim 3.

7. a genetically engineered host cell is characterized in that, it contains the described carrier of claim 6.

8. preparation method with polypeptide of people CLL-1 protein-active is characterized in that this method comprises:

(a) under the proteic condition of suitable expressing human CLL-1, cultivate the described host cell of claim 7;

(b) from culture, isolate polypeptide with people CLL-1 protein-active.

9. energy and the described people CLL-1 of claim 1 protein-specific bonded antibody.

10. nucleic acid molecule, it contains a successive 10-3018 Nucleotide in the described polynucleotide of claim 3.

11. whether there is the proteic method of CLL-1 in the test sample, it is characterized in that, comprising:

The described antibody of sample and claim 9 is contacted,

Observe whether form antibody complex, formed antibody complex and just represented to exist in the sample CLL-1 albumen.

12. the purposes of polypeptide as claimed in claim 1 is characterized in that, it is used to screen the agonist that promotes people CLL-1 protein-active, and perhaps screening suppresses the antagonist of people CLL-1 protein-active or is used to the peptide finger print identification.

13. the purposes of a nucleic acid molecule as claimed in claim 10 is characterized in that, it is used as primer and is used for pcr amplification reaction, perhaps is used for hybridization as probe, perhaps is used to make gene chip or microarray.

14. a pharmaceutical composition is characterized in that, it contains the described polypeptide of claim 1 and the pharmaceutically acceptable carrier of safe and effective amount.

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