CN1163506C - New human cell factor and its code sequence and use - Google Patents

Abstract

The present invention provides a novel human cell factor-protein CX1, polyribonucleotide for coding the polypeptide and a method for generating the polypeptide by a recombination technique. The present invention also discloses methods for treating various diseases by the polypeptide, such as anemia, hematopoiesis hypofunction, etc. The present invention also discloses an antagonist for resisting the polypeptide and therapeutical effects thereof; the present invention also discloses the applications of the polynucleotide for encoding the novel human protein CX1. The present invention also provides medical compositions containing the protein CX1.

Description

New human cell factor, its encoding sequence and purposes

The invention belongs to biotechnology and medical field, specifically, the present invention relates to the polynucleotide of new coding human cell factor CX1 (being also referred to as " CX1 albumen "), and the polypeptide of this polynucleotide encoding.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 and hematopoiesis related cytokine.

Cytokine is a class important protein in the animal body.Many cytokines all play an important role in vivo, for example tumour necrosis factor, Interferon, rabbit, interleukin-(IL2, IL-8, IL17, IL18 etc.), HGF, angiogenin, erythropoietin (EPO), GM-CSF etc.

In recent years, begun the Human Genome Project,, disclosed human life's the secret and the regularity of occurrence and development of disease, finally explored the effective ways of diagnosis of disease so that obtain human full gene group information.Along with the development of molecular biology and computer technology, information biology, large scale sequencing has become effective means (the Mao et al.Proc Natl Acad Sci USA.1998 that discloses the cellular gene expression spectrum and find the recruit; 95:8175).The structure in cDNA library is the basic and crucial of extensive random sequencing.The cDNA library mainly contains phage library and plasmid library two big classes.Phage library has the high outstanding advantage of titre, but also exist the higher shortcoming of plaque rate, utilize complementary expression of IPTG inductive LacZ to carry out in the white bacterium colony screening process of indigo plant, part is expressed the back unsettled clone of growth and may be suppressed, and causes clone's advantage to select; And it is sub also may to contain insertion in the part blue colonies.At present, the development of art technology has made the titre of plasmid library be enough to satisfy the requirement of extensive random sequencing.

Dendritic cell is an important full-time antigen presenting cell in the body, is the direct startup and the regulation and control person of body T cell-specific immunne response.In recent years, mechanism is offered in the processing of the differentiation and development of dendritic cell, antigen and the effect in tumour, infection, autoimmune disorder and transplant rejection has become field, immunologic forward position, seeking immune recruit from dendritic cell is a big research focus (Cao Xuetao etc. of present field of immunology, China's Journal of Immunology, 1998; 3:322, Banchereau et al.Nature.1998; 392:245).

Some well-known biotech company and research institutions competitively set foot in this field as DNAX, HGS, Immunex, Genetech etc. in the world.Dendritic cell recruit's research is significant to deeply illustrating migration in its differentiation and development, the body, biological action and molecule mechanism thereof, also may provide new thinking and means for the treatment of tumour, infection (HIV), transplant rejection and autoimmune disorder simultaneously.Adema etc. have found in human dendritic cell cDNA library can special chemotactic primary tape T cell CC chemokine DC-CK1, point out its may with DC to relevant (the Adema et al.Nature.1997 of the hormesis of Naive T cell uniqueness; 387:713).Anderson etc. compare by the homology of EST, from human dendritic cell, found TNF receptor superfamily newcomer RANK (Receptor Activator of NF-κ B), further be cloned into the part RANKL of this molecule by cloning by expression, and confirm that the interaction of RNAK and RANKL can effectively keep vigor (the Anderson et al.Nature.1997 of dendritic cell; 390:175).Lebecque etc. have found lysosome related membrane protein immune recruit (De Saint-Vis etal.Immunity.1998 such as (DC-LAMP) from human dendritic cell cDNA library; 9:326).

Because cytokine plays an important role aspect the normal physiological function keeping, and the dysfunction of cytokine is also closely related with some diseases (for example malignant tumour, anaemia etc.), therefore, significant for therapeutic purpose research and development new human cell factor and agonist/inhibitor thereof.

The purpose of this invention is to provide a kind of new human cell factor-CX1 protein polypeptide 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.

Another object of the present invention provides and contains the proteic pharmaceutical composition of CX1.

In a first aspect of the present invention, novel isolated CX1 protein 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, SEQ ID NO:3, SEQID NO:4 or SEQ ID NO:5 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 CX1 protein polypeptide of coding; (b) with polynucleotide (a) complementary polynucleotide.Preferably, this polynucleotide encoding has the polypeptide of aminoacid sequence shown in SEQ ID NO:2 or the SEQ ID NO:3.More preferably, the sequence of these polynucleotide is be selected from down group a kind of: the sequence that (a) has 45-587 position among the SEQ ID NO:1; (b) has the sequence of 105-587 position among the SEQ ID NO:1; (c) has the sequence of 1-704 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 CX1 protein-active, this method comprises: (a) under the proteic condition of suitable expressing human CX1, cultivate the above-mentioned host cell that is transformed or transduce; (b) from culture, isolate polypeptide with people CX1 protein-active.

In a fifth aspect of the present invention, provide and above-mentioned people CX1 protein polypeptide specificity bonded antibody.The nucleic acid molecule that can be used for detecting also is provided, and it contains a successive 10-704 Nucleotide in the above-mentioned polynucleotide.

In a sixth aspect of the present invention, simulation, promotion, the active compound of antagonism people CX1 protein polypeptide are provided, and the compound that suppresses the expression of people CX1 protein polypeptide.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 CX1 protein polypeptide.

In a seventh aspect of the present invention, the method that whether has the CX1 protein-active in the test sample is provided, a kind of method comprises: with sample and the reactive cells contacting of CX1, whether the propagation of observing the reactive cell of CX1 is promoted that the propagation of CX1 reacting cells is enhanced just represents to exist in the sample CX1 protein-active.Preferably, the reactive cell of this CX1 is the TF1 cell.Another kind method comprises: sample is contacted with the proteic specific antibody of CX1, observe whether form antibody complex, formed antibody complex and just represented to exist in the sample CX1 albumen.

In a eighth aspect of the present invention, a kind of disease relevant with people CX1 protein 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 screening and promote the active agonist of people's CX1 protein polypeptide, and perhaps screening suppresses people's active antagonist of CX1 protein polypeptide or is used to the peptide finger print identification.The proteic encoding sequence of people CX1 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 the people CX1 protein polypeptide of the present invention and the pharmaceutically acceptable carrier of safe and effective amount.These pharmaceutical compositions can be treated illnesss such as Immunological diseases, anaemia, cancer.

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.

Figure 1A is the aminoacid sequence of proteic cDNA sequence of inventor CX1 and total length.Wherein non-coding sequence is represented with lowercase, and encoding sequence is represented with capitalization." * " represents terminator codon." ↓ " marked the cleavage site of signal peptide.

Figure 1B is people CX1 albumen of the present invention and the proteic amino acid sequence homology comparison diagram of Ro 24-7472/000 (IL17).The top sequence is a people CX1 albumen, and the below sequence is a people IL17 albumen.Same amino acid represents with monocase amino acid that between two sequences similar amino acid is represented with ": " and ". ".

Fig. 2 is the hydrophobicity graphic representation that pair cell factor CX1 carries out the Kyte-doolitte hydrophobicity analysis.

Fig. 3 is expression plasmid pcDNA3.1/myc-his (-) structure iron.

Fig. 4 is to CX1 and CX1-His expression in the COS7 cells and supernatant of recombinant expression vector pcDNA-CX1 and pCX1-His gene transfection, carries out ³⁵The electrophorogram that the S metabolic marker detects.Wherein be successively from left to right: swimming lane 1, CX1-His; Swimming lane 2: the COS7 cell contrast supernatant of transfection empty carrier; Swimming lane 3, CX1; Swimming lane 4: the COS7 cell of transfection empty carrier.The left side is molecular weight size (kDa).

Fig. 5 A carries out CX1-His expression product in the culture supernatant behind recombinant expression plasmid pCX1-His gene transfection 293 cells ³⁵The electrophorogram that the S metabolic marker detects.Swimming lane mock is contrast; Swimming lane CX1-His is the CX1-His expression product.The left side is molecular weight size (kDa).

Fig. 5 B is the electrophorogram that CX1-His expression product in the culture supernatant behind recombinant expression plasmid pCX1-His gene transfection 293 cells is carried out the Western engram analysis.Swimming lane 1 and 3 is contrast, and swimming lane 2 is the CX1-His expression product.The left side is molecular weight size (kDa).

Fig. 6 is that the CX1-IgG fusion rotein to purifying carries out SDS-PAGE electrophoresis silver and dyes the electrophorogram of evaluation.The CX1-IgG fusion rotein is single band among the figure.The left side swimming lane is molecular weight marker (kDa).

Fig. 7 A has shown the proliferation function of the COS7 cells and supernatant of CX1 gene transfection to the TF1 cell.

Fig. 7 B has shown the proliferation function of 293 cells and supernatant of CX-His gene transfection to the TF1 cell.

Fig. 7 C has shown the proliferation function of CX1-IgG fusion rotein to the TF1 cell.

In the present invention, term " CX1 albumen ", " cell factor CX1 " are used interchangeably, and all refer to have the human cell factor CX1 of amino acid sequence of the mature form (SEQ ID NO :) of the total length form (SEQ ID NO:1) of human cell factor CX1 amino acid sequence or no signal peptide. They comprise the cell factor CX1 that contains or do not contain initial methionine.

As used herein, " separation " refers to that material separates (if crude, primal environment namely is natural surroundings) from its primal environment. Do not have separation and purification such as the polynucleotide under the native state in the active somatic cell and polypeptide, but same polynucleotide or polypeptide as from native state with in other materials that exist separately, then for separation and purification.

As used herein, " CX1 albumen or the polypeptide of separation " refers to that the CX1 polypeptide is substantially free of natural relative other albumen, lipid, carbohydrate or other material. Those skilled in the art can use the purified technology of protein purifying CX1 albumen of standard. Basically pure polypeptide can produce single master tape on non-reduced polyacrylamide gel. The purity of CX1 polypeptide can be used amino acid sequence analysis.

Polypeptide of the present invention can be restructuring polypeptide, natural polypeptides, synthetic polypeptide, the polypeptide of preferably recombinating. Polypeptide of the present invention can be the product of natural purifying, or the product of chemical synthesis, or uses the restructuring technology to produce from protokaryon or eucaryon host (for example, bacterium, yeast, higher plant, insect and mammalian cell). According to the used host of restructuring production decision, 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 CX1 albumen. As used herein, term " fragment ", " derivative " refer to basically keep the identical biological function of natural human CX1 albumen of the present invention or active polypeptide with " analog ". Polypeptide fragment of the present invention, derivative or analog can be that (i) has one or more conservative or substituted polypeptide of non-conservation amino acid residue (preferred conservative amino acid residue), and the amino acid residue of such replacement can be also can not encoded by genetic code, or (ii) in one or more amino acid residues, has a polypeptide of substituted radical, or (iii) mature polypeptide and another compound (such as the compound that prolongs the polypeptide half-life, polyethylene glycol for example) merges formed polypeptide, or (iv) additional amino acid sequence is fused to this peptide sequence and the polypeptide that forms (such as targeting sequencing or secretion sequence or be used for sequence or the proteinogen sequence of this polypeptide of purifying, or with the fusion of the formation of antigen I gG fragment). According to the instruction of this paper, these fragments, derivative and analog belong to the known scope of those skilled in the art.

In the present invention, term " people CX1 polypeptide " refers to have the SEQ ID NO. 2 of people CX1 protein active or the polypeptide of 3 sequences. This term also comprises having and variant forms people CX1 albumen identical function, SEQ ID NO.2 or 3 sequences. These variant forms comprise (but being not limited to): several (are generally 1-50, preferably 1-30, more preferably 1-20,1-10 best) amino acid whose disappearance, insertion and/or replacement, and add one or several and (be generally in 20 in that C end and/or N are terminal, preferably being in 10, more preferably is in 5) amino acid. For example, in the art, when replacing with the close or similar amino acid of performance, usually can not change the function of protein. Again such as, add the function that or several amino acid also can not change protein usually in that C end and/or N are terminal. This term also comprises active fragment and the reactive derivative of people CX1 albumen.

The variant form of this polypeptide comprises: homologous sequence, conservative variant, allelic variant, natural mutation, induced mutation body, under high or low stringency condition can with the coded albumen of the DNA of people CX1 DNA hybridization and the polypeptide or the albumen that utilize the antiserum of anti-human CX1 polypeptide to obtain. The present invention also provides other polypeptide, as comprises the fusion of people CX1 polypeptide or its fragment. Except the polypeptide of total length almost, the present invention has also comprised the soluble fragments of people CX1 polypeptide. Usually, this fragment have people CX1 polypeptide 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 analog of people CX1 albumen or polypeptide. The difference of these analogs and natural human CX1 polypeptide can be the difference on the amino acid sequence, also can be the difference that does not affect on the modified forms of sequence, perhaps haves both at the same time. These polypeptide comprise genetic variant natural or that induce. The induce variation body can obtain by various technology, as by radiation or be exposed to mutagens and produce random mutagenesis, also can pass through direct mutagenesis method or the biological technology of other known moleculars. Analog also comprises having the analog that is different from the amino acid whose residue of natural L-(such as D-amino acid), 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 acetylation or the 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 such as those in the synthetic and processing of polypeptide or further. This modification can be carried out glycosylated enzyme (such as mammiferous glycosylase or deglycosylating enzyme) and finishes by polypeptide is exposed to. Modified forms also comprises have the phosphorylated amino acid residue sequence of (such as phosphotyrosine, 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 CX1 albumen conservative variation polypeptide " refers to compare with the amino acid sequence of SEQ ID No.2 or 3, 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 variation polypeptide preferably carry out amino acid substitution 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	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, genomic DNA or artificial synthetic DNA. DNA can be strand or double-stranded. DNA can be coding strand or noncoding strand. The coding region sequence of encoding mature polypeptide can be identical with the coding region sequence shown in the SEQ ID NO:1 or the variant of degeneracy. As used herein, " variant of degeneracy " refers in the present invention encode and has the protein of SEQ ID NO:2 or SEQ ID NO:3, but with the differentiated nucleotide sequence of coding region sequence shown in the SEQ ID NO:1.

The polynucleotides of the mature polypeptide of coding SEQ ID NO:2 or SEQ ID NO:3 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 (with optional additional 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 the polypeptide of identical amino acid sequence or fragment, analog and the derivative of polypeptide with the present invention. The variant of these polynucleotides can be the allelic variant of natural generation or the variant that non-natural occurs. These nucleotide diversity bodies comprise and replace variant, deletion mutation body and insert variant. As known in the art, allelic variant is the replacement form of polynucleotides, and it may be replacement, disappearance or the insertion of one or more nucleotides, but can be from the function of the polypeptide that changes in fact its coding.

The invention still further relates to and above-mentioned sequence hybridization and two sequences between have at least 50%, preferably at least 70%, the polynucleotides of at least 80% homogeny more preferably. The present invention be more particularly directed under stringent condition and the interfertile polynucleotides of polynucleotides of the present invention. In the present invention, " stringent condition " refers to: (1) than the hybridization under LIS and the higher temperature and wash-out, such as 0.2 * SSC, and 0.1%SDS, 60 ℃; Or (2) hybridization the time is added with denaturant, such as 50% (v/v) formamide, 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 CX1.

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

People CX1 pyrenoids thuja acid 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 CX1 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 CX1 protein polypeptide of reorganization.In general following steps are arranged:

(1). with the proteic polynucleotide of coding people CX1 of the present invention (or varient), or transform or the transduction proper host cell with the recombinant expression vector that contains these polynucleotide;

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

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

Among the present invention, people CX1 albumen 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 CX1 encoding histone dna sequence dna 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 CX1 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 CX1 protein function as pharmacological agent CX1 protein function.For example, antibody can be used for activating or suppressing the proteic function of people CX1.The peptide molecule that can suppress or stimulate people CX1 protein function that can be used for seeking therapeutic value with the recombinant human CX1 protein screening peptide library of expressing.

On the other hand, the present invention also comprises people CX1 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 CX1 gene product or fragment.Preferably, refer to that those can combine with people CX1 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 CX1, comprise that also those do not influence the antibody of people CX1 protein function.The present invention also comprise those can with modify or without the people CX1 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 CX1 gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing human CX1 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 prepare (see people such as Kohler, Nature256; 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 CX1 protein function and the antibody that does not influence people CX1 protein function.Each antibody-like of the present invention can utilize the fragment or the functional zone of people CX1 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 CX1 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 CX1 can be used in the immunohistochemistry technology, detects the people CX1 albumen in the biopsy specimen.In addition, with the also available labelled with radioisotope of the protein bound monoclonal antibody of people CX1, 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 CX1 albumen.The antibody that gives suitable dosage can stimulate or block proteic generation of people CX1 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 CX1 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, attack the amino of antibody, exchange by disulfide linkage, toxin is incorporated on the antibody, this hybrid antibody can be used for killing the cell (tumour cell for example is as leukemia cell, chronic myelogenous leukemia cell K562, preceding marrow leukemia cell HL-60, Burkitt lymphomas cell Raji, knot rectal adenocarcinoma cell SW480, lung cell A549 and melanoma cell etc.) of people CX1 protein positive.

The production of polyclonal antibody can choose CX1 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 CX1 albumen interactional material takes place, as acceptor, inhibitor or antagonist etc. by various conventional screening methods.

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

Polypeptide of the present invention can be directly used in disease treatment, and for example, malignant tumour, anaemia, hemopoietic function hang down inferior.When using CX1 albumen of the present invention, also can use other treatment agent, for example EPO, GM-CSF, IL-3, G-CSF etc. simultaneously.

The present invention also provides a kind of pharmaceutical composition, and it contains CX1 protein polypeptide of the present invention 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.People CX1 albumen of the present invention can be made into the injection form, for example is prepared by ordinary method with the physiological saline or the aqueous solution that contains glucose and other assistant agents.Pharmaceutical composition such as tablet and capsule can be prepared by ordinary method.Pharmaceutical composition such as injection, solution, tablet and capsule should be made under aseptic condition.The dosage of activeconstituents is the treatment significant quantity, for example every day about 1 microgram/kg body weight-Yue 5 mg/kg body weight.In addition, polypeptide of the present invention also can use with the other treatment agent.

When making pharmaceutical composition, the CX1 albumen that is safe and effective amount is 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 CX1 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 CX1 of the proteic nothing expression of CX1 or unusual/non-activity.The CX1 albumen that the gene therapy vector (as virus vector) of reorganization can be designed to express variation is to suppress endogenic CX1 protein-active.Deriving from viral expression vector such as retrovirus, adenovirus, adeno-associated virus (AAV), hsv, parvovirus etc. can be used for the CX1 transgenosis to cell.The method that structure carries the recombinant viral vector of CX1 gene is found in existing document (Sambrook, et al.).Recombinant human CX1 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 CX1 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 CX1 obtains.During screening, must carry out mark to people CX1 protein molecular.

The invention still further relates to the diagnostic testing process of quantitative and detection and localization people CX1 protein level.These tests are known in the art, and comprise that FISH measures and radioimmunoassay.The people CX1 protein level that is detected in the test can be with laying down a definition the importance of people CX1 albumen in various diseases and be used to the disease of diagnosing CX1 albumen to work.

The method that whether has the CX1 protein-active in a kind of test sample is to utilize the reactive cell of CX1 to detect, it comprises: with sample and the reactive cells contacting of CX1, whether the propagation of observing the reactive cell of CX1 is promoted that the propagation of CX1 reacting cells is enhanced just represents to exist in the sample CX1 protein-active.In the present invention, " the reactive cell of CX1 (albumen) " refer to can be to CX1 proteic existence react, thereby cause breeding the cell of increase, erythroleukemia cell for example is as the TF1 cell.

Whether having the proteic method of CX1 in the another kind of detection test sample is to utilize the proteic specific antibody of CX1 to detect, and it comprises: sample is contacted with the CX1 protein specific antibody; Observe whether form antibody complex, formed antibody complex and just represented to exist in the sample CX1 albumen.

The proteic polynucleotide of CX1 can be used for the diagnosis and the treatment of CX1 protein related diseases.Aspect diagnosis, the proteic polynucleotide of CX1 can be used for detecting the proteic expression of CX1 CX1 abnormal exprssion whether or under morbid state.Can be used for the hybridization of biopsy specimen to judge the proteic abnormal expression of CX1 as CX1 protein D NA sequence.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 CX1 albumen and also can detect the proteic transcription product of CX1.

The sudden change that detects the CX1 gene also can be used for the disease of diagnosing CX1 albumen relevant.The form of CX1 protein mutation comprises that the point mutation compared with normal wild type CX1 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 CX1 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 PCR 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 inMan (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 mature 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:

tccaggcgggcagcagctgcaggctgaccttgcagcttggcgga ATG GAC TGG CCT CAC AAC CTG CTG TTT 71

CTT CTT ACC ATT TCC ATC TTC CTG GGG CTG GGC CAG CCC AGG AGC CCC AAA AGC AAG AGG 131

AAG GGG CAA GGG CGG CCT GGG CCC CTG GCC CCT GGC CCT CAC CAG GTG CCA CTG GAC CTG 191

GTG TCA CGG ATG AAA CCG TAT GCC CGC ATG GAG GAG TAT GAG AGG AAC ATC GAG GAG ATG 251

GTG GCC CAG CTG AGG AAC AGC TCA GAG CTG GCC CAG AGA AAG TGT GAG GTC AAC TTG GAG 311

CTG TGG ATG TCC AAC AAG AGG AGC CTG TCT CCC TGG GGC TAC AGC ATC AAC CAC GAC CCC 371

AGC CGT ATC CCC GTG GAC CTG CCG GAG GCA CGG TGC CTG TGT CTG GGC TGT GTG AAC CCC 431

TTC ACC ATG CAG GAG GAC CGC AGC ATG GTG AGC GTG CCG GTG TTC AGC CAG GTT CCT GTG 491

CGC CGC CGC CTC TGC CCG CCA CCG CCC CGC ACA GGG CCT TGC CGC CAG CGC GCA GTC ATG 551

GAG ACC ATC GCT GTG GGC TGC ACC TGC ATC TTC TGA attacctggcccagaagccaggccagcagccc 619

gagaccatcctccttgcacctttgtgccaagaaaggcctatgaaaagtaaacactgacttttgaaagcaaaaaaaaaaaa 699

aaaaa 704

The polynucleotide sequence total length that wherein comprises is 704 bases, and its open reading frame is positioned at the 45-587 position, coding 180 amino acid whose people CX1 albumen of total length (SEQ ID NO:2).

MDWPHNLLFL LTISIFLGLG QPRSPKSKRK GQGRPGPLAP GPHQVPLDLV 50

SRMKPYARME EYERNIEEMV AQLRNSSELA QRKCEVNLQL WMSNKRSLSP 100

WGYSINHDPS RIPVDLPEAR CLCLGCVNPF TMQEDRSMVS VPVFSQVPVR 150

RRLCPPPPRT GPCRQRAVME TIAVGCTCIF 180

Wherein, amino acid/11-20 is a signal peptide.Removed the mature C X1 albumen behind the signal peptide and contained 160 amino acid, sequence is shown in SEQ ID NO:3.

QPRSPKSKRK GQGRPGPLAP GPHQVPLDLV SRMKPYARME EYERNIEEMV 50

AQLRNSSELA QRKCEVNLQL WMSNKRSLSP WGYSINHDPS RIPVDLPEAR 100

CLCLGCVNPF TMQEDRSMVS VPVFSQVPVR RRLCPPPPRT GPCRQRAVME 150

TIAVGCTCIF 160

CX1 albumen has certain homology (16-18%) at amino acid levels and people, rat and mouse IL-17, HSV early gene 13 albumen.Clone the CX1 cDNA that contains full length coding region by PCR, insert the pcDNA3.1 carrier for expression of eukaryon, made up and expressed following proteic different expression plasmids: the fusion rotein CX1-His that (1) natural CX1, (2) CX1 C end and myc/His merge; And the fusion rotein CX1-IgG of (3) CX1 C end and the fusion of IgG Fc section.In eukaryotic cell, carry out the protein expression of multi-form (merge with non-fusion), and in African green monkey kidney cell strain COS7, carry out transient expression, carry out after after the transfection 48 hours that cells and supernatant detects and ³⁵The S metabolic analysis shows that cytokine CX1 gene can obtain expressing in eukaryotic cell, justacrine is to born of the same parents.

Application CX1 C end merges with myc/His and CX1 C end obtains different expression plasmids with the fusion of IgG Fc section, the CX1 recombinant expression vector is rotaring redyeing COS 7 cell, human embryonic kidney cell line 293 cells and Chinese hamster ovary cell Chinese hamster ovary celI respectively, all obtains effective expression.All can from the culture supernatant of transfectional cell, detect the CX1 fusion rotein (CX1-His) that C end contains myc epi-position and 6His with anti-myc antibody or anti-6His antibody, from the serum-free culture supernatant of the CHO stably express clone strain of transfection CX1-IgG gene, be purified to CX1-IgG fusion rotein about 50kD with the ProteinG affinity chromatography.

In order to inquire into the potential function of CX1, selected erythroleukemia cell strain TF1 cell to detect the hematopoiesis regulating and controlling effect of CX1.TF1 all has proliferative response (Murate et al.Exp Cell Res.208:35) to GM-CSF, IL-3 and EPO.Test shows that the fusion rotein CX1-IgG that different extent of dilution contain CX1 and CX1-His culture supernatant and purifying all has certain propagation promoter action to the TF1 cell.This shows that CX1 albumen is a kind of new cytokine.In addition, TF1 can be used for the CX1 biologic activity and detects as the reactive cell of CX1.

Test also shows, uses CX1-IgG separately to CD34 ⁺The proliferation and differentiation of bone marrow stem cell does not have obvious promoter action, but can promote differentiation of GM-CSF/G-CSF/IL-3 inductive grain system and the differentiation of the red system of IL-3/EPO inductive.Also preliminary observation promotes monocyte secretion NO, TNF and IL-6 to CX1.

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 CX1 albumen 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 cloned test kit (available from Gibco) with SuperScriptII cDNA fragment orientation is inserted on the multiple clone site of carrier, transform 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 the dna sequence dna of a cDNA clone SBBI30 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 SEQID NO:1), the new protein (shown in SEQ ID NO:2) of encoding.This protein is named as people CX1 albumen, and its encoding gene name is people CX1 gene.

Sequence SEQ ID NO:1 total length is 704bp, comprises 3 ' end non-coding region of 5 of 42bp ' end non-coding region and 117bp, and coding contains 180 amino acid whose polypeptide.

Contain the signal peptide of being made up of 20 amino acid according to Kyte-Doolitte hydrophobicity analysis and GCG software prediction N end, this shows that CX1 albumen is secretory protein.Mature C X1 albumen is made up of 160 amino acid, contains 8 halfcystines, may participate in the formation of intramolecularly and/or intermolecular disulfide bond; 55 Asn meet glycosylation consensus sequence NXS/T, are the potential glycosylation site.Calculating not in theory, the molecular weight of glycosylated ripe molecule is 18.1kD.

They are different with known for the BLAST analysis revealed, and protein level and people, rat and mouse IL-17, HSV early gene 13 albumen have homology (16-18%), and supposition may be a new cytokine, called after cytokine CX1.

Embodiment 2: with the proteic encoding sequence of RT-PCR method human cloning CX1

Be in logarithmic phase HL-60 cell total rna 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 CX1 is as follows: primer #261:5 '-ggaattc gccacc atg gac tgg cct cac aac-3 ' (SEQ ID NO:6), primer #354:5 '-gg ggt acc tca gaagat gca ggt gca gcc c-3 ' (SEQ ID NO:7).Primer #261 introduces gcc acc and is beneficial to the raising expression level before initiator codon ATG.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.The dna sequence analysis result shows that the DNA sequences encoding of this PCR product and the 45-587bp coding region shown in the SEQ ID NO:1 are identical.

The proteic Northern engram analysis of embodiment 3 CX1

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: the expression level of cytokine CX1 in tissues such as kidney, suprarenal gland, heart, skeletal muscle, liver and pancreas is higher, in tissues such as placenta, testis, ovary, peripheral blood leucocyte, colon and lung, be low expression level, in brain, spleen, thymus gland, small intestine and prostate gland, do not detect tangible positive signal; All there is mRNA in various degree to express at tumour cell such as people's lymphoblast leukemia cell Molt-4, chronic myelogenous leukemia cell K562, preceding marrow leukemia cell HL-60, Burkitt lymphomas cell Raji, knot rectal adenocarcinoma cell SW480, lung cell A549 and melanoma cell G361.

In addition, cytokine CX1 is wide expression in tumor tissues, points out in its generation in tumour, the evolution to play a role, but also may be the secondary performance of tumour cell gene expression regulation disorder.

Embodiment 4 cytokine CX1 recombinant expression vector pcDNA-CX1, the structure of pCX1-His and pCX1-Fc

Structure as shown in Figure 3 for carrier for expression of eukaryon pcDNA3.1/neo (Invitrogen company) used in the structure.It contains CMV promotor, SV40 replication origin, neomycin resistance gene, can carry out the transient expression and the stably express of goal gene.Downstream in its exogenous gene cloning site, the terminator codon that contains myc epi-position and 6 continuous Histidines (6his) encoding sequence and this frame, therefore can express C as required and hold the fusion rotein that is connected with myc epi-position and 6His, utilize 6His to carry out protein purification, utilize the myc epi-position to detect the fusion rotein of expressing.In addition, the two ends in exogenous gene cloning site are respectively the polyA signals that contains T7 promotor and Trobest BGH, and available T7 and BGH universal primer directly check order to main foreign gene.

(1) the expression vector pcDNA-CX1 of the natural cytokine CX1 of construction expression

Pcr amplification product with embodiment 2 is a template, the performing PCR amplifying target genes.Amplification CX1 primer is as shown in table 2, wherein comprises the terminator codon of CX1 in the pcDNA-CX1 antisense strand primer, and institute's amplification PCR products is designated as CX1-CQ, and size is 564bp; Because contain the terminator codon of CX1 itself in the proteic open reading frame of coding CX1, the myc epi-position and the 6His in its downstream can't translate, therefore expressed CX1 recombinant protein aminoacid sequence and natural CX1 are in full accord in theory, do not contain myc epi-position and 6His.Be used for the research of CX1 transient expression

The PCR product behind EcoRI and the KpnI double digestion, directly is connected into the carrier for expression of eukaryon pcDNA3.1/Myc-His (-) that same enzyme is cut behind column purification, the recombinant vectors of formation is designated as pcDNA-CX1.The gene order of its CX1 is through T7 and BGH primer order-checking conclusive evidence.

(2) structure of the expression vector pCX1-His of the fusion rotein CX1-His of the C-terminal of construction expression cytokine CX1 and myc/His fusion

By similar method construction of expression vector pCX1-His, difference is: the terminator codon of removing CX1 in the pCX1-His antisense strand primer (table 2), institute's amplification PCR products is designated as CX1-BN, estimate that size is 563bp, expressed recombinant protein c X1-His C end contains myc epi-position and 6His, and the theoretical molecular of maturation protein is 22kD.Be used for the research of CX1 stably express.

The PCR product behind EcoRI and the BamHI double digestion, directly is connected into the carrier for expression of eukaryon pcDNA3.1/Myc-His (-) that same enzyme is cut behind column purification, the recombinant vectors of formation is designated as pCX1-His.Gene order is through T7 and BGH primer order-checking conclusive evidence.

(3) structure of the expression vector pCX1-Fc of the fusion rotein CX1-IgG of the C-terminal of construction expression cytokine CX1 and IgG-Fc fragment fusion

The structure of pCX1-Fc needs to pass through the cDNA of RT-PCR clones coding human IgG1 CH2 and CH3 from B lymphoma cell strain Raji, wherein downstream primer contains the terminator codon of IgG, and amplification Fc primer is as shown in table 2.Amplified production is designated as IgG1 Fc, is expected to be 740bp.CX1 and Fc merge in same frame, the cDNA of CX1 is positioned at the Fc upstream, the expressed fusion protein precursor is 414 amino acid, wherein contain by 20 amino acid whose CX1 signal peptides, when therefore in eukaryotic cell, expressing, the ripe fusion rotein of secreting to born of the same parents is 395 amino acid (SEQ ID NO:5) in theory, and molecular weight is 46.8kD.

With the PCR product of above-mentioned Fc behind column purification, BamHI and KpnI double digestion.Mixes with CX1-His amplified production and EcoRI and the KnpI enzyme big fragment of carrier for expression of eukaryon pcDNA3.1/Myc-His (-) after cutting behind EcoRI and the BamHI double digestion then, the recombinant vectors that connects back formation is designated as pCX1-Fc.Gene order is through T7 and BGH primer order-checking conclusive evidence.

Table 2 is used for the Oligonucleolide primers sequence of pcr amplification

Product	Oligonucleotide (5 '-3 ')	Restriction enzyme site	The product size
				CX1-CQ	Justice is arranged: ggaattc gcc acc atg gac tgg cct cac aac(SEQ ID NO：6)	EcoRI	564bp
	Antisense: GG GGTACCTCAGAAGATGCAGGTGCAGCCC(SEQ ID NO：7)	KpnI
				CX1-BN	Justice is arranged: ggaattc gcc acc atg gac tgg cct cac aac(SEQ ID NO：6)	EcoRI	563bp
	Antisense: ggatcc GG GAA GAT GCA GGT GCA(SEQ ID NO：8)	BamHI
				IgG1 Fc	Justice is arranged: CG GGATCCGCCCAAATCTTCTGACAAAC(SEQ ID NO：9)	BamHI	740bp
	Antisense: GG GGTACCTCATTTACCCGGAGACAG(SEQ ID NO：10)	KpnI

Annotate: underscore is represented restriction enzyme site.

The eucaryon of embodiment 5 cytokine CX1 is recombinant expressed

Recombinant plasmid dna is mixed by a certain percentage room temperature effect 45 minutes with liposome; Deng transfectional cell wash twice the back mix with DNA.During transient expression, collected culture supernatant in transfection 48-72 hour; During stably express, added G418 in 72 hours and screen after transfection, obtain positive clone strain, culture supernatant is collected in the amplification back.

It is recombinant expressed to carry out the CX1 eukaryotic cell ³⁵During the S metabolic marker, collect the cell of logarithmic phase after the transfection, carry out cellular metabolism ³⁵S mixes mark, mixes 4 hours collection culture supernatant and carries out SDS-PAGE electrophoresis and radioautographic analysis.

The culture supernatant of 293 cytotostatic cloning by expression strains of transfection pCX1-His or control plasmid, the SDS-PAGE electrophoresis of row 15% behind centricon (molecular weight cut-off 10kD) ultrafiltration and concentration.

Earlier this recombinant expression plasmid transfection COS7 is carried out transient expression, and the expression of CX1 and CX1-His is carried out ³⁵The metabolic marker of S.After the transfection 48 hours, by ³⁵S is marked at and has detected the expression product of estimating in the COS7 culture supernatant, and wherein the proteic size of master tape CX1 is about 20kD, and CX1-His has the expression product (Fig. 4) of two kinds of different sizes of 23kD and 26kD.With pCX1-His plasmid transfection 293 cells, obtain the stably express clonal cell line through G418 screening, detect identical 5A and the 5B of the results are shown in Figure.Fig. 5 A carries out CX1-His expression product in the culture supernatant behind recombinant expression plasmid pCX1-His gene transfection 293 cells ³⁵The electrophorogram that the S metabolic marker detects.Swimming lane mock is the 293 cells contrast supernatant of the blank carrier of transfection; Swimming lane CX1-His is the CX1-His expression product.Fig. 5 B is the electrophorogram that CX1-His expression product in the culture supernatant behind recombinant expression plasmid pCX1-His gene transfection 293 cells is carried out the Western engram analysis.Electrophoresis result all shows, the master tape of fusion rotein CX1-His occurred.

The purifying of embodiment 6 cytokine CX1 fusion rotein CX1-IgG

Fusion expression plasmid pCX1Fc transfection CHO cell, after the G418 screening, the stable expression cell strain that obtains is amplification cultivation in HyQ CCM serum free medium (Hyclone Inc.).Collect its culture supernatant, with Protein G affinity column HiTrap (Pharmacia) purifying, rapid adjust pH to 7.6 behind citric acid (pH3.0) wash-out, row 15%SDS-PAGE electrophoresis and silver dye and detect its apparent molecular weight and purity of protein (Fig. 6), are single band.The CX1-IgG recombinant protein of Analysis and Identification filters-20 ℃ of preservations in back.

Embodiment 7 cytokine CX1 are to the proliferation function of erythroleukemia cell TF1

The fusion rotein CX1-IgG (500ng/ml) of the recombinant expressed supernatant of the recombinant expressed supernatant of cytokine CX1 to be measured, CX1-His fusion rotein, purifying and human GM-CSF standard substance 1: 2 doubling dilution of row in 96 well culture plates, each extent of dilution is established 3 multiple holes, every hole 50 μ l; Add the TF1 cell of logarithmic phase, contain 10%FCS in the final culture system.After 72 hours, mtt assay detects the proliferation function to TF1 in 37 ℃ of cultivations.

The result is shown in Fig. 7 A-C, and Fig. 7 A has shown the proliferation function of the COS7 cells and supernatant of CX1 gene transfection to the TF1 cell.Fig. 7 B has shown the proliferation function of 293 cells and supernatant of CX-His gene transfection to the TF1 cell.Fig. 7 C has shown the proliferation function of CX1-IgG fusion rotein to the TF1 cell.The result shows, finds that different extent of dilution contain CX1 and the CX1-His culture supernatant all has certain proliferation function (Fig. 7 A and B) to TF1.The CX1-IgG energy effective stimulus TF1 cell proliferation of purifying, effective dose pact＞2ng/ml, and CTLA-IgG contrast fusion rotein does not have obvious effect (Fig. 7 C) to the propagation of TF1.

Embodiment 8 cytokine CX1 are to marrow CD34 ⁺The proliferation function of hemopoietic stem cell

The breastbone that takes off from the patient flushes out medullary cell, through the lymphocyte separation medium gradient centrifugation, is suspended in perfect medium and cultivates 2 hours to remove stroma cell wherein; Add the MACS marking fluid, crosslinked antibiotin suspension (B) the 200 μ l of non-specific blocking antibody liquid (A1), the anti-CD34 monoclonal antibody of biotin labeling liquid (A2) and magnetic bead are through the sorting of magnetic cell separator column, the collection positive cell.Detect the expression of cell surface CD34 with identification of cell by flow cytometer (FACS calibur).

Be made into 1 * 10 with IMDM ⁴/ ml cultivates in 24 orifice plates (1ml/ hole), carries out the external evoked differentiation of grain system and red system respectively.Grain is that inductor is IL-3+GM-CSF+G-CSF; Red is that inductor is IL-3+EPO; The final concentration of somatomedin is IL-3 and GM-CSF 20ng/ml, and G-CSF is 100U/ml, EPO2U/ml.In 5%CO ₂, cultivated 3 days under 37 ℃ of conditions.Body in above-mentioned grain system and red system is induced the CX1-IgG fusion rotein that adds 100ng/ml in the system, observes it to CD34 ⁺The effect of the external evoked differentiation of bone marrow stem cell uses CTLA-IgG (100ng/ml) as its contrast simultaneously.

The 3rd day, adopt the methylcellulose gum method to carry out unicellular colony forming cell of grain (CFU-GM) and erythroid cells colony forming cell (CFU-E) cultivation.Add 20% foetal calf serum, 20% cell IMDM nutrient solution (contains through inductive CD34 ⁺Cell), 10%GM-CSF (100U/ml) and 50% methylcellulose gum (27g/L).Mixture is added 96 well culture plates, and 100 μ l/ holes are established 3 multiple holes, are put 37 ℃, saturated humidity CO for every group ₂Cultivate in the incubator.In cultivating the back the 7th day, under inverted microscope, count colony, each colony contains 40 cells at least.

FACS detects CD34 ⁺Cell purity＞88%.CX1 is separately to CD34 ⁺The in-vitro multiplication of bone marrow stem cell and the no obvious promoter action of differentiation, but can promote GM-CSF/G-CSF/IL-3 to CD34 ⁺The grain of bone marrow stem cell is an induction of differentiation, sees the following form 3.Control group CTLA-IgG fusion rotein does not have obvious promoter action, sees Table 4.CX1-IgG has significant promoter action to the differentiation of the red system of IL-3/EPO inductive.

Table 1 CX1-IgG promotes GM-CSF/G-CSF/IL-3 inductive CD34 ⁺The differentiation of bone marrow stem cell grain system

Groups CFU-GM(X±SD)

GM-CSF/IL-3/G-CSF 66.3±11.2

GM-CSF/IL-3/G-CSF/CTLA4-IgG 70.8±13.4

GM-CSF/IL-3/G-CSF/CX1-IgG 120.2±15.7 ^*

^*.p＜0.01, compared with the control

Table 2.CX1-IgG promotes IL-3/EPO inductive CD34 ⁺The differentiation of the red system of bone marrow stem cell

Groups CFU-E(X±SD)

IL-3/EPO 73.7±15.0

IL-3/EPO/CTLA4-IgG 77.3±16.0

IL-3/EPO/CX1-IgG 145.0±17.5 ^*

^*.p＜0.01, compared with the control

Embodiment 9: the generation of anti-people CX1 protein antibodies

The recombinant protein c X1-IgG that obtains among the embodiment 7 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 CX1 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 human cell factor, its encoding sequence and purposes

(iii) sequence number: 10

(2) information of SEQ ID NO:1:

(i) sequence signature:

(A) length: 704bp

(B) type: nucleic acid

(C) chain: two strands

(D) topological framework: linearity

(ii) molecule type: cDNA

(xi) sequence description: SEQ ID NO:1:

tccaggcggg cagcagctgc aggctgacct tgcagcttgg cggaATGGAC TGGCCTCACA 60

ACCTGCTGTT TCTTCTTACC ATTTCCATCT TCCTGGGGCT GGGCCAGCCC AGGAGCCCCA 120

AAAGCAAGAG GAAGGGGCAA GGGCGGCCTG GGCCCCTGGC CCCTGGCCCT CACCAGGTGC 180

CACTGGACCT GGTGTCACGG ATGAAACCGT ATGCCCGCAT GGAGGAGTAT GAGAGGAACA 240

TCGAGGAGAT GGTGGCCCAG CTGAGGAACA GCTCAGAGCT GGCCCAGAGA AAGTGTGAGG 300

TCAACTTGCA GCTGTGGATG TCCAACAAGA GGAGCCTGTC TCCCTGGGGC TACAGCATCA 360

ACCACGACCC CAGCCGTATC CCCGTGGACC TGCCGGAGGC ACGGTGCCTG TGTCTGGGCT 420

GTGTGAACCC CTTCACCATG CAGGAGGACC GCAGCATGGT GAGCGTGCCG GTGTTCAGCC 480

AGGTTCCTGT GCGCCGCCGC CTCTGCCCGC CACCGCCCCG CACAGGGCCT TGCCGCCAGC 540

GCGCAGTCAT GGAGACCATC GCTGTGGGCT GCACCTGCAT CTTCTGAatt acctggccca 600

gaagccaggc cagcagcccg agaccatcct ccttgcacct ttgtgccaag aaaggcctat 660

gaaaagtaaa cactgacttt tgaaagcaaa aaaaaaaaaa aaaa 704

(2) information of SEQ ID NO:2:

(i) sequence signature:

(A) length: 180 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ii) molecule type: polypeptide

(xi) sequence description: SEQ ID NO:2:

MDWPHNLLFL LTISIFLGLG QPRSPKSKRK GQGRPGPLAP GPHQVPLDLV 50

SRMKPYARME EYERNIEEMV AQLRNSSELA QRKCEVNLQL WMSNKRSLSP 100

WGYSINHDPS RIPVDLPEAR CLCLGCVNPF TMQEDRSMVS VPVFSQVPVR 150

RRLCPPPPRT GPCRQRAVME TIAVGCTCIF 180

(2) information of SEQ ID NO:3:

(i) sequence signature:

(A) length: 160 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ii) molecule type: polypeptide

(xi) sequence description: SEQ ID NO:3:

QPRSPKSKRK GQGRPGPLAP GPHQVPLDLV SRMKPYARME EYERNIEEMV 50

AQLRNSSELA QRKCEVNLQL WMSNKRSLSP WGYSINHDPS RIPVDLPEAR 100

CLCLGCVNPF TMQEDRSMVS VPVFSQVPVR RRLCPPPPRT GPCRQRAVME 150

TIAVGCTCIF 160

(2) information of SEQ ID NO:4:

(i) sequence signature:

(A) length: 191 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ii) molecule type: polypeptide

(xi) sequence description: SEQ ID NO:4:

QPRSPKSKRK GQGRPGPLAP GPHQVPLDLV SRMKPYARME EYERNIEEMV 50

AQLRNSSELA QRKCEVNLQL WMSNKRSLSP WGYSINHDPS RIPVDLPEAR 100

CLCLGCVNPF TMQEDRSMVS VPVFSQVPVR RRLCPPPPRT GPCRQRAVME 150

TIAVGCTCIF PDPSSVPSFL EQKLISEEDL NSAVDHHHHH H 191

(2) information of SEQ ID NO:5:

(i) sequence signature:

(A) length: 395 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ii) molecule type: polypeptide

(xi) sequence description: SEQ ID NO:5:

QPRSPKSKRK GQGRPGPLAP GPHQVPLDLV SRMKPYARME EYERNIEEMV 50

AQLRNSSELA QRKCEVNLQL WMSNKRSLSP WGYSINHDPS RIPVDLPEAR 100

CLCLGCVNPF TMQEDRSMVS VPVFSQVPVR RRLCPPPPRT GPCRQRAVME 150

TIAVGCTCIF PDPEPKSSDK THTSPPSPAP ELLGGPSVFL FPPKPKDTLM 200

ISRTLEVTCV VVDVSHEDLE VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV 250

VSVLTVLHQD WLNGKEYKCK VSNKALPAPI EKTISKAKGQ PREPQVYTLP 300

PSRDELTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYK TTPPVLDSDG 350

SFFLYSKLTV DKSRWQQGNV FSCSVMHEAL HNHYTQKSLS LSPGK 395

(2) information of SEQ ID NO:6

(i) sequence signature

(A) length: 31 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

(xi) sequence description: SEQ ID NO:6:

GGAATTCGCC ACCATGGACT GGCCTCACAA C 31

(2) information of SEQ ID NO:7

(i) sequence signature

(A) length: 30 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

(xi) sequence description: SEQ ID NO:7:

GGGGTACCTC AGAAGATGCA GGTGCAGCCC 30

(2) information of SEQ ID NO:8

(i) sequence signature

(A) length: 23 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

(xi) sequence description: SEQ ID NO:8:

GGATCCGGGA AGATGCAGGT GCA 23

(2) information of SEQ ID NO:9

(i) sequence signature

(A) length: 28 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

(xi) sequence description: SEQ ID NO:9:

CGGGATCCGC CCAAATCTTC TGACAAAC 28

(2) information of SEQ ID NO:10

(i) sequence signature

(A) length: 30 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

(xi) sequence description: SEQ ID NO:10:

GGGGTACCTC ATTTACCCGG AGACAG 26

Claims (11)

1. an isolated polypeptide is characterized in that, it is selected from down group:

(a) has the polypeptide of the aminoacid sequence of SEQ ID NO:2 or 3;

(b) disappearance, insertion or the replacement through one or several amino-acid residue of SEQ ID NO:2 or 3 aminoacid sequences formed, and have the propagation function that promotes erythroleukemia cell TF1 by (a) polypeptides derived.

2. polypeptide as claimed in claim 1 is characterized in that, this polypeptide is to have the polypeptide that is selected from down the group aminoacid sequence: SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5.

3. isolating polynucleotide is characterized in that, the described polypeptide of its coding claim 1.

4. polynucleotide as claimed in claim 3 is characterized in that, this polynucleotide encoding has the polypeptide of aminoacid sequence shown in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or the SEQ ID NO:5.

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 45-587 position among the SEQ ID NO:1;

(b) has the sequence of 105-587 position among the SEQ ID NO:1;

(c) has the sequence of 1-704 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. the preparation method of the described polypeptide of claim 1 is characterized in that, this method comprises:

(a) under expression condition, cultivate the described host cell of claim 7;

(b) from culture, isolate the described polypeptide of claim 1.

9. energy and the described polypeptid specificity bonded of claim 1 antibody.

10. whether there is the method for the described polypeptide of claim 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 described polypeptide.

11. a composition that promotes cell proliferation is characterized in that, it contains the described polypeptide of claim 1 and the acceptable carrier of significant quantity.

Images