CN1148381C - New human chemotaxis factor macrophage inflammatory protein, its coding sequence and use - Google Patents

Abstract

The present invention provides a novel human chemotactic factor-macrophage inflammatory protein (MIP)-2 gamma, polynucleotide which codes the polypeptide, and a method for generating the polypeptide by a recombination technique. The present invention also discloses an application of the polynucleotide which codes the novel human MIP-2 gamma protein. The present invention also discloses the expression situations of the polypeptide in normal tissues and various tumour cells, a method used for tumour diagnosis, and a curative method used for a plurality of diseases, such as neutrophilic granulocyte deficiency or hypofunction, tumour, autoimmune diseases, etc. The present invention also provides a medical composition which comprises MIP-2 gamma. The present invention also discloses an antagonist for resisting the polypeptide, and curative effect thereof.

Description

New human chemokine macrophage inflammatory protein, its encoding sequence and purposes

Technical field

The invention belongs to biotechnology and medical field, specifically, the present invention relates to new coding human chemokine macrophage inflammatory protein MIP-2 γ (macrophage inflammatory protein-2 γ, be also referred to as " chemokine MIP-2 γ " or " MIP-2 γ albumen ") polynucleotide, 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 be a kind of new with tumour, infect relevant chemokine.

Background technology

Chemokine and acceptor thereof are research fields that progress is very swift and violent, constantly have new chemokine and acceptor thereof to be found in recent years.Chemokine is to have conserved sequence and white corpuscle (neutrophil leucocyte, monocyte and lymphocyte etc.) is had the low molecular weight protein (LMWP) of chemotaxis, is a superfamily (Wardet al.Immunity, 1998,9: 1) in the cytokine.The discovery of chemokine makes much progress, and has become the focus in molecular immunology field.

Have 50 kinds of chemokines at present at least, its ripe bulk of molecule is 68～120 amino acid, and chemokine has conservative Cys structure.According to the characteristics of Cys structural domain in its structure, can be divided into four subfamilies at least, the chemokine of different families has different functional characteristicss.

(a) C-X-C family, i.e. α subfamily, its constructional feature is non-conserved amino acid at interval among N-terminal two Cys, comprises IL-8 etc., can the chemotactic neutrophil leucocyte, monocyte there is not chemotactic activity.

(b) C-C family, promptly the β subfamily is a family maximum in the chemokine, comprises more than 20 member in the people, and its constructional feature is that N-terminal two Cys arrange continuously, and main chemotactic monocyte and lymphocyte do not have chemotaxis to neutrophil leucocyte.The chemokine overwhelming majority who expresses in the dendritic cell (DC) belongs to this subfamily, as the DC-CK1 of specificity chemotactic tranquillization T cell.

(c) C subfamily claims γ family again, and member of lymphocyte chemotactic factor (LCF) is only arranged at present, only contains two among 4 Cys of common chemokine, can specificity chemotactic lymphocyte and NK cell.

(d) CX3C family, i.e. δ family, its constructional feature is three non-conserved amino acids at interval among N-terminal two Cys, and the T cell is had chemotactic activity.

Macrophage inflammatory protein (macrophage inflammatory protein abbreviates " MIP " as) is a kind of by mammalian cell, as scavenger cell and lymphocyte, and the albumen that after G-bacterium, lipopolysaccharides or concanavalin A etc. stimulate, produces.Therefore, but MIP may be subjected in infection, tumour, inflammation, grain system propagation and play a role in the diagnosis of autoimmune disorder and the treatment.

The cell expressing spectrum of chemokine is very wide, comprises mononuclear macrophage, endotheliocyte, mesentery cell, inoblast, keratinocyte and lymphocyte, dendritic cell etc.

Dendritic cell (DC) 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, the processing of the differentiation and development of dendritic cell, antigen is offered mechanism and the effect in tumour, infection, autoimmune disorder and transplant rejection has become immunologic forward position field (Cao Xuetao etc.China's Journal of Immunology.1998；3：322，Banchereau?et?al.Nature.1998；392：245)。Migration is a DC differentiation and maturation and to finish its angtigen presentation function necessary in the body of DC.Dendritic cell is carried out intravital directional migration, thereby is further reached maturity under the chemokine effect, finishes its angtigen presentation function.The expression of Chemokine Receptors is subjected to the regulation and control of DC differentiation, maturity state.Thereby the DC that makes the different stage of maturity to different chemokines have different reactivities (Delgado et al.Immunobiology 1998,198:490 and Dieu et al.J.Exp.Med.1998,188:373).Although DC expresses the CXC Chemokine Receptors, most of CXC chemokines such as IL-8, IP-10 and Gro-β there is no chemotaxis to DC.Only find that so far a kind of CXC chemokine SDF-1 has chemotaxis to DC.

DC is except expressing Chemokine Receptors, and chemokine is had beyond the reactivity, goes back the expression-secretion chemokine, regulates the chemotaxis of other immunocyte, participates in the formation of cytocerastic regulation and control of T and immunological tolerance.The chemokine of the last expression of DC comprises MIP-1 α, MIP-1 β, MIP-1 γ, PANTES, MCP-1, MIP-3 β and DC-CK1 etc.These chemokines belong to mostly CC chemokine family (Lore et al.J.Immunol.Methods1998,214:97).In addition, DC also expresses the CXC chemokine, as IL-8, and tool potential chemotactic neutrophil leucocyte ability.

The relation that chemokine and HIV infect is recent breakthrough progress.Think that now during the HIV cells infected, remove combining of its shell glycoprotein gp120 and CD4, it is the participation of Chemokine Receptors that accessory molecule also must be arranged; Chemokine can suppress HIV and enter cell.The molecule mechanism that chemokine suppresses HIV may be in this link of Chemokine Receptors.Chemokine Receptors such as CXCR4, the CCR3 that DC expresses and CCR5 and HIV enter DC relevant (Rubbert et al.J.Immunol.1998,160:3933).

Because chemokine plays a significant role in the regulation and control of differentiation, growth and the immune response of immunocyte, therefore more and more paid attention to by people.Dendritic cell is carried out intravital directional migration, thereby is further reached maturity under the chemokine effect, finishes its angtigen presentation function; Simultaneously, dendritic cell itself also produces, secretes chemokine, and meticulous adjusting comprises chemotactic in the body of other immunocyte of T cell, its startup and regulating effect in immune response of more effective execution.The Chemokine Receptors of dendritic cell expression simultaneously infects relevant with HIV.Therefore, by strengthening or block the chemotactic signal of dendritic cell, seek the diagnosis and the treatment New Policy tool significance of tumour, the immunological disease that infects, places oneself etc.

Yet, before the present invention, chemokine MIP-2 γ involved in the present invention was not still disclosed.

Summary of the invention

The purpose of this invention is to provide a kind of new human chemokine-be macrophage inflammatory protein MIP-2 γ (macrophage inflammatory protein-2 γ abbreviates " MIP-2 γ " as) protein polypeptide with and fragment, analogue and derivative.MIP-2 γ has chemotaxis to DC, and this helps further to understand the migration mechanism of DC.

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 MIP-2 γ.

In a first aspect of the present invention, novel isolated MIP-2 γ protein polypeptide is provided, this polypeptide is the people source, and 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 or SEQ ID NO:3 aminoacid sequence.

In a second aspect of the present invention, the polynucleotide of these polypeptide of separated 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 MIP-2 γ 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 41-376 position among the SEQ ID NO:1; (b) has the sequence of 143-376 position among the SEQ ID NO:1; (c) has the sequence of 1-463 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 MIP-2 γ protein-active, this method comprises: (a) under the proteic condition of suitable expressing human MIP-2 γ, cultivate the above-mentioned host cell that is transformed or transduce; (b) from culture, isolate polypeptide with people MIP-2 γ protein-active.

In a fifth aspect of the present invention, provide and above-mentioned people MIP-2 γ protein polypeptide specificity bonded antibody.The nucleic acid molecule that can be used for detecting also is provided, and it contains a successive 10-463 Nucleotide in the above-mentioned polynucleotide.

In a sixth aspect of the present invention, provide whether there is the proteic method of MIP-2 γ in the test sample, the antibody contact that sample is special to MIP-2 γ observes whether form antibody complex, has formed antibody complex and has just represented to exist in the sample MIP-2 γ albumen.

In a seventh 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 MIP-2 γ protein polypeptide, and perhaps screening suppresses people's MIP-2 active antagonist of γ protein polypeptide or is used to the peptide finger print identification.The proteic encoding sequence of people MIP-2 γ 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 eighth aspect of the present invention, provide a kind of screening or detection to express the relevant disease or the method for disease (especially tumour) susceptibility with people MIP-2 γ protein polypeptide.

In a ninth aspect of the present invention, provide a kind of and people MIP-2 γ protein polypeptide to express relevant dendritic cell and other immunocytes move in vivo, differentiation, sophisticated research strategy.

In a tenth aspect of the present invention, a kind of pharmaceutical composition is provided, it contains people MIP-2 γ protein polypeptide of the present invention or and antagonist, inhibitor and the pharmaceutically acceptable carrier of safe and effective amount.These pharmaceutical compositions can be treated illnesss such as Immunological diseases, tumour, acquired immune deficiency syndrome (AIDS).

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

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 MIP-2 γ 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 MIP-2 γ albumen of the present invention and people MIP-2 α and the proteic amino acid sequence homology comparison diagram of MIP-2 β.

Fig. 2 is to the expression of people MIP-2 γ in healthy tissues, carries out the electrophorogram of Northern hybridization.Wherein be pancreas, kidney, marrow flesh, liver, lungs, placenta, brain, heart, peripheral blood lymphocyte, colon, small intestine, ovary, testis, prostate gland, thymus gland and spleen from left to right.

Fig. 3 is to the expression of people MIP-2 γ in different tumor cell lines, carries out the electrophorogram that RT-PCR analyzes.Wherein be acute lymphoblastic leukemia cell strain Molt-4, skin T lymphoma cell strain Hut78, acute T chronic myeloid leukemia cell strain Jurkat, erythroleukemia cell strain K562, acute myeloid leukaemia cell strain HL-60, person monocytic cell's cell strain THP-1, the cell strain U937 of histocytic lymphoma and lung cancer cell line A549 from left to right.

Fig. 4 A is the structure iron of recombinant expression vector pcDNA3.1/Neo.

Fig. 4 B is in 293 cells and supernatant of recombinant expression vector transfection, the expression of people MIP-2 γ is carried out the electrophorogram of 35S metabolic marker detection.Wherein be successively from left to right: swimming lane MIP-2 γ is a people MIP-2 γ expression product; The contrast swimming lane is the 293 cells contrast supernatant of transfection empty carrier.The left side is molecular weight size (kDa).

Fig. 4 C is the electrophorogram that people MIP-2 γ expression product in the culture supernatant behind the recombinant expression plasmid rotaring redyeing 293 cell is carried out the Western engram analysis.Swimming lane 1 and 3 is contrast, and swimming lane 2 is a people MIP-2 γ expression product.The left side is molecular weight size (kDa).

Fig. 5 A is the chemotaxis graphic representations of 293 cell conditioned mediums of people MIP-2 γ gene transfection to fresh separated neutrophil leucocyte in the human peripheral.

Fig. 5 B is that 293 cell conditioned mediums of people MIP-2 γ gene transfection are to the lymphocytic chemotaxis graphic representation of fresh separated T in the human peripheral.

Fig. 5 C is that 293 cell conditioned mediums of people MIP-2 γ gene transfection are to the monocytic chemotaxis graphic representation of fresh separated in the human peripheral.

Fig. 5 D is the chemotaxis graphic representations of 293 cell conditioned mediums of people MIP-2 γ gene transfection to the dendritic cell in person monocytic cell source.

Summary of the invention

In the present invention, term " chemotactic factor (CF) MIP-2 γ ", " MIP-2 γ albumen " and " MIP-2 γ " are interchangeable Use, all refer to have human macrophage inflammatory protein 2 γ, it comprises having chemotactic factor (CF) MIP-2 γ amino acid sequence The people of the amino acid sequence of the mature form (SEQ ID NO:3) of total length form (SEQ ID NO:2) or no signal peptide becomes Change factor M IP-2 γ. They comprise the chemotactic factor (CF) MIP-2 γ that contains or do not contain initial methionine.

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

As used herein, " MIP-2 γ albumen or the polypeptide of separation " refers to that MIP-2 γ polypeptide is substantially free of the sky Right relative other albumen, lipid, carbohydrate or other material. Those skilled in the art can use the egg of standard White matter purification technique purifying MIP-2 γ albumen. Basically pure polypeptide can produce on non-reduced polyacrylamide gel Single master tape. Purity to MIP-2 γ polypeptide can be carried out amino acid analysis.

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

The present invention also comprises fragment, derivative and the analog of people MIP-2 γ albumen. As used herein, term " sheet Section ", " derivative " refer to basically keep natural human MIP-2 γ albumen of the present invention identical with " analog " Biological function or active polypeptide. Polypeptide fragment of the present invention, derivative or analog can be that (i) has one Or a plurality of conservative or substituted polypeptide of non-conservation amino acid residue (preferred conservative amino acid residue), and like this The amino acid residue of replacement can be also can be by the genetic code coding, or (ii) at one or more ammonia Base has the polypeptide of substituted radical in the sour residue, or (iii) mature polypeptide and another compound (such as the prolongation polypeptide The compound of half-life, for example polyethylene glycol) merge formed polypeptide, or (iv) additional amino acid sequence melts The polypeptide that is incorporated into this peptide sequence and forms (as targeting sequencing or secretion sequence or be used for this polypeptide of purifying sequence or The proteinogen sequence, 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 MIP-2 γ polypeptide " refers to have the SEQ ID NO. of people MIP-2 γ protein active The polypeptide of 2 or 3 sequences. This term also comprise have with people MIP-2 γ albumen identical function, SEQ ID NO.2 Or the variant form of 3 sequences. These variant forms comprise (but being not limited to): several (are generally 1-50, Good ground 1-30, more preferably 1-20,1-10 best) amino acid whose disappearance, insertion and/or replacement, and Add one or several (being generally in 20, preferably is in 10, more preferably in that C end and/or N are terminal Be 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 one or several amino acid in that C end and/or N are terminal Usually also can not change the function of protein. This term comprises that also active fragment and the activity of people MIP-2 γ albumen derive Thing.

The variant form of this polypeptide comprises: homologous sequence, conservative variant, allelic variant, natural sudden change Body, induced mutation body, under high or low stringency condition, can compile with the DNA of people MIP-2 γ protein D NA hybridization The albumen of code and the polypeptide or the albumen that utilize the antiserum acquisition of anti-people MIP-2 γ polypeptide. The present invention also carries Supply other polypeptide, as comprised the fusion of people MIP-2 γ polypeptide or its fragment. Many except total length almost Outside the peptide, the present invention has also comprised the soluble fragments of people MIP-2 γ polypeptide. Usually, this fragment has people MIP-2 γ The 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 60 continuous amino acids, best continuously amino at least about 70 Acid.

Invention also provides the analog of people MIP-2 γ albumen or polypeptide. These analogs and natural human MIP-2 γ albumen are many The difference of peptide 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 have both at the same time. These polypeptide comprise genetic variant natural or that induce. The induce variation body can be by various Technology obtains, as by radiation or be exposed to mutagens and produce random mutagenesis, and also can be by direct mutagenesis method or its The biological technology of his known molecular. Analog also comprises having and is different from the amino acid whose residue of natural L-(such as D-ammonia Base acid) analog, and have the similar of that non-natural exists or synthetic amino acid (such as β, gamma-amino acid) Thing. 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 modifying comprises: in the body or the chemically derived form of external polypeptide as Acetylation or carboxylated. Modify and also to comprise glycosylation, such as those in the synthetic and processing of polypeptide or further processing Carry out glycosylation modified in the step and polypeptide that produce. This modification can be carried out glycosylation by polypeptide is exposed to Enzyme (such as mammiferous glycosylase or deglycosylating enzyme) and finish. Modified forms also comprises having phosphorylation ammonia The sequence of the sour residue of base (such as phosphotyrosine, phosphoserine, phosphothreonine). Thereby also comprising being modified carries High its anti-proteolysis performance or optimized the polypeptide of solubility property.

In the present invention, " people MIP-2 γ albumen conservative variation polypeptide " refers to the amino acid with SEQ ID No.2 or 3 Sequence is compared, and has 10 at the most, and preferably at the most 8, more preferably at the most 5,3 amino acid at the most best Replaced by similar performance or close amino acid and form polypeptide. These conservative variation polypeptide are preferably according to table 1 Carry out amino acid substitution 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 with the identical or degeneracy of coding region sequence shown in the SEQ ID NO:1 Variant. As used herein, " variant of degeneracy " refers to that in the present invention coding has SEQ ID NO:2 Or the protein of 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: an encoding mature is many The coded sequence of peptide; 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 identical amino acid sequence with the present invention Polypeptide or fragment, analog and the derivative of polypeptide. The variant of these polynucleotides can be natural generation etc. The variant that position variant or non-natural take place. These nucleotide diversity bodies comprise replacement variant, deletion mutation body With the insertion variant. As known in the art, allelic variant is a kind of replacement form of polynucleotides, and it may Replacement, disappearance or the insertion of one or more nucleotides, but can be from the merit of the polypeptide that changes in fact its coding Energy.

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

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 MIP-2 γ.

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

People MIP-2 γ 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 from the library, obtain the cDNA of total length, 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 MIP-2 γ 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 MIP-2 γ protein polypeptide of reorganization.In general following steps are arranged:

(1). with the coding people proteic polynucleotide of MIP-2 γ 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 MIP-2 γ 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 MIP-2 γ 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, 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 PL promotor; 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 ₂Handle.If 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.

Recombinant polypeptide in the above methods can be in cell, extracellular or express or be secreted into the extracellular 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 (MIP-2 γ albumen LC) and other various liquid chromatography (LC) technology and these methods.

The people MIP-2 γ 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 MIP-2 γ protein function as pharmacological agent MIP-2 γ protein function.For example, antibody can be used for activating or suppressing the proteic function of people MIP-2 γ.The peptide molecule that can suppress or stimulate people MIP-2 γ protein function that can be used for seeking therapeutic value with the recombinant human MIP-2 γ protein screening peptide library of expressing.

On the other hand, the present invention also comprises people MIP-2 γ protein D NA 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 MIP-2 γ protein gene product or fragment.Preferably, refer to that those can combine with people MIP-2 γ protein 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 MIP-2 γ, comprise that also those do not influence the antibody of people MIP-2 γ protein function.The present invention also comprise those can with modify or without the people MIP-2 γ protein 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 MIP-2 γ protein gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing human MIP-2 γ albumen or its have antigenic segmental cell and can be used to immune animal and produce antibody.Antibody of the present invention also can be monoclonal antibody.This type of monoclonal antibody can utilize hybridoma technology to prepare that (see people such as Kohler, Nature 256; 495,1975; People such as Kohler, Eur.J.Immunol.6:511,1976; People such as Kohler, Eur.J.Immunol.6:292,1976; People such as Hammerling, In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981).Antibody of the present invention comprises the antibody that can block people MIP-2 γ protein function and the antibody that does not influence people MIP-2 γ protein function.Each antibody-like of the present invention can utilize the fragment or the functional zone of people CMIP-2 γ protein 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 MIP-2 γ protein 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 MIP-2 γ can be used in the immunohistochemistry technology, detects the people MIP-2 γ albumen in the biopsy specimen.In addition, with the also available labelled with radioisotope of the protein bound monoclonal antibody of people MIP-2 γ, 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 MIP-2 γ albumen.The antibody that gives suitable dosage can stimulate or block proteic generation of people MIP-2 γ or activity.

Antibody also can be used for designing the immunotoxin at a certain privileged sites in the body.As the monoclonal antibody of people MIP-2 γ 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 people MIP-2 γ positive cells.The production of polyclonal antibody can choose MIP-2 γ 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 MIP-2 γ 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, intravenously, subcutaneous, intracutaneous or topical.

Polypeptide of the present invention can be directly used in disease treatment, for example, and malignant tumour, neutrophil leucocyte shortage or hypofunction, autoimmune disorder etc.When using MIP-2 γ albumen of the present invention, also can use the other treatment agent simultaneously, the present invention also provides a kind of pharmaceutical composition, and it contains MIP-2 γ 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 MIP-2 γ 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 MIP-2 γ 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 MIP-2 γ 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 MIP-2 γ of the proteic nothing expression of MIP-2 γ or unusual/non-activity.The MIP-2 γ albumen that the gene therapy vector (as virus vector) of reorganization can be designed to express variation is to suppress endogenic MIP-2 γ protein-active.Deriving from viral expression vector such as retrovirus, adenovirus, adeno-associated virus (AAV), hsv, parvovirus etc. can be used for MIP-2 γ protein gene is transferred in the cell.The method that structure carries the recombinant viral vector of MIP-2 γ protein gene is found in existing document (Sambrook, et al.).Recombinant human MIP-2 γ protein gene can be wrapping in the liposome and then be transferred in the cell in addition.

Suppress the oligonucleotide (comprising sense-rna and DNA) of people MIP-2 γ protein 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 MIP-2 γ obtains.During screening, must carry out mark to people MIP-2 γ protein molecular.

The invention still further relates to the diagnostic testing process of quantitative and detection and localization people MIP-2 γ protein level.These tests are known in the art, and comprise that FISH measures and radioimmunoassay.The people MIP-2 γ protein level that is detected in the test can be with laying down a definition the importance of people MIP-2 γ albumen in various diseases and be used to the disease of diagnosing MIP-2 γ albumen to work.

Whether having the proteic method of MIP-2 γ in a kind of detection test sample is to utilize the proteic specific antibody of MIP-2 γ to detect, and it comprises: sample is contacted with MIP-2 γ protein specific antibody; Observe whether form antibody complex, formed antibody complex and just represented to exist in the sample MIP-2 γ albumen.

The proteic polynucleotide of MIP-2 γ can be used for the diagnosis and the treatment of MIP-2 γ protein related diseases.Aspect diagnosis, the proteic polynucleotide of MIP-2 γ can be used for detecting the proteic expression of MIP-2 γ MIP-2 γ abnormal exprssion whether or under morbid state.Can be used for the hybridization of biopsy specimen to judge the proteic abnormal expression of MIP-2 γ as MIP-2 γ 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 MIP-2 γ albumen and also can detect the proteic transcription product of MIP-2 γ.

The sudden change that detects MIP-2 γ protein gene also can be used for the disease of diagnosing MIP-2 γ albumen relevant.The form of MIP-2 γ protein mutation comprises that the point mutation compared with normal wild type MIP-2 γ protein D NA sequence, 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 MIP-2 γ 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 Inheritancein Man (can by with the online acquisition of Johns Hopkins University Welch Medical Library).Can pass through linkage analysis then, determine gene and navigated to relation between the disease on the chromosomal region already.

In an example of the present invention, a kind of isolating polynucleotide are provided, its coding has the polypeptide of aminoacid sequence shown in the SEQ ID NO:2.Polynucleotide of the present invention are isolated from human dendritic cell cDNA library.Its sequence is as follows:

gagctccggg?ccgccgctcc?gacgggccag?cgccctcccc?ATGTCCCTGC?TCCCACGCCG?60

CGCCCCTCCG?GTCAGCATGA?GGCTCCTGGC?GGCCGCGCTG?CTCCTGCTGC?TGCTGGCGCT?120

GTACACCGCG?CGTGTGGACG?GGTCCAAATG?CAAGTGCTCC?CGGAAGGGAC?CCAAGATCCG?180

CTACAGCGAC?GTGAAGAAGC?TGGAAATGAA?GCCAAAGTAC?CCGCACTGCG?AGGAGAAGAT?240

GGTTATCATC?ACCACCAAGA?GCGTGTCCAG?GTACCGAGGT?CAGGAGCACT?GCCTGCACCC?300

CAAGCTGCAG?AGCACCAAGC?GCTTCATCAA?GTGGTACAAC?GCCTGGAACG?AGAAGCGCAG?360

GTICTACGAA?GAATAGggtg?aaaaacctca?gaagggaaaa?ctccaaacca?gttgggagac?420

ttgtggcaaa?ggaactttgc?agattaaaaa?aaaaaaaaaa?aaa 463

The polynucleotide sequence total length that it comprises is 463 bases, and its open reading frame is positioned at the 41-376 position, coding 111 amino acid whose people MIP-2 γ albumen of total length (SEQ ID NO:2).

MSLLPRRAPP?VSMRLLAAAL?LLLLLALYTA?RVDGSKCKCS?RKGPKIRYSD?50

VKKLEMKPKY?PHCEEKMVII?TTKSVSRYRG?QEHCLHPKLQ?STKRFIKWYN?100

AWNEKRRFYE?E 111

Wherein, amino acid/11-34 is a signal peptide.Removed the sophisticated MIP-2 γ albumen behind the signal peptide and contained 77 amino acid, sequence is shown in SEQ ID NO:3.

SKCKCSRKGP?KIRYSDVKKL?EMKPKYPHCE?EKMVIITTKS?VSRYRGQEHC?50

LHPKLQSTKR?FIKWYNAWNE?KRRFYEE 77

The inventor will contain the cDNA of the MIP-2 γ of full length coding region, insert the pcDNA3.1 carrier for expression of eukaryon, make up the proteic carrier of expression MIP-2 γ, and express in rotaring redyeing 293 cell, carry out after after the transfection 48 hours that cells and supernatant detects and ³⁵The S metabolic analysis shows that chemokine MIP-2 γ gene can obtain expressing, and obtains the purifying protein of MIP-2 γ in eukaryotic cell.The Western engram analysis has proved the expression of MIP-2 γ.

In order to inquire into the proteic biologic activity of MIP-2 γ, fresh separated is carried out the chemotactic experiment from dendritic cell applying MIP-2 γ of human peripheral blood cell's neutrophil leucocyte, T lymphocyte, monocyte and cells of monocytic origin.The result shows that MIP-2 γ has chemotaxis to neutrophil leucocyte, but a little less than the chemotaxis to dendritic cell, neutrophil leucocyte and monocyte is not had chemotaxis.

Embodiment

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 MIP-2 γ albumen cDNA

Extract the total RNA of human dendritic cell with Trizol method (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 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 human chemokine MIP-2 γ or macrophage inflammatory protein 2 γ, its encoding gene called after human chemokine MIP-2 γ protein gene.

Sequence SEQ ID NO:1 total length is 463bp, comprises 3 ' end non-coding region of 5 of 40bp ' end non-coding region and 86bp.Open reading frame is positioned at the 41-376 position, and its coding contains 111 amino acid whose polypeptide (Fig. 1).

Find that according to Kyte-Doolitte hydrophobicity analysis and GCG software analysis the N end of MIP-2 γ contains the signal peptide of being made up of 34 amino acid, this shows that MIP-2 γ albumen is secretory protein.Sophisticated MIP-2 γ albumen is made up of 77 amino acid, and calculating not in theory, the molecular weight of glycosylated ripe molecule is 9.5kD.The structural domain that contains 4 conservative Cyc, wherein preceding two Cys residues are a non-conservative Methionin at interval, and this shows that MIP-2 γ has typical C XC chemokine constitutional features.

BLAST analysis revealed MIP-2 γ is different with known, the protein product of this molecule and MIP-2 α and MIP-2 β height homology, compare with MIP-2 α 34% identical, 38% homology; Compare with MIP-2 β 31% identical, 36% homology; Do not contain the ELR structural domain but compare with MIP-2 α/2 β, this discloses MIP-2 γ is a new chemokine (Figure 1B).

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

Extract the total RNA that is in logarithmic phase person monocytic cell cell strain THP-1 cell with Trizol (Gibco company), get the 6mg cell total rna and mix, carry out reverse transcription with 0.5 μ g Oligo-dT12-18.The reverse transcription system is 20 μ l, adds 80 μ l ddH20 after reaction finishes and dilutes.The used primer of pcr amplification MIP is as follows: adopted primer is arranged: 5 '-ggaattcgccatgtccctgctcc cacg-3 ' (SEQID NO:4), antisense primer: 5 '-gggtacctcattcttcgtagaacctg-3 ' (SEQ ID NO:5).There is adopted primer before initiator codon ATG, to introduce the EcoRI restriction enzyme site, before terminator codon, introduces the KpnI restriction enzyme site and be beneficial to the raising expression level.The PCR reaction volume is 50 μ l, wherein contain reverse transcription template 10 μ l, 0.4mM primer, 0.2mM 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 coding region dna sequence dna of this PCR product and the 41-376bp coding region shown in the SEQ ID NO:1 are identical.

The Northern engram analysis of embodiment 3 MIP-2 γ

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 * 106cpm/ml), and fully mixing was hybridized 2 hours in 68 ℃ to put rapid cooling back adding hybridization solution on ice.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 shows: chemokine MIP-2 γ comprises kidney, small intestine, brain, placenta, skeletal muscle in healthy tissues.In liver, spleen, thymus gland and the pancreas extensively and continuous expression is expressed in kidney the highest (Fig. 2).

The RT-PCR of embodiment 4 MIP-2 γ mRNA analyzes

Be in the logarithmic phase cell total rna with Trizol (Gibco company) extraction, cell strain to be measured comprises acute lymphoblastic leukemia cell strain Molt-4, skin T lymphoma cell strain Hut78, acute T chronic myeloid leukemia cell strain Jurkat, erythroleukemia cell strain K562, acute myeloid leukaemia cell strain HL-60, person monocytic cell's cell strain THP-1, the cell strain U937 of histocytic lymphoma and lung cancer cell line A549.

Get the 6mg cell total rna and mix, carry out reverse transcription with 0.5 μ g Oligo-dT12-18.The reverse transcription system is 20 μ l, adds 80 μ l ddH20 after reaction finishes and dilutes.The primer that pcr amplification MIP is used such as preceding.The PCR reaction volume is 50 μ l, wherein contain reverse transcription template 10 μ l, 0.4mM primer, 0.2mM dNTP and 1U ExTaq archaeal dna polymerase (Takara Inc.), the amplification parameter be 94 ℃ 30 seconds, 60 ℃ 30 seconds, 72 ℃ 45 seconds, the capable 2% agarose gel electrophoresis analysis of PCR product after 25 circulations, demonstration is except the THP-1 cell strain, and other tumor cell lines all do not have the expression (Fig. 3) of MIP-2 γ.This points out in the generation of MIP-2 γ in tumour, the evolution and may play a role, but also may be the secondary performance of tumour cell gene expression regulation disorder.

The structure of embodiment 5 cytokine MIP-2 γ recombinant expression vectors

Carrier for expression of eukaryon pcDNA3.1/neo (Invitrogen company) structure used in the structure is shown in Fig. 4 A.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, 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.

Pcr amplification product with embodiment 2 is a template, the performing PCR amplifying target genes.Amplification MIP-2 γ primer is as described in the embodiment 2.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 gene order of its MIP-2 γ is through T7 and BGH primer order-checking conclusive evidence.

Embodiment 6: the eucaryon of chemokine MIP-2 γ is recombinant expressed

Recombinant plasmid dna and liposome that embodiment 5 makes up are pressed 1: 5 mixed, room temperature effect 45 minutes; Deng transfectional cell wash twice the back mix with DNA, in transfection 48-72 hour the collection culture supernatant.

It is recombinant expressed to carry out MIP-2 γ 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 MIP-2 γ or control plasmid, the SDS-PAGE electrophoresis of row 15% behind centricon (molecular weight cut-off 10kD) ultrafiltration and concentration.

After the transfection 48 hours, by ³⁵S is marked at and has detected the expression product of estimating in 293 culture supernatant, and wherein the proteic size of MIP-2 γ is about 9kDa (Fig. 4 B).Expression product is carried out Western detect, the result also finds corresponding band (Fig. 4 C).

Embodiment 7: the generation of anti-people MIP-2 γ protein antibodies

The recombinant protein MIP-2 γ that obtains among the embodiment 6 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 MIP-2 γ protein gene translation product with it.Found that antibody can precipitate with MIP-2 γ albumen of the present invention specifically.

Embodiment 8: people MIP-2 γ albumen is to the chemotaxis of neutrophil leucocyte, T lymphocyte, monocyte, dendritic cell

Use Histopaque1077-1119 sedimentator (Sigma company) in centrifugal 30 minutes kinds of 700 * g to fresh peripheral blood, isolate neutrophil leucocyte and mononuclearcell.Use anti-CD14 and anti-CD3 again, MiniMACS magnetic bead partition method is isolated monocyte and lymphocyte from mononuclearcell.Separate back monocyte purity＞85%, T lymphocyte purity＞90%.Detect the chemotaxis of MIP-2 γ with the dendritic cell of peripheral blood neutrophil, T lymphocyte, monocyte and cells of monocytic origin.

The chemotactic experiment is to carry out at Boyden cell (NeuroProbe, Cabin John).The different dilution MIP-2 γ of 200ul are placed the cell bottom, and cell top adds 200ul (2 * 106/ml) different test cell.Up and down the chamber with the aperture of 5um (neutrophil leucocyte, lymphocyte, monocyte) or 3um (dendritic cell) be the polycarbonate leaching film of 13mm separate (Poretics, Livermore).The chemotactic cell is hatched 1 hour (neutrophil leucocyte), 2 hours (monocyte, dendritic cell) or 4 hours (lymphocyte) respectively for 37 ℃, takes off filter membrane, fixing, dyeing.Light microscopic is observed down, and chemotactic index represents that greater than 2 chemotaxis is arranged.(chemotactic index is experimental group chemotactic cell count and the ratio of using substratum chemotactic cell count).

The result is shown in Fig. 5 A-5D, show that MIP-2 γ has tangible chemotaxis to neutrophil leucocyte, T lymphocyte or monocyte then there is not chemotaxis, the chemotaxis of this and other CXC family is similar, but MIP-2 γ also has certain chemotaxis to the dendritic cell of cells of monocytic origin, also has vital role during prompting MIP-2 γ moves in the dendritic cell body, distributes.

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 chemokine macrophage inflammatory protein, its encoding sequence and purposes

(iii) sequence number: 5

(2) information of SEQ ID NO:1:

(i) sequence signature:

(A) length: 463bp

(B) type: nucleic acid

(C) chain: two strands

(D) topological framework: linearity

(ii) molecule type: cDNA

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

gagctccggg?ccgccgctcc?gacgggccag?cgccctcccc?ATGTCCCTGC?TCCCACGCCG?60

CGCCCCTCCG?GTCAGCATGA?GGCTCCTGGC?GGCCGCGCTG?CTCCTGCTGC?TGCTGGCGCT?120

GTACACCGCG?CGTGTGGACG?GGTCCAAATG?CAAGTGCTCC?CGGAAGGGAC?CCAAGATCCG?180

CTACAGCGAC?GTGAAGAAGC?TGGAAATGAA?GCCAAAGTAC?CCGCACTGCG?AGGAGAAGAT?240

GGTTATCATC?ACCACCAAGA?GCGTGTCCAG?GTACCGAGGT?CAGGAGCACT?GCCTGCACCC?300

CAAGCTGCAG?AGCACCAAGC?GCTTCATCAA?GTGGTACAAC?GCCTGGAACG?AGAAGCGCAG?360

GTTCTACGAA?GAATAGggtg?aaaaacctca?gaagggaaaa?ctccaaacca?gttgggagac?420

ttgtggcaaa?ggaactttgc?agartaaaaa?aaaaaaaaaa?aaa 463

(2) information of SEQ ID NO:2:

(i) sequence signature:

(A) length: 111 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ii) molecule type: polypeptide

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

MSLLPRRAPP?VSMRLLAAAL?LLLLLALYTA?RVDGSKCKCS?RKGPKIRYSD?50

VKKLEMKPKY?PHCEEKMVII?TTKSVSRYRG?QEHCLHPKLQ?STKRFIKWYN?100

AWNEKRRFYE?E 111

(2) information of SEQ ID NO:3:

(i) sequence signature:

(A) length: 77 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ii) molecule type: polypeptide

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

SKCKCSRKGP?KIRYSDVKKL?EMKPKYPHCE?EKMVIITTKS?VSRYRGQEHC?50

LHPKLQSTKR?FIKWYNAWNE?KRRFYEE 77

(2) information of SEQ ID NO:4

(i) sequence signature

(A) length: 27 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

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

GGAATTCGCC?ATGTCCCTGC?TCCCACG 27

(2) information of SEQ ID NO:5

(i) sequence signature

(A) length: 26 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

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

GGGTACCTCA?TTCTTCGTAG?AACCTG 26

Claims (12)

1. isolating people MIP-2 γ protein polypeptide is characterized in that it is selected from down group:

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

(b) replacement, disappearance or the interpolation through 1-10 amino-acid residue of SEQ ID NO:2 or 3 aminoacid sequences formed, and have neutrophil leucocyte chemotactic function 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 or SEQ ID NO:3.

3. a separated coding has the polynucleotide of the polypeptide of neutrophil leucocyte chemotactic function, it is characterized in that it contains a nucleotide sequence, and this nucleotide sequence is selected from:

(a) polynucleotide of polypeptide according to claim 1 of encoding;

(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 SEQ ID NO:2 or the SEQ ID NO:3.

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 41-376 position among the SEQ ID NO:1;

(b) has the sequence of 143-376 position among the SEQ ID NO:1;

(c) has the sequence of 1-463 position among the SEQ ID NO:1.

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

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

8. preparation method with polypeptide of people MIP-2 γ protein-active is characterized in that this method contains in steps:

(a) under the proteic condition of suitable expressing human MIP-2 γ, cultivate the described host cell of claim 7;

(b) from culture, isolate polypeptide with people MIP-2 γ protein-active.

9. energy and the described people MIP-2 of claim 1 γ protein-specific bonded antibody.

10. whether there is the proteic method of MIP-2 γ in a 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 MIP-2 γ albumen.

11. the purposes of polypeptide as claimed in claim 1 is characterized in that, it is used to screen the agonist that promotes people MIP-2 γ protein-active, and perhaps screening suppresses the antagonist of people MIP-2 γ protein-active or is used to the peptide finger print identification.

12. 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.

Images