CN1475503A

CN1475503A - New type human bone marrow substrate cell source growth inhibiting factor, its coded sequence and use

Info

Publication number: CN1475503A
Application number: CNA021365253A
Authority: CN
Inventors: 夏大静; 李楠; 王涛; 曹雪涛
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2002-08-16
Filing date: 2002-08-16
Publication date: 2004-02-18

Abstract

A bone marrow stromal cell-derived growth inhibitor (BDGI), its coding polynucleotide, the process for recombining said protein, and the application of said BDGI and its coding sequence in diagnosing and treating cancer are disclosed.

Description

The growth inhibiting factor of new type human bone marrow substrate cell source, its encoding sequence and purposes

Technical field

The invention belongs to biotechnology and medical field, specifically, the present invention relates to the growth inhibiting factor BDGI polypeptide of new coding human bone marrow substrate cell source, and polynucleotide polynucleotide encoding sequence.The invention still further relates to the purposes and the preparation of these polynucleotide and polypeptide.BDGI is a soluble proteins in a kind of cell relevant with cell growth inhibiting, has the function that suppresses the breast carcinoma cell strain growth.

Background technology

Marrow stromal cell (Bone Marrow Stromal Cell, BMSC) be the chief component of marrow hemopoiesis microenvironment, it is a functional colony that has the various kinds of cell component to constitute, comprise inoblast, scavenger cell and marrow endothelial cell etc., these cells effect such as directly contact by discharging cytokine, embrane-associated protein and cell with intercellular, regulate the differentiation and the growth of multipotential stem cell, in hematopoiesis and immunomodulatory, play a significant role.The functional defect of hematopoieticmicroenviron-ment can cause multiple diseases such as tumour, autoimmune disease, infection.Therefore, the research that derives from the recruit of BMSC is significant to differentiation and development, biological action and the molecule mechanism thereof of deeply illustrating BMSC, also may provide new thinking and means for the treatment of tumour, infection, transplant rejection and autoimmune disease etc. simultaneously.

Mammary cancer is the major malignant tumor of harm WomanHealth, and the whole world has 1,200,000 women that mammary cancer takes place every year approximately.In China, the sickness rate of mammary cancer is also rising year by year, and having jumped at its sickness rate of some big city is first in women's malignant tumour.The generation of discovering mammary cancer is relevant with the multiple factors such as expression of sudden change, somatomedin and the growth inhibiting factor of hormonal readiness, fertility, oncogene and cancer suppressor gene.Be accompanied by the variation of multiple hormone in the generation evolution of mammary cancer, and be subjected to the influence of these hormones.The morbidity of estradiol in the oestrogenic hormon and oestrone and mammary cancer has direct relation, and trihydroxy-oestrin and progesterone have been considered to provide protection, and prolactin then has promoter action in the breast cancer development process.The most relevant with the mammary cancer onset risk in the fertility factor is conceptional age first.Investigation find 30 years old just pregnant first later on person, the sickness rate of its mammary cancer be before 20 years old first pregnant person's 2-5 doubly, the danger of mammary cancer morbidity increases along with the postponement at primiparity age.The investigator finds that gestation can impel mammary gland cell to be broken up early by secreting some material such as growth inhibiting factor, reduces tumor susceptibility.

The multistage process that develops into of human tumor exists polygenic change in this process, so that tumour is the result that a kind of polygene changes.Modern age, RESEARCH ON CELL-BIOLOGY showed, cells in vivo is subjected to the adjusting of interior positivity of body and negativity two class adjustment signals in the process of propagation, differentiation and apoptosis.Equilibrated destruction can cause the generation of tumour between positivity signal (oncogene, somatomedin) and the negativity signal (cancer suppressor gene, growth inhibiting factor).Shi etc. think that the polygene that exists similar colorectal carcinoma in the mammary cancer forming process (to atypical hyperplasia, develop into carcinoma in situ from the hyperplasia of prostate of mammary tissue again, proceed to infiltrating cancer, metastasis) changes.

Somatomedin, growth inhibiting factor are low molecular peptide class or glycoprotein material, can stimulate or cell growth inhibiting by corresponding receptors bind with cell surface in vivo.Confirmed already that the balance between somatomedin and the growth inhibiting factor played an important role to the propagation of regulating mammary epithelial cell, existing lot of documents report somatomedin such as Urogastron (EGF) in nearly ten years, rhIGF-1 (IGF), transforming growth factor-alpha (TGF α) and fibroblast growth factor (FGF) etc. are being played the part of important role in causing the process of breast tumor, and newborn adenogenous growth inhibiting factor (MDGI), MDGI genes involved (MRG), growth inhibiting factor such as TGF β and igf binding protein 3 then have the effect that the potential breast-tumor resisting forms.Growth inhibiting factor such as MDGI, MRG equalenergy stimulus mammary epithelial cell differentiation, the local cells propagation that causes suppress.The unconventionality expression of somatomedin and growth inhibiting factor and the generation of tumour development are closely related, and disturbing the generation of somatomedin or the generation of effect link and raising growth inhibiting factor may be the New Policy that suppresses tumor growth.

In recent years, the research of breast cancer related gene has been become the focus in this field, newfound gene constantly increases, but the mechanism of action of its definite molecular mechanism and coded product waits further further investigation.

Therefore, this area presses for exploitation breast cancer related gene, especially growth inhibiting factor.

Summary of the invention

The growth inhibiting factor BDGI albumen that the purpose of this invention is to provide a kind of new human bone marrow substrate cell source 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 BDGI 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 selected from down group: the polypeptide that (a) has SEQ ID NO:2 aminoacid sequence; (b) SEQ ID NO:2 aminoacid sequence is formed through replacement, disappearance or the interpolation of one or more amino-acid residues, and have inhibition breast carcinoma cell strain (as MCF-7) growth function by (a) polypeptides derived.

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 BDGI 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 147-1577 position among the SEQ ID NO:1; (b) has the sequence of 1-1766 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 BDGI protein-active, this method comprises: (a) under the proteic condition of suitable expressing human BDGI, cultivate the above-mentioned host cell that is transformed or transduce; (b) from culture, isolate polypeptide with people BDGI protein-active.

In a fifth aspect of the present invention, provide and above-mentioned people BDGI polypeptid specificity bonded antibody.The nucleic acid molecule that can be used for detecting also is provided, and it contains a successive 10-1766 Nucleotide in the above-mentioned polynucleotide.

In a sixth aspect of the present invention, the compound of simulation, promotion, antagonism people BDGI polypeptide active is provided, and the compound that suppresses people BDGI 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 BDGI polypeptide.

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

In a eighth aspect of the present invention, a kind of disease relevant with people BDGI 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 BDGI polypeptide active, and perhaps screening suppresses the antagonist of people BDGI polypeptide active or is used to the peptide finger print identification.The proteic encoding sequence of people BDGI 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 BDGI 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, inflammation, neural system and cardiovascular diseases.

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

Description of drawings

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

Fig. 2 is a people BDGI RT-PCR expression analysis of the present invention, and prompting BDGI is expressed in the monocyte, dendritic cell in some tumour cell and human peripheral source.

Fig. 3 is the shown distribution of people BDGI Northern blot hybridization of the present invention.Results suggest BDGI is the molecule that a kind of tool particular organization distributes.

Fig. 4 has shown the growth-inhibiting of people BDGI to the MCF-7 breast cancer cell.

Embodiment

The inventor has obtained having the growth inhibiting factor (Bone MarrowStromal Cell-derived Growth Inhibitor abbreviates BDGI as) of the novel growth inhibiting factor-bone marrow substrate cell source that suppresses the growth of breast cancers effect from the extensive random sequencing of BMSC cDNA gene library.Finished the present invention on this basis.

BDGI is that to be separated to a kind of total length from normal people's marrow stromal cell (BMSC) be the 1766bp novel gene, contains complete open frame 1434bp, and its proteins encoded is 477 amino acid.Analyze this molecule shortage and stride film district and signal peptide, infer that it is the soluble protein of non-secretory.The assignment of genes gene mapping is in 16q24.1.A pregnant inductive growth inhibiting factor OKL38 height homology of this gene and (calendar year 2001) discovery recently, the OKL38 gene is high expression level in the mammary gland of gestation and lactation, and experimental result finds that the MCF-7 stably express strain of transfection OKL38 gene obviously reduces at external multiplication capacity and in the intravital tumorigenicity of nude mice.

Interesting is, the preliminary study of this new gene of being cloned into from BMSC gene library is found that it has the function of the inhibition breast cancer cell growth similar to OKL38.The Northern blot hybridization shows that it has the tissue distribution different with OKL38, predominant expression in tissues such as liver, testis and skeletal muscle.RT-PCR analysis revealed BDGI expresses higher in the dendritic cell in normal cell such as peripheral blood lymphocytes and peripheral blood lymphocytes source, expresses lowlyer in breast cancer cell line mcf-7, has expression in various degree in other tumour cell.

BDGI has the function that suppresses breast cancer cell propagation, and the discovery of new growth inhibiting factor gene will help further to illustrate the pathogenesis of mammary cancer, to the diagnosis of breast cancer and treated bigger pushing effect.Therefore, the growth inhibiting factor BDGI of the new human bone marrow substrate cell source of research and development is significant.

In the present invention, term " BDGI albumen ", " BDGI polypeptide " or " the growth inhibiting factor BDGI of bone marrow substrate cell source " are used interchangeably, and all refer to have the albumen or the polypeptide of the growth inhibiting factor BDGI aminoacid sequence (SEQ ID NO:2) of human bone marrow substrate cell source.

As used herein, " isolating " is meant that material separates (if natural substance, primal environment promptly is a natural surroundings) from its primal environment.Do not have separation and purification as 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, " isolating BDGI albumen or polypeptide " is meant that the BDGI 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 BDGI albumen of standard.Basically pure polypeptide can produce single master tape on non-reduced polyacrylamide gel.

Polypeptide of the present invention can be recombinant polypeptide, natural polypeptides, synthetic polypeptide, preferred recombinant polypeptide.Polypeptide of the present invention can be the product of natural purifying, or the product of chemosynthesis, or uses recombinant technology to produce from protokaryon or eucaryon host (for example, bacterium, yeast, higher plant, insect and mammalian cell).The host used according to the recombinant production scheme, 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 the proteic fragment of people BDGI, derivative and analogue.As used herein, term " fragment ", " derivative " are meant with " analogue " and keep identical biological function of natural human BDGI albumen of the present invention or active polypeptide basically.Polypeptide fragment of the present invention, derivative or analogue 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 transformation period, polyoxyethylene glycol for example) merges formed polypeptide, or (iv) additional aminoacid sequence is fused to this peptide sequence and the polypeptide that forms (as leader sequence or secretion sequence or be used for the sequence or the proteinogen sequence of this polypeptide of purifying, or with the fusion rotein of the segmental formation of antigen I gG).According to the instruction of this paper, these fragments, derivative and analogue belong to the known scope of those skilled in the art.

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

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

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

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

In the present invention, " people BDGI albumen conservative property variation polypeptide " refers to compare with the aminoacid sequence of SEQ ID NO:2, has 10 at the most, preferably at the most 8, more preferably at the most 5,3 amino acid is replaced by similar performance or close amino acid and is formed polypeptide at the most best.These conservative property variation polypeptide preferably carry out the amino acid replacement according to table 1 and produce.

Table 1

Initial residue	Representational replacement	The preferred replacement
Initial residue	Representational replacement	The preferred replacement	?Ala??(A)	Val；Leu；Ile	?Val
?Arg??(R)	Lys；Gln；Asn	?Lys	?Ala??(A)	Val；Leu；Ile	?Val
?Arg??(R)	Lys；Gln；Asn	?Lys	?Asn??(N)	Gln；His；Lys；Arg	?Gln
?Asp??(D)	Glu	?Glu	?Asn??(N)	Gln；His；Lys；Arg	?Gln
?Asp??(D)	Glu	?Glu	?Cys??(C)	Ser	?Ser
?Gln??(Q)	Asn	?Asn	?Cys??(C)	Ser	?Ser
?Gln??(Q)	Asn	?Asn	?Glu??(E)	Asp	?Asp
?Gly??(G)	Pro；Ala	?Ala	?Glu??(E)	Asp	?Asp
?Gly??(G)	Pro；Ala	?Ala	?His??(H)	Asn；Gln；Lys；Arg	?Arg
?Ile??(I)	Leu；Val；Met；Ala；Phe	?Leu	?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	?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	?Met??(M)	Leu；Phe；Ile	?Leu
?Phe??(F)	Leu；Val；Ile；Ala；Tyr	?Leu	?Pro??(P)	Ala	?Ala
?Ser??(S)	Thr	?Thr	?Pro??(P)	Ala	?Ala
?Ser??(S)	Thr	?Thr	?Thr??(T)	Ser	?Ser
?Trp??(W)	Tyr；Phe	?Tyr	?Thr??(T)	Ser	?Ser
?Trp??(W)	Tyr；Phe	?Tyr	?Tyr??(Y)	Trp；Phe；Thr；Ser	?Phe
?Val??(V)	Ile；Leu；Met；Phe；Ala	?Leu	?Tyr??(Y)	Trp；Phe；Thr；Ser	?Phe

Polynucleotide of the present invention can be dna form or rna form.Dna form comprises the DNA of cDNA, genomic dna or synthetic.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 varient of degeneracy.As used herein, " varient of degeneracy " is meant that in the present invention coding has the protein of SEQ ID NO:2, but with the differentiated nucleotide sequence of coding region sequence shown in the SEQ ID NO:1.

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

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

The invention still further relates to the varient of above-mentioned polynucleotide, its coding has the polypeptide of identical aminoacid sequence or fragment, analogue and the derivative of polypeptide with the present invention.The varient of these polynucleotide can be the allelic variant of natural generation or the varient that non-natural takes place.These nucleotide diversity bodies comprise and replace varient, deletion mutation body and insert varient.As known in the art, allelic variant is the replacement form of polynucleotide, and it may be replacement, disappearance or the insertion of one or more Nucleotide, but can be from not changing the function of its encoded polypeptides in fact.

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 BDGI.

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

People BDGI 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.Science1985 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 BDGI 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 BDGI polypeptide of reorganization.In general following steps are arranged:

(1). with the polynucleotide (or varient) of coding of the present invention people BDGI 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 BDGI 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 BioChem.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 BDGI DNA sequences encoding and suitable transcribing/the translate expression vector of control signal.These methods comprise (Sambroook, et al.Molecular Cloning, a LaboratoryManual, cold Spring 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 reorganization go into BDGI albumen or polypeptide is 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 BDGI protein function as pharmacological agent BDGI protein function.The peptide molecule that can suppress or stimulate people BDGI protein function that can be used for seeking therapeutic value with the recombinant human B DGI protein screening peptide library of expressing.

On the other hand, the present invention also comprises people BDGI 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 BDGI gene product or fragment.Preferably, refer to that those can combine with people BDGI 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 BDGI, comprise that also those do not influence the antibody of people BDGI protein function.The present invention also comprise those can with modify or without the people BDGI 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 BDGI gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing human BDGI 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.Imunol.B6:511,1976; 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 BDGI protein function and the antibody that does not influence people BDGI protein function.Each antibody-like of the present invention can utilize the fragment or the functional zone of people BDGI 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 BDGI 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 BDGI can be used in the immunohistochemistry technology, detects the people BDGI albumen in the biopsy specimen.In addition, with the also available labelled with radioisotope of the protein bound monoclonal antibody of people BDGI, 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 BDGI albumen.The antibody that gives suitable dosage can stimulate or block proteic generation of people BDGI 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 BDGI 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 BDGI protein positive.

The production of polyclonal antibody can choose BDGI 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 BDGI albumen interactional material takes place, as part, 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 tumour, inflammation, the treatment of neural system and cardiovascular diseases aspect.When using BDGI 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 BDGI 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 BDGI 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 BDGI 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 BDGI of the proteic nothing expression of BDGI or unusual/non-activity.The BDGI albumen that the gene therapy vector (as virus vector) of reorganization can be designed to express variation is to suppress endogenic BDGI protein-active.Deriving from viral expression vector such as retrovirus, adenovirus, adeno-associated virus (AAV), hsv, parvovirus etc. can be used for the BDGI transgenosis to cell.The method that structure carries the recombinant viral vector of BDGI gene is found in existing document (Sambrook, et al.).Recombinant human B DGI 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 BDGImRNA 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 BDGI obtains.During screening, must carry out mark to people BDGI protein molecular.

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

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

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

The sudden change that detects the BDGI gene also can be used for the disease of diagnosing BDGI albumen relevant.The form of BDGI protein mutation comprises that the point mutation compared with normal wild type BDGI 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 BDGI 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, MendelianInheritance in Man (can by with the online acquisition of Johns Hopkins University Welch MedicalLibrary).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 IDNO: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 1766 bases, and its open reading frame is positioned at the 147-1577 position, and the coding total length is 477 amino acid whose people BDGI albumen (SEQ ID NO:2).This BDGI albumen and pregnant inductive growth inhibiting factor OKL38 aminoacid sequence height homology, consistence can be up to 64%.The Northern blot hybridization shows that it has the tissue distribution different with OKL38, predominant expression in tissues such as liver, testis and skeletal muscle.RT-PCR analysis revealed BDGI expresses higher in the dendritic cell in normal cell such as peripheral blood lymphocytes and peripheral blood lymphocytes source, expresses lowlyer in breast cancer cell line mcf-7, has expression in various degree in other tumour cell.The research prompting of having carried out, BDGI has the function of the inhibition breast cancer cell growth similar to OKL38, infers that it is a novel growth inhibiting factor.Growth inhibiting factor is low molecular peptide class or glycoprotein material, in vivo can be by corresponding receptors bind cell growth inhibiting with cell surface.Confirmed already that the balance between somatomedin and the growth inhibiting factor played an important role to the propagation of regulating mammary epithelial cell.Therefore, BDGI albumen or its relevant antagonist, agonist etc. can be diseases such as treating tumour provides new immunodiagnosis and targeted therapy 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:ColdSpring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

Embodiment 1: the clone of people BDGI cDNA

Extract the total RNA of human bone marrow substrate cell with Trizol reagent (Life Technologies company).Then, from total RNA, separate poly (A) mRNA.Poly (A) mRNA after reverse transcription forms cDNA, is cloned test kit (Life Technologies) 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 the sequence of random choose clone's 5 ' end 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 the growth inhibiting factor BDGI of human bone marrow substrate cell source, the growth inhibiting factor BDGI gene of its encoding gene called after human bone marrow substrate cell source.

Sequence SEQ ID NO:1 total length is 1766bp, comprises 5 ' the end non-coding region of 146 bp and 3 ' the end non-coding region of 186 bp, and coding contains 477 amino acid whose polypeptide.Calculating not in theory, the molecular weight of glycosylated ripe molecule is about 52 kD.

They are different with known for the BLAST analysis revealed, and with people's gestation inductive growth inhibiting factor OKL38 height homology, consistence reaches 64%,, belong to novel growth inhibiting factor.

Hydrophobicity analysis prompting BDGI is a soluble proteins (Fig. 1) in the born of the same parents.

Embodiment 2: carry out the cell expressing analysis of people BDGI with the RT-PCR method

Extract total RNA of the clones such as MCF-7 be in logarithmic phase with Trizol reagent, get 5 μ g cell total rnas and 1 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 pcr amplification primer is as follows: adopted primer 5 ' TTGATGCCCTTCTACGCC (SEQ ID NO:3) is arranged, antisense primer 5 ' TGGCGAGGACCACGTTG (SEQ IDNO:4), simultaneously with β-actin as positive control.The PCR reaction volume is 50 μ l, wherein contains reverse transcription template 10 μ l, 0.5mM primer, 0.2mM dNTP and 1U rTaq archaeal dna polymerase (Takara), the amplification parameter be 95 ℃ 15 seconds, 57 ℃ 30 seconds, 72 ℃ 30 seconds, 28 circulations were extended 10 minutes for back 72 ℃.Capable 1.5% agarose gel electrophoresis of PCR product is tentatively confirmed.The dna sequence analysis result shows that the DNA sequences encoding of this PCR product and the 412-882 shown in the SEQ ID NO:1 are identical.

Expression analysis result (Fig. 2) shows, BDGI is high expression level in tumor cell line such as CaoV-3, K562, Daudi and dendritic cell, the low expression in clones such as PC-3, KG-1, HL-60, Molt-4.

Embodiment 3: the Northern engram analysis of people BDGI

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.

Northern blot hybridization result (Fig. 3) shows: expression (2.0kb) is in various degree arranged in healthy tissuess such as liver, testis, skeletal muscle, heart and spleen, and this shows that BDGI albumen is a kind of albumen of particular expression.

Embodiment 4: people BDGI protokaryon is recombinant expressed

In this embodiment, be template with the total mRNA of extractive dendritic cell, 5 ' and 3 ' the PCR Oligonucleolide primers held following with sequence carries out the RT-PCR amplification, obtains people BDGI DNA as inserting fragment.

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

5’-GTGGATCCATGAGCTCCTCCAGAAAGGAC-3’(SEQ?ID?NO：5)

This primer contains the restriction enzyme site of BamH I restriction enzyme, is the part encoding sequence of people BDGI after this restriction enzyme site;

3 ' end primer sequence is:

5’-GCGAATTCTGAGGGTGGCTTCCTGGTCT-3’(SEQ?ID?NO：6)

This primer contains the part encoding sequence of restriction enzyme site, translation termination and the people BDGI of EcoR I restriction enzyme.

With the PCR product purification that obtains after EcoR I enzyme cut and recombinate according to a conventional method with plasmid pGEM-3ZF again and be converted into the competence e. coli bl21, the picking positive colony is identified back purifying and order-checking (377 type sequenators of ABI company, BigDye Terminator test kit, PE company).The people BDGI cDNAEcoR I endonuclease bamhi of correct sequence is cloned into expression vector pGEX-2T (Pharmacia), forms carrier pGEX-2T-BDGI, then transformed into escherichia coli BL21.Positive colony is cut evaluation with EcoR I enzyme, and the forward clone cuts evaluation with BamH I enzyme, the capable 0.8% agarose gel electrophoresis analysis of product.Confirm through order-checking, inserted designed BDGI encoding sequence.

Choosing the positive BL21 clone who expresses BDGI 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, 1.8mM KH 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 the GST-BDGI fusion rotein.Molecular weight is about 78Kda, conforms to predictor.

In addition, replace SEQ ID NO:5 and 6 with following primer, repeat above-mentioned steps, obtain the fusion rotein of GST-BDGI fragment (4-94 amino acids): 5 '-GTGGATCCAGAAAGGACCACCTCG-3 ' (SEQ ID NO:7), 5 '-GCGAATTCGCGTAGAAGGGCATCAAAG-3 ' (SEQ ID NO:8).

Embodiment 5: anti-people BDGI production of antibodies

The recombinant protein GST-BDGI fusion rotein of acquisition among the embodiment 4 and the fusion rotein of GST-BDGI fragment (9-94 amino acids) are 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 BDGI gene translation product with it.Found that antibody can combine with albumen of the present invention specifically.

The cell growth inhibiting activity of embodiment 6:BDGI is observed

Total length BDGI plasmid DNA among the embodiment 1 with LipofectAMINE reagent (LifeTechnologies company) transfection breast cancer cell line mcf-7 cell, is contrasted as irrelevant with the pcDNA3.1 plasmid vector.The MTT that carried out cell proliferation after the transfection in 0,24,48 and 72 hour detects, and the result is expressed as the curve of OD value to the time.

Detected result (Fig. 4) shows that the expression of BDGI is suppressed the growth of breast cancer cell line mcf-7.

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＜110〉Inst. of Immunology, Zhejiang Univ.＜120〉GIF of new type human bone marrow substrate cell source, its coded sequence and purposes＜130〉024958＜160〉8＜170〉PatentIn version 3.1＜210〉1＜211〉1766＜212〉DNA＜213〉homo sapiens (Homo sapiens)＜220〉＜221〉CDS＜222〉(147) .. (1577)＜223〉＜400〉1ccacagggta atgggtgtcc gatgtcacgg gggactctgt gatccgtgtt cccctgaccc 60tcctagtgca caacttggcc gggctcactg ggctcctgca ccactgcctg tcaggtccgc 120tgccagcccc aagcccccca ccagcc atg agc tcc tcc aga aag gac cac ctc 173

Met?Ser?Ser?Ser?Arg?Lys?Asp?His?Leu

1???????????????5ggc?gcc?agc?agc?tca?gag?ccc?ctc?ccg?gtc?atc?att?gtg?ggt?aac?ggc??????221Gly?Ala?Ser?Ser?Ser?Glu?Pro?Leu?Pro?Val?Ile?Ile?Val?Gly?Asn?Gly10??????????????????15??????????????????20??????????????????25ccc?tct?ggt?atc?tgc?ctg?tcc?tac?ctg?ctc?tcc?ggc?tac?aca?ccc?tac??????269Pro?Ser?Gly?Ile?Cys?Leu?Ser?Tyr?Leu?Leu?Ser?Gly?Tyr?Thr?Pro?Tyr

30??????????????????35??????????????????40acg?aag?cca?gat?gcc?atc?cac?cca?cac?ccc?ctg?ctg?cag?agg?aag?ctc??????317Thr?Lys?Pro?Asp?Ala?Ile?His?Pro?His?Pro?Leu?Leu?Gln?Arg?Lys?Leu

45??????????????????50??????????????????55acc?gag?gcc?ccg?ggg?gtc?tcc?atc?ctg?gac?cag?gac?ctg?gac?tac?ctg??????365Thr?Glu?Ala?Pro?Gly?Val?Ser?Ile?Leu?Asp?Gln?Asp?Leu?Asp?Tyr?Leu

60??????????????????65??????????????????70tcc?gaa?ggc?ctc?gaa?ggc?cga?tcc?caa?agc?ccc?gtg?gcc?ctg?ctc?ttt??????413Ser?Glu?Gly?Leu?Glu?Gly?Arg?Ser?Gln?Ser?Pro?Val?Ala?Leu?Leu?Phe

75??????????????????80??????????????????85gat?gcc?ctt?cta?cgc?cca?gac?aca?gac?ttt?ggg?gga?aac?atg?aag?tcg????461Asp?Ala?Leu?Leu?Arg?Pro?Asp?Thr?Asp?Phe?Gly?Gly?Asn?Met?Lys?Ser90??????????????????95??????????????????100?????????????????105gtc?ctc?acc?tgg?aag?cac?cgg?aag?gag?cac?gcc?atc?ccc?cac?gtg?gtt????509Val?Leu?Thr?Trp?Lys?His?Arg?Lys?Glu?His?Ala?Ile?Pro?His?Val?Val

110?????????????????115?????????????????120ctg?ggc?cgg?aac?ctc?ccc?ggg?gga?gcc?tgg?cac?tcc?atc?gaa?ggc?tcc????557Leu?Gly?Arg?Asn?Leu?Pro?Gly?Gly?Ala?Trp?His?Ser?Ile?Glu?Gly?Ser

125?????????????????130?????????????????135atg?gtg?atc?ctg?agc?caa?ggc?cag?tgg?atg?ggg?ctc?ccg?gac?ctg?gag????605Met?Val?Ile?Leu?Ser?Gln?Gly?Gln?Trp?Met?Gly?Leu?Pro?Asp?Leu?Glu

140?????????????????145?????????????????150gtc?aag?gac?tgg?atg?cag?aag?aag?cga?aga?ggt?ctt?cgc?aac?agc?cgg????653Val?Lys?Asp?Trp?Met?Gln?Lys?Lys?Arg?Arg?Gly?Leu?Arg?Asn?Ser?Arg

155?????????????????160?????????????????165gcc?act?gcc?ggg?gac?atc?gcc?cac?tac?tac?agg?gac?tac?gtg?gtc?aag????701Ala?Thr?Ala?Gly?Asp?Ile?Ala?His?Tyr?Tyr?Arg?Asp?Tyr?Val?Val?Lys170?????????????????175?????????????????180?????????????????185aag?ggt?ctg?ggg?cat?aac?ttt?gtg?tcc?ggt?gct?gta?gtc?aca?gcc?gtg????749Lys?Gly?Leu?Gly?His?Asn?Phe?Val?Ser?Gly?Ala?Val?Val?Thr?Ala?Val

190?????????????????195?????????????????200gag?tgg?ggg?acc?ccc?gat?ccc?agc?agc?tgt?ggg?gcc?cag?gac?tcc?agc????797Glu?Trp?Gly?Thr?Pro?Asp?Pro?Ser?Ser?Cys?Gly?Ala?Gln?Asp?Ser?Ser

205?????????????????210?????????????????215ccc?ctc?ttc?cag?gtg?agc?ggc?ttc?ctg?acc?agg?aac?cag?gcc?cag?cag????845Pro?Leu?Phe?Gln?Val?Ser?Gly?Phe?Leu?Thr?Arg?Asn?Gln?Ala?Gln?Gln

220?????????????????225?????????????????230ccc?ttc?tcg?ctg?tgg?gcc?cgc?aac?gtg?gtc?ctc?gcc?aca?ggc?acg?ttc????893Pro?Phe?Ser?Leu?Trp?Ala?Arg?Asn?Val?Val?Leu?Ala?Thr?Gly?Thr?Phe

235?????????????????240?????????????????245gac?agc?ccg?gcc?cgg?ctg?ggc?atc?ccc?ggg?gag?gcc?ctg?ccc?ttc?atc????941Asp?Ser?Pro?Ala?Arg?Leu?Gly?Ile?Pro?Gly?Glu?Ala?Leu?Pro?Phe?Ile250?????????????????255?????????????????260?????????????????265cac?cat?gag?ctg?tct?gcc?ctg?gag?gcc?gcc?aca?agg?gtg?ggt?gcg?gtg????989His?His?Glu?Leu?Ser?Ala?Leu?Glu?Ala?Ala?Thr?Arg?Val?Gly?Ala?Val

270?????????????????275?????????????????280acc?ccg?gcc?tca?gac?cct?gtc?ctc?atc?att?ggc?gcg?ggg?ctg?tca?gcg????1037Thr?Pro?Ala?Ser?Asp?Pro?Val?Leu?Ile?Ile?Gly?Ala?Gly?Leu?Ser?Ala

285?????????????????290?????????????????295gcc?gac?gcg?gtc?ctc?tac?gcc?cgc?cac?tac?aac?atc?ccg?gtg?atc?cat????1085Ala?Asp?Ala?Val?Leu?Tyr?Ala?Arg?His?Tyr?Asn?Ile?Pro?Val?Ile?His

300?????????????????305?????????????????310gcc?ttc?cgc?cgg?gcc?gtg?gac?gac?cct?ggc?ctg?gtg?ttc?aac?cag?ctg????1133Ala?Phe?Arg?Arg?Ala?Val?Asp?Asp?Pro?Gly?Leu?Val?Phe?Asn?Gln?Leu

315?????????????????320?????????????????325ccc?aag?atg?ctg?tac?ccc?gag?tac?cac?aag?gtg?cac?cag?atg?atg?cgg????1181Pro?Lys?Met?Leu?Tyr?Pro?Glu?Tyr?His?Lys?Val?His?Gln?Met?Met?Arg330?????????????????335?????????????????340?????????????????345gag?cag?tcc?atc?ctg?tcg?ccc?agc?ccc?tat?gag?ggt?tac?cgc?agc?ctc????1229Glu?Gln?Ser?Ile?Leu?Ser?Pro?Ser?Pro?Tyr?Glu?Gly?Tyr?Arg?Ser?Leu

350?????????????????355?????????????????360ccc?agg?cac?cag?ctg?ctg?tgc?ttc?aag?gaa?gac?tgc?cag?gcc?gtg?ttc????1277Pro?Arg?His?Gln?Leu?Leu?Cys?Phe?Lys?Glu?Asp?Cys?Gln?Ala?Val?Phe

365?????????????????370?????????????????375cag?gac?ctc?gag?ggt?gtc?gag?aag?gtg?ttt?ggg?gtc?tcc?ctg?gtg?ctg????1325Gln?Asp?Leu?Glu?Gly?Val?Glu?Lys?Val?Phe?Gly?Val?Ser?Leu?Val?Leu

380?????????????????385?????????????????390gtc?ctc?atc?ggc?tcc?cac?ccc?gac?ctc?tcc?ttc?ctg?cct?ggg?gca?ggg????1373Val?Leu?Ile?Gly?Ser?His?Pro?Asp?Leu?Ser?Phe?Leu?Pro?Gly?Ala?Gly

395?????????????????400?????????????????405gct?gac?ttt?gca?gtg?gat?cct?gac?cag?ccg?ctg?agc?gcc?aag?agg?aac????1421Ala?Asp?Phe?Ala?Val?Asp?Pro?Asp?Gln?Pro?Leu?Ser?Ala?Lys?Arg?Asn410?????????????????415?????????????????420?????????????????425ccc?att?gac?gtg?gac?ccc?ttc?acc?tac?cag?agc?acc?cgc?cag?gag?ggc????1469Pro?Ile?Asp?Val?Asp?Pro?Phe?Thr?Tyr?Gln?Ser?Thr?Arg?Gln?Glu?Gly

430?????????????????435?????????????????440ctg?tac?gcc?atg?ggg?ccg?ctg?gcc?ggg?gac?aac?ttc?gtg?agg?ttt?gtg????1517Leu?Tyr?Ala?Met?Gly?Pro?Leu?Ala?Gly?Asp?Asn?Phe?Val?Arg?Phe?Val

445?????????????????450?????????????????455cag?ggg?ggc?gcc?ttg?gct?gtg?gcc?agc?tcc?ctg?cta?agg?aag?gag?acc????1565Gln?Gly?Gly?Ala?Leu?Ala?Val?Ala?Ser?Ser?Leu?Leu?Arg?Lys?Glu?Thr

460?????????????????465?????????????????470agg?aag?cca?ccc?taacactcgg?ccagacccgc?tggctcccag?gccctgagag??????????1617Arg?Lys?Pro?Pro

475gacagagatg accacatccc tgctggatgc aggacccgtc caaagatgcc ccggggaggg 1677gtgtcagccc acgttgctgg cctttggggt caagaggagt agggatccca ggctgccctg 1737gacttagacc agtgtctgag gtggtaaca 1766＜210〉2＜211〉477＜212〉PRT＜213〉homo sapiens (Homo sapiens)＜400〉2Met Ser Ser Ser Arg Lys Asp His Leu Gly Ala Ser Ser Ser Glu Pro1 5 10 15Leu Pro Val Ile Ile Val Gly Asn Gly Pro Ser Gly Ile Cys Leu Ser

20??????????????????25??????????????????30Tyr?Leu?Leu?Ser?Gly?Tyr?Thr?Pro?Tyr?Thr?Lys?Pro?Asp?Ala?Ile?His

35??????????????????40??????????????????45Pro?His?Pro?Leu?Leu?Gln?Arg?Lys?Leu?Thr?Glu?Ala?Pro?Gly?Val?Ser

50??????????????????55??????????????????60Ile?Leu?Asp?Gln?Asp?Leu?Asp?Tyr?Leu?Ser?Glu?Gly?Leu?Glu?Gly?Arg65??????????????????70??????????????????75??????????????????80Ser?Gln?Ser?Pro?Val?Ala?Leu?Leu?Phe?Asp?Ala?Leu?Leu?Arg?Pro?Asp

85??????????????????90??????????????????95Thr?Asp?Phe?Gly?Gly?Asn?Met?Lys?Ser?Val?Leu?Thr?Trp?Lys?His?Arg

100?????????????????105?????????????????110Lys?Glu?His?Ala?Ile?Pro?His?Val?Val?Leu?Gly?Arg?Asn?Leu?Pro?Gly

115?????????????????120?????????????????125Gly?Ala?Trp?His?Ser?Ile?Glu?Gly?Ser?Met?Val?Ile?Leu?Ser?Gln?Gly

130?????????????????135?????????????????140Gln?Trp?Met?Gly?Leu?Pro?Asp?Leu?Glu?Val?Lys?Asp?Trp?Met?Gln?Lys145?????????????????150?????????????????155?????????????????160Lys?Arg?Arg?Gly?Leu?Arg?Asn?Ser?Arg?Ala?Thr?Ala?Gly?Asp?Ile?Ala

165?????????????????170?????????????????175His?Tyr?Tyr?Arg?Asp?Tyr?Val?Val?Lys?Lys?Gly?Leu?Gly?His?Asn?Phe

180?????????????????185?????????????????190Val?Ser?Gly?Ala?Val?Val?Thr?Ala?Val?Glu?Trp?Gly?Thr?Pro?Asp?Pro

195?????????????????200?????????????????205Ser?Ser?Cys?Gly?Ala?Gln?Asp?Ser?Ser?Pro?Leu?Phe?Gln?Val?Ser?Gly

210?????????????????215?????????????????220Phe?Leu?Thr?Arg?Asn?Gln?Ala?Gln?Gln?Pro?Phe?Ser?Leu?Trp?Ala?Arg225?????????????????230?????????????????235?????????????????240Asn?Val?Val?Leu?Ala?Thr?Gly?Thr?Phe?Asp?Ser?Pro?Ala?Arg?Leu?Gly

245?????????????????250?????????????????255Ile?Pro?Gly?Glu?Ala?Leu?Pro?Phe?Ile?His?His?Glu?Leu?Ser?Ala?Leu

260?????????????????265?????????????????270Glu?Ala?Ala?Thr?Arg?Val?Gly?Ala?Val?Thr?Pro?Ala?Ser?Asp?Pro?Val

275?????????????????280?????????????????285Leu?Ile?Ile?Gly?Ala?Gly?Leu?Ser?Ala?Ala?Asp?Ala?Val?Leu?Tyr?Ala

290?????????????????295?????????????????300Arg?His?Tyr?Asn?Ile?Pro?Val?Ile?His?Ala?Phe?Arg?Arg?Ala?Val?Asp305?????????????????310?????????????????315?????????????????320Asp?Pro?Gly?Leu?Val?Phe?Asn?Gln?Leu?Pro?Lys?Met?Leu?Tyr?Pro?Glu

325?????????????????330?????????????????335Tyr?His?Lys?Val?His?Gln?Met?Met?Arg?Glu?Gln?Ser?Ile?Leu?Ser?Pro

340?????????????????345?????????????????350Ser?Pro?Tyr?Glu?Gly?Tyr?Arg?Ser?Leu?Pro?Arg?His?Gln?Leu?Leu?Cys

355?????????????????360?????????????????365Phe?Lys?Glu?Asp?Cys?Gln?Ala?Val?Phe?Gln?Asp?Leu?Glu?Gly?Val?Glu

370?????????????????375?????????????????380Lys?Val?Phe?Gly?Val?Ser?Leu?Val?Leu?Val?Leu?Ile?Gly?Ser?His?Pro385?????????????????390?????????????????395?????????????????400Asp?Leu?Ser?Phe?Leu?Pro?Gly?Ala?Gly?Ala?Asp?Phe?Ala?Val?Asp?Pro

405?????????????????410?????????????????415Asp?Gln?Pro?Leu?Ser?Ala?Lys?Arg?Asn?Pro?Ile?Asp?Val?Asp?Pro?Phe

420?????????????????425?????????????????430Thr?Tyr?Gln?Ser?Thr?Arg?Gln?Glu?Gly?Leu?Tyr?Ala?Met?Gly?Pro?Leu

435?????????????????440?????????????????445Ala?Gly?Asp?Asn?Phe?Val?Arg?Phe?Val?Gln?Gly?Gly?Ala?Leu?Ala?Val

450 455 460Ala Ser Ser Leu Leu Arg Lys Glu Thr Arg Lys Pro Pro465 470 475＜210〉3＜211〉18＜212〉DNA＜213〉＜400〉3ttgatgccct tctacgcc 18＜210〉4＜211〉17＜212〉DNA＜213〉＜400〉4tggcgaggac cacgttg 17＜210〉5＜211〉29＜212〉DNA＜213〉＜400〉5gtggatccat gagctcctcc agaaaggac 29＜210〉6＜211〉28＜212〉DNA＜213〉＜400〉6gcgaattctg agggtggctt cctggtct 28＜210〉7＜211〉24＜212〉DNA＜213〉＜400〉7gtggatccag aaaggaccac ctcg 24＜210〉8＜211〉27＜212〉DNA＜213〉＜400〉8gcgaattcgc gtagaagggc atcaaag 27。

Claims

1. isolating people supressor BDGI polypeptide of growing is characterized in that it comprises: polypeptide or its conservative property variation polypeptide or its active fragments or its reactive derivative with SEQID NO:2 aminoacid sequence.

2. polypeptide as claimed in claim 1 is characterized in that, this polypeptide is selected from down group:

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

(b) SEQ ID NO:2 aminoacid sequence is formed through replacement, disappearance or the interpolation of one or more amino-acid residues, and have suppress breast cancer cell line mcf-7 growth function by (a) polypeptides derived.

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 IDNO: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 147-1577 position among the SEQ ID NO:1;

(b) has the sequence of 1-1766 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, contains the described carrier of claim 6.

8. one kind has the grow preparation method of polypeptide of supressor BDGI polypeptide active of people, it is characterized in that this method comprises:

(a) under the condition that is fit to expressing human growth inhibiting factor BDGI polypeptide, cultivate the described host cell of claim 7;

(b) from culture, isolate and have the grow polypeptide of supressor BDGI polypeptide active of people.

9. an energy and the described people of the claim 1 supressor BDGI polypeptid specificity bonded antibody of growing.

10. 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.