CN1407900A

CN1407900A - Methods of treating or preventing cell, tissue, and organ damage using human myeloid progenitor inhibitory factor-1 (MPIF-1)

Info

Publication number: CN1407900A
Application number: CN00816608A
Authority: CN
Inventors: K·J·格泽戈祖斯基; C·A·罗森; V·帕德尔
Original assignee: Human Genome Sciences Inc
Current assignee: Human Genome Sciences Inc
Priority date: 1999-10-14
Filing date: 2000-10-13
Publication date: 2003-04-02
Also published as: MXPA02003801A; AU1083401A; WO2001026676A9; CA2387545A1; WO2001026676A1; EP1225911A1; JP2003516934A; KR20020057976A

Abstract

There are disclosed therapeutic compositions and methods using isolated nucleic acid molecules encoding a human myeloid progenitor inhibitory factor-1 (MPIF-1) polypeptide (previously termed MIP-3 and chemokine beta 8 (CK beta 8 or ckb-8)), as well as MPIF-1 polypeptide itself, as are vectors, host cells and recombinant methods for producing the same.

Description

Use CKBeta8 (MPIF-1) to send out the method for the treatment of or prevention cell, tissue and organ damage

Background of invention

Invention field

The present invention relates to use the new method of CKBeta8 (myeloid progenitor inhibitory factor-1, MPIF-1) polypeptide (being called in the past MIP-3 and chemokine beta 8 (CK β 8 or ckb-8)) and the polynucleotides that separate of coding MPIF-1. The invention still further relates to carrier, host cell and recombination method for the production of MPIF-1.

Correlation technique

Chemotactic factor (CF) (being also referred to as the intercrine cell factor) is structurally and functionally related cell factor subfamily. These bulks of molecule are 8-14kDa. Generally speaking, the homology that chemotactic factor (CF) is showed 20%-75% at amino acid levels is characterized as four conservative cysteine residues with two disulfide bond of formation. According to the arrangement of the first two cysteine residues, chemotactic factor (CF) is divided into two subfamilies, α and β. In the α subfamily, the first two cysteine is separated by an amino acid, thereby is called " C-X-C " subfamily. In the β subfamily, the first two cysteine is in adjacent position, thereby is called " C-C " subfamily. At least 8 different members of this family in the mankind, have been identified so far.

The intercrine cell factor is showed widely function. Their feature is the ability with the chemotactic migration that causes different cell types (comprising monocyte, neutrophil cell, T lymphocyte, basophilic granulocyte and fibroblast). Many chemotactic factor (CF)s have short scorching active, and relate to a plurality of steps in the inflammatory reaction. These activity comprise the adhesion that stimulates histamine release, lysosomal enzyme and leukotriene release, intensifier target immunocyte and endothelial cell, combination, the expression of inducing GA molecule and complement receptors and the respiratory burst that strengthens complement protein. Except relating to inflammation, some chemotactic factor (CF) is also showed other activity. For example, Monocyte chemoattractant protein-1 (MIP-1) can be contained the propagation of candidate stem cell, PF4 (PF-4) is the strong inhibitor of endothelial cell growth, interleukin-8 (IL-8) can promote the propagation of horn cell, and GRO is the autocrine growth factor of melanoma cells.

Diversity according to BA, chemotactic factor (CF) relates to many physiology and disease condition, comprise lymphocyte transportation, wound healing, hematopoietic regulation and immunology disorderly (such as allergy, asthma and arthritis), just not astonishing yet. The example of hematopoietic lineage instrumentality is MIP-1. MIP-1 is accredited as at first by proinflammatory cytokine endotaxin induction, that generated by macrophage. The MIP-1 that studies show that subsequently is by two kinds of differences but relevant protein, MIP-1 α and MIP-1 β, composition. MIP-1 α and MIP-1 β are the lymphocytic chemoattractants of macrophage, monocyte and T. What is interesting is that it is identical with MIP-1 β that the biochemical purification of multipotential stem cell mortifier (SCI) is disclosed SCI with subsequently sequence analysis. In addition, show that also MIP-1 β can resist the ability of MIP-1 α containment hemopoietic stem cell proliferation. Can suppose according to this discovery: the major physiological effect of MIP-1 is the hematopoiesis in the regulation and control marrow, and the inflammation function that proposes is less important. MIP-1 α blocks in the intermitotic ability of G2S the cell cycle relevant as the mode of action of stem cell mortifier with it. As if in addition, the inhibition of MIP-1 α is limited to the prematurity CFU-GM, and in fact it stimulate the CFU-GM in late period in the situation that has granulocyte macrophage colony stimulating factor (GM-CSF).

The use by oneself kDa major secretory protein of the RAW264.7 (Muridae macrophage tumor cell line) that lipopolysaccharides stimulates of Muridae MIP-1. Purified this protein, and find that it is by two kinds of related proteins, MIP-1 α and MIP-1 β, composition.

The gene of the human homologue that is likely MIP-1 α and MIP-1 β has been cloned by several groups. In all cases, separate cDNA by the library that makes up for the T cell RNA after activating.

MIP-1 protein detects in the wound inflammatory cell in early days, and demonstration induces the wound fibroblast to generate IL-1 and IL-6. In addition, after being expelled in the rabbit cerebrospinal fluid in being subcutaneously injected into mouse insole or in the pond, the natural MIP-1 of purifying (comprising MIP-1, MIP-1 α, MIP-1 beta polypeptides) causes acute inflammation (Wolpe and Cerami, FASEB J., 3:2565-2573,1989). Except these short scorching characteristics of MIP-1 (can be directly or indirectly), in the experiment mice model that adopts the aseptic wound groove, reclaimed the MIP-1 (people such as Fahey in the Earlier period of inflammation stage of wound healing, Cytokine, 2:92,1990). In addition, the PCT application US 92/05198 that submits to of Chiron company discloses in yeast as the template that generates mammalian macrophage inflammatory protein (MIP) and activated dna molecular.

Muridae MIP-1 α is different but closely-related cell factor with MIP-1 β. The partial purification mixture of these two kinds of protein affects the function of neutrophil cell, and causes local inflammation and heating. In yeast cells, express MIP-1 α, and be purified to homogeneous. Structural analysis has confirmed that MIP-1 α has secondary and the tertiary structure closely similar with PF4 (PF-4) and interleukin-8 (IL-8), and enjoys limited sequence homology. Verified, MIP-1 α is activated in vivo, can protect mouse stem cells to avoid containing subsequently the external of tritium thymidine and kill and wound. MIP-1 α also shows the propagation that can strengthen the ancestors' granular leukocyte macrophage colony of more finalizing the design and form cell response granulocyte macrophage colony stimulating factor people such as (, Cytokine, 4:76-82,1992) J.M.Clemens.

According to amino acid sequence homology, polypeptide MPIF-1 of the present invention (sometimes being also referred to as MIP-3 and CK β-8) is the newcomer of β chemotactic factor (CF) family.

Summary of the invention

On the one hand, the invention provides with total length or ripe MPIF-1 polypeptide and biologically have activity, useful in the diagnosis or in treatment useful fragment, analog and derivative prevent or treat the new method of cell, tissue and organ damage.

On the other hand, the invention provides treatment or the prevention method of the separation polynucleotides that use coding MPIF-1 polypeptide. The preferred animal origin of MPIF-1 of the present invention, more preferably origin of mankind.

What the present invention also provided MPIF-1 polypeptide and these polypeptide of coding separates polynucleotides (DNA or RNA), comprise mRNA, DNA, cDNA, genomic DNA, and activity is biologically being arranged and useful fragment, analog and derivative in diagnosis or treatment. The MPIF-1 polynucleotides

The present invention also provide comprise following polynucleotides or consisting of isolated nucleic acid molecule, described polynucleotide encoding has the MPIF-1 polypeptide of the coded amino acid sequence of the cDNA clone of ATCC preserved material numbering 75676 of the amino acid sequence shown in Fig. 1 (SEQ ID NO:2) or preservation on February 9 in 1994. The sequence of MPIF-1 clone by measuring preservation and definite nucleotide sequence is shown in Fig. 1 (SEQ ID NO:1), described sequence comprises the opening code-reading frame of the polypeptide of 120 amino acid residues of encoding, the targeting sequencing that wherein contains about 21 amino acid residues, the molecular weight of prediction maturation protein is about 11kDa (not glycosylation form) and about 11-14kDa (the glycosylation form depends on degree of glycosylation). The amino acid sequence of ripe MPIF-1 albumen is shown in Fig. 1 (SEQ ID NO:2).

Thereby, one aspect of the present invention provide comprise polynucleotides with the nucleotide sequence that is selected from lower group or consisting of isolated nucleic acid molecule: (1) coding has the nucleotide sequence of the MPIF-1 polypeptide of the complete amino acid sequence shown in Fig. 1 (SEQ ID NO:2); (2) coding has the complete amino acid sequence shown in Fig. 1 (SEQ ID NO:2) but the nucleotide sequence of the MPIF-1 polypeptide of the terminal methionine residues of disappearance N; (3) coding has the nucleotide sequence of the ripe MPIF-1 polypeptide of the 22-120 amino acids sequence among Fig. 1 (SEQ ID NO:2); (4) coding has the nucleotide sequence that the ATCC preserved material is numbered the MPIF-1 polypeptide of the coded complete amino acid sequence of 75676 contained cDNA clones; (5) coding has the nucleotide sequence that the ATCC preserved material is numbered the ripe MPIF-1 polypeptide of the coded amino acid sequence of 75676 contained cDNA clones; (6) with above (1), (2), (3), (4) or (5) in the nucleotide sequence of any nucleotide sequence complementation. MPIF-1 polynucleotides variant

The invention still further relates to the variant of polynucleotides mentioned above, described variant coding has the polypeptide of the derivation amino acid sequence shown in Fig. 1 (SEQ ID NO:2) or by fragment, analog and the derivative of the polypeptide of preservation clone's cDNA coding. The polynucleotides variant can be the variant that the non-natural of the allele variant of natural generation of polynucleotides or polynucleotides occurs. Homology MPIF-1 polynucleotides

Other embodiment of the present invention comprises isolated nucleic acid molecule, described isolated nucleic acid molecule comprise have with above (1), (2), (3), (4), (5) or (6) in the polynucleotides of any nucleotide sequence at least 95%, 96%, 97%, 98% or 99% same nucleotide sequence, perhaps under rigorous hybridization conditions with above (1), (2), (3), (4), (5) or (6) in the polynucleotides that can hybridize of polynucleotides. These polynucleotides that can hybridize or not with the polynucleotides that only have by A residue or the nucleotide sequence that only is comprised of the T residue under rigorous condition. Nucleic acid probe

According to another aspect of the present invention, also provide comprise length be enough to the nucleic acid molecules of MPIF-1 nucleotide sequence generation specific hybrid or consisting of nucleic acid probe. Recombinant vector, host cell and expression

The invention still further relates to the recombinant vector that comprises isolated nucleic acid molecule of the present invention, the host cell that comprises recombinant vector and generate these carriers and host cell and with they methods for the production of MPIF-1 polypeptide or peptide by recombinant technique. The MPIF-1 polypeptide

The present invention also provides the polypeptide of the separation MPIF-1 with the amino acid sequence that is selected from lower group: (1) has the amino acid sequence of the MPIF-1 polypeptide of 120 the amino acid whose complete sequence (comprising targeting sequencing) shown in Fig. 1 (SEQ ID NO:2); (2) have 120 the amino acid whose complete sequence (comprising targeting sequencing) shown in Fig. 1 (SEQ ID NO:2) but an amino acid sequence of the MPIF-1 polypeptide of the terminal methionine residues of disappearance N; (3) has the amino acid sequence of the ripe MPIF-1 polypeptide of the 22-120 amino acids sequence (not containing targeting sequencing) among Fig. 1 (SEQ ID NO:2); (4) has the amino acid sequence that the ATCC preserved material is numbered the MPIF-1 polypeptide of the coded complete amino acid sequence (comprising targeting sequencing) of 75676 contained cDNA clones; (5) has the amino acid sequence that the ATCC preserved material is numbered the ripe MPIF-1 polypeptide of the coded amino acid sequence of 75676 contained cDNA clones. Homology MPIF-1 polypeptide

Polypeptide of the present invention also comprises the homeopeptide that has with the same amino acid sequence of amino acid sequence described in (1), (2), (3), (4) or (5) above at least 95%, and has the polypeptide with above-mentioned amino acid sequence at least 95%, 96%, 97%, 98% or 99% same amino acid sequence. Carry polypeptide and the coded polynucleotide of MPIF-1 epi-position

This other embodiment on the one hand of the present invention relates to peptide or the polypeptide that the epi-position with MPIF-1 polypeptide is carried the amino acid sequence of part, and described MPIF-1 polypeptide has amino acid sequence described in above-mentioned (1), (2), (3), (4) or (5). Although the present invention also comprises the epi-position of any length (can reach and comprise the complete amino acid sequence of polypeptide of the present invention mentioned above) and carry polypeptide, the epi-position with MPIF-1 polypeptide of the present invention carries that the peptide of amino acid sequence of part or polypeptide comprise at least 6 of these polypeptide or 7, preferably at least 9, more preferably about at least 30 amino acid are to about 50 amino acid whose parts.

Another nucleic acid embodiment of the present invention relate to the epi-position that comprises coding MPIF-1 polypeptide carry part amino acid sequence polynucleotides or consisting of isolated nucleic acid molecule, described MPIF-1 polypeptide has the amino acid sequence in above-mentioned (1), (2), (3), (4) or (5). MPIF-1 antibody

According to another aspect of the present invention, provide the antibody for these polypeptide. In another embodiment, but the invention provides the separation antibody that specific binding has above the MPIF-1 polypeptide of amino acid sequence described in (1), (2), (3), (4) or (5).

But the present invention also provides the method for the antibody of the MPIF-1 polypeptide that the separation specific binding has amino acid sequence described herein. As described below, these antibody are useful in diagnosis or treatment. MPIF-1 antagonist and method

According to another aspect of the present invention, antagonist or the inhibitor of these polypeptide are provided, they can be used for suppressing the effect of these polypeptide, for example are used for the treatment of artery sclerosis, LADA and chronic inflammation and communicable disease, by inhibition, alpastic anemia and the myelodysplasia syndrome of allergy, eosinophilia's syndrome, silicosis, sarcoidosis, pneumonia disease, IL-1 and the TNF of histamine mediation. Perhaps, these polypeptide can be used for suppressing the generation of IL-1 and TNF-α, are used for the treatment of alpastic anemia, myelodysplasia syndrome, asthma and arthritis. Diagnostic test

According to another aspect of the present invention, provide for detection of the disease relevant with expression of polypeptides deficiency or over-expression with for detection of the diagnostic test of sudden change in the nucleotide sequence of these polypeptide of coding.

According to another aspect of the present invention, provide the polynucleotides that utilize these polypeptide or these polypeptide of encoding to be used for and scientific research, the synthetic external purpose relevant with the dna vector operation of DNA as research reagent, be used for the therapeutic agent of exploitation treatment human diseases and the method for diagnosticum.

The present invention also provide for the identification of can enhancer or inhibitor by the screening technique of the compound of the cell response of MPIF-1 polypeptid induction, comprise: make the cells contacting candidate compound of expressing the MPIF-1 polypeptide, measure cell response, and relatively cell response is replied with standard cell lines, and this standard is mensuration when contacting in the situation that lacks candidate compound; Thus, cell response is activator with respect to the rising indication compound of standard, and cell response is antagonist with respect to the reduction indication compound of standard.

For many disorders, thinking can be in the MPIF-1 gene expression that detects remarkable higher or lower level for " standard " MPIF-1 gene expression dose (namely taking from the tissue of the individuality with this disorder or the MPIF-1 expression in the body fluid) in some tissue of taking from the individuality with these disorders or the body fluid (such as serum, blood plasma, urine, synovia or spinal fluid). Thereby, the invention provides diagnostic method useful in disorderly diagnostic procedure, comprising: (1) measures individual cell or the MPIF-1 gene expression dose in the body fluid; (2) than its MPIF-1 gene expression dose and standard MPIF-1 gene expression dose, thus, the MPIF-1 gene expression dose of mensuration is disorderly with respect to rising or the reduction indication of standard expression. These disorders comprise leukaemia, chronic inflammation, autoimmune disease, solid tumor and from the toxicity of radiation and chemotherapy. Pharmaceutical composition

According to another aspect of the present invention, also provide to comprise the pharmaceutical composition that at least a MPIF-1 polynucleotides, probe, carrier, host cell, polypeptide, fragment, variant, derivative, epi-position carry part, antibody, antagonist or activator. Methods for the treatment of

According to another aspect of the present invention; the method that the polynucleotides of these polypeptide or these polypeptide of encoding is used for the treatment of purpose is provided, for example be used for chemotherapy process protection stem cell avoid chemotherapeutics infringement, eliminate that leukaemia, immune stimulatory reply, regulate and control hematopoiesis and lymphocyte transportation, treatment psoriasis, solid tumor, the enhancing host is for resistance and acute and chronically infected defence and stimulate wound healing.

Another aspect of the present invention relates to and is used for the treatment of the method that needs the individuality of MPIF-1 activity level in the rising body, comprises these individualities are used comprising the composition that the present invention who treats effective dose separates MPIF-1 polypeptide or its activator.

Another aspect of the present invention relates to and is used for the treatment of the method that needs reduce the individuality of MPIF-1 activity level in the body, comprises these individualities are used the composition that comprises the MPIF-1 antagonist for the treatment of effective dose.

In view of the teachings contained herein, these and other aspect of the present invention will be apparent to those skilled in the art.

The summary of figure

Following accompanying drawing illustration embodiment of the present invention, but be not intended to limit the scope of the present invention that claims comprise.

Fig. 1 has showed cDNA sequence (SEQ ID NO:1) and the corresponding derivation amino acid sequence (SEQ ID NO:2) of coding MPIF-1. Initial 21 targeting sequencings that amino acid represent is inferred. All bursts are by to being measured by the N terminal peptide of the protein of baculovirus expression.

Fig. 2 illustration the amino acid sequence homology between MPIF-1 (top) (SEQ ID NO:2) and the people MIP-1 α (bottom) (SEQ ID NO:36). Four characteristic cysteines that shown all chemotactic factor (CF)s.

Fig. 3 be in baculovirus expression system, express and three step purifying after the SDS-PAGE gel photograph of MPIF-1.

Fig. 4 A-4B. By the chemotactic factor assay method, use 48 holes trace slot devices (Neuro Probe company) to measure the chemotactic decoy activity of MPIF-1. Described in the guide of experiment flow such as manufacturer. For each MPIF-1 concentration of test, checked the migration in 5 high power fields. The result who shows has showed the mean value by twice independent experiment acquisition. Shown THP-1 (A) cell and human PBMC's (B) chemotactic decoy active.

Fig. 5. The cellular calcium concentration variation of replying MPIF-1 of having used Hitachi F-2000 fluorescent spectrophotometer assay. Add MPIF-1 by bacterial expression in the THP-1 cell that has loaded Indo-1 to final concentration 50nM, and level in the born of the same parents of monitoring calcium concentration.

Fig. 6. Low-density bone marrow cells in mice group coating (1,500 cell/plate) is specified chemotactic factor (CF) (100ng/ml) but comprised in the agar medium of IL-3 (5ng/ml), SCF (100ng/ml), IL-1 α (10ng/ml) and M-CSF (5ng/ml) comprising or do not contain. Shown in data display the mean value that is obtained by two independent experiments (each carries out double). Be coated with rear 14 days to colony count. The average percent of colony number when the colony number that generates under having the situation of chemotactic factor (CF) is stated as with respect to the chemotactic factor (CF) that lacks any interpolation.

Fig. 7 A-7B by HPP-CFC (A) and LPP-CFC (B) illustration MPIF-1 and the M-CIF impact on the formation of mouse bone marrow cells colony.

Fig. 8 illustration by the MPIF-1 of baculovirus expression and M-CIF on stimulating the bone marrow cell of fresh separated to form the impact of colony with M-CSF and SCF.

Fig. 9 illustration MPIF-1 and M-CIF IL-3 and SCF are stimulated the impact of lin-bone marrow cell group propagation and differentiation.

Figure 10 A-10B has shown that MPIF-1 and M-CIF are on being become the impact of Gr.1 and Mac-1 (surface markers) positive cell group by the bone marrow cell all living creatures who gets rid of pedigree. With the lin-cell only add that IL-3 (5ng/ml) and SCF (100ng/ml) (a) and also add MPIF-1 (50ng/ml) (b) or M-CIF (50ng/ml) growth medium (c) in be incubated. Then use the monoclonal antibody for marrow differentiation Gr.1, Mac-1, Sca-1 and CD45R surface antigen that cell is dyeed, and analyze by FACScan. Data are stated greatly the positive cell percentage in (Figure 10 A) and little (Figure 10 B) cell mass as.

Figure 11 illustration the existence of the MPIF-1 albumen bone marrow cell colony that can suppress to reply IL-3, M-CSF and GM-CSF form.

Figure 12. The dose response of MPIF-1 suppresses the bone marrow cell colony and forms. Separate and the processing cell such as Figure 13. Form in the experiment at the colony based on agar, in the situation that has IL-3, GM-CSF or M-CSF (5ng/ml), contains or do not contain MPIF-1 (1,10,50 and 100ng/ml), with the cell after the density coating processing of 1,000 cell/dish. Data are stated the percentage that colony forms the colony number that forms with respect to only with specificity factor the time as. Data are stated the mean value of a-type double dish as, error post indication standard deviation.

Figure 13. The expression of the RNA of coding MPIF-1 among the person monocytic cell. Separate total RNA by fresh monocyte through eluriating, and process with 100U/ml hu rIFN-γ, 100ng/ml LPS or the two. Will be from RNA (8 μ g) electrophoretic separation on 1.2% Ago-Gel of every kind of processing by electrophoresis, and transfer on the nylon membrane. By³²Detecting of the cDNA of P mark quantizes MPIF-1mRNA, and by optical densitometric method the band on the autoradiograph film that produces quantized.

Figure 14. The analysis of MPIF-1 amino acid sequence (SEQ ID NO:2). α, β, corner and curling district have been shown; Hydrophily and hydrophobicity; The amphiphilic district; Flex region; The antigenicity index; With surperficial probability. In " antigenicity index-Jameson-Wolf " figure, among 21-30 position, 31-44 position, 49-55 position, 59-67 position, 72-83 position, 86-103 position and 110-120 amino acids residue or Fig. 1 (SEQ ID NO:2) among Fig. 1 (SEQ ID NO:2) above-mentioned any scope or numerical value corresponding to shown in the high antigenicity zone of MPIF-1 albumen.

Figure 15 A-15B. (A) shown the marrow protection effect of MPIF-1 for the LPP-CFC cell killing of being induced by 5-FU. (B) shown the marrow protection effect of MPIF-1 for the LPP-CFC cell killing of being induced by Ara-C.

Figure 16 has shown the impact that mouse MPIF-1 preliminary treatment reduces the circulation WBC counting of being induced by 5-FU.

Figure 17 has shown the experimental design of the 3 groups of mouse (6 every group) that comprise following processing: the 1st group, and the 1st, 2 and 3 day pump pickle; The 2nd group, the 0th and 3 day injection 5-FU; The 3rd group, the 0th and 3 day injection 5-FU, the 1st, 2 and 3 day injection MPIF-1. The the 6th and 9 day results marrow is in order to measure HPP-CFC and LPP-CFC frequency with the former determination method of clone.

Figure 18 shown before for the second time taking 5-FU, use MPIF-1 on marrow in the impact of HPP-CFC and LPP-CFC frequency.

Figure 19 has shown the MPIF-1 variant. Use single-letter amino acid code has shown front 80 in 120 amino acid whose sequences of MPIF-1 (Fig. 1, SEQ ID NO:2), and wherein the feature of front 21 residue display sequences will can produce ripe wild-type protein after its excision. Saltant-1 and-6 comprises the methionine as the N terminal residue, and this is non-existent in wild type. Equally, front 4 amino acid (HAAG) of saltant-9 also are non-existent in wild type MPIF-1 albumen. Saltant-1 ,-6 and-9 corresponds respectively to SEQ ID NO:3,4 and 5. Saltant-2 is corresponding to the 46-120 amino acids residue among the SEQ ID NO:2. Saltant-3 is corresponding to the 45-120 amino acids residue among the SEQ ID NO:2. Saltant-4 is corresponding to the 48-120 amino acids residue among the SEQ ID NO:2. Saltant-5 is corresponding to the 49-120 amino acids residue among the SEQ ID NO:2. Saltant-7 is corresponding to the 39th-120 amino acids residue among the SEQ ID NO:2. Saltant-8 is corresponding to the 44-120 amino acids residue among the SEQ ID NO:2.

Figure 20 A-20B. Figure 20 A has shown the nucleotide sequence (SEQ ID NO:6) of people MPIF-1 splice variant cDNA. With this cDNA sequence, also shown the opening code-reading frame (SEQ ID NO:7) of 137 the amino acid whose protein of encoding with the single-letter amino acid code. Indicate terminal 21 targeting sequencings that amino acid represent is inferred of N of underscore. 18 the amino acid whose insetion sequences outstanding with italic do not exist in the MPIF-1 sequence, are unique for this splice variant. Figure 20 B has shown that the amino acid sequence of MPIF-1 variant (SEQ ID NO:7) and wild type MPIF-1 molecule (SEQ ID NO:2) compares.

Figure 21 has shown that the maximum calcium of realizing by MIP-1 α induces moves the 50% needed MPIF-1 mutant protein concentration of replying in the person monocytic cell.

Figure 22 A-22B. As described in the legend of Figure 21, in the person monocytic cell, measured and replied free calcium concentration variation in the born of the same parents that specify protein (100ng/ml).

Figure 23 has shown that the calcium that the MPIF-1 mutant makes MIP-1 α stimulate moves the ability (general introduction) that desensitizes in the person monocytic cell.

Figure 24 has shown the chemotactic response of human peripheral blood mononuclear cell (PBMC) to the MPIF-1 mutant. Number in the round parentheses has reflected that chemotactic stimulation surpasses the multiple of the background of observing in the prescribed concentration scope.

Figure 25 has shown that the MPIF-1 variant is in external growth to low multiplication potentiality colony forming cell (Low Proliferative Potential Colony-Forming Cells, LPP-CFC) and the impact of differentiation.

Figure 26 has shown that replying the stem cell that MPIF-1 uses in the normal mouse shifts.

Figure 27 has compared the impact of the 5-FU of two circulations being processed rear platelet recovery by the MPIF-1 of FACS Vantage method mensuration and G-CSF.

Gra.1 and the dual positive cell of Mac.1 recover in the blood after the 5-FU of two circulations is processed impact that Figure 28 has compared MPIF-1 and G-CSF.

Figure 29 has compared the impact that Gra.1 and the dual positive cell of Mac.1 in the rear marrow of 5-FU processing of two circulations is recovered by the MPIF-1 of FACS Vantage method mensuration and G-CSF.

HPC recovers in the marrow after the 5-FU of two circulations is processed impact that Figure 30 has compared MPIF-1 and G-CSF.

Figure 31 has shown the schematic diagram of the MPIF-1 Δ 23 cDNA coded sequences of pHE4-5 expression vector (SEQ ID NO:37) and subclone. Its indicating the position of kalamycin resistance marker gene, MPIF-1 Δ 23 coded sequences, oriC sequence and lacIq coded sequence.

Figure 32 has shown the general introduction for the production of the fermentation process of MPIF-1 Δ 23.

Figure 33 has shown the flow chart for the method that reclaims the MPIF-1 Δ 23 of being produced by method shown in Figure 32.

Figure 34 has shown for the process of purifying by the MPIF-1 Δ 23 of process production shown in Figure 32 and 33 and recovery.

Figure 35 has shown the nucleotide sequence (SEQ ID NO:38) of the controlling element of pHE promoter. Its indicating two lac operator sequences, SD sequence (S/D) and terminal HindIII and NdeI restriction site (italic).

Figure 36 A-36E has shown the complete nucleotide sequence (SEQ ID NO:37) of pHE4-5 carrier.

Figure 37 has shown that MPIF-1 protects intestines and stomach to avoid the damage of being induced by radiation in the short-term monitor procedure. Accept the radiation of sublethal dose (from¹³⁷2 * the 4.5gy in Cs source, interval 4 hours) processes the C57B1/6 female mice before or after. Shown in survival, situation and the body weight of date monitoring mouse. The data that show are that every group body weight percentage changes, be based on every mouse shown in the percentage of the body weight on date its body weight during with respect to the experiment beginning.

Figure 38 has shown that MPIF-1 protects intestines and stomach to avoid the damage of being induced by radiation in long-term monitor procedure. Accept the radiation of sublethal dose (from¹³⁷2 * the 4.5gy in Cs source, interval 4 hours) processes the C57B1/6 female mice before or after. Shown in survival, situation and the body weight of date monitoring mouse. The data that show are that every group body weight percentage changes, be based on every mouse shown in the percentage of the body weight on date its body weight during with respect to the experiment beginning. Curve is illustrated as two sections. Showed the variation in front 18 days for left section, right section body weight of showing all groups of closing day.

Figure 39 has shown that MPIF-1 protects intestines and stomach to avoid the damage of being induced by radiation in the short-term monitor procedure. Accept the radiation of sublethal dose (from¹³⁷2 * the 5.5gy in Cs source, interval 4 hours) processes the C57B1/6 female mice before or after. Shown in survival, situation and the body weight of date monitoring mouse. The data that show are that every group body weight percentage changes, be based on every mouse shown in the percentage of the body weight on date its body weight during with respect to the experiment beginning.

Figure 40 has shown that MPIF-1 protects intestines and stomach to avoid the damage of being induced by radiation in long-term monitor procedure. Accept the radiation of sublethal dose (from¹³⁷2 * the 5.5gy in Cs source, interval 4 hours) processes the C57B1/6 female mice before or after. Shown in survival, situation and the body weight of date monitoring mouse. The data that show are that every group body weight percentage changes, be based on every mouse shown in the percentage of the body weight on date its body weight during with respect to the experiment beginning.

Figure 41 has shown the therapeutic scheme schematic diagram that is used for for the Mice Body inner model of deadly protection of radiating.

Figure 42 has shown that MPIF-1 can strengthen the survival of the radiation mouse that causes death. Use logarithm classification nonparametric to carry out statistical analysis, tables of data is shown as the Kaplan-Meier survival curve. The 54th day finishes to test after radiation.

Figure 43 has shown the therapeutic scheme schematic diagram that is used for for the Mice Body inner model of the deadly protection of radiating in Asia.

Figure 44 has shown that MPIF can strengthen the recovery of the directed marrow of pedigree predecessor in the inferior radiation mouse that causes death.

Figure 45 has shown that MPIF can strengthen the recovery of multipotency myeloid progenitor in the inferior radiation mouse that causes death.

Figure 46 has shown that MPIF is in the impact of external propagation on people CD34+ CFU-GM.

Figure 47 has shown that MPIF is in the external impact that kills and wounds on being induced by cytotoxic agent.

Figure 48 has shown the general introduction of research in the MPIF body.

Figure 49 A-49B has shown that the MPIF preliminary treatment is in vivo on the impact of the toxicity of being induced by 5-FU in the myeloid progenitor. (A) MPIF is on the impact of total colony formation. (B) MPIF is on the impact of WBC recovery. The result is the mean value of 8 experiments.

Figure 50 has shown the chemotactic protectiveness impact of MPIF on many circulation treatments.

Figure 51 shown the MPIF scheme of taking medicine 5-FU is processed after marrow recover dynamic (dynamical) impact.

Figure 52 has shown the general introduction of multiple dosage toxicity research.

Figure 53 has shown the general introduction of the observed result of non-clinical toxicology research.

Figure 54 A-54B has shown that the pharmacokinetics behind intravenous or the subcutaneous administration MPIF compares pF. (A) 0-24 hour pharmacokinetics collection of illustrative plates. (B) 0-4 hour collection of illustrative plates. MPIF dosage is 20mg/kg.

Figure 55 has shown the differentiation that PBC forms among the human experimenter.

Figure 56 has shown among the Healthy People volunteer differentiation with respect to the average absolute monocyte count for the treatment of group.

Figure 57 has shown the MPIF concentration (ng/ml) among the healthy volunteer.

Figure 58 A-58E has shown the structure of MPIF-1. (A) and (B) superimposed about 30 kinds of simulated annealings (SA) structure of 77 residue average structures of MPIF-1 1-. (C) identical with A and B, just omitted terminal (67-77 position) residue of N terminal (1-10 position) and C in order to know. (D) with (E) use that the MOLMOL program produces, with the schematic diagram that is orientated identical MPIF-1 shown in the C figure people such as (, J.Mol.Graph., 14:29-42,1996) R.Koradi.

Figure 59 A-59F has shown distributing about the backbone atoms (A) of 30 kinds of simulated annealing structures of average structure and the atom rms of all heavy atoms (B) of the most suitable 11-66 position residue. The angle order parameter (S) and the accessible district of segmentation solvent that have also shown φ (C), φ (D) and χ 1 (E).

Figure 60 A-60G has shown MPIF-1's¹⁵The N dynamics data is as the function of residue. In figure A, B, C and D, shown respectively¹⁵N T ₁T ₂、T ₁/T ₂Ratio and NOE. Residual graph has shown by match¹⁵N T ₁T ₂, the kinetic parameter that calculates of NOE data; Order parameter, S²(E); Inner correlation time, Te (F); Change speed (G) with conformation.

Figure 61 A-61D has compared the structure of MPIF-1 and other CC chemotactic factor (CF) (MIP-1 β, HCC-2, RANTES and MCP-1). The 11-66 position residue of MPIF-1 is superimposed on the 11-67 position residue of the 10-65 position residue of 6-61 position residue, RANTES of 11-66 position residue, the HCC-2 of MIP-1 β and MCP-3. For clear, do not show N and C terminal residue.

Figure 62 has shown that the amino acid sequence of the relevant CC chemotactic factor (CF) with other of MPIF-1 compares. Conservative cysteine represents with runic. Conservative hydrophobic residue and conservative charged residue are described in embodiment 36.

Figure 63 A-63D has used the MOLMOL program display surface charge of MPIF-1 distribute people such as (, J.Mol.Graph., 14:29-42,1996) R.Koradi. Blueness and red display are used respectively in positively charged zone and electronegative zone. For clear, do not show 1-10 position and 69-77 position residue.

The detailed description of preferred embodiment

The invention provides the separation polynucleotide molecule or the polypeptide self that utilize coded polypeptide (is CKBeta8 (MPIF-1) polypeptide, be called in the past MIP-3 and chemokine beta 8 (CK β 8 or ckb-8)) diagnostic or therapeutic composition and method, also provide carrier, host cell and for the production of their restructuring or synthetic method. The MPIF-1 polynucleotides

According to one aspect of the present invention, the total length of the derivation amino acid sequence that coding has Fig. 1 (SEQ ID NO:2) or ripe MPIF-1 polypeptide are provided and by the isolating nucleic acid (polynucleotides) of the ripe MPIF-1 polypeptide of the cDNA clones coding of the ATCC preserved material numbering 75676 of preservation on February 9 in 1994. The address of U.S. typical case culture collecting center (American Type Culture Collection) is Patent Depository, 10801, University Boulevard, Manassas (Manassas), Virginia (Virginia), 20110-2209. Preservation the clone be contained in pBluescript SK (-) plasmid (Stratagene, LaJolla, CA).

The preserved material that this paper mentions is kept the clause according to the budapest treaty of the relevant microbial preservation international recognition that is used for proprietary program. These preserved materials provide just to making things convenient for those skilled in the art, but not assert the regulation needs preservation according to 35U.S.C. § 112. The amino acid sequence of contained polynucleotide sequence and coded polypeptide thereof in the preserved material is collected herein by reference, and when clashing with this paper sequence description, is as the criterion with the former. The production of preserved material, use or sale need license, and this paper does not authorize this license.

The polynucleotides of code book invention polypeptide are structurally relevant with the short scorching supergene " intercrine " that belongs to cell factor or chemotactic factor (CF) family. MPIF-1 is the homologue of MIP-1, and with MIP-1 α than with MIP-1 β homology more. The polynucleotides of coding MPIF-1 are derived from the aortic endothelial cell cDNA library, and comprise the opening code-reading frame of the polypeptide of 120 amino acid residues of encode, and described polypeptide is showed and many chemotactic factor (CF)s have remarkable homology. The highest coupling is and human macrophage inflammatory protein-1 α the homology of demonstration 36% and 66% similitude (Fig. 2).

Polynucleotides of the present invention can be the form of RNA or the form of DNA, and DNA comprises cDNA, genomic DNA and synthetic DNA. DNA can be two strands or strand, if strand then can be coding strand or non-coding (antisense) chain. The coded sequence of encoding mature polypeptide can be identical with coded sequence shown in Fig. 1 or the 20A (SEQ ID NO:1 or 6) or preservation clone's coded sequence; Perhaps, can be different coded sequences, because of the DNA of the redundancy of genetic code or degeneracy and Fig. 1 or 20A (SEQ ID NO:1 or 6) or the identical mature polypeptide of cDNA coding of preservation.

The mature polypeptide of code pattern 1 (SEQ ID NO:2) or can be comprised by the polynucleotides of the mature polypeptide of preservation cDNA coding: the coded sequence that only has mature polypeptide; The coded sequence of mature polypeptide and extra coded sequence are such as targeting sequencing or secretion sequence or proteinogen sequence; The coded sequence of mature polypeptide (with optional extra coded sequence) and non-coding sequence are such as 5 ' and/or 3 ' non-coding sequence of introne or mature polypeptide encoded sequence.

Thereby the polynucleotides that only comprise polypeptid coding sequence contained in term " polynucleotides of coded polypeptide ", and the polynucleotides that comprise extra coded sequence and/or non-coding sequence.

The invention still further relates to the variant of contained cDNA sequence among disclosed polynucleotide sequence in SEQ ID NO:1 or 6 or its complementary strand and/or the preservation clone.

" variant " refers to be different from MPIF-1 polynucleotides or polypeptide but keeps polynucleotides or the polypeptide of its intrinsic propesties. Generally speaking, variant is closely similar on the whole to MPIF-1 polynucleotides or polypeptide, and identical in many zones.

Except as otherwise noted, all definite nucleotide sequences are to use automation DNA sequencer (such as 373 types available from Applied Biosystems company) to measure by the sequence of measuring this paper dna molecular, and all amino acid sequences by the polypeptide of dna molecule encode that this paper determines are to predict by the dna sequence dna that translation is above determined. Therefore, as this area to any dna sequence dna of measuring by this automatic mode know like that, any nucleotide sequence of this paper mensuration all may contain some mistakes. Usually about at least 90% same by the automatic mode nucleotide sequence of measuring and the actual nucleotide sequence that is sequenced dna molecular, more generally about at least 95 %-about at least 99.9% are same. By other method, comprise manual dna sequencing method well-known in the art, can measure more accurately actual sequence. This area is also known, compare with actual sequence, single insertion in the determined nucleotide sequence or disappearance can cause the frameshit phenomenon in the nucleotide sequence translation, thereby from described insertion or missing point, will be fully different from the amino acid sequence of the dna molecular actual coding that is sequenced by determined nucleotide sequence coded derivation amino acid sequence.

Except as otherwise noted, this paper every kind " nucleotide sequence " listing represents with the sequence of deoxyribonucleotide (being abbreviated as A, G, C and T). Yet, " nucleotide sequence " of nucleic acid molecules or polynucleotides is the sequence of deoxyribonucleotide for dna molecular or polynucleotides, be corresponding ribonucleotide (A, G, C and U) sequence for RNA molecule or polynucleotides, wherein specify each thymine deoxyribotide (T) in the deoxyribonucleotide sequence to be substituted by uracil ribonucleotide (U). For example, have and use sequence SEQ ID NO:1 that the deoxyribonucleotide abbreviation lists or 6 RNA molecule, be intended to represent that each deoxyribonucleotide A, G in sequence SEQ ID NO:1 or 6 or C are by corresponding ribonucleotide A, G or C substitutes and each deoxyribonucleotide T has been substituted by ribonucleotide U RNA molecule.

Use information provided herein, such as the nucleotide sequence among Fig. 1, can Application standard clone and screening process (such as the flow process of cloning cDNA with mRNA as parent material) obtain the to encode nucleic acid molecules of the present invention of MPIF-1 polypeptide.

The invention still further relates to the variant of polynucleotides mentioned above, described polynucleotides variant coding have Fig. 1 (SEQ ID NO:2) the deduction amino acid sequence polypeptide or by fragment, analog and the derivative of the polypeptide of preservation clone's cDNA coding. The polynucleotides variant can be the variant that the non-natural of the allele variant of natural generation of polynucleotides or polynucleotides occurs.

The present invention also comprises the polynucleotides of the identical mature polypeptide that coding and the identical mature polypeptide shown in Fig. 1 (SEQ ID NO:2) or preservation clone's cDNA are coded, and the variant of these polynucleotides, the polypeptide of described variant code pattern 1 (SEQ ID NO:2) or by fragment, derivative or the analog of the polypeptide of preservation clone's cDNA coding. These nucleotide variants comprise disappearance variant, alternative variations and add or the insertion variant.

As mentioned above, the coded sequence that has of polynucleotides can be the allele variant of natural generation of coded sequence shown in Fig. 1 (SEQ ID NO:2) or preservation clone's coded sequence. As road known in the art, allele variant is a kind of replaceable form of polynucleotide sequence, and it can have the substituting of one or more nucleotides, disappearance or add, but does not basically change the function of coded polypeptide.

The present invention also comprises polynucleotides, wherein the coded sequence of mature polypeptide can merge the polynucleotide sequence that helps host cell expression and secrete polypeptide in identical reading frame, for example transports the targeting sequencing of polypeptide from transit cell with control as the secretion sequence functionating. Polypeptide with targeting sequencing is front albumen, thereby it may be cut the mature form that targeting sequencing forms polypeptide by host cell. The all right encoding proteins of polynucleotides is former, and it is that maturation protein adds extra N terminal amino acid residue. Maturation protein with former sequence is proteinogen, and it is the inactive form of protein. In case former sequence is cut, remaining is exactly activated maturation protein.

Thereby for example, polynucleotides of the present invention can encoding mature albumen, or the protein with former sequence, or not only had former sequence but also had the protein of presequence (targeting sequencing).

Polynucleotides of the present invention can also have the coded sequence that merges flag sequence in identical reading frame, and described flag sequence can be used for purifying polypeptide of the present invention. Be in the situation of bacterium the host, flag sequence can be the hexahistidine tag that is provided by the pQE-9 carrier, the mature polypeptide that merges with purifying and flag sequence; Or for example, when using mammalian hosts, during such as the COS-7 cell, flag sequence can be hemagglutinin (HA) label. The HA label is corresponding to the epi-position (people such as I.Wilson, Cell, 37:767,1984) derived from influenza virus hemagglutinin albumen.

Term " gene " refers to participate in generating the DNA section of polypeptide chain; It comprises before the code area and the intervening sequence (introne) between zone afterwards (targeting sequencing and tailer sequence) and each encode fragment (extron).

As mentioned above, nucleic acid molecules of the present invention can be the form of RNA, such as mRNA, or the form of DNA, comprise for example by clone or synthetic cDNA and the genomic DNA that generates. DNA can be two strands or strand. Single stranded DNA or RNA can be coding strand (being also referred to as sense strand), perhaps can be noncoding strands (being also referred to as antisense strand).

Term " separation " refers to material is taken out by its initial environment (such as natural surroundings, if the words of natural generation). For example, for the purposes of the present invention, polynucleotides or the polypeptide of the natural generation that exists in the surviving animals do not separate, and the identical polynucleotides that separate with some or all of coexisting substances in the natural system or DNA or polypeptide separate. These polynucleotides can be that the part of carrier and/or these polynucleotides or polypeptide can be the parts of composition, and remain separation, because these carriers or composition are not the parts of its natural surroundings. The isolation of RNA molecule comprises the interior or external rna transcription of the body of dna molecular of the present invention originally. Isolated nucleic acid molecule of the present invention also comprises synthetic these molecules that produce. Yet, for the purposes of the present invention, as member of library (such as genome or cDNA library) and not with clone that other member in library separates in the contained nucleic acid form of clone and other library member's homogeneous solution (as comprise) or chromosome preparation (such as the chromosome smear) upper contained nucleic acid or not with preparation in other separate nucleic acid the genomic DNA preparation (as complete, that shred and/or with after one or more restriction enzymes cuttings) nucleic acid of middle existence " do not separate ".

Isolated nucleic acid molecule of the present invention comprise the ORFs (ORF) that comprises MPIF-1 cDNA or consisting of dna molecular; Comprise ripe MPIF-1 albumen coded sequence or consisting of dna molecular; With comprise in fact from above-mentioned different sequence but because of still the encode dna molecular of MPIF-1 polypeptide of the degeneracy of genetic code. Certainly, genetic code is well-known in the art. Thereby those skilled in the art can the above-mentioned degeneracy variant of conventional preparation.

The invention still further relates to and sequence mentioned above can be hybridized the polynucleotides of (if having the homogeneity of at least 95 % between sequence). The present invention is specifically related to the polynucleotides that can hybridize with polynucleotides mentioned above under rigorous condition. When being used for this paper, term " rigorous condition " refers to only exist at least 95% between sequence, just hybridize during preferably at least 97% homogeneity. In a preferred embodiment, the coded polypeptide of the polynucleotides that can hybridize with polynucleotides mentioned above keeps with the cDNA of Fig. 1 (SEQ ID NO:1) or by the mature polypeptide of preservation cDNA coding substantially the same biological function or activity.

Perhaps, polynucleotides can have at least 20 bases, preferred 30 bases, and more preferably at least 50 bases, hybridization can occur and have homogeneity with polynucleotides of the present invention in it and polynucleotides of the present invention as mentioned above, and it may keep or retentive activity not. For example, can adopt these polynucleotides as the probe of SEQ ID NO:1 polynucleotides, for example for reclaiming polynucleotides or being used as diagnostic probe or being used as the PCR primer.

In one aspect of the method, the invention provides comprise following polynucleotides or consisting of isolated nucleic acid molecule, described polynucleotides under rigorous hybridization conditions with nucleic acid molecules of the present invention mentioned above (for example contained cDNA clones of ATCC preserved material 75676 (MPIF-1)) in the polynucleotides part can hybridize. " rigorous hybridization conditions " refer in the solution through shearing salmon sperm DNA that contains 50% formamide, 5x SSC (750mM NaCl, 75mM trisodium citrate), 50mM sodium phosphate (pH7.6), 5x DenhardtShi liquid, 10% dextran sulfate, 20 μ g/ml sex change in 42 ℃ of incubated overnight, subsequently in about 65 ℃ with 0.1x SSC cleaning filter membranes.

The polynucleotides that can hybridize with polynucleotides " part " refer to and about at least 15 nucleotides (nt), more preferably at least approximately 20nt, still more preferably at least approximately 30nt even the more preferably about polynucleotides (DNA or RNA) that can hybridize with reference to polynucleotides of 30-70nt. Also refer to and at least about 15nt, more preferably at least approximately 20nt, more preferably at least approximately 25nt, still more preferably at least approximately 30nt even the more preferably about polynucleotides that can hybridize with reference to polynucleotides of 30-70 (such as 30,35,40,45,50,55,60,65 and/or 70, other fragment length except this paper narration also is useful certainly) nt. As above discussion and hereinafter in more detail discussion, these polynucleotides can be used as diagnostic probe and primer.

Certainly, with reference polynucleotides (such as the cDNA of preservation clone's) major part (be the part of 50-750nt such as length) or even the polynucleotides that can hybridize with reference to polynucleotides of total length also can be used as probe of the present invention, be useful equally corresponding to the polynucleotides of the major part (if not all) of nucleotide sequence shown in the nucleotide sequence of preservation cDNA or the SEQ ID NO:1 or 6 (MPIF-1). For example, the polynucleotides of " length at least 20nt " partly refer to reference to 20 in the nucleotide sequence of polynucleotides or more continuous nucleotides. As mentioned above, thereby these parts can be used as the probe of conventional DNA hybridization technique or pass through polymerase chain reaction (PCR) amplified target sequence as primer in diagnosis, be described in for example " Molecular Cloning, A Laboratory Manual " (molecular cloning, laboratory manual), the 2nd edition, J.Sambrook, E.F.Fritsch and T.Maniatis compile, publishing house of cold spring harbor laboratory, the cold spring port, the New York, 1989 (listing in full it in this paper as a reference).

Since preservation MPIF-1 cDNA clone, and provide its nucleotide sequence of determining, therefore generating the polynucleotides that can hybridize with the part of MPIF-1 cDNA molecule will be routine work to those skilled in the art. For example, can simply use restriction endonuclease or shear the DNA parts that produce different sizes by ultrasonic wave processing MPIF-1 cDNA clone, they are exactly the polynucleotides that can hybridize with the part of MPIF-1 cDNA molecule.

Perhaps, can be according to the synthetic generation of known technology hybridization polynucleotides of the present invention. Certainly, the polynucleotides that only hybridization occur with the complementary series of polyA sequence (such as cDNA 3 ' terminal poly (A) sequence) or T or U residue are not included in for nucleic acid moiety of the present invention the polynucleotides of the present invention of hybridization occuring, because these polynucleotides all can be hybridized with any nucleic acid molecules that contains poly (A) sequence or its complement (such as actual what double-stranded cDNA clone that takes up an official post).

As mentioned above, the nucleic acid molecules of the present invention of coding MPIF-1 polypeptide can include but not limited to: the nucleic acid molecules of the amino acid sequence of self encoding mature polypeptide; The coded sequence of mature polypeptide and additional sequences is such as coding targeting sequencing or secretion sequence, such as front albumen, proteinogen or preproprotein sequence; The coded sequence of mature polypeptide, comprise or do not contain above-mentioned extra coded sequence, also has extra non-coding sequence, comprise such as but not limited to introne and 5 ' and 3 ' non-coding sequence, such as transcribing but the sequence of not translating, they transcribe, the ribosomes combination of mRNA processing (comprising montage and polyadenylation signal), mRNA and stable in play a role; The extra coded sequence of coding additional amino acid (such as additional functionality is provided). Thereby the sequence of coded polypeptide can merge flag sequence, helps the sequence of the peptide of purifying fused polypeptide such as coding. In this some preferred embodiment on the one hand of the present invention, marker amino acid sequence is six histidine peptides (such as the label that provides in pQE carrier (Qiagen company)) etc., many can the purchase by commercial sources. Such as people such as Gentz, Proc.NAtl.Acad.Sci.USA, described in the 86:821-824,1989, six histidines are convenient to the purifying of fusion. " HA " label is the peptide that another kind can be used for purifying, and it is corresponding to the epi-position derived from influenza virus hemagglutinin albumen, such as people such as Wilson, and Cell, 37:767 is described in 1984. As what hereinafter discuss, other these fusions are included in MPIF-1 polypeptide or the fragment of N end or C end fusion Fc.

The invention still further relates to the variant of nucleic acid molecules of the present invention, part, analog or the derivative of described variant coding MPIF-1 polypeptide. Variant can be natural generation, such as natural allele variant. " allele variant " refers to occupy one of several replaceable forms of the gene of specifying locus on the organism chromosome (" Gene V " (gene, the 5th edition), B.Lewin compiles, Oxford University Press, New York, 1994). Can generate with the induced-mutation technique that this area is known the variant that non-natural occurs.

These variants comprise by nucleotide substitution, disappearance or add the variant that generates. Substitute, lack or add and to relate to one or more nucleotides. Variant can change in code area, noncoding region or the two. Variation in the code area can cause conservative or nonconservative amino acid replacement, disappearance or interpolation. Especially preferred in these variants change is that silence substitutes, adds and disappearance, and they do not change characteristic and the activity of MPIF-1 polypeptide or its part. Same especially preferred in this is conservative substituting. Topnotch is the maturation protein of the cDNA clones coding of coding preservation as described herein or the nucleic acid molecules of mature amino acid sequence preferably. Homology MPIF-1 polynucleotides

The invention still further relates to the polynucleotides and the fragment thereof that have at least 95 % homogeneity with the polynucleotides of the polypeptide of the SEQ ID NO:2 that encodes, described fragment has at least 30 bases, preferably at least 50 bases. The invention still further relates to the coded polypeptide of these polynucleotides.

Other embodiment of the present invention comprise comprise have with the polynucleotides of following nucleotide sequences at least 80 %, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% same nucleotide sequence or consisting of isolated nucleic acid molecule: (1) coding has the MPIF-1 polypeptide of amino acid sequence (targeting sequencing that comprises prediction) of SEQ ID NO:2 or the nucleotide sequence of fragment; (2) coding have SEQ ID NO:2 amino acid sequence (targeting sequencing that comprises prediction) but without the MPIF-1 polypeptide of the terminal methionine residues of N or the nucleotide sequence of fragment; (3) nucleotide sequence of encoding mature MPIF-1 polypeptide (removing the full-length polypeptide of targeting sequencing); (4) coding has the nucleotide sequence of the full-length polypeptide of the coded complete amino acid sequence (comprising targeting sequencing) of the cDNA clone of preservation; (5) coding has the nucleotide sequence of the mature polypeptide of the coded amino acid sequence of the cDNA clone of preservation; Or the nucleotide sequence of any nucleotide sequence complementation in (6) and (1), (2), (3), (4) or (5).

Have with coding MPIF-1 polypeptide with reference to the nucleotide sequence polynucleotides of the nucleotide sequence of 95% " same " at least for example, the nucleotide sequence and the canonical sequence that mean these polynucleotides are same, just reach 5 point mutation in can comprising with reference to polynucleotide sequence described in per 100 nucleotides of nucleotide sequence of coded polypeptide. In other words, in order to obtain to have the polynucleotides with the same nucleotide sequence of reference nucleotide sequence at least 95%, can lack or with 5% nucleotides nearly in the another kind of nucleotide substitution canonical sequence, perhaps can in canonical sequence, insert the nucleotides that reaches canonical sequence nucleotides sum 5%. These sudden changes of canonical sequence can betide with reference to 5 ' or 3 ' terminal position of nucleotide sequence or any position between the terminal position, or intersperse among separately in the nucleotides of canonical sequence, or intersperse among in the canonical sequence with one or more continuous group forms.

In practical operation, use known computer program, such as Bestfit program (Wisconsin sequence analysis program package, Unix the 8th edition, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI53711), can conventional determine any specific nucleic acid molecules whether with for example nucleotide sequence shown in Figure 1 or preservation cDNA clone's nucleotide sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% same. Bestfit uses Smith and Waterman, Advances in Applied Mathematics, and 2:482-489, local homology's algorithm of 1981 is found out the best homology segment between two kinds of sequences. When determine with Bestfit or any other sequence alignment program specific sequence whether for example with canonical sequence 95% of the present invention with for the moment, arranging certainly of parameter should be so that calculate homogeneity percentage to the total length of reference nucleotide sequence, and allow to exist the nearly homology breach of canonical sequence nucleotides sum 5%.

In practical operation, use known computer program, can conventional determine that any specific nucleic acid molecules or polypeptide and nucleotide sequence of the present invention be whether at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% same. Be used for determining search sequence (query sequence, sequence of the present invention) method for optimizing of best overall match and between research sequence (subject sequence), be also referred to as the Compositive sequence comparison, can use based on people such as Brutlag, Comp.App.Biosci., 6:237-245, the FASTDB computer program of 1990 algorithm is determined. In sequence alignment, inquiry and research sequence all are dna sequence dnas. Can come comparison RNA sequence by U being transformed into T. The result of described Compositive sequence comparison recently explains with the homogeneity percentage. In the FASTDB of dna sequence dna comparison, the preferred parameter that is used for calculating homogeneity percentage is: matrix (Matrix)=Unitary, k-tuple=4, mispairing point penalty (Mismatch Penalty)=1, connect point penalty (Joining Penalty)=30, randomized grouping length (Randomization Group Length)=0, block score (Cutoff score)=1, breach point penalty (Gap Penalty)=5, breach size point penalty (Gap Size Penalty)=0.05, the length (selecting the shorter one) of window size (Window Size)=500 or indication nucleotide sequence.

If the research sequence is shorter than search sequence because of 5 ' or 3 ' disappearance (but not inner disappearance), then must carry out manual correction to the result. This is because the FASTDB program does not consider to study 5 ' and 3 ' brachymemma of sequence when calculating whole homogeneity percentage. The research sequence of 5 ' or 3 ' end brachymemma for relative search sequence, the search sequence base number that does not mate/compare that is positioned at research sequence 5 ' and 3 ' by calculating is recently proofreaied and correct homogeneity percentage as the percentage of search sequence total alkali radix. Whether nucleotides mates/compare is to be determined by the result of FASTDB sequence alignment. Then from the homogeneity percentage that uses designated parameter to calculate by above-mentioned FASTDB program, deduct this percentage, thereby obtain final homogeneity percentage score. This final score is exactly the value that relates among the present invention. When manual setting homogeneity percentage score, only calculate the FASTDB sequence alignment show do not mate/compare with search sequence, be positioned at research sequence 5 ' and 3 ' base base in addition.

For example, the search sequence of the research sequence of 90 bases of comparison and 100 bases is to determine homogeneity percentage. If disappearance betides research sequence 5 ' end, then the FASTDB comparison does not show front 10 the Mismatching/comparisons of 5 ' end. These 10 unpaired bases account for 10% (5 ' and 3 ' end does not mate base number/search sequence base sum) of sequence, therefore deduct 10% from the homogeneity percentage that the FASTDB program calculates. If remaining 90 bases are mated fully, then final homogeneity percentage is 90%. In another example, compare the research sequence of 90 bases and the search sequence of 100 bases. Lack in inside specifically, study like this 5 ' and 3 ' of sequence and hold the base of not mating with search sequence/comparing. In this case, the homogeneity percentage that calculates by FASTDB is not carried out manual correction. Moreover the base of only research sequence 5 ' and 3 ' not being mated with search sequence/comparing is carried out manual correction. For the purposes of the present invention, not carrying out the manual of other proofreaies and correct.

Preferably, program mentioned above is used for comparing polynucleotides of the present invention (canonical sequence) and the second sequence, and manual calculation homogeneity percentage. Homogeneity percentage is: the number of identical nucleotides between two kinds of sequences, the nucleotide residue sum divided by canonical sequence multiply by 100%. For example, for the 1-360 position nucleotides of measuring SEQ ID NO:1 and the homogeneity percentage of the second sequence, the number of counting mispairing nucleotides (being point mutation: insert, lack and substitute), and by deducting this number in 360 (the nucleotides numbers of canonical sequence) to obtain the number of identical nucleotides, the number that obtains divided by 360, then be multiply by 100%. If 1-5,18, the 201-210,300,302 of SEQ ID NO:1,318-328,330,336,341 and the 349-352 position exist mispairing (be point mutation: insert, disappearance and substitute), then homogeneity percentage will be 90% (5+1+10+1+1+11+1+1+1+3=36,360-36=324,324/360=0.9,0.9 * 100%=90%). Calculate the homogeneity percentage of polypeptide with similar approach.

Those of ordinary skills can understand, but because the variability of the cleavage site of targeting sequencing in order-checking error probability discussed above and the different known protein, the coded ripe MPIF-1 polypeptide of preservation cDNA comprises about 99 amino acid, but can be any situation in 75-120 the Amino Acid Range; The actual targeting sequencing of this protein is about 21 amino acid, but can be any situation in about 35 Amino Acid Ranges of about 15-.

The MPIF-1 variant can comprise change in code area, noncoding region or the two. Especially preferred polynucleotides variant comprises characteristic or the active change that causes silence to substitute, add or lack but do not change coded polypeptide. Preferred because the degeneracy of genetic code substitutes the nucleotide variants that produces by silence. In addition, also preferably with any combination mode substitute, disappearance or add 5-10,1-5 or 1-2 amino acid whose variant. Can generate for many reasons MPIF-1 polynucleotides variant, for example optimize codon for specific host and express (human mRNA's codon is become the preferred codon of bacterial host such as Escherichia coli).

The MPIF-1 variant of natural generation is called " allele variant ", refers to occupy on the organism chromosome one of several replaceable forms (" Gene II " (gene, the 2nd edition) of the gene of specifying locus, B.Lewin compiles, Wiley ﹠ Sons, New York, 1985). These allele variants can change at polynucleotides and/or polypeptide level, and are contained among the present invention. Perhaps, can be by induced-mutation technique or by directly synthesizing to generate the variant that non-natural occurs. Nucleic acid probe

These isolated molecules, particularly dna molecular can be used as probe, are used for gene mapping by Chromosomal in situ hybridization, by the Northern engram analysis for detection of the expression of MPIF-1 gene in human tissue. The invention still further relates to the fragment of isolated nucleic acid molecule described herein. Fragment with isolated nucleic acid molecule of nucleotide sequence shown in the nucleotide sequence of MPIF-1 cDNA of preservation or Fig. 1 or the 20A (SEQ ID NO:1 or 6) refers to that length is at least approximately 15nt, more preferably at least approximately 20nt, still more preferably at least approximately 30nt even more preferably at least about 40nt and can be used as the fragment of diagnostic probe described herein and primer. Certainly, according to the present invention also can use length as 50-500nt than large fragment, useful equally corresponding to the fragment of the major part (if not all) of nucleotide sequence shown in the nucleotide sequence of the MPIF-1 cDNA of preservation or Fig. 1 or the 20A (SEQ ID NO:1 or 6). For example, length refers to comprise 20 or the fragment of more continuous bases from nucleotide sequence shown in the nucleotide sequence of preservation cDNA or Fig. 1 or the 20A (SEQ ID NO:1 or 6) for the fragment of 20nt at least. Since preservation this gene, and provide nucleotide sequence shown in Fig. 1 or the 20A (SEQ ID NO:1 or 6), therefore generating these dna fragmentations will be routine work to those skilled in the art. For example, can simply produce the fragment of all size with restriction endonuclease cutting or ultrasonic shear. Perhaps can synthesize and produce these fragments.

The application relates to and nucleotide sequence 90% disclosed herein, 95%, 96%, 97%, 98% or 99% same nucleic acid molecules (having hereinafter polypeptide as the disclosed N of the m-n of SEQ ID NO:2 or 7 and/or C end disappearance amino acid sequence such as coding) at least, no matter and whether they encode and have the polypeptide of MPIF-1 functional activity. Even this is not have the polypeptide of MPIF-1 functional activity because specific nucleic acid molecules is not encoded, those skilled in the art still know how with the primer of this nucleic acid molecules as for example hybridization probe or polymerase chain reaction (PCR). The purposes of nucleic acid molecules of the present invention of polypeptide with MPIF-1 functional activity of not encoding comprises: (1) separates MPIF-1 gene or its allele or the splice variant in the cDNA library; (2) in situ hybridization of metaphase chromosome smear (such as " FISH ") is to provide the accurate chromosome mapping of MPIF-1 gene, example is seen the people such as Verma, " Human Chromosomes:A Manual of Basic Techniques " (human chromosomal: the basic technology guide), Pergamon publishing house, New York, 1988; (3) the Northern engram analysis is to detect the MPIF-1 mrna expression in the particular organization.

Yet, preferably having the nucleic acid molecules with nucleotide sequence at least 90% disclosed herein, 95%, 96 %, 97%, 98% or 99% same sequence, in fact described nucleic acid molecules is encoded and is had the polypeptide of MPIF-1 functional activity. " polypeptide with MPIF-1 functional activity " refers to the measurement according to specific Radioimmunoassay of vascular endothelial growth for example or biological assay, shows polypeptide similar with the functional activity of MPIF-1 polypeptide of the present invention (such as complete (total length) MPIF-1, ripe MPIF-1 and soluble M PIF-1 (as having contained sequence in the MPIF-1 ectodomain)) but activity that needn't be identical. For example, can come general measure MPIF-1 functional activity in conjunction with the ability of MPIF-1 part by measuring the MPIF-1 polypeptide. The ability of cell that can also be by measuring polypeptide (such as free or be expressed in the cognate ligand of cell surface) this polypeptide of abduction delivering is measured the MPIF-1 functional activity.

The fragment of full-length gene of the present invention can be used as the hybridization probe of cDNA library, to separate full-length cDNA and to separate other cDNA that has height sequence similarity or similar BA to this gene. Such probe preferably has at least 30 bases, and can comprise for example 50 or more base. Probe can be used for identifying the genomic clone of cloning and comprise the complete genome that comprises regulation and control and promoter region, extron and introne corresponding to the cDNA of total length transcript. The example of screening comprises by using the known dna sequence synthetic oligonucleotide probe to separate the code area of this gene. Use has with which member who determines this probe and library hybridizing through labeled oligonucleotide screening people cDNA, genomic DNA or mRNA library of gene complementation sequence of the present invention.

The invention still further relates to the polynucleotide passage of polynucleotides of the present invention. In the present invention, " polynucleotide passage " refers to short polynucleotides, the nucleotide sequence that it has: be the part of contained nucleotide sequence among the preservation clone, or the polypeptide of the cDNA coding among the coding preservation clone; The part of nucleotide sequence shown in SEQ ID NO:1 or 6 or its complementary strand, or the part of the polynucleotide sequence of the polypeptide of coding SEQ ID NO:2. The length of nucleotide fragments of the present invention is preferably at least approximately 15nt, more preferably at least approximately 20nt, still more preferably at least approximately 30nt even more preferably at least approximately 40nt, at least approximately 50nt, at least approximately 75nt or at least about 150nt. For example, the fragment of " length at least 20nt " refers to comprise from 20 or more continuous base of nucleotide sequence shown in contained cDNA sequence or SEQ ID NO:1 or 6 among the preservation clone. In this content, " approximately " comprises the numerical value of many or few several (5,4,3,2 or 1) nucleotides of numerical value and one or both ends of concrete narration. These nucleotide fragments are useful, include but not limited to as diagnostic probe described herein and primer. Certainly, preferred larger fragment (such as 50,150,500,600,2000 nucleotides).

，SEQ ID NO：1650-599、100-599、200-599、300-599、400-599、500-599 、600-1800、50-500、100-500、200-500、 300-500、400-500、50-400、100-400、200-400、300-400、50-300、100-300、200-300 、50-200、100-200、50-10025 、30、35、40 、45、50、 60、70、80、 90、100 。

In addition, the representative examples of polynucleotide passage of the present invention comprise contained cDNA among about 1-50 position of for example comprising SEQ ID NO:1 or 6,51-100 position, 101-150 position, 151-200 position, 201-250 position, 251-300 position, 301-350 position, 351-400 position, the 401st-450,451-500 position, 501-550 position or the 551st 's-terminal sequence or its complementary strand or the preservation clone or consisting of fragment. In this content, " approximately " comprises the scope of many or few several (5,4,3,2 or 1) nucleotides of scope and one or both ends of concrete narration. Preferably, these fragment codings have the polypeptide of BA. More preferably, these polynucleotides can be used as probe described herein or primer. The polynucleotides that the present invention also is encompassed under the rigorous hybridization conditions or can hybridizes with these nucleic acid molecules under the condition of low rigor, and by the polypeptide of these polynucleotide encodings. In the present invention, " polypeptide fragment " refers to as contained among the SEQ ID NO:2 or by the amino acid sequence of the part of the amino acid sequence of contained cDNA coding among the preservation clone. Carrier, host cell and protein expression

The invention still further relates to the carrier that comprises isolated nucleic acid molecule of the present invention, the genetically engineered host cell that comprises recombinant vector and produce the method for MPIF-1 polypeptide or its fragment by recombinant technique. The invention still further relates to and be used in the new expression vector of producing protein in the bacterial system. The example of these novel carriers is pHE4 serial carriers, particularly pHE4-5 carrier (Figure 31 and 36A-E).

The polynucleotides of code book invention protein can be connected the carrier that comprises selected marker, be used for increasing the host. As what hereinafter discuss in detail, generally speaking, in sediment, such as calcium phosphate precipitation, perhaps with the compound of electrically charged lipid in, plasmid vector is imported host cell. If carrier is virus, then can use suitable package cell line to carry out in vitro package, in the host cell of then transduceing.

The preferred carrier that comprises the cis acting control zone that is operatively connected polynucleotide of interest that uses in the practice of the present invention. The cis acting control zone comprises operator and enhancer sequence. When being used for this paper, term " operator " refers to control the nucleotide sequence of transcribing (usually being comprised of DNA) of contiguous nucleotide sequence. The operator sequence is usually derived from bacterial chromosome.

Can increase higher eucaryotic cells transcribing the nucleotide sequence of code book invention polypeptide by in carrier, inserting enhancer sequence. Enhancer is the cis-acting elements that promoter is worked to increase its transcriptional activity in specifying the host cell type, is typically about 10-300bp. The example of enhancer comprises the sub-enhancer of SV40 enhancer, cytomegalovirus early promoter that is positioned at origin of replication side in late period 100-270bp, polyomavirus enhancer and the adenovirus enhancer of origin of replication side in late period.

Can provide suitable trans-acting factor by host, complementary carrier or carrier self (after importing the host).

In some preferred embodiment of this point, it is specific expressed that carrier can provide, and it can be derivable and/or cell type-specific. The carrier that in these hosts, particularly preferably can be induced by the environmental factor (such as temperature and nourishing additive agent) of easy operating. The pHE4-5 carrier of describing in the preferred embodiment 30 is gone back in the expression of MPIF-1.

Can be used for other expression vector of the present invention and comprise the carrier of being derived by chromosome, episome and virus, such as the carrier of being derived by bacterial plasmid, bacteriophage, yeast episome, yeast chromosomal element, virus (such as baculoviral, papovavirus, vaccinia virus, adenovirus, fowlpox virus, pseudorabies virus and retrovirus), with united the carrier of deriving by it, such as clay and phasmid.

Can be by multiple flow process with suitable nucleotide sequence insertion vector. Usually, by flow process known in the art nucleotide sequence is inserted suitable restriction endonuclease site. These and other flow process should be known to those skilled in the art.

The nucleic acid Insert Fragment should be operatively connected suitable promoter, such as phageλ P_LPromoter, Escherichia coli lac, trp and tac promoter, SV40 early stage and late promoter, the promoter of retrovirus LTR and other promoter of the gene expression in known control protokaryon or eukaryotic or its virus. Those skilled in the art will know other suitable promoter. When being used for this paper, term " promoter " refers to that bottom line can be nucleotide sequence or nucleotide sequence group that the RNA polymerase effect provides binding site or initiation site. The expression construction also will comprise the site for transcription initiation, termination, and comprise the ribosome bind site for translation in transcriptional domain. The coded portion of the ripe transcript of being expressed by construction will be preferably comprises translation initiation codon in the starting point of polypeptide to be translated, comprises terminator codon (UAA, UGA or UAG) in the appropriate location of the terminal point of polypeptide to be translated. Carrier also can comprise the suitable sequence of expressing for strengthening.

When being used for this paper, phrase " is operatively connected " and refers to that nucleotide sequence links to each other with another kind of or other nucleotide sequences, thereby can change the function of sequence. For example, the protein coding sequence that is operatively connected with promoter/operator places the expression of protein coding sequence under the impact or control of these sequences. If inducing of promoter function caused transcribing of protein coding sequence mRNA, and, if the connection character (1) between these two kinds of nucleotide sequences does not cause the importing of frameshift mutation; And (2) do not hinder expression regulation sequence and instruct mRNA or protein expression, say that then two kinds of nucleotide sequences (such as protein coding sequence and the promoter region sequence that is connected to coded sequence 5 ' end) are operatively connected. Thereby if promoter can realize transcribing of nucleotide sequence, then promoter region namely is to be operatively connected nucleotide sequence.

When being used for this paper; phrase " cloning vector " refer to can be in host cell self-replicating and be characterized as plasmid or bacteriophage nucleic acid or other nucleotide sequence of or minority endonuclease enzyme recognition site; can cut these nucleotide sequences in these sites in confirmable mode and do not lose the basic biological function of carrier, thereby and can cause copying and cloning at these site montage nucleic acid. Cloning vector also can comprise the mark that is applicable to identify the cell that is cloned the carrier conversion. Mark is for example erythromycin and kalamycin resistance.

When being used for this paper, phrase " expression vector " refers to the carrier similar to cloning vector, and after being transformed into expression vector among the host, it can express wherein clone's structural gene. In expression vector, clone's structural gene (any purpose coded sequence) is placed under the control that can make some sequence that these genes express in specific host (can operate connection). For example in the pHE4-5 carrier, structural gene is operatively connected T5 bacteriophage promoter sequences and two lac operator sequences. Expression control sequenc will change, and can additionally comprise transcribe element (such as terminator sequence) and/or the translation element (such as the initial sum termination site).

As mentioned above, expression vector will preferably comprise at least a selected marker. These marks comprise dihyrofolate reductase or the neomycin resistance gene of cultivating for eukaryotic and tetracycline, kanamycins or the ampicillin resistance gene that is used for cultivating Escherichia coli and other bacterium. The representative examples of suitable host includes but not limited to bacterial cell, such as Escherichia coli, streptomycete and salmonella typhimurium cell; The fungal cell is such as yeast cells (such as saccharomyces cerevisiae or pichia pastoris (ATCC numbering 201178)); Insect cell is such as fruit bat S2 and Spodoptera Sf9 cell; Zooblast is such as CHO, COS, 293 and the Bowes melanoma cells; And plant cell. Know in this area for suitable culture medium and the condition of cultivating above-mentioned host cell.

Except expression vector the application in the present invention practice, the present invention also comprises and comprises operator that the nucleotide sequence with the coding destination protein is operatively connected and the new expression vector of promoter element. An example of this carrier is the pHE4-5 (SEQ ID NO:37) that hereinafter describes in detail with embodiment 14. The pHE4-5 carrier is preserved in U.S. typical case culture (the American Type Culture Collection of collecting center September 30 in 1997, Patent Depository, 10801 University Boulevard, Manassas, Virginia 20110-2209), the ATCC numbering 209311.

As general introduction in Figure 31 and 36, the composition of pHE4-5 carrier (SEQ ID NO:37) comprising: (1) is as nucleotide sequence (SEQ ID NO:27), (6) SD sequence, (7) lactose operon repressor GFP (lacIq) of the neomycin phosphotransferase gene of selected marker, (2) colibacillary origin of replication, (3) T5 bacteriophage promoter sequences, (4) two lac operator sequences, (5) coding MPIF-1 Δ 23. Origin of replication (oriC) is derived from pUC19 (LTI, Gaithersburg, MD). Promoter sequence and operator sequence are by synthetic preparation. The synthetic generation of nucleotide sequence is well known in the art. CLONTECH 95/96 catalogue, the 215-216 page or leaf, CLONTECH, 1020 East Meadow Circle, Palo Alto, CA 94303.

As mentioned above, the pHE4-5 carrier contains the lacIq gene. LacIq is allele (people such as E.Amann, Gene, 69:301-315,1988 of tightly regulating and control the lacI gene of lac operator; M.Stack, Gene, 51:255-267,1987). The LacIq gene code can and be blocked the aporepressor that downstream (namely 3 ') sequence is transcribed in conjunction with lac operator sequence. Yet when having lactose or some lactose analog (such as isopropyl B-D-thiogalactoside (IPTG)), lacIq gene outcome and lac operator are dissociated. Thereby, do not produce a large amount of MPIF-1 Δs 23 in the host cell comprising not inducing of pHE4-5 carrier. , after the material of adding IPTG and so on is induced these host cells, can cause the expression of MPIF-1 Δ 23 coded sequences.

The promoter of pHE4-5 carrier/operator sequence (SEQ ID NO:38) comprises T5 phage promoter and two lac operator sequences. Operator is positioned at 5 ' end of transcription initiation site and another is positioned at its 3 ' end. When these operators existed with the lacIq gene outcome, in the situation that lacks lac operon derivant such as IPTG, they closely suppressed downstream sequence. By adding lac operon derivant such as IPTG, can induce the expression that is operatively connected sequence that is positioned at lac operator downstream. The combination of lac derivant and lacIq protein causes it to break away from lac operator sequence, and the transcription initiation that is operatively connected sequence. The lac operon of gene expression is regulated and is summarized in T.Devlin, " TEXTBOOK OF BIOCHEMISTRY WITH CLINICAL CORRELATIONS " (biochemical and clinical correlation), the 4th edition, 1997, the 802-807 pages or leaves.

The pHE4 serial carrier comprises all the components except MPIF-1 Δ 23 coded sequences in the pHE4-5 carrier. The feature of pHE4 carrier comprises synthetic T5 phage promoter, lac operator and the SD sequence of optimization. In addition, these sequences are also by the separating of the best, thereby can tightly regulate and control to insert the expression of gene and produce high-caliber expression when inducing.

In being applicable to the known bacterium promoter of production protein of the present invention, comprise Escherichia coli lacI and lacZ promoter, T3 and T7 promoter, gpt promoter, λ P_RAnd P_LPromoter and trp promoter. Suitable eukaryotic promoter comprises promoter (such as the promoter of Rous sarcoma virus (RSV)) and the metallothionein promoter (such as Mouse Metallothionein-1 promoter) of CMV immediate early promoter, HSV thymidine kinase promoter, early stage and late period SV40 promoter, retrovirus LTR.

The pHE4-5 carrier also comprises the SD sequence that is positioned at AUG initiation codon 5 '. The SD sequence is the short sequence that is usually located at about 10 the nucleotides places of AUG upstream from start codon (namely 5 '). These sequences guide former nuclear ribosomal to AUG initiation codon place in essence.

Thereby, the invention still further relates to and be used in production protein expression carrier of the present invention in the bacterium. The example of this respect of the present invention is pHE4-5 carrier (SEQ ID NO:37) (ATCC numbering 20931) and variant thereof.

Other carrier that is preferred for expression protein of the present invention in bacterium comprises pQE70, pQE60 and pQE-9 (Qiagen); PBS carrier, pD10, Phagescript carrier, pBluescript carrier, pNH8A, pNH16a, pNH18A and pNH46A (Stratagene); Ptrc99a, pKK233-3, pDR540 and pRIT5 (Pharmacia). Preferred eukaryotic vector is pWLNEO, pSV2CAT, pOG44, pXT1 and pSG (Stratagene); PSVK3, pBPV, pMSG and pSVL (Pharmacia).

Other suitable carrier is apparent to those skilled in the art, comprises pBR322 (ATCC37017), pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, WI, the U.S.). These pBR322 " main chain " part and suitable promoter and structure sequence gang to be expressed. After transforming suitable host strain and host strain is cultured to suitable cell density, induce selected promoter by suitable means (such as variations in temperature or chemical induction), and cell is cultivated a period of time again.

In another embodiment, the present invention relates to comprise the host cell of construction mentioned above. Host cell can be higher eucaryotic cells (such as mammalian cell) or the eukaryotic (such as yeast cells) such as low; Perhaps host cell can be prokaryotic (such as bacterial cell). Can be by calcium phosphate transfection, by the transfection of DEAE-glucan mediation, by transfection, electroporation, transduction, infection or other method of cation lipid mediation construction is imported host cell. These methods are described in many standard laboratory guides, such as people such as Davis, and " Basic Methods In Molecular Biology " (molecular biology basic skills), 1986.

Can use well-known technology, such as infection, transduction, transfection, transposition, electroporation and conversion, the recombination to construct thing be imported host cell. Carrier can be for example bacteriophage, plasmid, virus or retroviral vector. Retroviral vector can be replication competent type or replication defect type. Under latter event, virus multiplication will only betide in the complementary host cell usually.

With carrier of the present invention host cell is carried out genetically engineered (transduction or conversion or transfection), described carrier can be for example cloning vector or expression vector. The form of carrier can be for example plasmid, virion, bacteriophage etc. Can suitably improved to activate promoter, selected to cultivate in the conventional nutrient medium of transformant or amplification MPIF-1 and gene the host cell through transforming. Condition of culture such as temperature, pH is those conditions of former host cell for selecting to express, and this should be apparent to those skilled in the art.

Can be with polynucleotides of the present invention for the production of polypeptide by recombinant technique. Thereby, for example, can in any of multiple expression vector (particularly carrier or plasmid), comprise described polynucleotides to express polypeptide. These carriers comprise chromosomal, achromosomal and synthetic nucleotide sequence, such as the derivative of SV40; Bacterial plasmid; Bacteriophage nucleic acid; Yeast plasmid; Unite the carrier of deriving by plasmid and bacteriophage nucleic acid, virus (such as vaccinia virus, adenovirus, fowlpox virus, Alphavirus and pseudorabies virus) nucleic acid. Yet as long as can copy in the host and survive, any other plasmid or carrier all can use.

As mentioned above, the carrier that comprises suitable nucleotide sequence mentioned above and suitable promoter or control sequence can be used for transforming suitable host, so that host expresses protein.

As the representative examples of suitable host, can mention: bacterial cell, such as Escherichia coli, streptomycete and salmonella typhimurium cell; The fungal cell is such as yeast; Insect cell is such as fruit bat and Sf9; Zooblast is such as CHO, COS and Bowes melanoma; Plant cell etc. In view of the teachings contained herein, the selection of suitable host cell should be known to those skilled in the art.

In particular, the present invention also comprises and comprises the above recombination to construct thing of broadly described one or more sequences. Construction comprises carrier, such as plasmid or expression vector, has wherein inserted sequence of the present invention with orientation forward or backwards. In aspect this embodiment preferred, construction also comprises regulating and controlling sequence, for example comprises the promoter that is operatively connected with described sequence. A large amount of suitable carrier and promoter are known to those skilled in the art, and can buy by commercial sources. Provide following carrier as example. Bacterium: pQE70, pQE60 and pQE-9 (Qiagen); PBS carrier, pD10, phagescript, psiX174, pBluescript SK, pbsks, pNH8A, pNH16a, pNH18A and pNH46A (Stratagene); Ptrc99a, pKK223-3, pDR540 and pRIT5 (Pharmacia). Eucaryon: pWLNEO, pSV2CAT, pOG44, pXT1 and pSG (Stratagene); PSVK3, pBPV, pMSG and pSVL (Pharmacia). Yet as long as can copy in the host and survive, any other plasmid or carrier all can use.

Can be in a usual manner with the construction in the host cell for the production of the gene outcome by the recombination sequence coding. Perhaps, also can be by the synthetic production of conventional peptide synthesizer polypeptide of the present invention.

Can be under the control of suitable promoter at the protein of mammalian cell, yeast, bacterium or other cells maturation. Use also can adopt cell free translation system to produce these protein derived from the RNA of nucleic acid construct thing of the present invention. The suitable clone and the expression vector that can be used for protokaryon and eucaryon host are described in the people such as Sambrook, " Molecular Cloning:A Laboratory Manual " (molecular cloning: laboratory manual), the 2nd edition, the cold spring port, New York, 1989 (being collected herein by reference).

Generally speaking, recombinant expression carrier will comprise origin of replication, allow the resistance marker (such as the TRP1 gene of colibacillary ampicillin resistance gene and saccharomyces cerevisiae) of transformed host cell and derived from the promoter of cance high-expression gene to instruct transcribing of downstream configurations sequence. This promoter can be derived from the operon of coding glycolytic ferment (such as glycerol 3-phosphate acid kinase (PGK)), α-factor, acid phosphatase or heat shock protein etc. Allos structure sequence and translation initiation and terminator sequence and preferably can mediate the targeting sequencing that the protein that is translated secretes to periplasmic space or the extracellular matrix and be assembled together by rights. Choose wantonly, the heterologous sequence codified comprises the fusion of the N Terminal Identification peptide of giving desired character, and described feature for example is that expressed recombinant protein stabilisation or purge process are simplified.

Expect protein and have the structural nucleic acid sequence of suitable translation initiation and termination signal to insert to be in the mode that can operate reading frame with functional promoter by encoding, can make up the useful expression vector for bacterium. Carrier can contain one or more Phenotypic Selection marks and origin of replication keeps and provides where necessary amplification in the host with what guarantee carrier. Although also can adopt other selection, the suitable prokaryotic hosts that be used for to transform comprises Escherichia coli, bacillus subtilis, salmonella typhimurium, and pseudomonas, streptomyces and staphylococcus a plurality of kinds.

General by centrifugal cell harvesting, by physics or chemical means smudge cells, and the CE that reservation produces is to be further purified.

Can be by comprising freeze thawing circulation, ultrasonic processing, Mechanical Crushing or come broken microbial cell for marking protein with any facilitated method of lysis agent that these methods are well-known to those skilled in the art.

Also can express recombinant protein with multiple mammalian cell culture system. The example of mammalian expression systems comprises Gluzman, Cell, and 23:175, the 1981 monkey kidney fibroblast COS-7 systems of describing, and can express other clone of compatibility carrier, for example C127,3T3, CHO, HeLa and bhk cell are. Mammalian expression vector will comprise origin of replication, suitable promoter and enhancer and any essential ribosome bind site, polyadenylation site, donor splicing site and acceptor site, transcription terminator and 5 ' flank non-transcribed sequence. The non-transcribed genetic elements of expectation can be provided with the nucleotide sequence derived from SV40 splice junction and polyadenylation site.

Also can be by following material recovery MPIF-1 polypeptide (preferred secreted form): by natural origin (comprise body fluid, tissue and cell, can be directly separate or cultivate) product of purifying; The product of chemical synthesis flow process; With the product that is generated by protokaryon or eucaryon host (comprising for example bacterium, yeast, higher plant, insect and mammalian cell) by recombinant technique. According to the host who adopts in the production procedure of grave group, the MPIF-1 polypeptide can be glycosylation or nonglycosylated. In addition, the MPIF-1 polypeptide also can comprise the methionine residues of initial modification, in that this is the result by the processing of host mediation in some cases. Thereby this area is well-known, in all eukaryotics by the terminal methionine of the N of translation initiation codon coding usually after the translation by any protein on efficiently excision. Although the terminal methionine of N is also excised by most protein in most of prokaryotics, this protokaryon excision processing is poor efficiency, depends on the covalently bound amino acid whose character of the terminal methionine of N.

Except containing the host cell that comprises vector construct described herein, the present invention also contain vertebrate originate from the origin of mammal particularly former generation, go down to posterity and the immortalization host cell, they can operate the genetic stocks (such as the heterologous polynucleotide sequence) that links to each other and can activate, change and/or increase endogenous MPIF-1 polynucleotides by transforming disappearance or having replaced endogenous genetic stocks (such as the MPIF-1 coded sequence) and/or comprised with MPIF-1 polynucleotides of the present invention. For example, can use technology known in the art, by homologous recombination allos control zone (such as promoter and/or enhancer) can be operated to link to each other with endogenous MPIF-1 polynucleotide sequence and (consult the U.S. Patent number 5,641,670 of for example awaring a certificate on June 24th, 1997; On September 26th, 1996 disclosed international publication number WO 96/29411; On August 4th, 1994 disclosed international publication number WO 94/12650; The people such as Koller, Proc.Natl.Acad.Sci.USA, 86:8932-8935,1989; With the people such as Zijlstra, Nature, 342:435-438,1989; Complete being collected herein by reference). The MPIF-1 polypeptide

The present invention also provides the separation MPIF-1 polypeptide with the amino acid sequence among the coded amino acid sequence of preservation cDNA or Fig. 1 (SEQ ID NO:2), perhaps comprise aforementioned polypeptides a part or consisting of peptide or polypeptide. Term " peptide " and " oligopeptides " are considered to synonym (as general approval), and when at least 2 amino acid whose chains needing in the context to indicate by the peptide bond coupling, these two terms can Alternate. Use " polypeptide " word to comprising 10 chains more than the amino acid residue herein. All oligopeptides of this paper and polypeptide formula or sequence are that amino terminal is to carboxyl terminal from left to right. The present invention also provide comprise the coded peptide sequence of polynucleotides of the present invention or consisting of protein.

The variant of the coded peptide sequence of disclosed peptide sequence among the SEQ ID NO:2 and/or preservation clone is also contained in the present invention.

" variant " refers to be different from MPIF-1 polynucleotides or polypeptide but keeps polynucleotides or the polypeptide of its fundamental property. Generally speaking, variant is closely similar on the whole to MPIF-1 polynucleotides or polypeptide, and same in many zones.

Preferably, polynucleotide passage coding of the present invention is showed the polypeptide of MPIF-1 functional activity. The polypeptide of showing MPIF-1 " functional activity " refers to that polypeptide can show one or more known functions relevant with total length (complete) MPIF-1 protein activity. These functional activities include but not limited to BA, antigenicity (in conjunction with (or with the competition of the MPIF-1 polypeptide in conjunction with) ability to anti-MPIF-1 antibody), immunogenicity (generation can in conjunction with the ability of the antibody of MPIF-1 polypeptide), and MPI-1 polypeptide of the present invention forms polymeric ability and in conjunction with the ability of acceptor or the part of MPIF-1 polypeptide.

Can measure by several different methods the functional activity of MPIF-1 polypeptide and fragment, variant, derivative and analog.

For example, a kind of measure in conjunction with or with the total length MPIF-1 polypeptide competition embodiment in conjunction with the ability of anti-MPIF-1 antibody in, the panimmunity determination method that can use this area to know includes but not limited to use competitiveness and the noncompetitive mensuration system of measuring the technology such as determination method, gel diffusion precipitation reaction, immune diffusion measurement method, original position immunoassay (such as using collaurum, enzyme or labelled with radioisotope), Western trace, precipitation reaction, agglutination assay (such as gel agglutination assay, hemagglutination determination method), complement fixation determination method, immunofluorescence assay, albumin A determination method and immunoelectrophoresis determination method such as radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), sandwich immunoassay method, immune radiating. In one embodiment, the antibody combination is to detect by the mark that detects on the primary antibodie. In another embodiment, primary antibodie is to detect by detecting two combinations anti-or reagent and primary antibodie. In another embodiment, two is anti-through mark. The several different methods for detection of the combination in the immunoassay is known in this area, and comprises within the scope of the invention.

In another embodiment, if identification of M PIF-1 acceptor or part, or in the multimerization ability of assessing polypeptide fragment of the present invention, variant or derivative, can measure combination by for example method well-known in the art, such as reduction and irreducibility gel chromatography, protein affinity chromatography and imprinting. See the people such as E.Phizicky, Microbiol.Rev., 59:94-123,1995. In another embodiment, can measure MPIF-1 related with the physiological of its Binding Capacity (signal transduction).

In addition, the determination method known of determination method as herein described (seeing embodiment) and other this area can routine be applied to measure MPIF-1 polypeptide and fragment, variant, derivative and analog and causes the ability that the MPIF-1 associated biomolecule is learned active (no matter external or body in). Other method is known to those skilled in the art, and is comprised within the scope of the invention.

MPIF-1 albumen of the present invention or its fragment can be monomer or polymer (being dimer, tripolymer, the tetramer and senior polymer). Therefore, the present invention relates to monomer and polymer, their preparation of MPIF-1 albumen of the present invention and comprise their composition (preferred pharmaceutical compositions). In a particular, polypeptide of the present invention is monomer, dimer, tripolymer or the tetramer. In other embodiments, polymer of the present invention is at least dimer, at least tripolymer or the tetramer at least.

The polymer that the present invention is contained can be with aggressiveness or different aggressiveness. When being used for this paper, term refers to only comprise the polymer (comprising MPIF fragment, variant and fusion, as described herein) of MPIF albumen of the present invention with aggressiveness. These can comprise the MPIF albumen with identical or different peptide sequence with aggressiveness. In a particular, of the present invention is the polymer that only comprises the MPIF albumen with identical peptide sequence with aggressiveness. In another embodiment, of the present invention is the polymer that comprises the MPIF albumen with different peptide sequences with aggressiveness. In a particular, polymer of the present invention is homodimer (as comprising the MPIF albumen with identical or different peptide sequence) or homotrimer (as comprising the MPIF albumen with identical or different peptide sequence). In other embodiments, homopolymer of the present invention is at least homodimer, at least homotrimer or the same at least tetramer.

When being used for this paper, the different aggressiveness of term refers to also comprise the polymer of heterologous protein (protein that namely only comprises the peptide sequence that does not correspond to MPIF coded by said gene peptide sequence) except MPIF albumen of the present invention. In a particular, polymer of the present invention is heterodimer, heterotrimer or the different tetramer. In other embodiments, homopolymer of the present invention is at least homodimer, at least homotrimer or the same at least tetramer.

Polymer of the present invention can be hydrophobicity, hydrophily, ionic and/or covalently bound result, and/or can be the indirect joint that forms by for example liposome. Thereby in one embodiment, polymer of the present invention (such as homodimer or homotrimer) forms in solution each other at protein of the present invention. In another embodiment of the invention, heteromultimeric of the present invention (such as heterotrimer or the different tetramer) is that the antibody for polypeptide of the present invention formed when (comprising the antibody for the allogeneic polypeptide sequence in the fusion of the present invention) in protein contact solution of the present invention. In another embodiment, polymer of the present invention be by and the covalent bond of MPIF albumen of the present invention and/or the covalent bond between the MPIF albumen of the present invention form. This covalent bond may relate to peptide sequence or the ATCC preserved material narrated among the SEQ ID NO:2 and number contained one or more amino acid residues in the coded polypeptide of 75676 contained cDNA clones. In one case, covalent bond is the crosslinked of (they interact in natural (being natural generation) polypeptide) between the cysteine in the peptide sequence of protein. In another case, covalent bond is the result of chemistry or reorganization operation. Perhaps, this covalent bond may relate to contained one or more amino acid residues in the allogeneic polypeptide sequence in the MPIF fusion. In one embodiment, covalent bond is (to consult such as U.S. Patent number 5,478,925) between the heterologous sequence contained in the fusion of the present invention. In a particular embodiment, covalent bond is between the heterologous sequence contained in the MPIF-Fc fusion of the present invention (as described herein).

In another embodiment, MPIF polypeptide of the present invention and epi-position thereof have been carried segment composition heterologous antigen (such as polypeptide, carbohydrate, phosphatide or nucleic acid).

In a specific embodiment, heterologous antigen is immunogene. In a more particular embodiment, heterologous antigen is gp120 albumen or its fragment of HIV. The polynucleotides of these polypeptide of encoding are also contained in the present invention.

The chemical technology that can use this area to know produces polymer of the present invention. For example, the linkers that can know with this area and linkers length optimization technology come the chemical crosslinking expectation to be contained in protein (consulting such as U.S. Patent number 5,478,925 complete being collected herein by reference) in the polymer of the present invention. In addition, one or more intermolecular cross-linkings that the technology that can use this area to know forms between cysteine residues produce polymer of the present invention, described cysteine is arranged in the peptide sequence of protein that expectation is contained in polymer and (consults such as U.S. Patent number 5,478,925, complete being collected herein by reference). In addition, can be by adding cysteine or biotin and conventionally modify polypeptide of the present invention to the C end of protein and peptide sequence or N being terminal, the method that this area is known can be applicable to produce the polymer of the protein that comprises one or more modifications like this and (consults such as U.S. Patent number 5,478,925, complete being collected herein by reference). In addition, the technology that this area is known can be applicable to produce and comprises expectation and be contained in the liposome (consulting such as U.S. Patent number 5,478,925 complete being collected herein by reference) of the protein component in the polymer of the present invention.

The genetic engineering technology that perhaps, can use this area to know produces polymer of the present invention. In one embodiment, use fusion protein technology restructuring described herein or that this area is known to produce the protein (consulting such as U.S. Patent number 5,478,925 complete being collected herein by reference) that is contained in the polymer of the present invention. In a particular, be prepared as follows the polynucleotides of code book invention homodimer: the sequence that the polynucleotide sequence of code book invention polypeptide is connected coding joint polypeptide, then further connect synthetic polynucleotides, the described polynucleotide encoding in the other direction polypeptide translation product (shortage targeting sequencing) of (terminal terminal to N by C) (is consulted such as U.S. Patent number 5,478,925, complete being collected herein by reference). In another embodiment, recombinant technique described herein or that this area is known is applied to produce recombinant polypeptide of the present invention, it comprises membrane spaning domain and can mix in the liposome by the film reconfiguration technique and (consult such as U.S. Patent number 5,478,925, complete being collected herein by reference).

In addition, can use the technology chemical synthesis protein of the present invention that this area knows (consult such as Creighton, 1983, " Proteins:Structures and Molecular Principles " (protein: structure and molecular principle), W.H.Freeman ﹠ Co., New York; With the people such as M.Hunkapiller, Nature, 310:105-111,1984). For example, can use the synthetic peptide corresponding to MPIF polypeptide fragment of the present invention of peptide synthesizer. In addition, if necessary, can be with nonclassical amino acid or chemical amino acid analogue as an alternative or add and import the MPIF peptide sequence. Nonclassical amino acid includes but not limited to D-isomers, 2,4-diamino-butanoic, α-aminoacid, 4-Aminobutanoicacid, Abu, 2-amino-butyric acid, g-Abu, e-Ahx, 6-aminocaprolc acid, Aib, 2-aminoisobutyric acid, 3-alanine, ornithine, nor-leucine, norvaline, hydroxyproline, methyl amimoacetic acid, citrulling, homotype citrulling, cystine, tert-butyl group glycine, tert-butyl group alanine, phenylglycine, Cyclohexylalanine, Beta-alanine, fluorine amino acid, design amino acid such as Beta-methyl amino acid, Ca-methylamino acid, Na-methylamino acid and the common amino acid analogue of common amino acid. In addition, amino acid can be D type (dextrorotation) or L-type (left-handed).

The induced-mutation technique that can use this area to know generates the variant that non-natural occurs, include but not limited to (be consulted such as people such as Carter by oligonucleotide mediated mutagenesis, Alanine-scanning, PCR mutagenesis, direct mutagenesis, Nucl.Acids Res., 13:4331,1986; With people such as Zoller, Nucl.Acids Res., 10:6487,1982), cassette mutagenesis (is consulted such as people such as Wells Gene, 34:315,1985), restricted selection mutagenesis (is consulted such as people such as Wells, Philos.Trans.R.Soc.London Ser A, 317:415,1986).

The present invention also is included among the translation process or the MPIF polypeptide of different modifying afterwards, such as the derivatization of glycosylation, acetylation, phosphorylation, amidatioon, known protection/blocking groups, proteolysis cutting, with being connected of antibody molecule or other cell ligand etc. Can carry out any in many chemical modifications by known technology, include but not limited to cyanogen bromide, trypsase, chymotrypsin, papain, V8 protease, NaBH₄The specificity chemical cleavage; Acetylation, formylated, oxidation, reduction; Metabolic synthesizes in the situation of tunicamycin existing; Deng.

The present invention also comprises other posttranslational modification, comprises that N-for example connects or processing, chemical part that O-connects carbohydrate chain, N end or C end adhering on amino acid backbone, N-connects or O-connects carbohydrate chain chemical modification and the terminal methionine residues of N are expressed interpolation or the disappearance that causes because of prokaryotic host cell. Also available detectable label modified polypeptide such as enzyme, fluorescein, isotope or affinity labeling, thereby can detect and isolated protein.

The present invention also provides the chemical modification derivative of MPIF, and they can provide extra advantage, such as solubility, stability and the circulation timei of improving polypeptide, or reduces immunogenicity (consulting U.S. Patent number 4,179,337). The chemical part that is used for deriving can be selected from water-soluble polymer, such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethyl cellulose, glucan, polyvinyl alcohol etc. Also can be at intramolecular random site or intramolecular precalculated position modified polypeptide, and can comprise one, two, three or the chemical part that adheres to more.

Polymer can be any molecular weight, and can be branch or unbranched. For polyethylene glycol, preferred molecular weight be between the about 100kDa of about 1kDa-(term " approximately " is illustrated in the polyethylene glycol preparation, and some molecule can be higher or low than described molecular weight) so that operate and manufacturing. Also can use other big or small molecule, this depends on the treatment curve (for example the slowly-releasing duration of expectation, the effect (if any) to BA, easy degree, antigenic degree or the shortage processed and polyethylene glycol are to other known action of therapeutic protein or analog) of expectation. For example, the mean molecule quantity of polyethylene glycol can be about 200,500,1000,1500,2000,2500,3000,3500,4000,4500,5000,5500,6000,6500,7000,7500,8000,8500,9000,9500,10000,10500,11000,11500,12000,12500,13000,13500,14000,14500,15000,15500,16000,16500,17000,17500,18000,18500,19000,19500,20000,25000,30000,35000,40000,50000,55000,60000,65000,70000,75000,80000,85000,90000,95000 or 100000kDa.

As mentioned above, polyethylene glycol can have branched structure. The polyethylene glycol of branch is described in for example U.S. Patent number 5,643,575; The people such as Morpurgo, Appl.Biochem. Biotechnol., 56:59-72,1996; The people such as Vorobjev, Nucleosides Nucleotides, 18:2745-2750,1999; With the people such as Caliceti, Bioconjug. Chem., 10:638-646,1999, be collected herein by reference.

For the consideration on the impact of the functional of protein or antigenicity domain, peg molecule (or other chemical part) should be attached to protein. Those skilled in the art can utilize multiple adherence method, for example EP 0401,384, (coupling PEG to G-CSF) is collected herein by reference, also can consult the people such as Malik, Exp.Hematol., 20:1028-1035,1992 (reported and used the trifluoro ethyl sulfonic chloride to the PEGization of GM-CSF). For example, can be by amino acid residue through reactive group (such as free amino or carboxyl) covalent bond polyethylene glycol. Reactive group be activated polyethylene glycol molecule combinative those. Amino acid residue with free amine group can comprise lysine residue and N terminal amino acid residue; Amino acid residue with free carboxy can comprise asparagicacid residue, glutaminic acid residue and C terminal amino acid residue. Mercapto groups also can be used as reactive group and is used for adhering to peg molecule. For therapeutic purposes, preferably adhere at the amino place, such as adhering at N end or lysine group place.

As mentioned above, can make polyethylene glycol be attached to protein by being connected with any of many amino acid residues. For example, can make polyethylene glycol be connected protein by forming covalent bond with lysine, histidine, aspartic acid, glutamic acid or cysteine residues. The amino acid residue (such as lysine, histidine, aspartic acid, glutamic acid, cysteine and combination thereof) that can adopt one or more reactive chemistry methods that polyethylene glycol is attached to surpass one type in particular amino acid residue (such as lysine, histidine, aspartic acid, glutamic acid or cysteine) in the protein or the protein.

People may be desirably in the terminally chemically modified protein of N especially. Use polyethylene glycol as the illustration of the present composition, can select multiple peg molecule (by molecular weight, branch etc.), peg molecule and the ratio of protein (or peptide) molecule in reactant mixture, pending pegylation reaction type, and obtain the method for the terminal pegylated protein of selected N. The method (if necessary namely, the single Pegylation of this part and other partly being separated) that obtains the terminal polyethylene glycol chemical preparation of N can be by the terminal Pegylation material of purifying N in the pegylated protein molecule colony. Can realize that to the terminal chemical modification of the selective N of protein, it has utilized the differential responses of available dissimilar primary amino radical groups (lysine and N are terminal) in specified protein is derived by the reduction alkanisation. Under suitable reaction condition, can basically realize with carboxylic polymer at the terminal selective derivatization protein of N.

As mentioned above, can realize by multiple means the Pegylation of protein of the present invention. For example, can make polyethylene glycol directly or be attached to protein by getting involved joint. Be described in the people such as Delgado, Crit.Rev. Thera.Drug Carrier Sys., 9:249-304,1992 for the non junction system that polyethylene glycol is attached to protein; The people such as Francis, Intern. J.of Hematol., 68:1-18,1998; U.S. Patent number 4,002,531; U.S. Patent number 5,349,052; WO 95/06058; With WO 98/32466, be collected herein by reference.

A kind of system that need not to get involved joint for polyethylene glycol directly is attached to the gal4 amino acid residue adopts trifluoro second sulfonylation MPEG, and it is to use trifluoro ethyl sulfonic chloride (ClSO₂CH ₂CF ₃) modify mono methoxy polyethylene glycol (MPEG) and generate. After protein and trifluoro second sulfonylation MPEG reacted, polyethylene glycol directly was attached to the amido of protein. Thereby, the present invention includes by protein of the present invention and the peg molecule with 2,2,2-HFC-143a sulfonyl and react and the protein that generates-polyethylene glycol conjugate.

Can also use many different intervention joints that polyethylene glycol is attached to protein. For example, U.S. Patent number 5,612,460 (complete being collected herein by reference) disclose the urethane joint that is used for connecting polyethylene glycol and protein. Can also be by protein and such as MPEG-succinimide succinate, with 1, MPEG, the MPEG-2 of the activation of 1 '-carbonyl dimidazoles, the reaction of the compounds such as 4,5-trichlorophenyl carbonic ester, the p-nitrophenyl carbonate of MPEG-and multiple MPEG-succinate derivative generates protein-polyethylene glycol conjugate (wherein polyethylene glycol is attached to protein by joint). Many other polyethyleneglycol derivatives and the chemical reaction method that are used for polyethylene glycol is attached to protein have been described among the WO 98/32466 (complete being collected herein by reference). The pegylated protein product that uses the listed chemical reaction method of this paper to generate belongs within the scope of the present invention.

The number (being substitution level) that is attached to the polyalkylene glycol moiety on each protein of the present invention also can change. For example, pegylated protein of the present invention can connect average 1,2,3,4,5,6,7,8,9,10,12,15,17,20 or more peg molecule. Similar, the scope of average substitution level can be such as 1-3,2-4,3-5,4-6,5-7,6-8,7-9,8-10,9-11,10-12,11-13,12-14,13-15,14-16,15-17,16-18,17-19 or 18-20 polyalkylene glycol moiety/protein molecule. Such as people such as Delgado, Crit.Rev.Thera.Drug Carrier Sys., 9:249-304 has discussed the method that is used for measuring substitution level in 1992.

" polypeptide with MPIF-1 activity " refers to the measurement according to the particular biological determination method, shows with the active similar of MPIF-1 albumen of the present invention (full-length proteins or preferred maturity albumen) but the polypeptide of activity that needn't be identical. Can by

embodiment

9,10 and Fig. 5 in listed determination method measure the MPIF-1 protein active. For example, can measure the MPIF-1 protein active with disclosed external marrow protection determination method among the embodiment 9 hereinafter.

In brief, separate the cell mass (Lin that exhausts pedigree by mouse bone marrow cells^-Cell), and having cytokine profiles and contain or do not contain in the situation of MPIF-1 be incubated. After 48 hours, one group of every kind of culture is accepted 5-FU, then continue insulation 24 hours, measure at that time the number of the low multiplication potentiality colony forming cell (Lpp-CFC) of survival by any suitable former determination method of clone that those skilled in the art will know that. Exist in the situation of MPIF-1, the LPP-CFC of large percentage (such as 〉=30-50%, such as 〉=40%) has been subject to protection and the cytotoxicity that avoids being induced by 5-FU; And lacking MPIF-1, existing in the situation of irrelevant protein, observe seldom (＜5%) to the protection of LPP-CFC. In this determination method, exist in the situation of MPIF-1, the colony forming cell of high proliferation potentiality (HPP-CFC) can additionally be protected and the cytotoxicity that avoids being induced by 5-FU, but quite different in some cases. When LPP-CFC was not protected, HPP-CFC was not protected usually.

Also can by disclosed Asia among the embodiment 16-18 cause death and deadly model, embodiment 19 in disclosed cytoprotection method measure the MPIF-1 protein active.

Thereby " polypeptide with MPIF-1 protein active " is included in the polypeptide of showing the MPIF-1 activity in the determination method mentioned above. Although level of activity needn't be identical with MPIF-1 albumen, but preferably, " polypeptide with MPIF-1 protein active " will show that comparing basically similar activity with MPIF-1 albumen (is that candidate's polypeptide will be showed with respect to reference MPIF-1 protein active and the activity of Yan Genggao, or active be not less than the about 1/20th of the latter, preferably be not less than about 1/10th).

The invention still further relates to the MPIF-1 polypeptide of the coded amino acid sequence of the have Fig. 1 derivation amino acid sequence of (SEQ ID NO:2) or preservation cDNA, and fragment, analog and derivative.

Term " fragment ", " derivative " and " analog " refer to basically keep the biological function identical with described polypeptide or active polypeptide during at the polypeptide that is used for Fig. 1 (SEQ ID NO:2) or by the polypeptide of preservation cDNA coding. Thereby analog comprises proteinogen, and it can produce activated mature polypeptide by excision proteinogen part and be activated.

Polypeptide of the present invention can be recombinant polypeptide, natural polypeptides or synthetic polypeptide, preferred recombinant polypeptide.

The polypeptide of Fig. 1 (SEQ ID NO:2) or have fragment, derivative or the analog of the polypeptide of preservation cDNA coding to be: (1) wherein one or more amino acid residues are guarded or non-conservative amino acid residue (preferred conservative amino acid residues) substitutes and also replaced amino acid residue yes or no by the type of the amino acid residue of genetic code coding, or (2) wherein one or more amino acid residues comprise substituent type, or (3) wherein mature polypeptide merged another kind of compound, type such as the compound (for example polyethylene glycol) that improves the polypeptide half life, or (4) wherein mature polypeptide merged additional amino acid, such as leading or secretion sequence or be used for the sequence of purifying mature polypeptide or the type of proteinogen sequence. In view of the teachings contained herein, think that these fragments, derivative and analog belong within those skilled in the art's the scope.

Any MPIF-1 polypeptide all can be used for generating fusion. For example, the MPIF-1 polypeptide can be used as the antigenicity label after merging the second protein. By with the combination of MPIF-1, can be used for direct-detection the second protein for the antibody of MPIF-1 polypeptide preparation. In addition, because secretary protein is according to transporting signal targeting to cell position, so the MPIF-1 polypeptide can be used as targeted molecular after merging other oroteins.

The example that can merge the domain of MPIF-1 polypeptide not only comprises the allos burst, also comprises other allos functional areas. Fusion needs not to be directly, but can be undertaken by joint sequence.

Preferably provide polypeptide of the present invention with unpack format, preferably be purified to homogeneous.

Polypeptide of the present invention comprises the polypeptide (particularly mature polypeptide) of SEQ ID NO:2, and the polypeptide that has at least 95% similitude (still more preferably at least 95% homogeneity) with the polypeptide of SEQ ID NO:2, the fragment that also comprises these polypeptide, described fragment have at least 25 amino acid of polypeptide, at least 30 amino acid, at least 35 amino acid, at least 40 amino acid, at least 45 amino acid, more preferably at least 50 amino acid, at least 55 amino acid, at least 60 amino acid, at least 65 amino acid, at least 70 amino acid, at least 75 amino acid, at least 80 amino acid, at least 85 amino acid, at least 90 amino acid, at least 95 amino acid and at least 100 amino acid.

Protein (polypeptide) fragment can be " dissociating ", or is contained in the larger polypeptide, and wherein said fragment consists of a part or a zone, most preferably as single continuum. The representative examples of polypeptide fragment of the present invention comprise for example comprise by about 1-20 position, the 21st-40,41-60 position, 61-80 position, 81-100 position, 101-120 position, 121-140 position, 141-160 position, the 161st main coding district end amino acid or consisting of fragment. In addition, the length of polypeptide fragment can be about 20,30,40,50,60,70,80,90,100,110,120,130,140 or 150 amino acid. In this content, " approximately " comprises scope or the numerical value of concrete narration, and many or few several (5,4,3,2 or 1) amino acid whose scope or the numerical value in one or both ends. The polynucleotides of these polypeptide of encoding are also contained in the present invention.

Even cause forfeiture or the modification of one or more biological functions of protein from the one or more amino acid of the N of protein end disappearance, still can keep other functional activity, for example BA, multimerization ability, in conjunction with the ability of MPIF-1 part. For example, when being removed by N end when being no more than complete or mature polypeptide residue most of, the MPIF-1 mutain of brachymemma generally can keep induces and/or in conjunction with the ability of the antibody of the complete or mature form of this polypeptide of identification. Other method that can be easy to know by conventional method as herein described and this area determines whether the specific polypeptide that lacks complete polypeptide N terminal residue keeps these immunologic competences. Lacked possible some biology or the immunologic competence of still keeping of MPIF-1 mutain of the N terminal amino acid residue of greater number. In fact, usually can cause immune response by few peptide that forms to 6 MPIF-1 amino acid residues.

Therefore, polypeptide fragment comprises secreting type MPIF-1 albumen and mature form thereof. Other preferred polypeptide fragment comprises by amino or carboxyl terminal or the two and lacks secreting type MPIF-1 albumen and the mature form thereof of a series of continuous residues. For example, can be by the amino acid of arbitrary number in 1-60 scope of aminoterminal disappearance of secreting type MPIF-1 or its mature form. Similarly, can be by the amino acid of arbitrary number in the carboxy terminal deletion 1-30 scope of secreting type MPIF-1 or its mature form. In addition, any combination of above-mentioned aminoterminal and carboxy terminal deletion also is preferred. Similarly, the encode polynucleotides of these polypeptide fragments also are preferred.

As road known in the art, the amino acid sequence by a peptide species relatively and conserved amino acid thereof substitute the sequence with the second polypeptide, can measure " similitude " between two peptide species.

Certainly, because the degeneracy of genetic code, those of ordinary skills can recognize immediately, have and will the encode polypeptide of " having the MPIF-1 protein active " of a large amount of nucleic acid molecules of nucleotide sequence at least 90%, 95%, 96%, 97% shown in the nucleotide sequence of preservation cDNA (ATCC 75676) or Fig. 1 or the 20A (SEQ ID NO:1 or 6), 98% or 99% identical sequence. In fact, the identical polypeptide because the degeneracy variant of these nucleotide sequences is all encoded, so those skilled in the art even need not to carry out above-mentioned comparative test and namely know this point. Those of skill in the art also will appreciate that for these nucleic acid molecules that are not the degeneracy variant, a great deal of is also encoded and had the polypeptide of MPIF-1 protein active. This is because those of skill in the art can grasp fully amino acid replacement (being substituted by another aliphatic amino acid such as an aliphatic amino acid) unlikely or can not the appreciable impact protein function.

In practical operation, use known computer program, can conventional determine that the coded amino acid sequence of the amino acid sequence of any specific polypeptide and for example SEQ ID NO:2 or the cDNA among the preservation clone is whether at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% same. Be used for determining the method for optimizing of best overall match between search sequence (sequence of the present invention) and research sequence, also claim the Compositive sequence contrast, can use based on people such as Brutlag Comp.Appl.Biosci., 6:237-245, the FASTDB computer program of 1990 algorithm is determined. In sequence alignment, inquiry all is nucleotide sequence or amino acid sequence with the research sequence. The preferred parameter that uses in the comparison of FASTDB amino acid is: matrix=PAM 0, k-tuple=2, mispairing point penalty=1, connect point penalty=20, randomized grouping length=0, block minute=1, window size=sequence length, breach point penalty=5, breach size point penalty=0.05, window size=500 or indication length amino acid sequence (selecting the shorter one).

If the research sequence is held because of N or C end disappearance (but not inner disappearance) than the search sequence weak point, then must be carried out manual correction to the result. This is because the FASTDB program does not consider to study N end and the brachymemma of C end of sequence when calculating whole homogeneity percentage. The research sequence of N end or the brachymemma of C end for relative search sequence, the search sequence residue number that does not mate/compare that is positioned at research sequence N end and C end by calculating is recently proofreaied and correct homogeneity percentage as the percentage of the total residue number of search sequence. Whether residue mates/compare is to be determined by the result of FASTDB sequence alignment. Then from the homogeneity percentage that uses designated parameter to calculate by above-mentioned FASTDB program, deduct this percentage, thereby obtain final homogeneity percentage score. This final homogeneity percentage score is exactly employed value among the present invention. For manual setting homogeneity percentage score, only consider the residue that does not mate/compare with search sequence, is in research sequence N end or C end. That is, only consider to be positioned in the search sequence residue position beyond the N end of research sequence or the C end residue.

For example, the search sequence of the research sequence of 90 amino acid residues of comparison and 100 residues is to determine homogeneity percentage. Disappearance betides research sequence N end, so the FASTDB comparison shows that N holds front 10 residues not mate/compare. These 10 unpaired residues account for 10% (N end and C end do not mate residue number/search sequence residue sum) of sequence, therefore deduct 10% from the homogeneity percentage that the FASTDB program calculates. If remaining 90 residues mate fully, then final homogeneity percentage is 90%. In another example, the research sequence of 90 residues and the search sequence of 100 residues compare. Lack in inside specifically, so that the residue that the N end of research sequence and C end do not mate with search sequence/compare. In this case, the homogeneity percentage that calculates by FASTDB is not carried out manual correction. Moreover only comparison shows that the residue that the N end that is positioned at the research sequence and C do not mate with search sequence/compare outside holding carries out manual correction to FASTDB. For the purposes of the present invention, not carrying out the manual of other proofreaies and correct.

Preferably, program mentioned above is used for comparing polypeptide of the present invention (canonical sequence) and the second sequence, and manual calculation homogeneity percentage. Homogeneity percentage is: the number of same amino acid between two kinds of sequences, the amino acid residue sum divided by canonical sequence multiply by 100 %. For example, for the 1-100 amino acids of measuring SEQ ID NO:2 and the homogeneity percentage of the second sequence, the amino acid whose number of counting mispairing (being point mutation: insert, lack and substitute), and by deducting this number in 100 (amino acid numbers of canonical sequence) to obtain the number of same amino acid, the number that obtains divided by 100, then be multiply by 100%. If SEQ ID NO:2 the 1st, 5,21-23,41 and the 96-100 position have mispairing (be point mutation: the insertion of single amino acids, disappearance and substitute), then homogeneity percentage will be 90% (1+1+3+1+4=10,100-10=90,90/100=0.9,0.9 * 100%=90%).

For example, guidance about the amino acid replacement that how to generate the phenotype silence is provided in the people such as J.U.Bowie, " Deciphering the Message in Protein Sequences:Tolerance to Amino Acid Substitutions " (deciphering the information in the protein sequence: to the tolerance of amino acid replacement), Science, 247:1306-1310,1990, wherein the author points out to study amino acid sequence has two kinds of main policies to the tolerance that changes.

The first strategy utilizes in the evolutionary process natural selection to the tolerance of amino acid replacement. By comparing the amino acid sequence in the different plant species, can identify conserved amino acid. These conserved amino acids are likely important for the function of protein. On the contrary, to indicate these positions be not most important for the function of protein to the amino acid position that substitutes of natural selection tolerance. Thereby, can modify the position of tolerance amino acid replacement and the BA of Protein requirement still.

The genetic engineering of the second strategy use changes to identify to the vital zone of protein function at the ad-hoc location importing amino acid of clone gene. For example, can use direct mutagenesis or alanine scanning mutagenesis (each the residue place in molecule imports single alanine mutation) (Cunningham and Wells, Science, 244:1081-1085,1989). Then can be active to the mutant molecules test organisms that produces.

State that as the author these two kinds of strategies have disclosed protein amino acid replacement is had surprising tolerance. The author has pointed out also which amino acid of some position in protein changes and has been likely permission. For example, most of hidden (in the tertiary structures of protein) amino acid residue needs non-polar sidechain, and the feature of surface side chains is generally seldom guarded. In addition, the conserved amino acid of tolerance substitutes and comprises substituting of aliphatic or hydrophobic amino acid Ala, Val, Leu and Ile; Hydroxyl residue Ser and Thr substitute; Acidic residues Asp and Glu substitute; Amide residues Asn and Gln substitute; Alkaline residue Lys, Arg and His substitute; Aromatic residue Phe, Tyr and Trp substitute; Small-sized amino acid Ala, Ser, Thr, Met and Gly substitute.

Polypeptide fragment of the present invention or part can be synthetic for generating corresponding full-length polypeptide by peptide; Therefore, fragment can be used as intermediate for generating full-length polypeptide. Polynucleotide passage of the present invention or part can be used for synthetic total length polynucleotides of the present invention.

For the protein with translation is secreted in endoplasmic, periplasmic space or the born of the same parents' external environment, suitable secretion signal can be mixed the polypeptide of expression. Described signal can be endogenous for this polypeptide, also can be external source.

Form that can be modified (such as fusion) is expressed polypeptide, and polypeptide not only can comprise secretion signal, also can comprise other allos functional areas. For example, can add extra amino acid region (particularly charged amino acid) to improve polypeptide in host cell, in the purge process or subsequently processing and the stability in the storage process and persistence in that the N of polypeptide is terminal. Also can in polypeptide, add peptide moiety so that purifying. Before the final preparation of polypeptide, can excise these zones. In polypeptide, add peptide moiety with cause secretion or outside secrete, improve stability and be convenient to purifying etc., this is to know and the technology of routine in this area. Preferred fusion comprises the allos district from immunoglobulin (Ig), and this zone can be used for solubilized protein matter. For example, EP-A 464,533 (Canadian application 2045869 of the same clan) discloses the fusion that comprises the various parts of immunoglobulin molecules constant region and another kind of human protein or its part. Under many circumstances, the Fc part in the fusion is very favourable for treatment and diagnostic uses, thereby causes for example improvement of pharmacokinetic properties (EP-A 0232,262). On the other hand, for some application, need to after with described advantageous manner expression, detection and purified fusion protein, excise the Fc part. When Fc partly becomes the obstacle for the treatment of and diagnostic uses, when fusion is used as the antigen of immunity, be exactly like this for example. For example in drug development, will identify such as the human proteins such as hIL-5 acceptor and Fc partial fusion the antagonist of hIL-5 to carry out the high flux screening determination method. Consult the people such as D.Bennett, Journal of Molecular Recognition, 8:52-58,1995; With the people such as K.Johanson, The Journal of Biological Chemistry, 270 (16): 9459-9471,1995.

Can by well-known method by reclaiming and purifying MPIF-1 albumen in the recombinant cell culture thing, comprise ammonium sulfate or ethanol precipitation, acid extractants, anion or cation-exchange chromatography, cellulose phosphate chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and agglutinin chromatography. Most preferably, adopt high performance liquid chroma-tography (HPLC) to carry out purifying. Polypeptide of the present invention comprises the product of natural purifying, the product of chemical synthesis flow process and the product that is generated by protokaryon or eucaryon host (comprising for example bacterium, yeast, higher plant, insect and mammalian cell) by recombinant technique. According to the host who adopts in the recombinant production flow process, polypeptide of the present invention can be glycosylation or nonglycosylated. In addition, polypeptide of the present invention also can comprise initial modification methionine residues, in that this is the result by the processing of host mediation in some cases. The MPIF-1 polypeptide variants

This area will recognize that some amino acid sequences of MPIF-1 polypeptide can change, and the structure of not appreciable impact protein or function. If comprise these differences in the sequence, should remember to exist in the protein to determine active most important zone. Generally speaking, might substitute the residue that forms tertiary structure, as long as use the residue of exercising similar functions just passable. In other cases, if change the non-important area that betides protein, then the type of residue may be fully unessential.

Thereby the present invention also comprises the MPIF-1 polypeptide variation that shows basic MPIF-1 polypeptide active or comprise MPIF-1 albumen zone (all protein fragments as discussed below). These mutant comprise that disappearance, insertion, inversion, repetition and type substitute (for example principle is to substitute another kind of hydrophily residue with a kind of hydrophily residue, but not substitutes the strong-hydrophobicity residue with the strongly hydrophilic residue). Little variation or these " neutrality " amino acid replacements are usually very little on the impact of activity.

What be commonly referred to be that conserved amino acid substitutes is exchange between aliphatic amino acid Ala, Val, Leu and the Ile; Exchange between hydroxyl residue Ser and the Thr; Exchange between acidic residues Asp and the Glu; Exchange between amide residues Asn and the Gln; Exchange between alkaline residue Lys and the Arg; Exchange between aromatic residue Phe and the Tyr.

In addition, interested especially is with another kind of charged amino acid or with the neutral charged amino acid of amino acid replacement. This may cause the characteristic of protein to be improved, and reduces such as assembling. Prevent from assembling is high expectations. The gathering of protein not only can cause activity decreased, and bring problem can for the preparation of pharmaceutics preparation, because they may have immunogenicity (people such as Pinckard, Clin.Exp.Immunol., 2:331-340,1967; The people such as Robbins, Diabetes, 36:838-845,1987; The people such as Cleland, Crit.Rev. Therapeutic Drug Carrier Systems, 10:307-377,1993).

Amino acid whose substitute can also change with the combination of cell surface receptor selective. The people such as Ostade, Nature, 361:266-268,1993 described some TNF α sudden change cause TNF α only optionally with the combination of one of two kinds of known TNF acceptors.

Be described in detail as mentioned, can be people such as J.U.Bowie, " Deciphering the Message in Protein Sequences:Tolerance to Amino Acid Substitutions " (deciphering the information in the protein sequence: to the tolerance of amino acid replacement), Science, which amino acid 247:1306-1310 find in 1990 (the seeing Table 1) about to change the guidance that is likely phenotype reticent (be unlikely function is had significant adverse effect).

As mentioned above, advantageous variant is inappreciable, and is alternative such as the folding or active conserved amino acid of not appreciable impact protein.

Table 1: conserved amino acid substitutes
Table 1: conserved amino acid substitutes		Aromatic series	Phenylalanine tryptophan tyrosine
Hydrophobic	Leucine isoleucine valine	Aromatic series	Phenylalanine tryptophan tyrosine
Hydrophobic	Leucine isoleucine valine	Polarity	The glutamine asparagine
Alkalescence	The Arginine Lysine histidine	Polarity	The glutamine asparagine
Alkalescence	The Arginine Lysine histidine	Acid	Aspartic acid glutamic acid
Little	Alanine serine threonine methionine glycine	Acid	Aspartic acid glutamic acid

Certainly, those of skill in the art can be according to the number that comprises above and hereinafter described those several factors determines amino acid replacement. Generally speaking, according to purpose, the alternative number of any appointment MPIF-1 polypeptide or its mutant is no more than 50,40,30,20,10,5 or 3. Concrete MPIF-1 amino acid replacement has hereinafter been described.

Another aspect of the present invention also comprises amino acid replacement. Interested especially is that the conserved amino acid of not appreciable impact protein folding substitutes. Above table 1 has been listed the alternative example of conserved amino acid that those skilled in the art will know that.

In addition, the interested especially charged amino acid of also useful another kind or with the neutral charged amino acid of amino acid replacement. This may cause the characteristic of protein to be improved, and reduces such as assembling. Prevent from assembling is high expectations. The gathering of protein not only can cause activity decreased, and bring problem can for the preparation of pharmaceutics preparation, because they may have immunogenicity (people such as Pinckard, Clin.Exp.Immunol., 2:331-340,1967; The people such as Robbins, Diabetes, 36:838-845,1987; The people such as Cleland, Crit.Rev. Therapeutic Drug Carrier Systems, 10:307-377,1993).

MPIF-1 albumen can comprise one or several amino acid replacement, disappearance or add, itself or from natural sudden change or from manual operation. Hereinafter provide some in the amino acid sequence shown in Fig. 1 (SEQ ID NO:2) preferably to suddenly change. Wherein name is: for example Ala (21) Met refers to substitute the Ala that is positioned at the 21st of Fig. 1 (SEQ ID NO:2) with Met.

Ala(21)Met Asp(53)Ser

Thr(24)Ala Asp(53)Thr

Lys(25)Asn Asp(53)Met

Asp(26)Ala Ser(51)Gly

Asp(45)Ala Ser(34)Gly

Asp(45)Gly Glu(30)Gln

Asp(45)Ser Glu(28)Gln

Asp(45)Thr Pro(60)Thr

Asp(45)Met Ser(70)Ala

Asp(53)Ala

Asp(53)Gly

For example, by substituting specific amino acids with conserved amino acid, the fixed point variation can generated in MPIF-1 on the amino acid levels. Preferred conservative sudden change comprises: for example, preferred mutual benefit and replacement comprises: substitute M1 with A, G, I, L, S, T or V; Substitute K2 with H or R; Substitute V3 with A, G, I, L, S, T or M; Substitute S4 with A, G, I, L, T, M or V; Substitute V5 with A, G, I, L, S, T or M; Substitute A6 with G, I, L, S, T, M or V; Substitute A7 with G, I, L, S, T, M or V; Substitute L8 with A, G, I, S, T, M or V; Substitute S9 with A, G, I, L, T, M or V; Substitute L11 with A, G, I, S, T, M or V; Substitute M12 with A, G, I, L, S, T or V; Substitute L13 with A, G, I, S, T, M or V; Substitute V14 with A, G, I, L, S, T or M; Substitute T15 with A, G, I, L, S, M or V; Substitute A16 with G, I, L, S, T, M or V; Substitute L17 with A, G, I, S, T, M or V; Substitute G18 with A, I, L, S, T, M or V; Substitute S19 with A, G, I, L, T, M or V; Substitute Q20 with N; Substitute A21 with G, I, L, S, T, M or V; Substitute R22 with H or K; Substitute V23 with A, G, I, L, S, T or M; Substitute T24 with A, G, I, L, S, M, V; Substitute K25 with H or R; Substitute D26 with E; Substitute A27 with G, I, L, S, T, M or V; Substitute E28 with D; Substitute T29 with A, G, I, L, S, M or V; Substitute E30 with D; Substitute F31 with W or Y; Substitute M32 with A, G, I, L, S, T or V; Substitute M33 with A, G, I, L, S, T or V; Substitute S34 with A, G, I, L, T, M or V; Substitute K35 with H or R; Substitute L36 with A, G, I, S, T, M or V; Substitute L38 with A, G, I, S, T, M or V; Substitute E39 with D; Substitute N40 with Q; Substitute V42 with A, G, I, L, S, T or M; Substitute L43 with A, G, I, S, T, M or V; Substitute L44 with A, G, I, S, T, M or V; Substitute D45 with E; Substitute R46 with H or K; Substitute F47 with W or Y; Substitute H48 with K or R; Substitute A49 with G, I, L, S, T, M or V; Substitute T50 with A, G, I, L, S, M or V; Substitute S51 with A, G, I, L, T, M or V; Substitute A52 with G, I, L, S, T, M or V; Substitute D53 with E; Substitute I56 with A, G, L, S, T, M or V; Substitute S57 with A, G, I, L, T, M or V; Substitute Y58 with F or W; Substitute T59 with A, G, I, L, S, M or V; Substitute R61 with H or K; Substitute S62 with A, G, I, L, T, M or V; Substitute I63 with A, G, L, S, T, M or V; Substitute S66 with A, G, I, L, T, M or V; Substitute L67 with A, G, I, S, T, M or V; With A, G, I, S, T, M, V or alternative L68; Substitute E69 with D; Substitute S70 with A, G, I, L, T, M or V; Substitute Y71 with F or W; Substitute F72 with W or Y; Substitute E73 with D; Substitute T74 with A, G, I, L, S, M or V; Substitute N75 with Q; Substitute S76 with A, G, I, L, T, M or V; Substitute E77 with D; Substitute S79 with A, G, I, L, T, M or V; Substitute K80 with H or R; Substitute G82 with A, I, L, S, T, M or V; Substitute V83 with A, G, I, L, S, T or M; Substitute I84 with A, G, L, S, T, M or V; Substitute F85 with W or Y; Substitute L86 with A, G, I, S, T, M or V; Substitute T87 with A, G, I, L, S, M or V; Substitute K88 with H or R; Substitute K89 with H or R; Substitute G90 with A, I, L, S, T, M or V; Substitute R91 with H or K; Substitute R92 with H or K; Substitute F93 with W or Y; Substitute A95 with G, I, L, S, T, M or V; Substitute N96 with Q; Substitute S98 with A, G, I, L, T, M or V; Substitute D99 with E; Substitute K100 with H or R; Substitute Q101 with N; Substitute V102 with A, G, I, L, S, T or M; Substitute Q103 with N; Substitute V104 with A, G, I, L, S, T or M; Substitute M106 with A, G, I, L, S, T or V; Substitute R107 with H or K; Substitute M108 with A, G, I, L, S, T or V; Substitute L109 with A, G, I, S, T, M or V; Substitute K110 with H or R; Substitute L111 with A, G, I, S, T, M or V; Substitute D112 with E; Substitute T113 with A, G, I, L, S, M or V; Substitute R114 with H or K; Substitute I115 with A, G, L, S, T, M or V; Substitute K116 with H or R; Substitute T117 with A, G, I, L, S, M or V; Substitute R118 with H or K; Substitute K119 with H or R; Substitute N120 with Q.

Can be to this specification of the conventional screening of construction is described and this area is known activity or the function that produces. Preferably, for the construction that produces, a kind of MPIF-1 activity or function raise and/or reduce, and all the other MPIF-1 activity or function are kept. More preferably, for the construction that produces, surpass the rising of a kind of MPIF-1 activity or function and/or reduction, and all the other MPIF-1 activity or function are kept.

Except conserved amino acid substitutes, the MPIF-1 variant comprises substituting of (1) one or more non-conservative amino acid residues, wherein replaced amino acid residue can yes or no by the amino acid residue of genetic code coding, or (2) are one or more comprises substituting of substituent amino acid residue, or (3) mature polypeptide has merged another kind of compound, such as the compound (for example polyethylene glycol) that improves polypeptide stability and/or solubility, or (4) polypeptide merged additional amino acid, such as IgG Fc corresponding circle of sensation peptide or leading or secretion sequence or be convenient to the sequence of purifying. In view of the teachings contained herein, think that these polypeptide variants belong within those skilled in the art's the scope.

For example, but comprise with the be improved protein of (such as assemble reducing) of the charged amino acid whose MPIF-1 polypeptide variants formation characteristic of other electrically charged or neutral amino acid replacement. Because the immunogenicity of aggregation, the gathering of protein had both reduced activity, had increased again removing (people such as Pinckard, Clin.Exp.Immunol., 2:331-340,1967; The people such as Robbins, Diabetes, 36:838-845,1987; The people such as Cleland, Crit.Rev. Therapeutic Drug Carrier Systems, 10:307-377,1993).

，MPIF-1：，：D、E、H、K、R、N、Q、F、W、Y、P、CM1；D、E、 A、G、I、L、S、T、M、V、N、Q、F、W、Y、P、CK2；D、E、 H、K、R、N、Q、F、W、Y、P、CV3；D、E、H、K、R、N、Q、 F、W、Y、P、CS4；D、E、H、K、R、N、Q、F、W、Y、P、C V5；D、E、H、K、R、N、Q、F、W、Y、P、CA6；D、E、 H、K、R、N、Q、F、W、Y、P、CA7；D、E、H、K、R、N、Q、 F、W、Y、P、CL8；D、E、H、K、R、N、Q、F、W、Y、P、C S9；D、E、H、K、R、A、G、I、L、S、T、M、V、N、Q、F、W、 Y、PC10；D、E、H、K、R、N、Q、F、W、Y、P、CL11； D、E、H、K、R、N、Q、F、W、Y、P、CM12；D、E、H、K、 R、N、Q、F、W、Y、P、CL13；D、E、H、K、R、N、Q、F、W、 Y、P、CV14；D、E、H、K、R、N、Q、F、W、Y、P、C T15；D、E、H、K、R、N、Q、F、W、Y、P、CA16；D、E、H、 K、R、N、Q、F、W、Y、P、CL17；D、E、H、K、R、N、Q、F、 W、Y、P、CG18；D、E、H、K、R、N、Q、F、W、Y、P、C S19；D、E、H、K、R、A、G、I、L、S、T、M、V、F、W、Y、P、 CQ20；D、E、H、K、R、N、Q、F、W、Y、P、CA21； D、E、A、G、I、L、S、T、M、V、N、Q、F、W、Y、P、CR22； D、E、H、K、R、N、Q、F、W、Y、P、CV23；D、E、H、K、 R、N、Q、F、W、Y、P、CT24；D、E、A、G、I、L、S、T、M、 V、N、Q、F、W、Y、P、CK25；H、K、R、A、G、I、L、S、T、 M、V、N、Q、F、W、Y、P、CD26；D、E、H、K、R、N、Q、F、 W、Y、P、CA27；H、K、R、A、G、I、L、S、T、M、V、N、Q、 F、W、Y、P、CE28；D、E、H、K、R、N、Q、F、W、Y、P、 CT29；H、K、R、A、G、I、L、S、T、M、V、N、Q、F、W、Y、P、 CE30；D、E、H、K、R、A、G、I、L、S、T、M、V、N、Q、P、 CF31；D、E、H、K、R、N、Q、F、W、Y、P、CM32； D、E、H、K、R、N、Q、F、W、Y、P、CM33；D、E、H、K、R、 N、Q、F、W、Y、P、CS34；D、E、A、G、I、L、S、T、M、V、 N、Q、F、W、Y、P、CK35；D、E、H、K、R、N、Q、F、W、Y、 P、CL36；D、E、H、K、R、A、G、I、L、S、T、M、V、N、Q、 F、W、Y、CP37；D、E、H、K、R、N、Q、F、W、Y、P、C L38；H、K、R、A、G、I、L、S、T、M、V、N、Q、F、W、Y、P、 CE39；D、E、H、K、R、A、G、I、L、S、T、M、V、F、W、Y、 P、CN40；D、E、H、K、R、A、G、I、L、S、T、M、V、N、Q、 F、W、Y、CP41；D、E、H、K、R、N、Q、F、W、Y、P、C V42；D、E、H、K、R、N、Q、F、W、Y、P、CL43；D、E、 H、K、R、N、Q、F、W、Y、P、CL44；H、K、R、A、G、I、L、 S、T、M、V、N、Q、F、W、Y、P、CD45；D、E、A、G、I、L、 S、T、M、V、N、Q、F、W、Y、P、CR46；D、E、H、K、R、A、 G、I、L、S、T、M、V、N、Q、P、CF47；D、E、A、G、I、L、 S、T、M、V、N、Q、F、W、Y、P、CH48；D、E、H、K、R、N、 Q、F、W、Y、P、CA49；D、E、H、K、R、N、Q、F、W、Y、P、 CT50；D、E、H、K、R、N、Q、F、W、Y、P、CS51； D、E、H、K、R、N、Q、F、W、Y、P、CA52；H、K、R、A、G、 I、L、S、T、M、V、N、Q、F、W、Y、P、CD53；D、E、H、K、 R、A、G、I、L、S、T、M、V、N、Q、F、W、Y、PC54；D、E、 H、K、R、A、G、I、L、S、T、M、V、N、Q、F、W、Y、PC55； D、E、H、K、R、N、Q、F、W、Y、P、CI56；D、E、H、K、 R、N、Q、F、W、Y、P、CS57；D、E、H、K、R、N、Q、A、G、 I、L、S、T、M、V、P、CY58；D、E、H、K、R、N、Q、F、W、 Y、P、CT59；D、E、H、K、R、A、G、I、L、S、T、M、V、N、 Q、F、W、Y、CP60；D、E、A、G、I、L、S、T、M、V、N、Q、 F、W、Y、P、CR61；D、E、H、K、R、N、Q、F、W、Y、P、 CS62；D、E、H、K、R、N、Q、F、W、Y、P、CI63；D、 E、H、K、R、A、G、I、L、S、T、M、V、N、Q、F、W、Y、CP64； D、E、H、K、R、A、G、I、L、S、T、M、V、N、Q、F、W、Y、P C65；D、E、H、K、R、N、Q、F、W、Y、P、CS66；D、E、 H、K、R、N、Q、F、W、Y、P、CL67；D、E、H、K、R、N、Q、 F、W、Y、P、CL68；H、K、R、A、G、I、L、S、T、M、V、N、 Q、F、W、Y、P、CE69；D、E、H、K、R、N、Q、F、W、Y、P、 CS70；D、E、H、K、R、N、Q、A、G、I、L、S、T、M、V、P、 CY71；D、E、H、K、R、A、G、I、L、S、T、M、V、N、Q、P、 CF72；H、K、R、A、G、I、L、S、T、M、V、N、Q、F、W、Y、 P、CE73；D、E、H、K、R、N、Q、F、W、Y、P、CT74； D、E、H、K、R、A、G、I、L、S、T、M、V、F、W、Y、P、C N75；D、E、H、K、R、N、Q、F、W、Y、P、CS76；H、K、R、 A、G、I、L、S、T、M、V、N、Q、F、W、Y、P、CE77；D、E、 H、K、R、A、G、I、L、S、T、M、V、N、Q、F、W、Y、PC78； D、E、H、K、R、N、Q、F、W、Y、P、CS79；D、E、A、G、 I、L、S、T、M、V、N、Q、F、W、Y、P、CK80；D、E、H、K、 R、A、G、I、L、S、T、M、V、N、Q、F、W、Y、CP81；D、E、 H、K、R、N、Q、F、W、Y、P、CG82；D、E、H、K、R、N、Q、 F、W、Y、P、CV83；D、E、H、K、R、N、Q、F、W、Y、P、 CI84；D、E、H、K、R、A、G、I、L、S、T、M、V、N、Q、P、 CF85；D、E、H、K、R、N、Q、F、W、Y、P、CL86； D、E、H、K、R、N、Q、F、W、Y、P、CT87；D、E、A、G、I、 L、S、T、M、V、N、Q、F、W、Y、P、CK88；D、E、A、G、I、 L、S、T、M、V、N、Q、F、W、Y、P、CK89；D、E、H、K、R、 N、Q、F、W、Y、P、CG90；D、E、A、G、I、L、S、T、M、V、 N、Q、F、W、Y、P、CR91；D、E、A、G、I、L、S、T、M、V、 N、Q、F、W、Y、P、CR92；D、E、H、K、R、A、G、I、L、S、 T、M、V、N、Q、P、CF93；D、E、H、K、R、A、G、I、L、S、 T、M、V、N、Q、F、W、Y、PC94；D、E、H、K、R、N、Q、F、 W、Y、P、CA95；D、E、H、K、R、A、G、I、L、S、T、M、V、 F、W、Y、P、CN96；D、E、H、K、R、A、G、I、L、S、T、M、 V、N、Q、F、W、Y、CP97；D、E、H、K、R、N、Q、F、W、Y、 P、CS98；H、K、R、A、G、I、L、S、T、M、V、N、Q、F、W、 Y、P、CD99；D、E、A、G、I、L、S、T、M、V、N、Q、F、W、 Y、P、CK100；D、E、H、K、R、A、G、I、L、S、T、M、V、F、 W、Y、P、CQ101；D、E、H、K、R、N、Q、F、W、Y、P、C V102；D、E、H、K、R、A、G、I、L、S、T、M、V、F、W、Y、P、 CQ103；D、E、H、K、R、N、Q、F、W、Y、P、CV104； D、E、H、K、R、A、G、I、L、S、T、M、V、N、Q、F、W、Y、P C105；D、E、H、K、R、N、Q、F、W、Y、P、CM106；D、 E、A、G、I、L、S、T、M、V、N、Q、F、W、Y、P、CR107；D、 E、H、K、R、N、Q、F、W、Y、P、CM108；D、E、H、K、R、N、 Q、F、W、Y、P、CL109；D、E、A、G、I、L、S、T、M、V、N、 Q、F、W、Y、P、CK110；D、E、H、K、R、N、Q、F、W、Y、P、 CL111；H、K、R、A、G、I、L、S、T、M、V、N、Q、F、W、Y、 P、CD112；D、E、H、K、R、N、Q、F、W、Y、P、CT113； D、E、A、G、I、L、S、T、M、V、N、Q、F、W、Y、P、CR114； D、E、H、K、R、N、Q、F、W、Y、P、CI115；D、E、A、G、 I、L、S、T、M、V、N、Q、F、W、Y、P、CK116；D、E、H、K、 R、N、Q、F、W、Y、P、CT117；D、E、A、G、I、L、S、T、M、 V、N、Q、F、W、Y、P、CR118；D、E、A、G、I、L、S、T、M、 V、N、Q、F、W、Y、P、CK119；D、E、H、K、R、A、G、I、L、 N120。

In addition, can be as mentioned above substitute and surpass an amino acid (such as 2,3,4,5,6,7,8,9 and 10) with substituting amino acid (conservative or nonconservative). Alternative amino acid residue can be present in total length, maturation or the proteinogen form of MPIF-1 albumen, and has hereinafter N end and the C end deletion mutant of listed general formula m-n.

Another embodiment of the invention relates to the polypeptide of the amino acid sequence that comprises the MPIF-1 polypeptide, the amino acid sequence that described MPIF-1 polypeptide has comprises at least one amino acid replacement, but be no more than 50 amino acid replacements, even more preferably no more than 40 amino acid replacements, even more preferably no more than 30 amino acid replacements, even more preferably no more than 20 amino acid replacements. Certainly, with the order that preference increases, the height preferred polypeptide has such amino acid sequence, and it comprises the amino acid sequence of MPIF-1 polypeptide, but wherein comprises at least one, but is no more than 10,9,8,7,6,5,4,3,2 or 1 amino acid replacements. In specific embodiments, add in the amino acid sequence of Fig. 1 or its fragment (mature form as described herein and/or other fragment), substitute and/or the number of disappearance is 1-5,5-10,5-25,5-50,10-50 or 50-150, preferred conserved amino acid substitutes.

As mentioned above, the MPIF-1 polypeptide can comprise the coded amino acid sequence of the amino acid sequence of SEQ ID NO:2 or 7 or preservation cDNA clone or consisting of, just wherein comprise one or more amino acid replacements, disappearance or interpolation. Similarly, as described above, the MPIF-1 polypeptide can with the coded polypeptide at least 80% of the polypeptide of SEQ ID NO:2 or 7 or preservation cDNA clone, 85 %, 90%, 92%, 95%, 96%, 97%, 98% or 99% same. In one embodiment, these polypeptide variants also have the structure identical or substantially the same with MPIF-1 or its at least one zone. This structure can be the solution structure (seeing embodiment 37) of for example measuring by NMR. The polynucleotides of these polypeptide of encoding are also contained in the present invention.

For example, the MPIF-1 polypeptide can comprise one or several amino acid and change (substitute, lack or add), but does not change at specific residue or position. May get rid of substitute, disappearance or add beyond the position comprise conserved residues, such as four Cys residues of the Cys54 that is arranged in SEQ ID NO:2 (Cys11 of embodiment 37), Cys55 (Cys12 of embodiment 37), Cys78 (Cys35 of embodiment 37), Cys94 (Cys51 of embodiment 37). Conserved residues also comprises the Cys65 (Cys22 of embodiment 37) that is arranged in SEQID NO:2 and two Cys residues of Cys105 (Cys62 of embodiment 37). Any of these positions or its combination may be not replaced, disappearance or add. Preferably, the MPIF-1 variant comprises the substituting of any place of not being positioned at these six Cys residues of MPIF-1, disappearance or adds.

May comprise the conservative residue that substitutes by not reformed other residue, such as Ile56 (Ile13 among the embodiment 37), Tyr58 (Tyr15 among the embodiment 37), Arg61 (Arg18 among the embodiment 37) and the Ile63 (Ile20 among the embodiment 37) of SEQ ID NO:2. In these positions any or its combination may be not replaced, disappearance or add. Preferably, the MPIF-1 variant comprises, and is not in these residues any substitute, disappearance or adds. Perhaps, these conservative residues that substitute can change by conservative substituting.

May comprise the conservative residue that substitutes by not reformed other residue, such as Ile56 (Ile13 among the embodiment 37), Tyr58 (Tyr15 among the embodiment 37), Arg61 (Arg18 among the embodiment 37), Thr74 (Thr31 among the embodiment 37) and the Thr87 (Thr44 among the embodiment 37) of SEQ ID NO:2. In these positions any or its combination may be not replaced, disappearance or add beyond. Preferably, the MPIF-1 variant comprises and is not in these residues any substitute, disappearance or adds. Perhaps, these conservative residues that substitute can change by conservative substituting.

May comprise the conservative residue that substitutes by not reformed other residue, such as Ile56 (Ile13 among the embodiment 37), Tyr58 (Tyr15 among the embodiment 37), Arg61 (Arg18 among the embodiment 37), Ile63 (Ile20 among the embodiment 37), Thr74 (Thr31 among the embodiment 37) and the Thr87 (Thr44 among the embodiment 37) of SEQ ID NO:2. In these positions any or its combination may be not replaced, disappearance or add. Preferably, the MPIF-1 variant comprises and is not in the substituting of any place in these residues, disappearance or adds. Perhaps, these conservative residues that substitute can change by conservative substituting.

Conservative alternative residue also comprises Ile63 (Ile20 among the embodiment 37), Leu68 (Leu25 among the embodiment 37), Tyr71 (Tyr28 among the embodiment 37), Phe72 (Phe29 among the embodiment 37), Val83 (Val40 among the embodiment 37), Ile84 (Ile41 among the embodiment 37), Phe85 (Phe42 among the embodiment 37), Phe93 (Phe50 among the embodiment 37), Ala95 (Ala52 among the embodiment 37), Val102 (Val59 among the embodiment 37), Met106 (Met63 among the embodiment 37) and the Leu109 (Leu66 among the embodiment 37) of SEQ ID NO:2. In these positions any or its combination may be not replaced, disappearance or add. Preferably, the MPIF-1 variant comprises and is not in the substituting of any place in these residues, disappearance or adds. Perhaps, these conservative residues that substitute can change by conservative substituting.

Other conservative alternative residue also comprises Ile56 (Ile13 among the embodiment 37), Tyr58 (Tyr15 among the embodiment 37), Arg61 (Arg18 among the embodiment 37), Thr74 (Thr31 among the embodiment 37) and the Gly82 (Gly39 among the embodiment 37) of SEQ ID NO:2. In these positions any or its combination may be not replaced, disappearance or add. Preferably, the MPIF-1 variant comprises and is not in the substituting of any place in these residues, disappearance or adds. Perhaps, these conservative residues that substitute can change by conservative substituting.

Other conservative alternative residue also comprises Pro64 (Pro21 among the embodiment 37) and the Pro97 (Pro54 among the embodiment 37) of SEQ ID NO:2. In these positions any or its combination may be not replaced, disappearance or add. Preferably, the MPIF-1 variant comprises and is not in the substituting of any place in these residues, disappearance or adds. Perhaps, these conservative residues that substitute can change by conservative substituting.

Other conservative alternative residue also comprises the Gln101 (Gln58 among the embodiment 37) of SEQ ID NO:2. Preferably, not replaced, the disappearance of this residue or add. Preferably, it is guarded replacement. More preferably, with the amino acid replacement that lacks the large volume side chain it. Thereby, preferably use except the amino acid replacement the Trp for example it.

Other conservative alternative residue also comprises Arg61 (Arg18 among the embodiment 37), Lys88 (Lys45 among the embodiment 37) and the Arg91 (Arg48 among the embodiment 37) of SEQ ID NO:2. In these positions any or its combination may be not replaced, disappearance or add. Preferably, the MPIF-1 variant comprises and is not in the substituting of any place in these residues, disappearance or adds. Perhaps, these conservative residues that substitute can change by conservative substituting.

Other conservative alternative residue also comprises Lys89 (Lys46 among the embodiment 37), Lys100 (Lys57 among the embodiment 37), Arg107 (Arg64 among the embodiment 37) and the Lys110 (Arg67 among the embodiment 37) of SEQ ID NO:2. In these positions any or its combination may be not replaced, disappearance or add. Preferably, the MPIF-1 variant comprises and is not in the substituting of any place in these residues, disappearance or adds. Perhaps, these conservative residues that substitute can change by conservative substituting.

Except above-mentioned preferred compositions, the conservative and conservative preferred compositions that substitutes residue comprises Thr74 (Thr31 among the embodiment 37), Gly82 (Gly39 among the embodiment 37), Tyr58 (embodiment 37 in Tyr15), Cys54 (Cys11 among the embodiment 37), Cys55 (Cys12 among the embodiment 37) and the Cys94 (embodiment 37 in Cys51) of SEQ ID NO:2. Preferably, the MPIF-1 variant comprises and is not in the substituting of any place in these residues, disappearance or adds. Perhaps, these residues can change by conservative substituting.

Preferably, the MPIF-1 variant comprises except at least one or all above-mentioned conservative residue amino acid outside that substitutes and changes. Perhaps, at least one or all above-mentioned conservative residues that substitutes can change by conservative substituting.

Preferably, the MPIF-1 variant comprises amino acid and comprises except at least one or all above-mentioned conserved residues (being one or more Cys residues) and at least one or all conservative residue (being one or more all the other above-mentioned residues) amino acid outside that substitutes and change. Perhaps, at least one or all conservative residues that substitutes can change by conservative substituting. The MPIF-1 splice variant

In addition, identified and characterized the variant of MPIF-1. Several in these analogs comprise the amino terminal brachymemma. In addition, identified and characterized the MPIF-1 analog (Figure 20, SEQ ID NO:7) that is obviously produced by other splice site. Embodiment 11 discloses the BA of these MPIF-1 analogs. The sequence of these analogs is shown in Figure 19 (SEQ ID NO:3,4 and 5, and 46-120 position, 45-120 position, 120 of 48-, 49-120 position, 39-120 position and 44-120 amino acids residue among the SEQ ID NO:2).

On the other hand, the present invention includes 137 amino acid replacements in the amino acid whose MPIF-1 splice variant. For example, conservative substituting is included among the SEQ ID NO:7 with the alternative M1 of A, G, I, L, S, T or V; Substitute K2 with H or R; Substitute V3 with A, G, I, L, S, T or M; Substitute S4 with A, G, I, L, T, M or V; Substitute V5 with A, G, I, L, S, T or M; Substitute A6 with G, I, L, S, T, M or V; Substitute A7 with G, I, L, S, T, M or V; Substitute L8 with A, G, I, S, T, M or V; Substitute S9 with A, G, I, L, T, M or V; Substitute L11 with A, G, I, S, T, M or V; Substitute M12 with A, G, I, L, S, T or V; Substitute L13 with A, G, I, S, T, M or V; Substitute V14 with A, G, I, L, S, T or M; Substitute T15 with A, G, I, L, S, M or V; Substitute A16 with G, I, L, S, T, M or V; Substitute L17 with A, G, I, S, T, M or V; Substitute G18 with A, I, L, S, T, M or V; Substitute S19 with A, G, I, L, T, M or V; Substitute Q20 with N; Substitute A21 with G, I, L, S, T, M or V; Substitute R22 with H or K; Substitute V23 with A, G, I, L, S, T or M; Substitute T24 with A, G, I, L, S, M, V; Substitute K25 with H or R; Substitute D26 with E; Substitute A27 with G, I, L, S, T, M or V; Substitute E28 with D; Substitute T29 with A, G, I, L, S, M or V; Substitute E30 with D; Substitute F31 with W or Y; Substitute M32 with A, G, I, L, S, T or V; Substitute M33 with A, G, I, L, S, T or V; Substitute S34 with A, G, I, L, T, M or V; Substitute K35 with H or R; Substitute L36 with A, G, I, S, T, M or V; Substitute L38 with A, G, I, S, T, M or V; Substitute E39 with D; Substitute N40 with Q; Substitute V42 with A, G, I, L, S, T or M; Substitute L43 with A, G, I, S, T, M or V; Substitute L44 with A, G, I, S, T, M or V; Substitute D45 with E; Substitute M46 with A, G, I, L, S, T or V; Substitute L47 with A, G, I, S, T, M or V; Substitute W48 with F or Y; Substitute R49 with H or K; Substitute R50 with H or K; Substitute K51 with H or R; Substitute I52 with A, G, L, S, T, M or V; Substitute G53 with A, I, L, S, T, M or V; Substitute Q55 with N; Substitute M56 with A, G, I, L, S, T or V; Substitute T57 with A, G, I, L, S, M or V; Substitute L58 with A, G, I, S, T, M or V; Substitute S59 with A, G, I, L, T, M or V; Substitute H60 with K or R; Substitute A61 with G, I, L, S, T, M or V; Substitute A62 with G, I, L, S, T, M or V; Substitute G63 with A, I, L, S, T, M or V; Substitute F64 with W or Y; Substitute H65 with K or R; Substitute A66 with G, I, L, S, T, M or V; Substitute T67 with A, G, I, L, S, M or V; Substitute S68 with A, G, I, L, T, M or V; Substitute A69 with G, I, L, S, T, M or V; Substitute D70 with E; Substitute I73 with A, G, L, S, T, M or V; Substitute S74 with A, G, I, L, T, M or V; Substitute Y75 with F or W; Substitute T76 with A, G, I, L, S, M or V; Substitute R78 with H or K; Substitute S79 with A, G, I, L, T, M or V; Substitute I80 with A, G, L, S, T, M or V; Substitute S83 with A, G, I, L, T, M or V; Substitute L84 with A, G, I, S, T, M or V; Substitute L85 with A, G, I, S, T, M or V; Substitute E86 with D; Substitute S87 with A, G, I, L, T, M or V; Substitute Y88 with F or W; Substitute F89 with W or Y; Substitute E90 with D; Substitute T91 with A, G, I, L, S, M or V; Substitute N92 with Q; Substitute S93 with A, G, I, L, T, M or V; Substitute E94 with D; Substitute S96 with A, G, I, L, T, M or V; Substitute K97 with H or R; Substitute G99 with A, I, L, S, T, M or V; Substitute V100 with A, G, I, L, S, T or M; Substitute I101 with A, G, L, S, T, M or V; Substitute F102 with W or Y; Substitute L103 with A, G, I, S, T, M or V; Substitute T104 with A, G, I, L, S, M or V; Substitute K105 with H or R; Substitute K106 with H or R; Substitute G107 with A, I, L, S, T, M or V; Substitute R108 with H or K; Substitute R109 with H or K; Substitute F110 with W or Y; Substitute A112 with G, I, L, S, T, M or V; Substitute N113 with Q; Substitute S115 with A, G, I, L, T, M or V; Substitute D116 with E; Substitute K117 with H or R; Substitute Q118 with N; Substitute V119 with A, G, I, L, S, T or M; Substitute Q120 with N; Substitute V121 with A, G, I, L, S, T or M; Substitute M123 with A, G, I, L, S, T or V; Substitute R124 with H or K; Substitute M125 with A, G, I, L, S, T or V; Substitute L126 with A, G, I, S, T, M or V; Substitute K127 with H or R; Substitute L128 with A, G, I, S, T, M or V; Substitute D129 with E; Substitute T130 with A, G, I, L, S, M or V; Substitute R131 with H or K; Substitute I132 with A, G, L, S, T, M or V; Substitute K133 with H or R; Substitute T134 with A, G, I, L, S, M or V; Substitute R135 with H or K; Substitute K136 with H or R; And/or substitute N137 with Q.

，137：SEQ ID NO： 2D、E、H、K、R、N、Q、F、W、Y、P、CM1；D、E、A、G、 I、L、S、T、M、V、N、Q、F、W、Y、P、CK2；D、E、A、G、 I、L、S、T、M、V、N、Q、F、W、Y、P、CV3；D、E、H、K、 R、N、Q、F、W、Y、P、CS4；D、E、H、K、R、N、Q、F、W、 Y、P、CV5；D、E、H、K、R、N、Q、F、W、Y、P、CA6； D、E、H、K、R、N、Q、F、W、Y、P、CA7；D、E、H、K、R、 N、Q、F、W、Y、P、CL8；D、E、H、K、R、N、Q、F、W、Y、 P、CS9；D、E、H、K、R、A、G、I、L、S、T、M、V、N、Q、 F、W、Y、PC10；D、E、H、K、R、N、Q、F、W、Y、P、C L11；D、E、H、K、R、N、Q、F、W、Y、P、CM12；D、E、 H、K、R、N、Q、F、W、Y、P、CL13；D、E、H、K、R、N、Q、 F、W、Y、P、CV14；D、E、H、K、R、N、Q、F、W、Y、P、 CT15；D、E、H、K、R、N、Q、F、W、Y、P、CA16；D、 E、H、K、R、N、Q、F、W、Y、P、CL17；D、E、H、K、R、N、 Q、F、W、Y、P、CG18；D、E、H、K、R、N、Q、F、W、Y、P、 CS19；D、E、H、K、R、A、G、I、L、S、T、M、V、F、W、Y、 P、CQ20；D、E、H、K、R、N、Q、F、W、Y、P、CA21； D、E、A、G、I、L、S、T、M、V、N、Q、F、W、Y、P、CR22； D、E、H、K、R、N、Q、F、W、Y、P、CV23；D、E、H、K、 R、N、Q、F、W、Y、P、CT24；D、E、A、G、I、L、S、T、M、 V、N、Q、F、W、Y、P、CK25；H、K、R、A、G、I、L、S、T、 M、V、N、Q、F、W、Y、P、CD26；D、E、H、K、R、N、Q、F、 W、Y、P、CA27；H、K、R、A、G、I、L、S、T、M、V、N、Q、 F、W、Y、P、CE28；D、E、H、K、R、N、Q、F、W、Y、P、 CT29；H、K、R、A、G、I、L、S、T、M、V、N、Q、F、W、Y、P、 CE30；D、E、H、K、R、A、G、I、L、S、T、M、V、N、Q、Y、 P、CF31；D、E、H、K、R、N、Q、F、W、Y、P、CM32； D、E、H、K、R、N、Q、F、W、Y、P、CM33；D、E、H、K、 R、N、Q、F、W、Y、P、CS34；D、E、A、G、I、L、S、T、M、 V、N、Q、F、W、Y、P、CK35；D、E、H、K、R、N、Q、F、W、 Y、P、CL36；D、E、H、K、R、A、G、I、L、S、T、M、V、N、 Q、F、W、Y、CP37；D、E、H、K、R、N、Q、F、W、Y、P、 CL38；H、K、R、A、G、I、L、S、T、M、V、N、Q、F、W、Y、P、 CE39；D、E、H、K、R、A、G、I、L、S、T、M、V、F、W、Y、 P、CN40；D、E、H、K、R、A、G、I、L、S、T、M、V、N、Q、 F、W、Y、CP41；D、E、H、K、R、N、Q、F、W、Y、P、C V42；D、E、H、K、R、N、Q、F、W、Y、P、CL43；D、E、 H、K、R、N、Q、F、W、Y、P、CL44；H、K、R、A、G、I、L、 S、T、M、V、N、Q、F、W、Y、P、CD45；D、E、H、K、R、N、 Q、F、W、Y、P、CM46；D、E、H、K、R、N、Q、F、W、Y、P、 CL47；D、E、H、K、R、A、G、I、L、S、T、M、V、N、Q、P、 CW48；D、E、A、G、I、L、S、T、M、V、N、Q、F、W、Y、P、 CR49；D、E、A、G、I、L、S、T、M、V、N、Q、F、W、Y、P、 CR50；D、E、A、G、I、L、S、T、M、V、N、Q、F、W、Y、P、 CK51；D、E、H、K、R、N、Q、F、W、Y、P、CI52； D、E、H、K、R、N、Q、F、W、Y、P、CG53；D、E、H、K、R、 A、G、I、L、S、T、M、V、N、Q、F、W、Y、CP54；D、E、H、 K、R、A、G、I、L、S、T、M、V、F、W、Y、P、CQ55；D、E、 H、K、R、N、Q、F、W、Y、P、CM56；D、E、H、K、R、N、Q、 F、W、Y、P、CT57；D、E、H、K、R、N、Q、F、W、Y、P、 CL58；D、E、H、K、R、N、Q、F、W、Y、P、CS59；D、 E、A、G、I、L、S、T、M、V、N、Q、F、W、Y、P、CH60；D、 E、H、K、R、N、Q、F、W、Y、P、CA61；D、E、H、K、R、N、 Q、F、W、Y、P、CA62；D、E、H、K、R、N、Q、F、W、Y、P、 CG63；D、E、H、K、R、A、G、I、L、S、T、M、V、N、Q、P、 CF64；D、E、A、G、I、L、S、T、M、V、N、Q、F、W、Y、P、 CH65；D、E、H、K、R、N、Q、F、W、Y、P、CA66； D、E、H、K、R、N、Q、F、W、Y、P、CT67；D、E、H、K、R、 N、Q、F、W、Y、P、CS68；D、E、H、K、R、N、Q、F、W、Y、 P、CA69；H、K、R、A、G、I、L、S、T、M、V、N、Q、F、W、 Y、P、CD70；D、E、H、K、R、A、G、I、L、S、T、M、V、N、 Q、F、W、Y、PC71；D、E、H、K、R、A、G、I、L、S、T、M、 V、N、Q、F、W、Y、PC72；D、E、H、K、R、N、Q、F、W、Y、 P、CI73；D、E、H、K、R、N、Q、F、W、Y、P、CS74； D、E、H、K、R、A、G、I、L、S、T、M、V、N、Q、P、CY75； D、E、H、K、R、N、Q、F、W、Y、P、CT76；D、E、H、K、 R、A、G、I、L、S、T、M、V、N、Q、F、W、Y、CP77；D、E、 A、G、I、L、S、T、M、V、N、Q、F、W、Y、P、CR78；D、E、 H、K、R、N、Q、F、W、Y、P、CS79；D、E、H、K、R、N、Q、 F、W、Y、P、CI80；D、E、H、K、R、A、G、I、L、S、T、M、 V、N、Q、F、W、Y、CP81；D、E、H、K、R、A、G、I、L、S、 T、M、V、N、Q、F、W、Y、PC82；D、E、H、K、R、N、Q、F、 W、Y、P、CS83；D、E、H、K、R、N、Q、F、W、Y、P、C L84；D、E、H、K、R、N、Q、F、W、Y、P、CL85；H、K、 R、A、G、I、L、S、T、M、V、N、Q、F、W、Y、P、CE86；D、 E、H、K、R、N、Q、F、W、Y、P、CS87；D、E、H、K、R、A、 G、I、L、S、T、M、V、N、Q、P、CY88；D、E、H、K、R、A、 G、I、L、S、T、M、V、N、Q、P、CF89；H、K、R、A、G、I、 L、S、T、M、V、N、Q、F、W、Y、P、CE90；D、E、H、K、R、 N、Q、F、W、Y、P、CT91；D、E、H、K、R、A、G、I、L、S、 T、M、V、F、W、Y、P、CN92；D、E、H、K、R、N、Q、F、W、 Y、P、CS93；H、K、R、A、G、I、L、S、T、M、V、N、Q、F、 W、Y、P、CE94；D、E、H、K、R、A、G、I、L、S、T、M、V、 N、Q、F、W、Y、PC95；D、E、H、K、R、N、Q、F、W、Y、P、 CS96；D、E、A、G、I、L、S、T、M、V、N、Q、F、W、Y、P、 CK97；D、E、H、K、R、A、G、I、L、S、T、M、V、N、Q、F、 W、Y、CP98；D、E、H、K、R、N、Q、F、W、Y、P、C G99；D、E、H、K、R、N、Q、F、W、Y、P、CV100；D、E、 H、K、R、N、Q、F、W、Y、P、CI101；D、E、H、K、R、A、G、 I、L、S、T、M、V、N、Q、P、CF102；D、E、H、K、R、N、Q、 F、W、Y、P、CL103；D、E、H、K、R、N、Q、F、W、Y、P、 CT104；D、E、A、G、I、L、S、T、M、V、N、Q、F、W、Y、P、 CK105；D、E、A、G、I、L、S、T、M、V、N、Q、F、W、Y、P、 CK106；D、E、H、K、R、N、Q、F、W、Y、P、CG107； D、E、A、G、I、L、S、T、M、V、N、Q、F、W、Y、P、CR108； D、E、A、G、I、L、S、T、M、V、N、Q、F、W、Y、P、CR109； D、E、H、K、R、A、G、I、L、S、T、M、V、N、Q、P、CF110； D、E、H、K、R、A、G、I、L、S、T、M、V、N、Q、F、W、Y、P C111；D、E、H、K、R、N、Q、F、W、Y、P、CA112；D、 E、H、K、R、A、G、I、L、S、T、M、V、F、W、Y、P、CN113； D、E、H、K、R、A、G、I、L、S、T、M、V、N、Q、F、W、Y、C P114；D、E、H、K、R、N、Q、F、W、Y、P、CS115；H、 K、R、A、G、I、L、S、T、M、V、N、Q、F、W、Y、P、CD116； D、E、A、G、I、L、S、T、M、V、N、Q、F、W、Y、P、CK117； D、E、H、K、R、A、G、I、L、S、T、M、V、F、W、Y、P、C Q118；D、E、H、K、R、N、Q、F、W、Y、P、CV119；D、E、 H、K、R、A、G、I、L、S、T、M、V、F、W、Y、P、CQ120；D、 E、H、K、R、N、Q、F、W、Y、P、CV121；D、E、H、K、R、A、 G、I、L、S、T、M、V、N、Q、F、W、Y、PC122；D、E、H、K、 R、N、Q、F、W、Y、P、CM123；D、E、A、G、I、L、S、T、M、 V、N、Q、F、W、Y、P、CR124；D、E、H、K、R、N、Q、F、W、 Y、P、CM125；D、E、H、K、R、N、Q、F、W、Y、P、C L126；D、E、A、G、I、L、S、T、M、V、N、Q、F、W、Y、P、C K127；D、E、H、K、R、N、Q、F、W、Y、P、CL128；H、 K、R、A、G、I、L、S、T、M、V、N、Q、F、W、Y、P、CD129； D、E、H、K、R、N、Q、F、W、Y、P、CT130；D、E、A、G、 I、L、S、T、M、V、N、Q、F、W、Y、P、CR131；D、E、H、K、 R、N、Q、F、W、Y、P、CI132；D、E、A、G、I、L、S、T、M、 V、N、Q、F、W、Y、P、CK133；D、E、H、K、R、N、Q、F、W、 Y、P、CT134；D、E、A、G、I、L、S、T、M、V、N、Q、F、W、 Y、P、CR135；D、E、A、G、I、L、S、T、M、V、N、Q、F、W、 Y、P、CK136；/D、E、H、K、R、A、G、I、L、S、T、M、 V、F、W、Y、P、CN137。

In order to improve or change the feature of MPIF-1 polypeptide, can adopt protein engineering. Can use the recombinant DNA technology that those skilled in the art will know that to generate new protein. Mutain and disappearance or fusion can show the stability such as the active or increase that strengthens. In addition, the productive rate that they can be higher carries out purifying, and shows better dissolubility at least under some purifying and condition of storage. Hereinafter listed the further example of the sudden change that can make up. MPIF-1 amino terminal and carboxyl-terminal deletion

By the carboxyl-terminal deletion 8-10 amino acid residue by protein, interferon gamma shows that active raising can reach 10 times (7:199-216,1988 for the people such as D  beli, J.of Biotechnology). The people such as Ron, J.Biol.Chem., 268 (4): 2984-2988, even 1993 reported

forfeiture amino terminal

3,8 or 27 amino acid residues and still had the modified KGF albumen of heparin binding activity. Those skilled in the art will know that many other examples.

Particularly, the N of MPIF-1 polypeptide end disappearance can be described as general formula m-120, and wherein m is the integer of 2-115, and corresponding to the amino acid residue position of in SEQ ID NO:2, having identified. In particular, the invention provides coding comprise following residue amino acid sequence or consisting of the polynucleotides of polypeptide: the K2-N120 of SEQ ID NO:2, V3-N120, S4-N120, V5-N120, A6-N120, A7-N120, L8-N120, S9-N120, C10-N120, L11-N120, M12-N120, L13-N120, V14-N120, T15-N120, A16-N120, L17-N120, G18-N120, S19-N120, Q20-N120, A21-N120, R22-N120, V23-N120, T24-N120, K25-N120, D26-N120, A27-N120, E28-N120, T29-N120, E30-N120, F31-N120, M32-N120, M33-N120, S34-N120, K35-N120, L36-N120, P37-N120, L38-N120, E39-N120, N40-N120, P41-N120, V42-N120, L43-N120, L44-N120, D45-N120, R46-N120, F47-N120, H48-N120, A49-N120, T50-N120, S51-N120, A52-N120, D53-N120, C54-N120, C55-N120, I56-N120, S57-N120, Y58-N120, T59-N120, P60-N120, R61-N120, S62-N120, I63-N120, P64-N120, C65-N120, S66-N120, L67-N120, L68-N120, E69-N120, S70-N120, Y71-N120, F72-N120, E73-N120, T74-N120, N75-N120, S76-N120, E77-N120, C78-N120, S79-N120, K80-N120, P81-N120, G82-N120, V83-N120, I84-N120, F85-N120, L86-N120, T87-N120, K88-N120, K89-N120, G90-N120, R91-N120, R92-N120, F93-N120, C94-N120, A95-N120, N96-N120, P97-N120, S98-N120, D99-N120, K100-N120, Q101-N120, V102-N120, Q103-N120, V104-N120, C105-N120, M106-N120, R107-N120, M108-N120, L109-N120, K110-N120, L111-N120, D112-N120, T113-N120, R114-N120, I115-N120. The polynucleotides of these polypeptide of encoding are also contained in the present invention.

Similarly, as described above, even cause one or more protein function loss of activity or modified by the one or more amino acid of the C of protein end disappearance, still can keep other functional activity, such as BA, multimerization ability, in conjunction with the ability of MPIF-1 acceptor. For example, when removing the major part that is no more than complete or mature polypeptide residue by C end, the MPIF-1 mutain of brachymemma generally can keep induces and/or in conjunction with the ability of the antibody of the complete or mature form of this polypeptide of identification. Other method that can be easy to know by conventional method as herein described and this area determines whether the specific polypeptide that lacks complete peptide C terminal residue keeps these immunologic competences. Lacked possible some biology or the immunologic competence of still keeping of MPIF-1 mutain of the C terminal amino acid residue of greater number. In fact, usually can cause immune response by few peptide that forms to 6 MPIF-1 amino acid residues.

Therefore, the present invention also provides by the carboxyl-terminal deletion of MPIF-1 polypeptid acid sequence shown in Fig. 1 (SEQ ID NO:2) polypeptide of one or more residues, be described as general formula 1-n, wherein n is the integer of 6-119, and corresponding to the amino acid residue position of in SEQ ID NO:2, having identified. In particular, the invention provides coding comprise following residue amino acid sequence or consisting of the polynucleotides of polypeptide: the A27-K119 of SEQ ID NO:2; A27-R118; A27-T117; A27-K116; A27-I115; A27-R114; A27-T113; A27-D112; A27-L111; A27-K110; A27-L109; A27-M108; A27-R107; A27-M106; A27-C105; A27-V104; A27-Q103; A27-V102; A27-Q101; A27-K100; A27-D99; A27-S98; A27-P97; A27-N96; A27-A95; A27-C94; A27-F93; A27-R92; A27-R91; A27-G90; A27-K89; A27-K88; A27-T87; A27-L86; A27-F85; A27-I84; A27-V83; A27-G82; A27-P81; A27-K80; A27-S79; A27-C78; A27-E77; A27-S76; A27-N75; A27-T74; A27-E73; A27-F72; A27-Y71; A27-S70; A27-E69; A27-L68; A27-L67; A27-S66; A27-C65; A27-P64; A27-I63; A27-S62; A27-R61; A27-P60; A27-T59; A27-Y58; A27-S57; A27-I56; A27-C55; A27-C54; A27-D53; A27-A52; A27-S51; A27-T50; A27-A49; A27-H48; A27-F47; A27-R46; A27-D45; A27-L44; A27-L43; A27-V42; A27-P41; A27-N40; A27-E39; A27-L38; A27-P37; A27-L36; A27-K35; A27-S34; A27-M33; A27-M32; A27-F31; A27-E30; A27-T29; A27-E28; M1-D26; M1-K25; M1-T24; M1-V23; M1-R22; M1-A21; M1-Q20; M1-S19; M1-G18; M1-L17; M1-A16; M1-T15; M1-V14; M1-L13; M1-M12; M1-L11; M1-C10; M1-S9; M1-L8; M1-A7. The polynucleotides of these polypeptide of encoding are also contained in the present invention.

In addition, can add burst to these C end constructions. For example, can be with the 1-26 amino acids of SEQ ID NO:2; The 2-26 amino acids of SEQ ID NO:2; The 3-26 amino acids of SEQ ID NO:2; The 4-26 amino acids of SEQ ID NO:2; The 5-26 amino acids of SEQ ID NO:2; The 6-26 amino acids of SEQ ID NO:2; The 7-26 amino acids of SEQ ID NO:2; The 8-26 amino acids of SEQ ID NO:2; The 9-26 amino acids of SEQ ID NO:2; The 10-26 amino acids of SEQ ID NO:2; The 11-26 amino acids of SEQ ID NO:2; The 12-26 amino acids of SEQ ID NO:2; The 13-26 amino acids of SEQ ID NO:2; The 14-26 amino acids of SEQ ID NO:2; The 15-26 amino acids of SEQ ID NO:2; The 16-26 amino acids of SEQ ID NO:2; The 17-26 amino acids of SEQ ID NO:2; The 18-26 amino acids of SEQ ID NO:2; The 19-26 amino acids of SEQ ID NO:2; 20-26 amino acids of SEQ ID NO:2; The 21-26 amino acids of SEQ ID NO:2; The 22-26 amino acids of SEQ ID NO:2; The 23-26 amino acids of SEQ ID NO:2; The 24-26 amino acids of SEQ ID NO:2; The 25-26 amino acids of SEQID NO:2; The 26th amino acids of SEQ ID NO:2 is added on the above N end of listed every kind of C end construction.

In addition, can unite any above listed N or C end disappearance to generate all MPIF-1 polypeptide of disappearance of N and C end. The present invention also provides by amino terminal and carboxyl terminal and has all lacked one or more amino acid whose polypeptide, and they can be described as the m-n position residue with SEQ ID NO:2, and wherein n and m are integers mentioned above. The polynucleotides of these polypeptide of encoding are also contained in the present invention.

Other preferred polypeptide fragment comprises the amino acid sequence of following residue or consisting of M1-T15; K2-A16; V3-L17; S4-G18; V5-S19; A6-Q20; A7-A21; L8-R22; S9-V23; C10-T24; L11-K25; M12-D26; L13-A27; V14-E28; T15-T29; A16-E30; L17-F31; G18-M32; S19-M33; Q20-S34; A21-K35; R22-L36; V23-P37; T24-L38; K25-E39; D26-N40; A27-P41; E28-V42; T29-L43; E30-L44; F31-D45; M32-R46; M33-F47; S34-H48; K35-A49; L36-T50; P37-S51; L38-A52; E39-D53; N40-C54; P41-C55; V42-I56; L43-S57; L44-Y58; D45-T59; R46-P60; F47-R61; H48-S62; A49-I63; T50-P64; S51-C65; A52-S66; D53-L67; C54-L68; C55-E69; I56-S70; S57-Y71; Y58-F72; T59-E73; P60-T74; R61-N75; S62-S76; I63-E77; P64-C78; C65-S79; S66-K80; L67-P81; L68-G82; E69-V83; S70-I84; Y71-F85; F72-L86; E73-T87; T74-K88; N75-K89; S76-G90; E77-R91; C78-R92; S79-F93; K80-C94; P81-A95; G82-N96; V83-P97; I84-S98; F85-D99; L86-K100; T87-Q101; K88-V102; K89-Q103; G90-V104; R91-C105; R92-M106; F93-R017; C94-M108; A95-L109; N96-K110; P97-L111; S98-D112; D99-T113; K100-R114; Q101-I115; V102-K116; Q103-T117; V104-R118; C105-K119; M106-N120. These polypeptide fragments can keep the BA of MPIF-1 polypeptide of the present invention, and can be used for generating antibody, and seeing below further describes. The polynucleotides of these polypeptide fragments of encoding are also contained in the present invention.

The present invention also comprises the nucleotide sequence of the polypeptide that the part of the complete MPIF-1 amino acid sequence of coding forms, wherein complete MPIF-1 amino acid sequence is to number cDNA clones coding contained in 75676 by the ATCC preserved material, wherein said part does not comprise arbitrary integer of amino acid residue of about 110 amino acids of aminoterminal 1-of complete amino acid sequence, or arbitrary integer of the amino acid residue of c-terminus the 1st-about 110 amino acids, or arbitrary combination of above-mentioned aminoterminal and carboxy terminal deletion, described complete amino acid sequence is to number cDNA clones coding contained in 75676 by the ATCC preserved material. The polynucleotides of all above-mentioned deletion mutation type polypeptide forms of encoding are also contained in the present invention.

The invention still further relates to the protein that comprises with the MPIF-1 peptide sequence at least 90%, 95%, 96%, 97% of the listed m-n of this paper, 98% or 99% same polypeptide. In preferred embodiments, the application relates to the protein that comprises with the polypeptide at least 90%, 95%, 96%, 97% of the amino acid sequence with specific MPIF-1N described herein and C terminal deletion, 98% or 99% same polypeptide. The polynucleotides of these polypeptide of encoding are also contained in the present invention.

The particularly preferred MPIF-1 polypeptide that has hereinafter shown the amino acid sequence shown in (SEQ ID NO:2) that has Fig. 1:

Val(23)---Asn(120) Val(23)---Lys(119)

Thr(24)---Asn(120) Thr(24)---Arg(118)

Lys(25)---Asn(120) Lys(25)---Thr(117)

Asp(26)---Asn(120) Asp(26)---Lys(116)

Ala(27)---Asn(120) Ala(27)---Ile(115)

Glu(28)---Asn(120) Glu(28)---Arg(114)

Thr(29)---Asn(120) Thr(29)---Thr(113)

Glu(30)---Asn(120) Thr(29)---Asp(112)

Phe(31)---Asn(120) Thr(29)---Leu(111)

Met(32)---Asn(120) Thr(29)---Lys(110)

Met(33)---Asn(120) Met(33)---Leu(109)

Ser(34)---Asn(120) Ser(34)---Met(108)

Lys(35)---Asn(120) Ser(34)---Arg(107)

Leu(36)---Asn(120) Ser(34)---Met(106)

Pro(37)---Asn(120) Ser(34)---Cys(105)

Leu(38)---Asn(120) Ser(34)---Val(104)

Glu(39)---Asn(120) Ser(34)---Gln(103)

Asn(40)---Asn(120) Ser(34)---Val(102)

Pro(41)---Asn(120) Ser(34)---Gln(101)

Val(42)---Asn(120) Ser(34)---Lys(100)

Leu(43)---Asn(120) Ser(34)---Asp(99)

Leu(44)---Asn(120) Ser(34)---Ser(98)

Asp(45)---Asn(120) Ser(34)---Pro(97)

Arg(46)---Asn(120) Ser(34)---Asn(96)

Phe(47)---Asn(120) Ser(34)---Ala(95)

His(48)---Asn(120) Ser(34)---Cys(94)

Ala(49)---Asn(120) Ser(34)---Phe(93)

Thr(50)---Asn(120) Ser(34)---Arg(92)

Ser(51)---Asn(120) Ser(34)---Arg(91)

Ala(52)---Asn(120) Ser(34)---Gly(90)

Asp(53)---Asn(120) Ser(34)---Lys(89)

Ser(34)---Ile(84)

Ser(34)---Ser(79)

Ser(34)---Asn(75)

Ser(34)---Phe(72)

Ser(34)---Leu(68)

Thereby, on the one hand, the invention provides the N end deletion mutant of MPIF-1. These mutant comprise those comprise amino acid sequence shown in Fig. 1 (SEQ ID NO:2) or consisting of and front at least 22 N terminal amino acid residues (namely lacking at least Met1-Arg22) of disappearance Fig. 1 (SEQ ID NO:2) but be no more than the mutant of front 60 N terminal amino acid residues. Perhaps, disappearance comprises front at least 22 N terminal amino acid residues of Fig. 1 (SEQ ID NO:2) but is no more than front 53 N terminal amino acid residues. Perhaps, disappearance comprises front at least 33 N terminal amino acid residues of Fig. 1 (SEQ ID NO:2) but is no more than front 53 N terminal amino acid residues. Perhaps, disappearance comprises that front at least 37 N terminal amino acid residues of Fig. 1 (SEQ ID NO:2) (namely lack at least Met1-Pro37) but are no more than front 53 N terminal amino acid residues. Perhaps, disappearance comprises front at least 48 N terminal amino acid residues of Fig. 1 (SEQ ID NO:2) but is no more than front 53 N terminal amino acid residues.

Scope except MPIF-1 N mentioned above end deletion mutant the invention still further relates to all combinations of scope mentioned above, such as front at least 22 N terminal amino acid residues of disappearance Fig. 1 (SEQ ID NO:2) but be no more than front 48 N terminal amino acid residues; The disappearance Fig. 1 (SEQ ID NO:2) front at least 37 N terminal amino acid residues but be no more than front 48 N terminal amino acid residues; The disappearance Fig. 1 (SEQ ID NO:2) front at least 22 N terminal amino acid residues but be no more than front 37 N terminal amino acid residues; The disappearance Fig. 1 (SEQ ID NO:2) front at least 22 N terminal amino acid residues but be no more than front 33 N terminal amino acid residues; The disappearance Fig. 1 (SEQ ID NO:2) front at least 33 N terminal amino acid residues but be no more than front 37 N terminal amino acid residues; The disappearance Fig. 1 (SEQ ID NO:2) front at least 33 N terminal amino acid residues but be no more than front 48 N terminal amino acid residues.

On the other hand, the invention provides the C end deletion mutant of MPIF-1. Preferably, the N terminal amino acid residue of described MPIF-1 C end deletion mutant is that the amino acid residue Met1 of Fig. 1 (SEQ ID NO:2) is to Arg22. These mutant comprise comprise amino acid sequence shown in Fig. 1 (SEQ ID NO:2) or consisting of and the disappearance Fig. 1 (SEQ ID NO:2) at least last C terminal amino acid residue (Asn120) but be no more than last 52 C terminal amino acid residues mutant (as the disappearance Fig. 1 (SEQ ID NO:2) amino acid residue Glu69-Asn120). Perhaps, disappearance comprises last at least 10 or 15 C terminal amino acid residues of Fig. 1 (SEQ ID NO:2) but is no more than last 52 C terminal amino acid residues. Perhaps, disappearance comprises last at least 20 C terminal amino acid residues of Fig. 1 (SEQ ID NO:2) but is no more than last 52 C terminal amino acid residues. Perhaps, disappearance comprises last at least 30 C terminal amino acid residues of Fig. 1 (SEQ ID NO:2) but is no more than last 52 C terminal amino acid residues. Perhaps, disappearance comprises last at least 36 C terminal amino acid residues of Fig. 1 (SEQ ID NO:2) but is no more than last 52 C terminal amino acid residues. Perhaps, disappearance comprises last at least 41 C terminal amino acid residues of Fig. 1 (SEQ ID NO:2) but is no more than last 52 C terminal amino acid residues. Perhaps, disappearance comprises last at least 45 C terminal amino acid residues of Fig. 1 (SEQ ID NO:2) but is no more than last 52 C terminal amino acid residues. Perhaps, disappearance comprises last at least 48 C terminal amino acid residues of Fig. 1 (SEQ ID NO:2) but is no more than last 52 C terminal amino acid residues.

Scope except C mentioned above end deletion mutant the invention still further relates to all combinations of scope mentioned above, such as at least last C terminal amino acid residue of disappearance Fig. 1 (SEQ ID NO:2) but be no more than last 48 C terminal amino acid residues; The disappearance Fig. 1 (SEQ ID NO:2) at least last C terminal amino acid residue but be no more than last 45 C terminal amino acid residues; The disappearance Fig. 1 (SEQ ID NO:2) at least last C terminal amino acid residue but be no more than last 41 C terminal amino acid residues; The disappearance Fig. 1 (SEQ ID NO:2) at least last C terminal amino acid residue but be no more than last 36 C terminal amino acid residues; The disappearance Fig. 1 (SEQ ID NO:2) at least last C terminal amino acid residue but be no more than last 10 C terminal amino acid residues; The disappearance Fig. 1 (SEQ ID NO:2) last at least 10 C terminal amino acid residues but be no more than last 20 C terminal amino acid residues; The disappearance Fig. 1 (SEQ ID NO:2) last at least 10 C terminal amino acid residues but be no more than last 30 C terminal amino acid residues; The disappearance Fig. 1 (SEQ ID NO:2) last at least 10 C terminal amino acid residues but be no more than last 36 C terminal amino acid residues; The disappearance Fig. 1 (SEQ ID NO:2) last at least 20 C terminal amino acid residues but be no more than last 30 C terminal amino acid residues; Etc.; Etc.; Etc.

Also have an aspect, the present invention also to provide terminal by N and the C end the two all lack the MPIF-1 deletion mutant of amino acid residue. These mutant comprise all combinations of N end deletion mutant mentioned above and C end deletion mutant. These mutant comprise those comprise amino acid sequence shown in Fig. 1 (SEQ ID NO:2) or consisting of and front at least 22 N terminal amino acid residues of disappearance Fig. 1 (SEQ ID NO:2) but be no more than front 52 N terminal amino acid residues and at least last C terminal amino acid residue of disappearance Fig. 1 (SEQ ID NO:2) but be no more than the mutant of last 52 C terminal amino acid residues. Perhaps, disappearance can comprise front at least 33,37 or 48 N terminal amino acid residues of Fig. 1 (SEQ ID NO:2) but be no more than front 52 N terminal amino acid residues, and comprises last at least 10,20,30,36,41,45 or 48 C terminal amino acid residues of Fig. 1 (SEQ ID NO:2) but be no more than last 52 C terminal amino acid residues. The present invention also comprises all combinations of scope mentioned above.

The particularly preferred fragment of the present invention comprises that those are characterized as the fragment of MPIF-1 structure or functional character. The alpha-helix of the amino acid residue that these fragments comprise comprises complete (being total length) MPIF-1 (SEQ ID NO:2) and alpha-helix form district (α district), beta sheet and beta sheet form district (β district), corner and corner form district's (corner regions), curling and curl into district (curling district), hydrophilic area, hydrophobic region, α amphiphilic district, β amphiphilic district, surface formation distinguishes and high antigenicity exponential region (evaluation that namely comprises 4 or a plurality of default parameters according to use Jameson-Wolf program has the continuous amino acid that is greater than or equal to 1.5 antigenicity index). Fig. 4 has listed some preferably zone, include but not limited to, the zone of the above-mentioned type of differentiating by amino acid sequence shown in the analysis chart 1 (SEQ ID NO:2), these favored area comprise: α district, β district, corner regions and the curling district of Garnier-Robson prediction; α district, β district, corner regions and the curling district of Chou-Fasman prediction; Hydrophilic area and the hydrophobic region of Kyte-Doolittle prediction; Eisenberg α and β amphiphilic district; The Emini surface forms the district; With the high antigenicity of Jameson-Wolf exponential region, they are to use the default parameter of these computer programs to predict. The polynucleotides of these polypeptide of encoding are also contained in the present invention.

In other embodiments, the functional characteristic of polynucleotide encoding MPIF-1 of the present invention. In this respect preferred embodiment of the present invention comprise the α spiral that comprises MPIF-1 and α spiralization district (α district), β-pleated sheet and β-pleated sheet form district (β district), corner and corner form district's (corner regions), curling and curl into district (curling district), hydrophilic area, hydrophobic region, α amphiphilic district, β amphiphilic district, flex region, surperficial formation is distinguished and the fragment of high antigenicity exponential region.

Listed other dominant area among the embodiment 37.

As mentioned above, the data of the structure of the representative MPIF-1 that lists of Figure 14 or functional character are to use various modes and algorithm to be produced by the DNA*STAT that is set as default parameter.

The data of showing among Figure 14 in preferred embodiments, can be used for determining to show among the MPIF-1 zone of the former begetting power of high resistance. Can be determined in described environment, can in the initiating process of immune response, antigen recognizing occur in high antigenicity zone by the data of showing by selecting to represent the numerical value in the polypeptide zone that in environment, might be exposed to the polypeptide surface.

Listed above-mentioned favored area includes but not limited to the zone of the above-mentioned type identified by listed amino acid sequence in the analysis chart 1 among Figure 14. As listed among Figure 14, these favored area include but not limited to Garnier-Robson α district, β district, corner regions and curling district; Chou-Fasman α district, β district and curling district; Kyte-Doolittle hydrophilic area and hydrophobic region; Eisenberg α and β amphiphilic district; Karplus-Schulz flex region, Emini surface form the district; With the high antigenicity of Jameson-Wolf exponential region.

Highly preferred fragment is those fragments that comprise the MPIF-1 zone of several architectural characteristics of associating (such as above several or listed characteristic hereinafter) in this respect.

In one embodiment, MPIF-1 variant and/or fragment can have the structure identical or substantially the same with MPIF-1 or its at least one zone, for example by the solution structure (seeing embodiment 37) of NMR spectrum (NMR) mensuration or the structure of measuring by other technology. Thereby, MPIF-1 variant and/or fragment can have the different amino acid sequence of amino acid sequence of cloning coded polypeptide from amino acid sequence or the preservation cDNA of SEQ ID NO:2 or 7, but still have and MPIF-1 or its at least one regional identical or substantially the same structure. The polynucleotides of these polypeptide of encoding are also contained in the present invention.

MPIF-1 variant and/or fragment can have the structure identical or substantially the same with at least one zone of MPIF-1. MPIF-1 zone comprises the N end-rings of describing among the embodiment 37,310 rings, article one, β chain, first III type corner, second β chain, I type corner, article three, β chain, second III type corner, with the α spiral, i.e. the 56-63 position of SEQ ID NO:2 (the 13-20 position among the embodiment 37), 64-67 position (the 21-24 position among the embodiment 37), 70-74 position (the 27-31 position among the embodiment 37), 75-81 position (the 32-38 position among the embodiment 37), 82-87 position (the 39-44 position among the embodiment 37), 88-90 position (the 45-47 position among the embodiment 37), 91-95 position (the 48-52 position among the embodiment 37), 96-98 position (the 53-55 position among the embodiment 37), 99-109 position (the 56-66 position among the embodiment 37) amino acid. The MPIF-1 zone also comprises shown in the 44-120 position (the 1-77 position among the embodiment 37), 44-53 position (the 1-10 position among the embodiment 37), 54-109 position (the 11-66 position among the embodiment 37), 54-105 (the 11-62 position among the embodiment 37), 56-109 position (the 13-66 position among the embodiment 37), 106-120 position (the 63-77 position among the embodiment 37), 110-120 position (the 67-77 position among the embodiment 37) amino acid, Figure 14 of SEQ ID NO:2 regional and described herein or by predictable other zone of amino acid sequence analysis. MPIF-1 polypeptide variants and/or fragment can have with one of these MPIF-1 zones or make up identical or substantially the same structure.

For MPIF-1 variant and/or fragment with structure identical or substantially the same with surpassing MPIF-1 zone, these structures can be connected with each other. In one embodiment, these structures are not connected with each other, and namely they are separated by one or more amino acid residues. Preferably, these structures and those MPIF-1 structures are compared. Preferably, these structure superpositions are on those MPIF-1 structures. In preferred embodiments, these structures have the toward each other structure identical with counter structure among the MPIF-1 (being identical tertiary structure).

For example, MPIF-1 variant and/or fragment can have the structure identical or substantially the same with the N end-rings of MPIF-1, article one β chain, second β chain, the 3rd β chain and α spiral. Thereby, in preferred combination, MPIF-1 variant and/or fragment have the structure identical or substantially the same with 56-63 position (the 13-20 position among the embodiment 37), 70-74 position (the 27-31 position among the embodiment 37), 82-87 position (the 39-44 position among the embodiment 37), the 91st-95 (the 48-52 position among the embodiment 37) and 99-109 position (the 56-66 position among the embodiment 37) amino acid of SEQ ID NO:2.

As another example, MPIF-1 variant and/or fragment have the structure identical or substantially the same with the N end-rings of MPIF-1,310 rings, article one β chain, first III type corner, second β chain, I type corner, the 3rd β chain, second III type corner and α spiral. Thereby MPIF-1 variant and/or fragment have the structure identical or substantially the same with 56-63 position (the 13-20 position among the embodiment 37), 64-67 position (the 21-24 position among the embodiment 37), the 70th-74 (the 27-31 position among the embodiment 37), 75-81 position (the 32-38 position among the embodiment 37), 82-87 position (the 39-44 position among the embodiment 37), 88-90 position (the 45-47 position among the embodiment 37), 91-95 position (the 48-52 position among the embodiment 37), 96-98 position (the 53-55 position among the embodiment 37), 99-109 position (the 56-66 position among the embodiment 37) amino acid of SEQ ID NO:2.

In preferred embodiments, MPIF-1 variant and/or fragment have the structure identical or substantially the same with 56-109 position (the 13-66 position among the embodiment 37) amino acid of SEQ ID NO:2.

In another embodiment, the MPIF-1 polypeptide can comprise MPIF-1 one or more zones amino acid sequence or consisting of. For the amino acid sequence that comprises two or more zones or consisting of polypeptide, described each zone can be connected with each other. In one embodiment, described zone is not connected with each other, and namely they are separated by one or more amino acid residues. Preferably, the amino acid sequence of these amino acid sequences and MPIF-1 respective regions is compared, thus use with MPIF-1 in the amino acid residue of similar number they are separated.

In another embodiment, MPIF-1 variant and/or fragment comprise amino acid and change (substitute, lack and insert) in one or more above-mentioned zones, but do not contain variation in one or more other zones. The polynucleotides of these polypeptide of encoding are also contained in the present invention.

Other preferred polypeptide fragment is BA MPIF-1 fragment. Biological active fragment is that those show fragment similar with the MPIF-1 polypeptide active but activity that needn't be identical. The BA of fragment can comprise the unexpected activity that the expectation that makes moderate progress is active and decrease. The polynucleotides of these polypeptide fragments of encoding are also contained in the present invention.

Yet the public can obtain many polynucleotide sequences (such as the EST sequence) by sequence library. In these sequences some are relevant with SEQ ID NO:1 or 6, and just can openly obtain before the present invention. Preferably, these related polynucleotides are specifically got rid of beyond the scope of the invention. Listing each correlated series may be trouble. Therefore, preferably by the present invention get rid of be comprise the nucleotide sequence described by general formula a-b or consisting of one or more polynucleotides, wherein a is the arbitrary integer between the 1-349 of SEQ ID NO:1, b is an integer between the 15-363, and a and b be corresponding to the position of nucleotide residue shown in the SEQ ID NO:1, and b is more than or equal to a+14. Amino terminal and the carboxyl-terminal deletion of 137 amino acid whose splice variants of MPIF-1

As mentioned above, the present invention also provides people MPIF-1 splice variant. Figure 20 A (SEQ ID NO:6 and 7) has shown cDNA sequence and 137 amino acid whose sequences. Use eukaryotic expression system, the present invention has found 3 kinds of N end deletion mutants of this MPIF-1 splice variant, comprises His60-Asn137, Met46-Asn137 and Pro54-Asn137. Thereby, on the other hand, the invention provides MPIF-1 splice variant N end deletion mutant. These mutant comprise those comprise amino acid sequence shown in Figure 20 A (SEQ ID NO:7) or consisting of and front at least 45 N terminal amino acid residues of disappearance Figure 20 A (SEQ ID NO:7) but be no more than the mutant of front 59 N terminal amino acid residues. Perhaps, disappearance will comprise front at least 53 N terminal amino acid residues of Figure 20 A (SEQ ID NO:7) but be no more than front 59 N terminal amino acid residues. Perhaps, disappearance will comprise front at least 45 N terminal amino acid residues of Figure 20 A (SEQ ID NO:7) but be no more than front 53 N terminal amino acid residues.

Other N end disappearance of 137 amino acid whose splice variant polypeptide of the present invention (SEQ ID NO:7) comprise the amino acid sequence that comprises following residue or consisting of polypeptide: the K2-N137 of SEQ ID NO:7; V3-N137; S4-N137; V5-N137; A6-N137; A7-N137; L8-N137; S9-N137; C10-N137; L11-N137; M12-N137; L13-N137; V14-N137; T15-N137; A16-N137; L17-N137; G18-N137; S19-N137; Q20-N137; A21-N137; R22-N137; V23-N137; T24-N137; K25-N137; D26-N137; A27-N137; E28-N137; T29-N137; E30-N137; F31-N137; M32-N137; M33-N137; S34-N137; K35-N137; L36-N137; P37-N137; L38-N137; E39-N137; N40-N137; P41-N137; V42-N137; L43-N137; L44-N137; D45-N137; M46-N137; L47-N137; W48-N137; R49-N137; R50-N137; K51-N137; I52-N137; G53-N137; P54-N137; Q55-N137; M56-N137; T57-N137; L58-N137; S59-N137; H60-N137; A61-N137; A62-N137; G63-N137; F64-N137; H65-N137; A66-N137; T67-N137; S68-N137; A69-N137; D70-N137; C71-N137; C72-N137; I73-N137; S74-N137; Y75-N137; T76-N137; P77-N137; R78-N137; S79-N137; I80-N137; P81-N137; C82-N137; S83-N137; L84-N137; L85-N137; E86-N137; S87-N137; Y88-N137; F89-N137; E90-N137; T91-N137; N92-N137; S93-N137; E94-N137; C95-N137; S96-N137; K97-N137; P98-N137; G99-N137; V100-N137; I101-N137; F102-N137; L103-N137; T104-N137; K105-N137; K106-N137; G107-N137; R108-N137; R109-N137; F110-N137; C111-N137; A112-N137; N113-N137; P114-N137; S115-N137; D116-N137; K117-N137; Q118-N137; V119-N137; Q120-N137; V121-N137; C122-N137; M123-N137; R124-N137; M125-N137; L126-N137; K127-N137; L128-N137; D129-N137; T130-N137; R131-N137; Or I132-N137.

Equally, the C of 137 amino acid whose splice variant polypeptide of the present invention (SEQ ID NO:7) end disappearance comprises the polypeptide of the amino acid sequence that comprises following residue: the M1-K136 of SEQ ID NO:7; M1-R135; M1-T134; M1-K133; M1-I132; M1-R131; M1-T130; M1-D129; M1-L128; M1-K127; M1-L126; M1-M125; M1-R124; M1-M123; M1-C122; M1-V121; M1-Q120; M1-V119; M1-Q118; M1-K117; M1-D116; M1-S115; M1-P114; M1-N113; M1-A112; M1-C111; M1-F110; M1-R109; M1-R108; M1-G107; M1-K106; M1-K105; M1-T104; M1-L103; M1-F102; M1-I101; M1-V100; M1-G99; M1-P98; M1-K97; M1-S96; M1-C95; M1-E94; M1-S93; M1-N92; M1-T91; M1-E90; M1-F89; M1-Y88; M1-S87; M1-E86; M1-L85; M1-L84; M1-S83; M1-C82; M1-P81; M1-I80; M1-S79; M1-R78; M1-P77; M1-T76; M1-Y75; M1-S74; M1-I73; M1-C72; M1-C71; M1-D70; M1-A69; M1-S68; M1-T67; M1-A66; M1-H65; M1-F64; M1-G63; M1-A62; M1-A61; M1-H60; M1-S59; M1-L58; M1-T57; M1-M56; M1-Q55; M1-P54; M1-G53; M1-I52; M1-K51; M1-R50; M1-R49; M1-W48; M1-L47; M1-M46; M1-D45; M1-L44; M1-L43; M1-V42; M1-P41; M1-N40; M1-E39; M1-L38; M1-P37; M1-L36; M1-K35; M1-S34; M1-M33; M1-M32; M1-F31; M1-E30; M1-T29; M1-E28; M1-A27; M1-D26; M1-K25; M1-T24; M1-V23; M1-R22; M1-A21; M1-Q20; M1-S19; M1-G18; M1-L17; M1-A16; M1-T15; M1-V14; M1-L13; M1-M12; M1-L11; M1-C10; M1-S9; M1-L8; Or M1-A7.

Preferably provide polypeptide of the present invention with unpack format, and preferred basically pure. Can be by Smith and Johnson, Gene, 67:31-40, the one-step method of describing in 1988 is the recombinant production form of purifying MPIF-1 polypeptide basically.

Polypeptide of the present invention comprise by preservation cDNA coding and comprise targeting sequencing polypeptide (being full-length proteins), by preservation cDNA coding and without the polypeptide (being ripe albumen) of leading sequence, comprise Fig. 1 (SEQ ID NO:2) of targeting sequencing polypeptide, comprise targeting sequencing but without the polypeptide of Fig. 1 (SEQ ID NO:2) of N end methionine residues, without the polypeptide of Fig. 1 (SEQ ID NO:2) of leading sequence, and have at least 80%, 85%, 90%, 92 % or 95% similitude with aforementioned polypeptides, the more preferably polypeptide of at least 96%, 97%, 98% or 99% similitude still. Other polypeptide of the present invention comprises and the polypeptide 80%, 85% of the coded polypeptide of preservation cDNA or Fig. 1 (SEQ ID NO:2), 90% or 95% same at least, more preferably at least 96%, 97%, 98% or 99% same polypeptide still also comprises at least 30 amino acid of these polypeptide, more preferably at least 50 amino acid whose fragments.

" similitude percentage " between two peptide species refers to by using Bestfit program (Wisconsin sequence analysis program package, Unix the 8th edition, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711) and be used for measuring the amino acid sequence of default setting comparison two peptide species of similitude and the similitude score that produces. Bestfit uses Smith and Waterson, Advances in Applied Mathematics, and 2:482-489, local homology's algorithm of 1981 find out the best section of similitude between two kinds of sequences.

Have with the MPIF-1 polypeptide with reference to the amino acid sequence polypeptide of the amino acid sequence of 95% " same " at least for example, mean can comprise 5 amino acid whose changes of as many as in per 100 amino acid with reference to amino acid sequence of MPIF-1 polypeptide except this peptide sequence, the amino acid sequence of this polypeptide is identical with canonical sequence. In other words, in order to obtain having the polypeptide with the same amino acid sequence of reference amino acid sequence at least 95%, can lack or with the amino acid residue of as many as 5% in the another kind of amino acid replacement canonical sequence, perhaps can in canonical sequence, insert the amino acid of arbitrary number of as many as canonical sequence amino acid residue sum 5%. These changes of canonical sequence can betide with reference to the amino of amino acid sequence or carboxyl terminal position, also can betide any position between the described terminal position, these changes or intersperse among separately in the residue of canonical sequence, or intersperse among in the canonical sequence with one or more continuous group forms.

In practical operation, use known computer program, such as Bestfit program (Wisconsin sequence analysis program package, Unix the 8th edition, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI53711), can conventional determine that the coded amino acid sequence of any specific polypeptide and amino acid sequence shown in for example Fig. 1 (SEQ ID NO:2) or preservation cDNA clone is whether at least 95%, 96%, 97%, 98% or 99% same. When determine with Bestfit or any other sequence alignment program specific sequence whether for example with canonical sequence 95% of the present invention with for the moment, arranging certainly of parameter should be so that calculate homogeneity percentage to the total length of reference amino acid sequence, and allow to exist the nearly homology breach of canonical sequence amino acid residue sum 5%.

Use the well-known method of those skilled in the art, polypeptide of the present invention can be used as molecular weight standard and is used for SDS-PAGE gel or molecular sieve gel filtration column.

As hereinafter describing in detail, polypeptide of the present invention also can be used for preparing polyclone and monoclonal antibody, and they can be used for the determination method of detection MPIF-1 protein expression hereinafter described, or is used as activator and the antagonist of energy enhancer or inhibitor MPIF-1 protein function. In addition, to can be used for yeast two-hybrid system also be the MPIF-1 protein-binding protein of candidate's activator of the present invention and antagonist with " catching " to these polypeptide. Yeast two-hybrid system is described in Fields and Song, Nature, 340:245-246,1989. The MPIF-1 epi-position is carried polypeptide

On the other hand, the invention provides comprise the part of carrying epi-position in the polypeptide of the present invention or consisting of peptide or polypeptide. The epi-position of this polypeptide portion is immunogenicity or the antigenic epitopes of polypeptide of the present invention. " immunogenicity epi-position " is defined as the fragment that causes antibody response when whole protein is immunogene in the protein. Think that these immunogenicity epi-positions are confined to a few locations on the molecule. On the other hand, the zone that can be combined with antibody in the protein molecule is defined as " antigenic epitopes ". The number of immunogenicity epi-position is less than the number of antigenic epitopes usually in the protein. Consult such as people such as Geysen Proc.Natl.Acad.Sci.USA, 81:3998-4002,1993.

Can generate by any conventional means the fragment of performance epi-position function. Consult for example R.A.Houghten, Proc.Natl.Acad.Sci.USA, 82:5131-5135,1985; Also be described in U.S. Patent number 4,631,211.

For the peptide that carries antigenic epitopes (namely comprising the zone that to be combined with antibody in the protein molecule) or the selection of polypeptide, this area is well-known, and the relatively short synthetic peptide of a simulated albumin matter sequence part can cause the antiserum that the protein with partial simulation can react usually. Consult for example J.G.Sutcliffe, T.M.Shinnick, N.Green and R.A.Learner, Science, 219:660-666,1983.

Often with the protein primary sequence peptide that can cause proteins react serum is described, can identify these peptides by the simple chemical rule of a cover, they both had been not limited to the immundominance zone (being the immunogenicity epi-position) of whole protein, also were not limited to amino or carboxyl terminal. Extremely hydrophobic peptide and contain 6 or still less the peptide of residue usually can not effectively induce can be in conjunction with the antibody of institute's simulated albumin matter; The peptide of growing, the peptide that especially contains proline residue is normally effective. The people such as Sutcliffe, the same, the 661st page. For example, in the 20 kinds of peptides design of these guides, that comprise 8-39 the residue that covers influenza virus hemagglutinin HA1 polypeptide chain sequence 75%, there are 18 kinds to induce the antibody that can react with HA1 albumen or intact virus; From 12 kinds in 12 kinds of peptides of MulV polymerase with from 18 kinds of antibody of all having induced precipitable separately protein in 18 kinds of peptides of rabies virus glycoprotein.

Therefore, but the present invention carries the peptide of antigenic epitopes and the antibody that polypeptide can be used for preparing specific binding polypeptide of the present invention, comprises monoclonal antibody. Thereby most of hybridomas that the splenocyte of the donor of the peptide immunity of carrying epitope by merging to use by oneself obtains can be secreted the antibody that reacts with native protein usually. The people such as Sutcliffe, the same, the 663rd page. Antibody with the peptide that carries antigenic epitopes or polypeptide preparation can be used for detecting the protein of simulating, and can be used for following the trail of the whereabouts in a plurality of zones of protein precursor of translating rear processing for the antibody of different peptides. The protein that peptide and anti-peptide antibody can be used for simulating multiple qualitatively or quantitatively determines method, competition assay for example because verified even small peptide (such as about 9 amino acid) all can be in immunoprecipitation assay combination and the larger peptide of displacement. Consult such as people such as Wilson Cell, 37:767-778,1984, the 777 pages. Anti-peptide antibody of the present invention also can be used for the protein that purifying is simulated, for example by using method well-known in the art to carry out adsorption chromatography.

Carry the peptide of antigenic epitopes and polypeptide according to the present invention of above-mentioned guide design and preferably contain contained in the polypeptid acid sequence of the present invention at least 7, more preferably at least 9, about about 30 amino acid whose sequences of 15-most preferably. Yet, comprise the major part (containing about 50 amino acid of about 30-) of polypeptid acid sequence of the present invention or as many as and comprise the whole amino acid sequences of polypeptide of the present invention any length or consisting of peptide or polypeptide, think that also the present invention carries peptide or the polypeptide of epi-position, and can be used for inducing the antibody that can react with the protein of simulating. Preferably, select to carry the amino acid sequence of peptide of epi-position so that good dissolubility (namely this sequence comprises relatively hydrophilic residue, preferably avoids highly hydrophobic sequence) to be provided in aqueous solvent; The sequence that particularly preferably comprises proline residue.

In the present invention, antigenic epitopes preferably comprises at least 4, at least 5, at least 6, at least 7, more preferably at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, about about 30 amino acid whose sequences of 15-most preferably. Comprise immunogenicity or antigenic epitopes or consisting of the length of preferred polypeptide be at least 10,15,20,25,30,35,40,45,50,55,60,65,70,75,80,85,90,95 or 100 amino acid residues.

Can be used for generating the antigenic polypeptide of MPIF-1 specific antibody or the non-limiting example of peptide comprises: comprise SEQ ID NO:2 the approximately the about 31 amino acids residues of 21-or consisting of polypeptide; Comprise SEQ ID NO:2 the approximately the about 44 amino acids residues of 31-or consisting of polypeptide; The polypeptide that comprises about about 55 amino acids residues of 49-of SEQ ID NO:2; The polypeptide that comprises about about 67 amino acids residues of 59-of SEQ ID NO:2; The polypeptide that comprises about about 83 amino acids residues of 72-of SEQ ID NO:2; The polypeptide that comprises about about 103 amino acids residues of 86-of SEQ ID NO:2; The polypeptide that comprises about about 120 amino acids residues of 110-of SEQ ID NO:2. As mentioned above, the inventor has determined that the aforementioned polypeptides fragment is the antigenicity zone of MPIF-1 albumen.

Other antigenic polypeptide or the peptide that can be used for generating the MPIF-1 specific antibody comprise that N end mentioned above and C hold deletant.

The present invention also provides the epitope Fragments of 137 amino acid whose MPIF-1 splice variants (SEQ ID NO:7). In particular, the invention provides the polypeptide of the amino acid sequence with following residue: the M1-T15 of SEQ ID NO:7; K2-A16; V3-L17; S4-G18; V5-S19; A6-Q20; A7-A21; L8-R22; S9-V23; C10-T24; L11-K25; M12-D26; L13-A27; V14-E28; T15-T29; A16-E30; L17-F31; G18-M32; S19-M33; Q20-S34; A21-K35; R22-L36; V23-P37; T24-L38; K25-E39; D26-N40; A27-P41; E28-V42; T29-L43; E30-L44; F31-D45; M32-M46; M33-L47; S34-W48; K35-R49; L36-R50; P37-K51; L38-I52; E39-G53; N40-P54; P41-Q55; V42-M56; L43-T57; L44-L58; D45-S59; M46-H60; L47-A61; W48-A62; R49-G63; R50-F64; K51-H65; I52-A66; G53-T67; P54-S68; Q55-A69; M56-D70; T57-C71; L58-C72; S59-I73; H60-S74; A61-Y75; A62-T76; G63-P77; F64-R78; H65-S79; A66-I80; T67-P81; S68-C82; A69-S83; D70-L84; C71-L85; C72-E86; I73-S87; S74-Y88; Y75-F89; T76-E90; P77-T91; R78-N92; S79-S93; I80-E94; P81-C95; C82-S96; S83-K97; L84-P98; L85-G99; E86-V100; S87-I101; Y88-F102; F89-L103; E90-T104; T91-K105; N92-K106; S93-G107; E94-R108; C95-R109; S96-F110; K97-C111; P98-A112; G99-N113; V100-P114; I101-S115; F102-D116; L103-K117; T104-Q118; K105-V119; K106-Q120; G107-V121; R108-C122; R109-M123; F110-R124; C111-M125; A112-L126; N113-K127; P114-L128; S115-D129; D116-T130; K117-R131; Q118-I132; V119-K133; Q120-T134; V121-R135; C122-K136; Or M123-N137.

By being used for generating any conventional method of peptide or polypeptide, comprise the recombination method that uses nucleic acid molecules of the present invention, can generate peptide and polypeptide that the present invention carries epi-position. For example, the amino acid sequence that carries short epitopes can be merged with larger polypeptide, this polypeptide is taken on carrier in the process of recombinant production and purifying, be used for generating anti-peptide antibody in immunologic process. Can also synthesize the peptide that carries epi-position with known chemical synthesis process. For example, Houghten has described a kind of straightforward procedure of synthetic big figure peptide, represent 248 kinds of 13 different residue peptide (R.A.Houghten of 10-20mg of the single amino acids variant of a section of HA1 polypeptide such as Preparation and identification within the time that was less than for 4 weeks (by ELISA type binding), solid phase is synthesized the universal method of big figure peptide fast: the specificity of the antigen-antibody interaction on each amino acid levels, Proc.Natl. Acad.Sci.USA, 82:5131-5135,1985). The method of this " simultaneously synthetic a plurality of peptides (SMPS) " is further described in the U.S. Patent number 4,631,211,1986 of authorizing the people such as Houghten. In the method, will be included in for each resin of the synthetic a plurality of peptides of solid phase the permeable bag of solvent separately, with many identical repeating step included in the optimum utilization solid phase method. Fully artificial method can be carried out synthetic (people such as Houghten, the same, the 5134th page) of 500-1000 or more kinds of peptides simultaneously.

Preferred nucleic acid fragment of the present invention comprises that the epi-position in the coding MPIF-1 albumen carries the nucleic acid molecules of part.

Particularly, these MPIF-1 nucleic acid fragments of the present invention comprise the nucleic acid molecules of the following polypeptide of encoding: comprise SEQ ID NO:2 the approximately the about 31 amino acids residues of 21-or consisting of polypeptide; The polypeptide that comprises about about 44 amino acids residues of 31-of SEQID NO:2; The polypeptide that comprises about about 55 amino acids residues of 49-of SEQ ID NO:2; The polypeptide that comprises about about 67 amino acids residues of 59-of SEQ ID NO:2; The polypeptide that comprises about about 83 amino acids residues of 72-of SEQ ID NO:2; The polypeptide that comprises about about 103 amino acids residues of 86-of SEQ ID NO:2; The polypeptide that comprises about about 120 amino acids residues of 110-of SEQ ID NO:2; Or wherein any scope or numerical value.

The present inventor has determined that the aforementioned polypeptides fragment is the antigenicity zone of MPIF-1 albumen. Be used for determining that other this class epi-position method partly of carrying of MPIF-1 albumen is described in detail in hereinafter.

According to method well-known in the art, include but not limited to immunity in the body, external immunity and phage display method, the polypeptide that carries epi-position of the present invention can be used for inducing antibody. Consult such as people such as Sutcliffe, see above; The people such as Wilson see above; With the people such as Bittle, J.Gen.Virol., 66:2347-2354,1985. If immunity in the use body, available free peptide immune animal; Yet, can be by peptide coupling macromolecular carrier (such as keyhole  hemocyanin (KLH) or tetanus toxoid) be strengthened tiring of anti-peptide antibody. For example, the peptide that can use the joint of a maleimide benzoyl-N-hydroxy-succinamide ester (MBS) will contain cysteine residues is coupled on the carrier, and can use bridging agent more commonly used (such as glutaraldehyde) that other peptide is coupled on the carrier. For example by in the peritonaeum and/or intracutaneous injection contain the emulsion of about 100 μ g peptides or carrier protein and Freunds adjuvant, with the peptide immune animal of free or coupling carrier, such as rabbit, rat and mouse. May need several times booster shots, about two weeks of interval for example, providing with the anti-peptide antibody of tiring, it for example can carry out the ELISA determination method by the free peptide that use is adsorbed onto solid phase surface and detect. Can improve by the selection of anti-peptide antibody and tire the antibody of for example selecting according to the peptide on the method absorption solid support well-known in the art and wash-out from the anti-peptide antibody of immune serum.

Can identify that according to the method that this area is known the present invention carries the peptide of immunogenicity epi-position, namely when whole protein is immunogene, cause those parts of antibody response in the protein. For example, people's (seeing above) such as Geysen discloses the method that is used on solid support fast hundreds of peptide that synthetic purity simultaneously is enough to react at enzyme-linked immunosorbent assay. Then, need not they and holder are separated the interaction that can detect easily synthetic peptide and antibody. Like this, those of ordinary skills can conventional evaluation carry the peptide of the immunogenicity epi-position of expectation protein. For example, the people such as Geysen orient the important epi-position of immunology in this protein by all 208 kinds six possible peptides of overlapping whole 213 the amino acid whose sequences of covering foot and mouth disease virus outer membrane protein of a synthetic cover with 7 amino acid whose resolution ratio. Then, a complete synthetic cover substitutes peptide, and wherein each position is used all 20 amino acid replacements successively in epi-position, determines the specific specific amino acids of giving with antibody response. Thereby, can conventionally prepare the peptide analogues that the present invention carries the peptide of epi-position by this method. The U.S. Patent number 4,708,781 of authorizing Geysen (1987) has also been described the method for the peptide of identifying the immunogenicity epi-position of carrying expectation protein.

Also have, authorize the U.S. Patent number 5 of Geysen (1990), 194,392 have described the universal method that detects or measure monomer (amino acid or other compound) sequence, and described monomer is the epi-position topology equivalent (be mimotope (mimotope)) complementary with the particular complementary position (antigen binding site) of purpose antibody. Say that more generally the U.S. Patent number 4,433,092 of authorizing Geysen (1989) has been described and detected or the method for definite sequence monomer, described monomer is the part topology equivalent with the ligand binding site complementation of specific purpose acceptor. Similarly, the U.S. Patent number 5,480,971 of authorizing the people's such as R.A.Houghten (1996) relevant full alkanisation oligo peptide discloses linear C₁-C ₇Series and the library of the full alkanisation oligopeptides of-alkyl and this peptide, and use this oligopeptides series and library to measure the method for sequence of the full alkanisation oligopeptides of preferential binding purpose acceptor molecule. Thereby, also can conventionally prepare the non-peptide analogues that the present invention carries the peptide of epi-position by these methods.

The complete content of every part of file that " polypeptide and peptide " this part is quoted is collected herein by reference.

Those skilled in the art will understand, and MPIF-1 polypeptide of the present invention mentioned above and epi-position thereof are carried part can partly unite the formation chimeric polyeptides with the constant region of immunoglobulin (Ig) (IgG). These fusions are convenient to purifying and are shown that half life increases in the body. For example, the chimeric protein that is comprised of each domain of the first two domain of people CD4 polypeptide and mammalian immune globulin heavy chain or constant region of light chain namely shows this phenomenon, and (EPA 394,827; The people such as Traunecker, Nature, 331:84-86,1988). The fusion that has the dimeric structure that is connected by disulfide bond because of IgG part aspect combination and other molecule that neutralizes than the more effective (people such as Fountoulakis of independent monomeric form MPIF-1 albumen or protein fragments, J.Biochem., 270:3958-3964,1995).

Those skilled in the art will understand, and as mentioned above, comprise immunogenicity or antigenic epitopes or consisting of polypeptide of the present invention can merge allogeneic polypeptide sequence. For example, polypeptide of the present invention can merge the constant domain of immunoglobulin (Ig) (IgA, Ige, IgG, IgM) or its part (CH1, CH2, CH3, comprise their any combination all or in part) and produces chimeric polyeptides. These fused proteins are convenient to purifying and are shown that half life increases in the body. For example, the chimeric protein that is comprised of each domain of the first two domain of people CD4 polypeptide and mammalian immune globulin heavy chain or constant region of light chain namely shows this phenomenon, and (EPA 394,827; The people such as Traunecker, Nature, 331:84-86,1988). The fusion that has the dimeric structure that is connected by disulfide bond because of IgG part aspect combination and other molecule that neutralizes than independent monomeric form polypeptide or its fragment is more effective (consults such as people such as Fountoulakis, J.Biochem., 270:3958-3964,1995). The nucleic acid of the above-mentioned epi-position of encoding also can be used as detection and the purifying that epitope tag and genes of interest recombinate to help expressed polypeptide.

Can be by gene shuffling, primitive reorganization, extron reorganization and/or codon reorganization (being referred to as " DNA reorganization ") technology one-tenth in next life other fusion of the present invention. DNA reorganization can be used for regulating the activity corresponding to the polypeptide of SEQ ID NO:2, effectively generates thus activator and the antagonist of polypeptide. Usually consult U.S. Patent number 5,605,793,5,811,238,5,830,721,5,834,252 and 5,837,458, and the people such as Patten, Curr.Opinion Biotechnol., 8:724-733,1997; S.Harayama, Trends biotechnol., 16 (2): 76-82,1998; The people such as Hansson, J.Mol.Biol., 287:265-276,1999; And M.M.Lorenzo and R.Blasco, Biotechniques, 24 (2): 308-313,1998 (with these patents and deliver thing and be collected herein by reference). In one embodiment, can by DNA reorganize to realize corresponding to SEQ ID NO:1 or 6 and the polynucleotides of corresponding polypeptide change. DNA reorganization comprises by homology or site-specific restructuring two or more DNA sections is assembled into expectation molecule corresponding to polynucleotides SEQ ID NO:1 of the present invention or 6. In another embodiment, can before restructuring, carry out random mutagenesis through fallibility PCR, random nucleotide insertion or other method, thereby change polynucleotides and corresponding polypeptide corresponding to SEQ ID NO:1 or 6. In another embodiment, can with corresponding to the coded polynucleotide of SEQ ID NO:1 or 6 or by one or more elements of the polypeptide of its coding, primitive, section, partly, one or more elements of domain, fragment etc. and one or more heterologous molecule, primitive, section, partly, the restructuring such as domain, fragment. The purification and separation of polypeptide

By comprising the method for ammonium sulfate or ethanol precipitation, acid extractants, anion or cation-exchange chromatography, cellulose phosphate chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and agglutinin chromatography, reclaimed and purifying MPIF-1 by the recombinant cell culture thing. As required, can use protein again folding step to realize the configuration of maturation protein. At last, can adopt high performance liquid chroma-tography (HPLC) for last purification step.

Polypeptide of the present invention can be the product of natural purifying, the product of chemical synthesis flow process or the product that is generated by protokaryon or eucaryon host (bacterium, yeast, higher plant, insect and the mammalian cell for example cultivated) by recombinant technique. According to the host who adopts in the recombinant production flow process, polypeptide of the present invention can be by mammal or other eucaryote carbohydrate glycosylation, or nonglycosylated. Polypeptide of the present invention also can comprise initial methionine residues.

In addition, can use the technology chemical synthesis polypeptide of the present invention that this area knows (consult such as Creighton, 1983, " Proteins:Structures and Molecular Principles " (protein: structure and molecular principle), W.H.Freeman ﹠ Co., New York; With the people such as M.Hunkapiller, Nature, 310:105-111,1984). For example, can use the synthetic peptide corresponding to MPIF polypeptide fragment of the present invention of peptide synthesizer. In addition, if necessary, can be with nonclassical amino acid or chemical amino acid analogue as an alternative or add and import the MPIF-1 peptide sequence. Nonclassical amino acid includes but not limited to D-isomers, 2,4-diamino-butanoic, α-aminoacid, 4-Aminobutanoicacid, Abu, 2-amino-butyric acid, g-Abu, e-Ahx, 6-aminocaprolc acid, Aib, 2-aminoisobutyric acid, 3-alanine, ornithine, nor-leucine, norvaline, hydroxyproline, methyl amimoacetic acid, citrulling, homotype citrulling, cystine, tert-butyl group glycine, tert-butyl group alanine, phenylglycine, Cyclohexylalanine, Beta-alanine, fluoroamino acid, design amino acid such as Beta-methyl amino acid, Ca-methylamino acid, Na-methylamino acid and the common amino acid analogue of common amino acid. In addition, amino acid can be D type (dextrorotation) or L-type (left-handed).

The present invention is encompassed among the translation process or the MPIF polypeptide of different modifying afterwards, is connected such as the derivatization of glycosylation, acetylation, phosphorylation, amidatioon, known protection/blocking groups, proteolysis cutting, with antibody molecule or other cell ligand etc. Can carry out any in many chemical modifications by known technology, include but not limited to cyanogen bromide, trypsase, chymotrypsin, papain, V8 protease, NaBH₄The specificity chemical cleavage; Acetylation, formylated, oxidation, reduction; The metabolic that carries out in the situation of tunicamycin is synthetic existing; Deng.

Other posttranslational modification is also contained in the present invention, comprises that N-for example connects or processing, chemical part that O-connects carbohydrate chain, N end or C end adhering on amino acid backbone, N-connects or O-connects the chemical modification of carbohydrate chain and the terminal methionine residues of N and expresses because of prokaryotic host cell and add or lack. Also available detectable label modified polypeptide such as enzyme, fluorescein, isotope or affinity labeling, thereby can detect and isolated protein.

The present invention also provides the chemical modification derivative of polypeptide of the present invention, and they can provide extra advantage, such as solubility, stability and the circulation timei of improving polypeptide, or reduces immunogenicity (consulting U.S. Patent number 4,179,337). The chemical part that is used for deriving can be selected from water-soluble polymer, such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethyl cellulose, glucan, polyvinyl alcohol etc. Also can be at intramolecular random site or intramolecular precalculated position modified polypeptide, and can comprise one, two, three or the chemical part that adheres to more.

Polymer can be any molecular weight, and can be branch or unbranched. For polyethylene glycol, preferred molecular weight be between the about 100kDa of about 1kDa-(term " approximately " is illustrated in the polyethylene glycol preparation, and some molecule can be higher or low than described molecular weight) so that operate and manufacturing. Also can use other big or small molecule, this depends on the treatment curve (for example slowly-releasing duration, the effect (if any) to BA, easy degree, antigenic degree and the shortage processed and polyethylene glycol are to other known action of therapeutic protein or analog) of expectation.

For the consideration on the impact of the function of protein or antigenicity domain, peg molecule (or other chemical part) should be attached to protein. Those skilled in the art can utilize multiple adherence method, for example EP 0401,384, (coupling PEG to G-CSF) is collected herein by reference, also can consult the people such as Malik, Exp.Hematol., 20:1028-1035,1992 (reported and used the trifluoro ethyl sulfonic chloride to the PEGization of GM-CSF). For example, can be by amino acid residue through reactive group (such as free amino or carboxyl) covalent bond polyethylene glycol. Reactive group be activated polyethylene glycol molecule combinative those. Amino acid residue with free amine group can comprise lysine residue and N terminal amino acid residue; Amino acid residue with free carboxy can comprise asparagicacid residue, glutaminic acid residue and C terminal residue. Mercapto groups also can be used as reactive group and is used for adhering to peg molecule. For therapeutic purposes preferably in the adhering to of amino, such as adhering at N end or lysine group place.

People may be desirably in the terminally chemically modified protein of N especially. Use polyethylene glycol as the illustration of the present composition, can select multiple peg molecule (as according to molecular weight, branch etc.), peg molecule and the ratio of protein (or polypeptide) molecule in reactant mixture, pending pegylation reaction type, and obtain the method for the terminal pegylated protein of selected N. The method (if necessary namely, the single Pegylation of this part and other partly being separated) that obtains the terminal polyethylene glycol chemical preparation of N can be by by purifying N end Pegylation material in the pegylated protein molecule colony. Can realize to the terminal chemical modification of the selective N of protein that by the reduction alkanisation this method has been utilized can be for the differential responses of the dissimilar primary amino radical groups (lysine and N are terminal) that utilize in specified protein is derived. Under suitable reaction condition, can basically realize with the polymer that contains carbonyl at the terminal selective derivatization protein of N. Antibody

Can prepare the MPIF-1 protein specific antibody that uses among the present invention for complete MPIF-1 albumen or its antigenic polypeptide fragment, described albumen or fragment can be passed animal system (such as rabbit or mouse) by being with carrier protein (white such as clear chela), if perhaps its sufficiently long words (about at least 25 amino acid) then need not carrier.

When being used for this paper, term " antibody (Ab) " or " monoclonal antibody (Mab) " be intended to comprise can specific binding MPIF-1 albumen complete molecule and antibody fragment (such as Fab and F (ab) '₂Fragment). Fab and F (ab) '₂Fragment lacks the Fc fragment of complete antibody, faster and may have the non-specific tissue bond lower than complete antibody people such as (, J.Nucl.Med., 24:316-325,1983) Wahl by the speed of loop cleaning. Thereby these fragments are preferred.

Polypeptide, their fragment, other derivative or analog or the cell of expressing them can be used as immunogene and are used for generating antibody for them. These antibody can be for example polyclone or monoclonal antibody. The present invention also comprises the product of chimeric, strand, humanized antibody and Fab fragment or Fab expression library. Can generate with the several different methods that this area is known these antibody and fragments.

The invention still further relates to comprise peptide sequence shown in SEQ ID NO:2 or 7 or preservation clone the coded peptide sequence of contained cDNA epi-position or consisting of polypeptide fragment. The polynucleotides (such as disclosed sequence in SEQ ID NO:1 or 6) of these epi-positions of encoding are also contained in the present invention, and the nucleotide sequence of the complementary strand of the polynucleotides of these epi-positions of encoding. And the polynucleotides that under rigorous hybridization conditions or low rigorous condition, can hybridize with complementary strand.

In the present invention, " epi-position " refers to that (especially in human body) has antigenicity or immunogenic polypeptide fragment in animal. The preferred embodiments of the invention relate to comprise epi-position or consisting of polypeptide fragment, and the polynucleotides of this fragment of encoding. In the protein molecule antibody can in conjunction with zone definitions be " antigenic epitopes ". On the contrary, " immunogenicity epi-position " is defined as the part that causes antibody response in the protein. Consult such as people such as Geysen Proc. Natl.Acad.Sci.USA, 81:3998-4002,1993.

But antigenic epitopes can be used for for example preparing the antibody of specific binding epi-position, comprises that monoclonal antibody (consults such as people such as Wilson Cell, 37:767-778,1984; The people such as Sutcliffe, Science, 219:660-666,1983).

Similar, according to method well-known in the art, the immunogenicity epi-position can be used for such as inducing antibody (to consult such as people such as Sutcliffe, see above; The people such as Wilson see above; The people such as Chow, Proc.Natl.Acad.Sci.USA, 82:910-914, defective year part; With the people such as Bittle, J.Gen.Virol., 66:2347-2354,1985). Preferred immunogenicity epi-position comprises secretary protein. The immunogenicity epi-position can be with carrier protein (such as albumin) passs animal system (such as rabbit and mouse), if perhaps sufficiently long words (about at least 25 amino acid) then can need not carrier. Yet, comprise and few show namely that to 8-10 amino acid whose immunogenicity epi-position be enough to cause at least can be in conjunction with the antibody (as in the Wstern trace) of the linear epitope in the sex change polypeptide.

But the invention still further relates to antibody and the T cell antigen receptor (TCR) of specific binding polypeptide of the present invention. Antibody of the present invention comprises that IgG (comprises IgG₁、IgG ₂、IgG ₃, and IgG₄), IgA (comprises IgA₁And IgA₂), IgD, IgE, IgM and IgY. When being used for this paper, term " antibody (Ab) " is intended to comprise complete antibody (comprising the strand complete antibody) and Fab thereof. Most preferably, antibody is human antigen's binding antibody fragment of the present invention, includes but not limited to Fab, Fab ' and F (ab ')₂, the Fv (sdFv) that connects of Fd, scFv (scFv), single-chain antibody, disulfide bond and comprise V_LOr V_HThe fragment of domain. Antibody can from any animal origin, comprise birds and mammal. Preferably, antibody is from people, mouse, rabbit, goat, cavy, camel, horse or chicken.

Fab (comprising single-chain antibody) can only comprise the variable region or also comprise following domain complete or part: hinge area, CH₁、CH ₂, and CH₃Domain. The present invention also comprises variable region and hinge area, CH₁、CH ₂, and CH₃Any combination of domain. But the present invention also comprises the monoclonal, polyclone of specific binding polypeptide of the present invention, chimeric, humanization and human monoclonal and people's polyclonal antibody. The present invention also comprises the anti-idiotype for antibody of the present invention.

Antibody of the present invention can be monospecific, bispecific, tri-specific or more much higher heavy specific. Multiple specific antibody can have specificity for the different epi-positions of polypeptide of the present invention, or can all have specificity for polypeptide of the present invention and for allos component (such as heterologous polypeptide or solid support material). Consult for example WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; The people such as A.Tutt, J.Immunol., 147:60-69,1991; U.S. Patent number 5,573,920,4,474,893,5,601,819,4,714,681,4,925,648; The people such as S.A.Kostelny, J.Immunol., 148:1547-1553,1992.

Antibody of the present invention can be described or illustrate by epi-position or the part of polypeptide of the present invention, and described epi-position or part can be by antibody recognition or specific bindings. Epi-position or polypeptide portion can as described hereinly be illustrated, for example and C terminal position terminal by N, the size by the contiguous amino acid residue or as show and figure in listed illustrating. But the antibody of any epi-position of specific binding the present invention or polypeptide also can foreclose. Therefore, but the present invention includes the antibody of specific binding polypeptide of the present invention, and allow these antibody are foreclosed.

Antibody of the present invention also can be described or illustrate by their cross reactivity. Comprise not the antibody in conjunction with any other analog, orthoevolution homologue or the homologue of polypeptide of the present invention. The present invention does not also comprise in conjunction with having with polypeptide of the present invention and is lower than 95%, is lower than 90%, is lower than 85%, is lower than 80%, is lower than 75%, is lower than 70%, is lower than 65%, is lower than 60%, is lower than 55%, is lower than 50% homogeneity the antibody of the polypeptide of (use this area know calculate with method described herein). The present invention also comprises only in conjunction with the antibody by the coded polypeptide of the polynucleotides that can hybridize with polynucleotides of the present invention under rigorous hybridization conditions (as described herein). Antibody of the present invention also can be described or illustrate by their binding affinity. Preferred binding affinity comprises that those dissociation constants or Kd are lower than 5 * 10^-6M、10 ^-6M、5×10 ^-7M、10 ^-7M、5×10 ^-8M、 10 ^-8M、5×10 ^-9M、10 ^-9M、5×10 ^-10M、10 ^-10M、5×10 ^-11M、10 ^-11M、5×10 ^-12M、10 ^-12M、 5×10 ^-13M、10 ^-13M、5×10 ^-14M、10 ^-14M、5×10 ^-15M or 10^-15The affinity of M.

Antibody of the present invention for example can be used for, and includes but not limited to the method for purifying, detection and the target polypeptide of the present invention known this area, comprises diagnosis and methods for the treatment of in external and the body. For example, antibody can be used in the immunoassay, measures the level of polypeptide of the present invention in biological sample with quantitative and qualitative analysis. Consult such as people such as Harlow, and " Antibodies:A Laboratory Mamual " (antibody: lab guide), publishing house of cold spring harbor laboratory, the 2nd edition, 1988 (being collected herein by reference).

Antibody of the present invention can use separately or unite with other component. Antibody can be further merges heterologous polypeptide at N or the terminal restructuring of C, or chemically conjugated polypeptide or other component (comprise covalency with non-covalent puting together). For example, antibody of the present invention can be recombinated and be merged or be conjugated in molecule and the effector molecules molecule (such as heterologous polypeptide, medicine or toxin) that can be used as mark in the detection assay method. Consult for example WO 92/08495; WO 91/14438; WO 89/12624; U.S. Patent number 5,314,995; With EP 396,387.

Can prepare antibody of the present invention by any proper method that this area is known. For example, polypeptide of the present invention or its antigenicity fragment can be applied to animal, generate the serum that contains polyclonal antibody to induce. Term " monoclonal antibody " is not limited to the antibody by the hybridoma technology generation. Term " monoclonal antibody " refers to derived from the monospecific polyclonal antibody of (comprising any eucaryon, protokaryon or phage clone), rather than refers to generate its method. Can prepare monoclonal antibody with the multiple technologies that this area is known, comprise the use of hybridoma, restructuring and display technique of bacteriophage.

Hybridoma technology comprises that this area is known and the people such as Harlow, and " Antibodies:A Labotatory Manual " (antibody: lab guide), publishing house of cold spring harbor laboratory, the 2nd edition, 1988; The people such as Hammerling, " Monoclonal Antibodies and T Cell Hybridomas " (monoclonal antibody and T quadroma), 563-681 page or leaf, Elsevier, New York, the technology of instruction in 1981 (being collected herein by reference described document is complete). Can use such as papain (produce Fab fragment) or trypsase (produce F (ab ')₂Fragment) generates Fab and F (ab ') by proteolysis cutting₂Fragment.

Perhaps, can be by using recombinant DNA technology and display technique of bacteriophage or the synthetic chemistry method by the method known with this area prepares antibody of the present invention. For example, can prepare antibody of the present invention with the multiple phage display method that this area is known. In the phage display method, the functional antibodies domain is illustrated in the surface of the phage particle that carries their polynucleotide sequence of coding. By antigen, normally with solid phase surface or pearl in conjunction with or the antigen of catching directly select, selected to have the bacteriophage of expectation binding characteristic by library or combinatorial antibody storehouse (such as people or mouse). The bacteriophage of using in these methods is normally carried and phage gene III or Fab, the Fv of gene VIII protein restructuring fusion or the filobactivirus in disulfide bond stabilisation Fv antibody structure territory, comprises fd and M13. The example that can be used for generating the phage display method of antibody of the present invention is included in the method for describing in the following document: the people such as Brinkman, J. Immunol.Methods, 182:41-50,1995; The people such as Ames, J.Immunol. Methods, 184:177-186,1995; The people such as Kettleborough, Eur.J. Immunol., 24:952-958,1994; The people such as Persic, Gene, 187:9-18,1997; The people such as Burton, Advances in Immunology, 57:191-280,1994; PCT/GB91/01134; WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; With U.S. Patent number 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727 and 5,733,743 (being collected herein by reference described document is complete).

Described in above-mentioned document, after bacteriophage is selected, can be by bacteriophage separation antibody code area, and for the Fab that generates complete antibody (comprising human antibodies) or any other expectation, and in host's (comprising mammalian cell, insect cell, plant cell, yeast and bacterium) of any expectation, express. For example, also can adopt for restructuring and generate Fab, Fab ' and F (ab ')₂The technology of fragment, the method for using this area to know is such as the method for describing in following document: WO 92/22324; The people such as Mullinax, BioTechniques, 12 (6): 864-869,1992; The people such as Sawai, AJRI, 34:26-34,1995; With the people such as Better, Science, 240:1041-1043, disclosed method in 1988 (being collected herein by reference described document is complete).

The example that can be used for generating the technology of scFv and antibody comprises U.S. Patent number 4,946,778 and 5,258,498; The people such as Huston, Methods in Enzymology, 203:46-88,1991; The people such as Shu, PNAS, 90:7995-7999,1993; With the people such as Skerra, Science, 240:1038-1040, the technology of describing in 1988. For some purposes, comprise in the body of antibody in the people and to use and the vitro detection determination method, preferably use chimeric, humanization or people's antibody. The method that is used for the generation chimeric antibody is known in this area. Consult such as people such as Morrison Science, 229:1202,1985; The people such as Oi, BioTechniques, 4:214,1986; The people such as Gillies, J.Immunol.Methods, 125:191-202; With U.S. Patent number 5,807,715. Can use several different methods to make the antibody humanization, (EP 239,400 to comprise the CDR transplanting; WO 91/09967; U.S. Patent number 5,530,101; With 5,585,089), inlay or resurfacing (EP 592,106; EP 519,596; E.A.Padlan, Molecular Immunology, 28 (4/5): 489-498,1991; The people such as Studnicka, Protein Engineering, 7 (6): 805-814,1994; The people such as Roguska, PNAS, 91:969-973,1994) and chain reorganization (U.S. Patent number 5,565,332). Can generate people's antibody by the several different methods that this area is known, comprise phage display method mentioned above. Also can consult U.S. Patent number 4,444,887,4,716,111,5,545,806 and 5,814,318; With WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735 and WO 91/10741 (being collected herein by reference described document is complete).

The present invention comprises also that restructuring is merged or the antibody of chemically conjugated (comprise covalency with non-covalent puting together) polypeptide of the present invention. Antibody can have specificity to the antigen except polypeptide of the present invention. For example, by polypeptide of the present invention being merged or puts together specific antibody for the specific cells surface receptor, antibody be used in external or in vivo with polypeptide target of the present invention to particular cell types. The method of using this area to know merges or the antibody of puting together polypeptide of the present invention also can be used in vitroimmunoassay method and purification process. Consult such as people such as Harbor, see above; WO 93/231232; EP 439,095; The people such as Naramura, Immunol.Lett., 39:91-99,1994; U.S. Patent number 5,474,981; The people such as Gillies, PNAS, 89:1428-1432,1992; The people such as Fell, J.Immunol., 146:2446-2452,1991 (being collected herein by reference described document is complete).

The present invention also comprises the composition that comprises the polypeptide of the present invention that merges with antibody structure territory except the variable region or put together. For example, polypeptide of the present invention can merge or put together antibody Fc district or its part. The antibody moiety that merges with polypeptide of the present invention can comprise hinge area, CH₁Domain, CH₂Domain and CH₃Any combination of domain or complete structure territory or its part. The method of using this area to know can merge polypeptide of the present invention or put together above-mentioned antibody moiety with half life in the body that improves polypeptide or be used for immunoassay. Polypeptide also can merge or put together above-mentioned antibody moiety to form polymer. For example, the Fc part of fusion polypeptide of the present invention can form dimer by the disulfide bond between the Fc part. Can generate by the partial fusion with polypeptide and IgA and IgM higher polymer form. Be used for polypeptide of the present invention and antibody moiety fusion or the method for puting together are known in this area. Consult for example U.S. Patent number 5,336,603,5,622,929,5,359,046,5,349,053,5,447,851,5,112,946; EP 307,434, EP 367,166; WO 96/04388, WO 91/06570; The people such as Ashkenazi, PNAS, 88:10535-10539,1991; The people such as Zheng, J.Immunol., 154:5590-5600,1995; With the people such as Vil, PNAS, 89:11337-11341,1992 (being collected herein by reference described document is complete).

The invention still further relates to the activator of taking on polypeptide of the present invention or the antibody of antagonist. For example, the present invention includes the interactional antibody that can partly or entirely destroy receptor/ligand and polypeptide of the present invention. Comprising receptor specific antibody and ligand specificity's antibody. Also comprise the receptor specific antibody that does not stop ligand binding but stop receptor activation. Can measure receptor activation (being the signal conduction) by other technology that technology described herein or this area are known. Also comprise the receptor specific antibody that not only stops ligand binding but also stop receptor activation. Similarly, but wherein also comprise binding partner and stop part and the neutralizing antibody of receptors bind, but and binding partner stop thus receptor activation but do not stop the antibody of ligand binding acceptor. But the antibody that also comprises activated receptor. These antibody can be taken on the activator of all or part BA of being realized by ligand-mediated receptor activation. Antibody can be elaborated into activator or the antagonist of the BA that comprises activity specific described herein. Can generate with the technology that this area is known above-mentioned antibody agonist. Consult for example WO 96/40281; U.S. Patent number 5,811,097; The people such as Deng, Blood, 92 (6): 1981-1988,1998; The people such as Chen, Cancer Res., 58 (16): 3668-3678,1998; The people such as Harrop, J.Immunol., 161 (4): 1786-1794,1998; The people such as Zhu, Cancer Res., 58 (15): 3209-3214,1998; The people such as Yoon, J.Immunol., 160 (7): 3170-3179,1998; The people such as Prat, J.Cell. Sci., 111 (Pt2): 237-247,1998; The people such as Pitard, J.Immunol.Methods, 205 (2): 177-190,1997; The people such as Liautard, Cytokine, 9 (4): 233-241,1997; The people such as Carlson, J.Biol.Chem., 272 (17): 11295-11301,1997; The people such as Taryman, Neuron, 14 (4): 755-762,1995; The people such as Muller, Srtructure, 6 (9): 1153-1167,1998; The people such as Bartunek, Cytokine, 8 (1): 14-20,1996 (being collected herein by reference all documents are complete).

As mentioned above, in addition, can use the well-known technology of those skilled in the art, utilization (is consulted for example Greenspan and Bona for the anti-idiotype of antibody generation " simulation " polypeptide of the present invention of polypeptide of the present invention, FASEB J., 7 (5): 437-444,1989; And Nissinoff, J.Immunol., 147 (8): 2429-2438,1991). For example, can in conjunction with and the competitive antibody that suppresses polypeptide multimerization and/or polypeptide of the present invention and ligand binding can be used for generating " simulation " polypeptide polymerization and/or binding structural domain thereby can be in conjunction with in also and the anti-idiotype of polypeptide and/or its part. These neutrality anti-idiotypes or its Fab fragment can be used for therapeutic scheme with in and polypeptide ligand. For example, these anti-idiotypes can be used for blocking thus its BA in conjunction with polypeptide of the present invention and/or in conjunction with its ligand/receptor.

Can be by polypeptide being injected directly in the animal or obtaining the antibody that generates for polypeptide or its body inner recipient corresponding to sequence of the present invention by polypeptide being applied to animal (preferred non-human animal). But the antibody Binding peptide self that then so obtains. Like this, even only also can be used for generating can be in conjunction with the antibody of whole natural polypeptides for the sequence of coded polypeptide fragment. Then these antibody can be used for separating described polypeptide by the tissue of expressing polypeptide.

In order to prepare monoclonal antibody, can use any technology that the antibody that is produced by the continuous cell line culture can be provided. Hybridoma technology (Kohler and Milstein for example, Nature, 256:495-497,1975), three knurl technology, human B-lymphocyte hybridoma technology (people such as Kozbor, Immunology Today, 4:72,1983) and be used for to generate EBV hybridoma technology (people such as Cole, " Monoclonal Antibodies and Cancer Therapy " (monoclonal antibody and cancer therapy), the Alan R.Liss company of human monoclonal antibodies, the 77-96 page or leaf, 1985).

The technology (U.S. Patent number 4,946,778) that describe to be used for generates single-chain antibody can be used for generating the single-chain antibody for immunogenic polypeptide product of the present invention.

Can be prepared by a number of procedures antibody of the present invention. For example, can use the cell of expressing MPIF-1 albumen or its antigenicity fragment to animal and generate the serum that contains polyclonal antibody to induce. In a preferred method, preparation and purifying MPIF-1 preparation make it be substantially free of natural pollutant. Then these preparations are imported animals and have polyclonal antiserum than high specific acitivity with generation.

In most preferred method, antibody of the present invention is monoclonal antibody (or its MPIF-1 protein combination fragment). Useful hybridoma technology prepares these monoclonal antibodies (people such as Kohler, Nature, 256:495,1975; The people such as Kohler, Eur.J.Immunol., 6:511,1976; The people such as Kohler, Eur.J.Immunol., 6:292,1976; The people such as Hammerling, " Monoclonal Antibodies and T Cell Hybridomas " (monoclonal antibody and T quadroma), 563-681 page or leaf, Elsevier, New York, 1981). Generally speaking, these flow processs comprise with the MPIF-1 proteantigen or more preferably express the cellular immunity animal (preferred mouse) of MPIF-1 albumen. Can identify suitable cell by them in conjunction with the ability of anti-MPIF-1 protein antibodies. Can in any suitable culture medium for tissue culture, cultivate these cells; Yet, preferred cultured cell in the amended EagleShi culture medium of EarleShi, add 10% hyclone (in about 56 ℃ of deactivations) in the described culture medium, and add about 10 μ g/l nonessential amino acid, about 1000U/ml penicillin and about 100 μ g/ml streptomysins. Extract the splenocyte of these mouse and make it and merge with suitable myeloma cell line. Can adopt any suitable myeloma cell line according to the present invention; Yet, preferably adopt parent myeloma cell lines (SP20), they can be obtained by American type culture collection (Rockville, Maryland). After the fusion, in the HAT culture medium, selectively keep the hybridoma of generation, then such as people such as Wands, Gastroenterology, 80:225-232,1981 describedly clone by limiting dilution assay. Then measure the hybridoma that obtains by this selection, to identify that secrete can be in conjunction with the clone of the antibody of MPIF-1 proteantigen.

Perhaps, can generate in using two step flow processs of anti-idiotype can be in conjunction with other antibody of MPIF-1 proteantigen. This method has been utilized the following fact: namely antibody self also is antigen, and therefore might obtain can be in conjunction with the antibody of another kind of antibody. According to this method, the MPIF-1 protein specific antibody is used for immune animal, preferred mouse. Then the splenocyte of this animal is used for generating hybridoma, and the clone of screening hybridoma to identify that the antibody that generated can be blocked by the MPIF-1 proteantigen in conjunction with the ability of MPIF-1 protein specific antibody. These antibody comprise the anti-idiotype for the MPIF-1 protein specific antibody, and can be used for immune animal to induce other MPIF-1 protein specific antibody of generation.

Can understand, can use Fab and F (ab ') according to method disclosed herein₂Other fragment with antibody. Common use such as papain (producing the Fab fragment) or pepsin (generation F (ab ')₂Fragment) generates these fragments by proteolysis cutting. Perhaps, can be by using recombinant DNA technology or generating MPIF-1 protein combination fragment by the synthetic chemistry method.

May preferably use " humanization " chimeric mAb. Can generate these antibody with the genetic constructs of being derived by the hybridoma that generates monoclonal antibody mentioned above. The method that is used for the generation chimeric antibody is known in this area. Summary is consulted Morrison, Science, 229:1202,1985; The people such as Oi, BioTechniques, 4:214,1986; The people such as Cabilly, U.S. Patent number 4,816,567; The people such as Taniguchi, EP 171,496; The people such as Morrison, EP 173,494; The people such as Neuberger, WO 86/01533; The people such as Robinson, WO 87/02671; The people such as Boulianne, Nature, 312:643,1984; The people such as Neuberger, Nature, 314:268,1985.

Hereinafter provide other label that is applicable to MPIF-1 protein specific antibody of the present invention. The example of suitable enzyme labeling thing comprises malic dehydrogenase, staphylococcal nuclease, δ-5-steroids isomerase, YAD, alpha-phosphate glycerol dehydrogenase, phosphotriose isomerase, peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, β-galactosidase, ribalgilase, urase, catalase, glucose-6-phosphate dehydrogenase (G6PD), glucoamylase and acetylcholinesterase.

The example of suitable radioisotopic tracer comprises³H、 ¹¹¹In、 ¹²⁵I、 ¹³¹I、 ³²P、 ³⁵S、 ¹⁴C、 ⁵¹Cr、 ⁵⁷To、 ⁵⁸Co、 ⁵⁹Fe、 ⁷⁵Se、 ¹⁵²Eu、 ⁹⁰Y、 ⁶⁷Cu、 ²¹⁷Ci、 ²¹¹At、 ²¹²Pb、 ⁴⁷Sc、 ¹⁰⁹Pd, etc. When using in-vivo imaging,¹¹¹In is preferred isotope, because it has avoided liver pair¹²⁵I or¹³¹The problem of the dehalogenation of I labeled monoclonal antibody. In addition, this radioactivity element has more favourable γ radiant (people such as Perkins, Eur.J.Nucl. Med., 10:296-301,1985 for imaging; The people such as Carasquillo, J.Nucl.Med., 28:281-287,1987).

The example of suitable non radioactive isotope label comprises¹⁵⁷Gd、 ⁵⁵Mn、 ¹⁶²Dy、 ⁵²Tr and⁵⁶Fe。

The example of suitable fluorescent marker comprises¹⁵²Eu label, fluorescein-labelled thing, isothiocyanic acid label, rhodamine label, phycoerythrin label, phycocyanin label, allophycocyanin label, phthalic aldehyde label and fluorescamine label.

The example of suitable tagged toxin substrate comprises diphtheria toxin, ricin (WA) and cholera toxin.

The example of chemiluminescent labels comprises luminal label, different luminal label, fragrant acridinium ester label, imidazoles label, acridinium salt label, oxalate label, luciferin label, luciferase label and aequorin label.

The example of nuclear magnetic resonance contrast medium comprises the heavy metal nucleon, such as Gd, Mn and Fe. The people such as Kennedy, Clin.Chim.Acta, 70:1-31,1976 and the people such as Schurs, Clin.Chim.Acta, 81:1-40,1977 provide and have been used for typical technology that label mentioned above is combined with antibody. The coupling technology of mentioning among the latter is glutaraldehyde method, periodate method, dimaleimide method, a maleimide benzyl-N-hydroxyl-succinimide ester method (all these methods are collected herein by reference). Chromosome is measured

Nucleic acid molecules of the present invention also can be used for Chromosome Identification. Ad-hoc location on the single human chromosome of sequence-specific target also can be hybridized with it. In addition, the present specific site that needs to identify on the chromosome. The chromosome marking reagent based on actual sequence data (Repeat Polymorphism) that can be used at present the marker chromosome position is less. The important first step that those sequences and disease related gene are interrelated is according to the present invention DNA to be plotted on the chromosome.

In some preferred embodiment in this respect, cDNA disclosed herein is used for the genomic DNA of clone MPIF-1 GFP. This can realize that with well-known technology and library they can obtain by commercial sources usually. Then use the well-known technology that is used for this purpose that genomic DNA is used for the mapping of native staining body. Usually, according to the old process that is used for chromosome mapping, may need some tests and error to identify the genomic probe of the in situ hybridization signal that can produce.

In brief, sequence can be plotted on the chromosome by prepared PCR primer (preferred 15-25bp) by cDNA. Can utilize the cDNA Computer Analysis to select fast primer, the zone that described primer is crossed in genomic DNA is no more than an extron, thereby can not make the amplification procedure complicated. Then with the PCR screening of these primers for the body cell heterozygote that contains single human chromosome. Only have those heterozygotes that contain corresponding to the people's gene of primer can produce amplified fragments.

The PCR of body cell heterozygote mapping is that specific DNA is arranged in fast method on the specific chromosome. Identical Oligonucleolide primers is used for the present invention, uses from the plate in specific chromosome part or large genomic clone storehouse and can realize inferior location. Can be similar be used for being plotted on other mapping strategy on its chromosome comprise in situ hybridization, use streaming sorting chromosome through mark carry out prescreen and by with make up the hybridization of chromosome specific cDNA library and carry out preselected.

Can utilize cDNA clone and FISH (FISH) step of metaphase chromosome smear that accurate chromosome mapping is provided. This technology can be used and be as short as 50 or the probe of 60bp from cDNA. The summary of this technology is consulted the people such as Verma, and " Human Chromosomes:A Manual of Basic Techniques " (human chromosomal: the basic fundamental guide), Pergamon publishing house, New York, 1988.

In case sequence is plotted on accurate chromosome position, physical location and the genome data of sequence on chromosome can be interrelated. These data can be at for example V.McKusick, finds among the Mendelian Inheritance In Man (human Mendelian inheritance can be obtained by the Johns Hopkins Welch of university medical library by network). Then identify the gene of identical chromosomal region and the relation between the disease of being plotted on by linkage analysis (the common heredity of physically adjoining gene).

Then, the essential difference of determining cDNA between the ill and not ill individuality or genome sequence. If observe sudden change in some or all of ill individualities, and all do not observe sudden change in any normal individuality, this sudden change just may be the triggering factor of disease so.

According to the resolution ratio of up-to-date physical mapping and gene mapping technology, the cDNA that accurately is positioned the disease association chromosomal region can be in 50-500 the potential Disease-causing gene. Wherein supposition mapping resolution ratio is 1Mb, and a gene is 20kb.

Ill and diseased individuals not relatively generally include the structural change of at first seeking in the chromosome, such as disappearance or the transposition that can be found out by the chromosome smear or use the PCR based on this cDNA sequence to detect. At last, existing and will suddenling change of need to checking order the gene complete from several individualities to confirm to suddenly change makes a distinction with polymorphism.

The invention still further relates to by suppress in vivo MPIF-1 with antisense technology and can utilize antisense technology to form by triple helix or come controlling gene to express by antisense DNA or RNA, these two kinds of methods are all based on the combination of polynucleotides and DNA or RNA. For example, 5 ' coded portion of the polynucleotides of code book invention polypeptide being used for design length is the antisense rna oligonucleotide of about 10 1 40 base-pairs. (triple helix is consulted the people such as Lee to the DNA oligonucleotides of the regional complementarity that participation is transcribed in design and the gene, Nucl.Acids Res., 6:3073,1979; The people such as Cooney, Science, 241:456,1988; With the people such as Dervan, Science, 251:1360,1991), prevent thus transcribing and generating of MPIF-1. Antisense rna oligonucleotide and mRNA are hybridized in vivo, and blocking-up mRNA molecule is translated into MPIF-1 albumen, and (antisense participates in Okano, J. Neurochem., 56:560,1991; " Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression " (oligonucleotides is as the antisense inhibitor of gene expression), CRC publishing house, Boca Raton, FL, 1988).

Perhaps, can oligonucleotides mentioned above be delivered to cell by the flow process of this area, so that antisense RNA or DNA can express in vivo, thereby suppress the generation of MPIF-1 in mode mentioned above.

Therefore, antisense construct thing for MPIF-1 can be used for treating the disorder of being induced or being strengthened by MPIF-1, for example other case of artery sclerosis, autoimmunity (such as multiple sclerosis and IDD) and chronic inflammation and communicable disease, other chronic inflammation disease of allergy, rheumatoid arthritis, silicosis, sarcoidosis, idiopathic pulmonary fibrosis and lung by the histamine mediation, the too much syndrome of idiopathic eosinophil, endotoxin shock, the allergy by the histamine mediation, the heating of prostaglandin dependent/non-dependent and alpastic anemia and marrow failure. Antagonist, activator and method

The present invention also provides and has been used for screening compounds with the antagonist of evaluation chemokine polypeptides of the present invention and the method for activator. Activator is to have the compound that the biological function similar to polypeptide maybe can strengthen the polypeptide function, and antagonist these functions capable of blocking. Can following mensuration chemotaxis: will be subject to the cell that polypeptide chemotactic of the present invention lures and be coated on the filter top, the aperture of described filter be enough to allow cell to pass (about 5 μ m). The solution of potential agonist is placed the bottom of groove, and suitable control medium is placed the top compartment, measure thus the concentration gradient of activator by the counting that in time migration is entered or passes the cell of perforated membrane.

When measuring antagonist, chemokine polypeptides of the present invention is placed kerve, and add potential antagonist to measure the chemotaxis that whether stops cell.

Perhaps, mammalian cell or film preparation insulation in the situation that has this compound through mark chemokine polypeptides (such as radioactivity) of polypeptide receptor will be expressed. Then can measure this interactional ability of this compounds block. When measuring activator by this way, will there be chemotactic factor (CF), and measure the ability of activator self and acceptor interaction.

But the example of potential MPIF-1 antagonist comprises antibody or (being in some cases) oligonucleotides of Binding peptide. Another example of potential antagonist is the dominant negative mutant of polypeptide. Dominant negative mutant is can be in conjunction with the acceptor of wild type peptide but fail to keep the polypeptide of BA.

Using the antisense construct thing of antisense technology preparation also is potential antagonist. Can utilize antisense technology to form by triple helix or come controlling gene to express by antisense DNA or RNA, these two kinds of methods are all based on the combination of polynucleotides and DNA or RNA. For example, 5 ' coded portion of the polynucleotides of code book invention polypeptide being used for design length is the antisense rna oligonucleotide of about 10-40 base-pair. (triple helix is consulted the people such as Lee to the DNA oligonucleotides of the regional complementarity that participation is transcribed in design and the gene, Nucl.Acids Res., 6:3073,1979; The people such as Cooney, Science, 241:456,1988; With the people such as Dervan, Science, 251:1360,1991), prevent thus transcribing and generating of chemokine polypeptides. Antisense rna oligonucleotide and mRNA are hybridized in vivo, and blocking-up mRNA molecule is translated into polypeptide, and (antisense participates in Okano, J. Neurochem., 56:560,1991; " Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression " (oligonucleotides is as the antisense inhibitor of gene expression), CRC publishing house, Boca Raton, FL, 1988). Antisense RNA or DNA oligonucleotides mentioned above can be delivered to cell, so that can express the generation with the chemokine inhibiting polypeptide in vivo.

Another kind of potential chemokine antagonists is this polypeptide has lost biological function but still thereby identification and Binding peptide acceptor effectively seal the peptide derivant of acceptor, and they can be the natural or synthetic modification analogs of polypeptide. The example of peptide derivant includes but not limited to little peptide or peptide sample molecule.

Antagonist can be used for treating the disorder of being induced or being strengthened by MPIF-1, for example autoimmunity and chronic inflammation and infectious diseases. The example of autoimmune disease comprises multiple sclerosis and IDD.

Antagonist also can by preventing raising and activate and being used for the treatment of communicable disease of mononuclear macrophage, comprise silicosis, sarcoidosis, idiopathic pulmonary fibrosis. They also can be used for the treatment of the too much syndrome of idiopathic eosinophil by generation and the migration that prevents eosinophil. Also can treat endotoxin shock to the prevention effect of macrophage migration and chemokine polypeptides of the present invention generation by antagonist.

Antagonist also can be used for the treatment of artery sclerosis by preventing the monocyte infiltration in the arterial wall.

Antagonist also can be used to treat for disorderly by allergy and the immunology of histamine mediation by the mast cell that suppresses to be induced by chemotactic factor (CF) and basophil degranulation and histamine release, comprises allergy in late period, chronic urticaria and atopic dermatitis. Also can treat the allergy by the IgE mediation, such as allergic asthma, rhinitis and eczema.

Antagonist also can be by preventing that monocyte is attracted to impingement to be used for the treatment of chronic and acute inflammation. They also can be used for regulating and control the normal macrophage group of lung because in chronic and acute inflammatory tuberculosis and the lung mononuclear macrophage compile relevant.

Antagonist also can be used for the treatment of rheumatoid arthritis by preventing from monocyte is attracted in patient's knuckle synovia. Monocytic inflow and activation all have important effect in degeneration and struvite arthropathic pathogenesis.

Antagonist can be used for intervening the harmful cascade that mainly is attributed to IL-1 and TNF, and this has prevented the biosynthesis of other struvite cell factor. Like this, antagonist can be used for preventing inflammation. The prostaglandin dependent/non-dependent that antagonist also can be used for suppressing to be induced by chemotactic factor (CF) generates heat.

Antagonist also can be used for treating the case of marrow failure, for example alpastic anemia and myelodysplastic syndrome.

Antagonist also can be by preventing that the eosinophil accumulation is used for the treatment of asthma and allergy in the lung. Antagonist also can be used for the upper subcutaneous basilar memebrane fibrillatable for the treatment of, and this is the notable feature of asthma lung.

Activator. The MPIF-1 activator comprises any little molecule with activity similar to any one or more polypeptide described herein. For example, the MPIF-1 activator can be used for strengthening the MPIF-1 activity. For example, be used for strengthening the marrow protective effect of being induced by MPIF-1 in the patient body of accepting chemotherapy or bone-marrow transplantation. Disease diagnosis and prognosis

Just as discussed below, some disease or disorder may be with relevant with the level rising of the mRNA of coding MPIF-1 albumen than the MPIF-1 albumen of corresponding " standard " mammal (mammal that does not namely suffer from the same species of this disease or disorder). In addition, think in from mammiferous some body fluid (such as serum, blood plasma, urine and spinal fluid) of suffering from disease or disorder, can detect than raising from its MPIF-1 protein level of the mammiferous serum of same species of not suffering from this disease or disorder. Thereby, the invention provides diagnostic method, be included in the expression of the gene of measuring coding MPIF-1 albumen in mammalian cell or the body fluid, and this gene expression dose compared with standard MPIF-1 gene expression dose, wherein gene expression dose exists some disease or disorder with respect to the standard indication that raises to some extent.

Make disease or disorderly diagnosis according to conventional method after, the present invention can be used as prognostic indicator, shows that the patient that MPIF-1 gene expression strengthens will have relatively poor clinical effectiveness than the patient who expresses this gene with reduced levels.

" measure the expression of the gene of coding MPIF-1 albumen " and refer to directly (as by measuring or estimating absolute protein matter level or mRNA level) or relative (as by with second part of biological sample in MPIF-1 level or mRNA level compare) qualitative or quantitative measurement or estimate the level of MPIF-1 albumen in first part of biological sample or the level of the mRNA of the MPIF-1 albumen of encoding.

Preferably, measure or estimate first part of MPIF-1 protein level or mRNA level in the biological sample, and compare with standard MPIF-1 protein level or mRNA level, described standard is taken from the second part of biological sample that obtains by not suffering from this disease or disorderly individuality. To understand as this area, in case known standard MPIF-1 protein level or mRNA level, it can be reused as comparative standard.

" biological sample " refers to by individuality, clone, tissue culture or comprises any biological sample that other source of MPIF-1 albumen or mRNA obtains. Biological sample comprises the mammalian body fluid (such as serum, blood plasma, urine, synovia and spinal fluid) of the ripe MPIF-1 albumen that comprises secretion, also comprises ovary, prostate, heart, placenta, pancreas, ascites, muscle, skin, body of gland, kidney,liver,spleen, lung, bone, marrow, eyes, peripheral nerve, nervous centralis, breast and umbilical cord tissue. Be used for being obtained to organize the method for vivisection sample and body fluid to be well known in the art by mammal. When requiring biological sample to comprise mRNA, organizing the vivisection sample is preferred source.

The present invention can be used for detecting the disease in the mammal. Particularly, the present invention can be used for the panimmunity System Dependent disorder in diagnosis or the treatment mammal (preferred people). These disorders comprise any out of control of tumour, cancer and immune cell function, include but not limited to autoimmunity, arthritis, leukaemia, lymthoma, immune containment, pyemia, wound healing, acute and chronic infection, the immunity by cell-mediated, humoral immunity, IBD, marrow containment, etc. Preferred mammal comprises monkey, ape, cat, dog, ox, pig, horse, rabbit and people. People particularly preferably.

Can use any appropriate technology to separate total cell RNA by biological sample, such as Chomczynski and Sacchi, Anal.Biochem., 162:156-159, a step guanidine thiocyanate-benzene phenol-chloroform method of describing in 1987. Then use any appropriate technology to measure the level of the mRNA of coding MPIF-1 albumen. These methods comprise the reverse transcription (RT-PCR) of Northern engram analysis, S1 nuclease mapping, PCR (PCR), association aggregation PCR and the reverse transcription (RT-LCR) of associating ligase chain reaction.

Can be such as people such as Harada, Cell, 63:303-312 carries out the Northern engram analysis described in 1990. In brief, prepare total RNA by biological sample as mentioned above. For carrying out the Northern trace, with RNA sex change in suitable buffer solution (such as glyoxal/methyl-sulfoxide/sodium phosphate buffer), carry out agarose gel electrophoresis, and transfer on the nitrocellulose filter. Make RNA connect filter membrane by the UV joint after, with filter membrane prehybridization in the solution that contains formamide, SSC, DenhardtShi liquid, sex change salmon sperm dna, SDS and sodium phosphate buffer. Use according to any appropriate method (such as³²The multiple initiation dna marker of P system (Amersham)) the MPIF-1 cDNA of mark is as probe. After hybridization was spent the night, cleaning filter membranes also exposed to X-ray film. Above described the cDNA as probe according to the present invention in the part, length is preferably at least 15bp.

Can be such as people such as Fujita, Cell, 49:357-367 carries out the S1 mapping described in 1987. For the dna probe for the preparation of S1 mapping, with the sense strand of cDNA mentioned above as template for the synthesis of the antisense DNA through mark. Then use suitable restriction endonuclease digestion antisense DNA to generate other dna probe of desired length. These antisense probes can be used for making the protected band corresponding to said target mrna (mRNA of the MPIF-1 albumen of namely encoding) to develop. Can carry out as mentioned above the Northern engram analysis.

Preferably, use the people such as Makino, Technique, 2:295-301, the RT-PCR method of describing in 1990 is measured the level of the mRNA of coding MPIF-1 albumen. By this method, the initial concentration linear correlation of the radioactivity of " amplicon " and said target mrna in the polyacrylamide gel band. In brief, this method comprises and adds the total RNA that is separated by biological sample in the reactant mixture that contains RT primer and suitable buffer. After being incubated for primer annealing, mixture can replenish RT buffer solution, dNTP, DTT, RNA enzyme inhibitor and reverse transcriptase. , use and through labeled primer the RT product is carried out PCR after being incubated for the reverse transcription of realizing RNA. Perhaps, not labeled primer, but in the PCR reactant mixture, comprise through mark dNTP. Can in the DNA thermal cycler, carry out pcr amplification according to conventional art. After suitably the wheel number increases with realization, the PCR reactant mixture is carried out electrophoresis at polyacrylamide gel. Behind gel drying, use imaging analysis instrument that the radioactivity of suitable band (corresponding to the mRNA of coding MPIF-1 albumen) is carried out quantitatively. RT and PCR reaction composition and condition, reagent and gel strength and labeling method are well known in the art. The variation of RT-PCR method will be apparent for those of skill in the art.

The described use of part and the design any Oligonucleolide primers pair of said target mrna of reverse transcription that will increase as mentioned.

Can measure MPIF-1 protein level in the biological sample with the method that any this area is known. Be preferred for measuring the technology that is based on antibody of the MPIF-1 protein level in the biological sample. For example, can come MPIF-1 protein expression in the research organization with classical immunohistology method. In these methods, specific recognition is provided by primary antibodie (polyclone or monoclonal), and that secondary detection system can utilize that fluorescence, enzyme or other put together is two anti-. The result has obtained the immunohistology dyeing to the tissue block that is used for pathological examination. Can also be with for example urea and neutral detergent extracting tissue, thus MPIF-1 albumen discharged for Western trace or round dot/narrow line determination method (people such as M.Jalkanen, J.Cell.Biol., 101:976-985,1985; The people such as M.Jalkanen, J.Cell.Biol., 105:3087-3096,1987). In based on this technology of using the cation solid phase, can realize the quantitative of MPIF-1 albumen as standard with separating MPIF-1 albumen. This technology also can be applicable to body fluid. Use these samples, the molar concentration of MPIF-1 albumen will help to different body fluid (as serum, blood plasma, urine, spinal fluid, etc.) standard value of MPIF-1 protein content is set. Then can be set with the numerical value from healthy individual normally manifesting of MPIF-1 protein content, and the numerical value that can be obtained by experimenter to be measured itself and those compares.

Other method based on antibody that can be used for detecting MPIF-1 gene expression comprises immunoassay, such as enzyme linked immunological absorption measurement method (ELISA) and radioimmunoassay (RIA). For example, MPIF-1 protein-specific monoclonal antibody can be used as immunosorbent and enzyme labelled probe for detection of with quantize MPIF-1 albumen. Can come the MPIF-1 protein content that exists in the calculation sample with the amount that exists in the linear regression computerized algorithm reference standard preparation. In another kind of ELISA determination method, can be with two kinds of not homospecific monoclonal antibodies for detection of the MPIF-1 albumen in the body fluid. In this determination method, a kind of antibody is as immunosorbent, and is another kind of as enzyme labelled probe.

Above-mentioned technology can " step " or " two steps " determination method be carried out basically. Determination method comprises makes MPIF-1 albumen contact immobilized antibody " one step ", and makes the mixture contact through the antibody of mark, need not between twice contact to clean. " two steps " mensuration rule is included in the mixture contact was cleaned before the antibody of mark. If suitable, also can adopt other conventional method. Usually the expectation a kind of composition that will measure system is fixed on the holder, thus so that other composition of system can contact this composition and be easy to separates with sample.

Suitable enzyme labeling thing includes but not limited to those from the label of oxidizing ferment group, oxidizing ferment by with the generation of the catalytic reaction hydrogen peroxide of substrate. Glucose oxidase is particularly preferred, because its good stability, and substrate (glucose) is easy to obtain. Can measure by the concentration of measuring the hydrogen peroxide that is formed by enzyme labelled antibody/substrate reactions the activity of oxidizing ferment label. Except enzyme, other suitable label comprises radio isotope, such as iodine (¹²⁵I、 ¹²¹I), carbon (¹⁴C), sulphur (³⁵S), tritium (³H), indium (¹¹²In) and technetium (^99mTc), and fluorescent marker, such as fluorescein, rhodamine and biotin.

The polynucleotides of polypeptide of the present invention and these polypeptide of coding can be used as research reagent and are used for and scientific research, the synthetic external purpose relevant with the dna vector operation of DNA, and are used for the therapeutic agent of exploitation treatment human diseases and the purpose of diagnosticum. For example, MPIF-1 can be used for amplification prematurity HPC, for example granulocyte, macrophage or monocyte by temporarily preventing differentiation. These bone marrow cells can be in vitro culture.

The hybridization probe that the fragment of total length MPIF-1 gene can be used as cDNA library for separating of full-length gene with separate other gene that has high sequence similarity or similar BA to this gene. Yet preferably, this probe comprises at least 30 bases, for example can comprise 50 or polybase base more. The complete genome genomic clone of (comprising regulation and control and promoter region, extron and introne) can also be cloned and comprise to this probe for the identification of the cDNA corresponding to the total length transcript. The example of screening comprises by coming synthetic oligonucleotide probe with known dna sequence, and then the code area of isolated genes. To have gene of the present invention complementary series be used for screening people cDNA, genomic DNA or mRNA library through labeled oligonucleotide, hybridize to determine which member and probe in the library.

The invention still further relates to MPIF-1 as the part of diagnostic test and for detection of the disease relevant with the existence that suddenlys change in the nucleotide sequence or the purposes of disease susceptibility. These diseases are relevant with the expression deficiency of chemokine polypeptides.

Can detect the individuality that carries sudden change in the MPIF-1 gene at dna level by multiple technologies. Can be obtained by patient's cell diagnostic nucleic acid, such as coming autoblood, urine, saliva, organizing the cell of vivisection sample and autopsy. Genomic DNA can be directly used in detection, or before analysis by using PCR people such as (, Nature, 324:163-166,1986) Saiki to carry out enzymatic amplification. RNA or cDNA also can be used for identical purpose. For example, the PCR primer of nucleic acid complementation with coding MPIF-1 can be used for identifying and analyzing MPIF-1 and sudden change. For example, can detect deletion and insertion by amplified production than the size variation of normal genotype. Can identify point mutation by the DNA after will increasing with hybridizing through radiolabeled MPIF-1 RNA or radiolabeled MPIF-1 antisense dna sequence. The sequence of mating fully and the duplex of mispairing can be made a distinction by RNaseA digestion or the difference by melting temperature.

Can carry out testing based on the heredity of dna sequence dna difference by the variation that detects the dna fragmentation electrophoretic mobility at the gel that contains or do not contain denaturant. Can observe the insertion of becoming estranged of foreword Lieque by high-resolution gel electrophoresis. On sex change formamide gradient gel, can distinguish not homotactic dna fragmentation, in described gel, according to specifically unwinding or the part melting temperature of different dna fragmentations, its migration is arrested in the diverse location of gel and (consults such as people such as Myers, Science, 230:1242,1985).

By protecting determination method or chemical cracking method (such as people such as Cotton, PNAS USA, 85:4397-4401,1985) also can disclose the sequence variation of ad-hoc location such as the nuclease of RNA enzyme and S1 protective effect.

Thereby, can realize the detection of specific dna sequence by the methods such as Southern trace such as hybridization, the effect of RNA enzyme protection, chemical cracking, direct dna sequencing or use restriction enzyme (such as RFLP (RFLP)) and genomic DNA.

Except more conventional gel electrophoresis and dna sequencing, also can detect sudden change by the original position analysis.

The invention still further relates to the diagnostic test that changes for detection of MPIF-1 protein level in the various tissues, because than the existence of its protein overexpression detectable disease of Normal group tissue samples or disease susceptibility, for example tumour. Be well-known to those skilled in the art for detection of the determination method derived from MPIF-1 protein level in host's the sample, comprise radioimmunoassay, competition binding assay, Western engram analysis, ELISA determination method and " sandwich " determination method. ELISA determination method people such as (, Current Protocols in Immunology, the 1 (2), the 6th chapter, 1991) Coligan at first comprises preparation for the specific antibody of MPIF-1 antigen, preferred monoclonal antibody. Also prepare in addition the report antibody for this monoclonal antibody. Adhere to detectable reagent at report antibody, such as radioactivity, fluorescence or (in this example) horseradish peroxidase. Taken a sample by the host, and sample is placed and can be incubated in conjunction with the solid support (such as the polystyrene dish) of sample protein is upper. Then by being incubated with any vacant protein binding site on the covering disk with nonspecific proteins matter (such as BSA). Then, monoclonal antibody is incubated in dish, this moment, monoclonal antibody was attached to any MPIF-1 albumen that adheres on the polystyrene dish. With all unconjugated monoclonal antibodies of buffer solution flush away. This moment, the report antibody that will link to each other with horseradish peroxidase placed dish, made report antibody and in conjunction with any monoclonal antibody combination of MPIF-1. Then the unconjugated report antibody of flush away. Then add peroxidase substrate in dish, when comparing with calibration curve, the amount of colour developing is exactly the tolerance of the amount of the MPIF-1 albumen that exists in patient's sample of designated volume in the fixed time.

Also can adopt competitive assays, wherein will be attached to solid support for the specific antibody of MPIF-1, and make through the MPIF-1 of mark with derived from host's sample flow and cross this solid support, the amount of the label that detects by for example liquid scintillation chromatography is relevant with the amount of protein in the sample.

" sandwich " determination method is similar to the ELISA determination method. In " sandwich " determination method, make MPIF-1 flow through solid support, and in conjunction with the antibody that is attached on the solid support. Then make the second antibody in conjunction with MPIF-1. Then make the third antibody of specificity for the second antibody through mark flow through this solid support and in conjunction with the second antibody, determine again the size of amount.

The invention provides the method for the acceptor of identifying chemokine polypeptides. Can be by the several different methods that those skilled in the art will know that, such as part elutriation and FACS sorting people such as (, Current Protocols in Immunology, the 1 (2), the 5th chapter, 1991) Coligan, come the gene of identification code acceptor. The preferred expression cloning method that adopts is wherein prepared the RNA of polyadenylation by the cell to this responding property of polypeptide, the cDNA library that RNA is thus made up is divided into some groups, and is used for rotaring redyeing COS cell or to other cell of this polypeptide anergy. To be exposed to polypeptide through mark through transfectional cell what glass slide was grown. Can come labeling polypeptide by multiple means, comprise iodine labeling or comprise the recognition site of locus specificity protein kinase. After fixing and being incubated, slide is carried out radioautographic analysis. Identify positive group, prepare subgroup, use the Asia of repeating to hive off and again again transfection of screening process, final acquisition coding is inferred the monospecific polyclonal of acceptor.

The alternative approach of identifying as acceptor can light is affine to be connected with carrying out through the cell membrane of labeling polypeptide and expressed receptor molecule or extract formulation. Analyze the resolution cross-linked material by PAGE, and X-ray film is exposed. Cutting-out contain polypeptide receptor through labeled complex, be separated into fragments of peptides, and carry out the protein microsequencing. To be used for by the amino acid sequence that microsequencing obtains design one cover degenerate oligonucleotide probe, with the screening cDNA library, thereby identify the gene that coding is inferred acceptor. Therapeutic agent

Polypeptide of the present invention can be used for panimmunity regulation and control and struvite function, also has the numerous disease situation. MPIF-1 belongs to chemotactic factor (CF) family, so it is the chemotactic decoy of leucocyte (such as monocyte, neutrophil cell, T lymphocyte, eosinophil, basophilic granulocyte etc.).

The Northern engram analysis shows that MPIF-1 mainly expresses in the tissue of hematopoietic origin. The therapeutic of MPIF-1/diagnostic application

MPIF-1 is presented in the regulation and control of immune response and inflammation and plays a significant role. Figure 13 has shown that lipopolysaccharides can induce the person monocytic cell to express MPIF-1. Therefore, as replying that induced by endotoxin exists, therefore monocytes MPIF-1 can adopt and use the immune response that MPIF-1 comes modulate host. MPIF-1 can be used as anti-inflammatory agent.

As shown in Figure 4, MPIF-1 is significant to the chemotactic attractive activity of THP-1 cell (A) and PBMC (B). MPIF-1 also induces significant calcium migration in the THP-1 cell, show that MPIF-1 has biological impact to monocyte. In addition, MPIF-1 produces the dose dependent chemotaxis in the person monocytic cell and the calcium migration is replied.

In addition, polypeptide of the present invention can be used for antitumor therapy, can cause the tumour decline because evidence suggests the cell of the expression chemotactic factor (CF) that is expelled in the tumour, for example in the treatment of Kaposi sarcoma. But MPIF-1 inducing cell TNF secretion-α, this is be used to the known factor that makes the tumour decline, in this case, this protein can be used for the induced tumor decline. MPIF-1 also can be used for inducing the person monocytic cell to secrete other tumour and cancer inhibitor, such as IL-6, IL-1 and G-CSF. Also have, but MPIF-1 passes through invasion and the activation of their chemotactic activity stimulation of host defence (tumoricidal) cell (such as cytotoxic T cell and macrophage), like this, MPIF-1 can be used for treating solid tumor. The marrow protection

This polypeptide can be used for suppressing propagation and the differentiation of hematopoietic cell, thereby is used in the chemotherapy process infringement that the protection stem cell avoids chemotherapeutics. Fig. 6 and 7 illustrations the MPIF-1 colony that can suppress low multiplication potentiality colony forming cell (LPP-CFC) form. But Fig. 8 illustration the M-CIF specificity suppress to be formed by the colony that M-CSF stimulates, can not and MPIF-1 is scarce. Because MPIF-1 significantly suppresses growth and/or the differentiation of bone marrow cell, therefore this anti-proliferative effect can allow to use the more chemotherapeutics of high dose, thereby obtains more effective chemotherapeutic treatment.

The MPIF-1 polypeptide can be used for suppressing leukaemia's propagation in treatment to the inhibition of typing CFU-GM subgroup (for example granulocyte and monocytes/macrophages).

In addition, the inventor finds that MPIF-1 and variant thereof (such as MPIF-1 Δ 23) can be in external inhibition people's marrow and granulocyte predecessor's propagation and differentiation. Similar, zooscopy shows that for example MPIF-1 Δ 23 is in external and in vivo all growths of the low multiplication potentiality colony forming cell (LPP-CFC) of specificity inhibition and granulocyte/monocyte typing CFU-GM. These discoveries point out that MPIF-1 has the therapeutic application as chemoprotectant, can protect early stage myeloid progenitor to avoid the cytotoxic effect of Common Chemotherapy medicine.

Because MPIF-1 and variant thereof have the ability of selective inhibition myeloid progenitor, so MPIF-1 can be used for treating bone marrow proliferative diseases, such as essential thrombocythemia (ET), polycythemia vera (PV) or agnogenic myeloid metaplasia (AMM), they are closely-related clinically. Each disorder all is to be produced by the gain mutation of single candidate stem cell, and it gives the filial generation of this stem cell with growth vigor. These disorderly Pathological Physiology are different, namely have the excessive generation of different cell types. In PV, red blood cell, granulocyte and megacaryocyte excessively generate. In ET, according to definition, blood platelet and leucocyte excessively generate. And AMM also shows thrombocythemia or leukocytosis except myelofibrosis.

Remove red blood cell by bloodletting, can stablize PV patient thus. Yet, do not have suitable therapy for the platelet levels that raises among the ET patient. After deliberation several marrow containment therapies reduce the risk of thrombocythemia. Use the treatment of radiophosphorus, hydroxycarbamide, alkylating agent (busulfan and Chlorambucil), interferon or anagrelide all to show significant side effect. Particularly, in every kind of marrow containment therapy (except anagrelide), the risk of acute leukemia has all raise. Anagrelide is promising therapy. Yet, consider the bad reaction of anagrelide, but also not determine its chronic toxicity potentiality. Because the inconvenience that cost, side effect and stomach and intestine are used outward thinks that at present interferon is two gamma therapies. These discoveries are pointed out, still extremely need the therapy to these diseases.

Studies have shown that the inhibition to the blood platelet progenitor cell proliferation in the body that the mouse of then processing with 5-FU with 23 preliminary treatment of MPIF-1 Δ is carried out.

MPIF-1 is also contained in the present invention and variant is united the purposes that other marrow is contained therapy and reagent.

In Fig. 9,10 and 11, remove used cytophyletic committed cell, and make cell colony contact M-CIF or the MPIF-1 of acquisition. M-CIF causes that Mac-1 positive cell colony reduces almost 50%, and this is consistent with the result of Fig. 8. Fig. 8 shows that M-CIF can induce the inhibition to M-CSF responsiveness colony forming cell. As shown in figure 11, the MPIF-1 CFU-GM that can suppress to finalize the design is replied IL-3, GM-CSF and M-CSF and is formed the ability of colony. In addition, as shown in figure 12, the dose response of MPIF-1 shows that can suppress colony forms. This inhibition may be owing to the specific inhibition by the differentiation signal of these factors mediation, perhaps owing to the cytotoxic effect to CFU-GM. In addition,

embodiment

9 and 10 has proved that MPIF-1 has for marrow prolection in the Cytotoxic external and body of chemotherapeutics. Thereby MPIF-1 can be used as the patient that marrow protectant is used for accepting chemotherapy.

As mentioned above, a kind of major complications that is caused by chemotherapy and radiation is the destruction to non-pathologic cell type. The invention provides by containing that individual proliferation of bone marrow cells protects marrow to avoid radiating method with the infringement of chemotherapeutics. These methods comprise uses the MPIF-1 of marrow containment amount as the part of radiotherapy or chemotherapy regimen: macrophage inflammatory protein-1 α (MIP-1 α), MIP-2 α (MIP-2 α), platelet factor 4 (PF4), interleukin-8 (IL-8), macrophage chemotactic and activity factor (MCAF) and macrophage inflammatory protein GAP-associated protein GAP-2 (MRP-2) separately or with one or more chemotactic factor (CF)s that are selected from lower group to individuality. Marrow of the present invention containment composition can provide marrow protection effect thus, and can with bone marrow cell is had dysgenic therapy and unites use. This is because marrow of the present invention containment composition places at a slow speed recurrent state with bone marrow cell, and the protection of the cellular damage that causes for the chemotherapy by for example radiotherapy or use cell cycle active medicine (such as cytarabine, hydroxycarbamide, 5-FU and Arc-C) is provided thus. In case by individual System Cleaning chemotherapeutics, will expect to use marrow stimulant (such as interleukin-11 (IL-11), hematopoietin (EPO), GM-CSF, G-CSF, stem cell factor (SCF) and TPO (TPO)) for example to stimulate to be subject to rapid amplifying and the differentiation of the CFU-GM of MPIF-1 protection.

Embodiment 13 has proved that MPIF-1 gives the ability of marrow protection in the body under having the situation of chemotherapeutics. Embodiment 13 shows, before using chemotherapeutics, individuality used MPIF-1 and accelerated hematoblastic recovery in the blood, in addition still like this after the 5-FU in a plurality of cycles treatment. Listed experiment also proves among the embodiment 13, and the MPIF-1 treatment in the 5-FU therapeutic process in a plurality of cycles can cause the fast quick-recovery of granulocyte. In addition, the structure of embodiment 13 illustrates that also MPIF-1 and G-CSF bring into play additive effect when using altogether.

As mentioned above, the inventor finds that MPIF-1 and variant thereof are showed strong external containment to the low multiplication potentiality colony forming cell (LPP-CFC) from marrow. LPP-CFC is the dual intensity HPC that produces granulocyte and monocyte pedigree. MPIF-1 also reversible inhibition by people CD34⁺The colony of stem cell-derived granulocyte and monocyte colony forming cell forms. Inventor's external chemoproection experiment shows that MPIF-1 Δ 23 these HPCs of protection avoid the cytotoxic effect of medicine 5 FU 5 fluorouracil (5-FU), cytarabine and taxol Taxo1 .

MPIF-1 variant (Δ 23) the in vivo use in the chemotherapy model shows that it causes myeloid progenitor and neutrophil cell and blood platelet peripheral cell group's faster quick-recovery. In addition, shown in

embodiment

10 and 13, using of MPIF-1 causes reducing and thrombocytopenic recovery acceleration with neutrocyte in the animal used as test of 5-FU processing. Thereby MPIF-1 and variant thereof have shortened the hypoplastic bone marrow relevant with chemotherapeutics, granulocyte reduces and the thrombocytopenic time, have reduced thus the possibility that infects among the patient who accepts these reagent treatments.

Thereby, the present invention relates to for the protection of myeloid progenitor and accelerate blood platelet and method that granulocyte recovers, comprise the individuality of the therapy of accepting preferentially to kill cell in the division (such as radiotherapy or use the treatment of cell cycle active medicine) is used MPIF-1. To be enough to the providing amount for marrow protective effect in the body of the treatment of preferentially killing cell in the division and reagent to use MPIF-1. " use MPIF-1 " and refer to treat analog or its combination that effective dose is used MPIF-1, MPIF-1. Hereinafter discussed the mode of administration of MPIF-1 in detail.

Can be before the treatment of preferentially killing cell in the division, afterwards or among use MPIF-1. In preferred embodiments, before radiotherapy or dosed cells cycle active medicine, use MPIF-1, and the propagation of enough MPIF-1 containments of time bone marrow cell. The present invention comprises that also MPIF-1 is used for preferentially killing at many wheels the purposes of protection bone marrow cell in the therapy of division cell. In this case, can single dose or the different time points in therapy or therapeutic scheme use MPIF-1 with multi-agent.

As mentioned above, MPIF-1 can use separately, perhaps unites one or more marrow stimulants. The marrow stimulant is used for accepting radiotherapy or using the individuality bone marrow cell of the treatment of cell cycle active medicine to reduce the rear propagation that stimulates bone marrow cell at present in the art. Consult such as people such as V.Kannan, Int.J.Radiat.Oncol.Biol.Phys., 37:1005-1010,1997; The people such as M.Engelhardt, Bone Marrow Transplant, 19:529-537,1997; The people such as S.Vadhan-Raj, Ann.Intern.Med., 126:673-681,1997; The people such as L.Harker, Blood, 89:155-165,1997; The people such as R.Basser, Lancet, 348:1279-1281,1996; The people such as A.Grossman, Blood, 88:3363-3370,1996; The people such as M.Gordon, Blood, 87:3615-3624,1996. For example, can be in killing division use MPIF-1 before the therapy of cell, and after the course for the treatment of of this therapy or among use one or more marrow stimulants. In this case, MPIF-1 will protect bone marrow cell to avoid the infringement of therapy, and then using of marrow stimulant will cause protected bone marrow cell group amplification.

Usually the patient who accepts the chemotherapeutics treatment is used the marrow stimulant with the treatment effective dose. Dosage formulation and mode of administration can change with many factors, comprise the situation of individuality to be treated, cell to be stimulated, treatment stage and the employed marrow stimulant of chemotherapy regimen. For example, GM-CSF and G-CSF are effectively at the dosage of about 1 μ g/kg body weight and 5-10 μ g/kg body weight in treatment respectively, and can use every day by hypodermic injection. Consult such as people such as V.Kannan, Int.J.Radiat.Oncol.Biol.Phys., 37:1005-1010,1997; The people such as M.Engelhardt, Bone Marrow Transplant, 19:529-537,1997; The people such as I.Sniecinski, Blood, 89:1521-1528,1997. Can be by hypodermic injection, use IL-11 every day with the dosage of as many as 100 μ g/kg body weight. Consult such as people such as M.Gordon, see above. Yet, think that the IL-11 dosage that is lower than 10 μ g/kg body weight also is effective in reducing the decrease of platelet of being induced by chemotherapy. Can use TPO with the dosage range of 0.3-2.5 μ g/kg body weight by intravenous injection. Consult such as people such as S.Vadhan-Raj Ann.Intern.Med., 126:673-681,1997; The people such as L.Harker, Blood, 89:155-165,1997. Those skilled in the art will recognize that optimal dose prescription and mode of administration will change along with many factors (comprising those factors mentioned above). Dosage formulation and the mode of administration of other marrow stimulant are known in this area.

Also can be along with above about dosage formulation and the described factor of mode of administration and change as the marrow stimulant time of application of a therapeutic scheme part that comprises the therapy of preferentially killing cell in the division. Delivered openly individuality has been used the marrow stimulant as the many reports that comprise the therapeutic scheme part of radiotherapy or cell cycle active medicine. For example, the people such as S.Vadhan-Raj see above, and have reported to use chemotherapeutics front 3 all intravenous single doses and give TPO. The people such as C.Papadimitrou, Cancer, 79:2391-2395,1997 and the people such as R.Whitehead, J.Clin. Oncol., 15:2414-2419,1997 have reported and have been included in the embolic chemotherapy of using chemotherapeutics in a few courses for the treatment of in week. In each of these situations, after the first day that uses the chemotherapeutics treatment with last day before a plurality of time points use multi-agent G-CSF. The people such as M.Gordon see above and the people such as M.Michael, Am.J.Clin.Oncol., and 20:259-262,1997 have reported the similar usage of IL-11 and GM-CSF. Yet, those skilled in the art will recognize that the Best administration time of marrow stimulant will be along with the marrow stimulant of concrete use and application conditions and changed.

Thereby this area is known in order to alleviate the therapy of preferentially killing cell in the division to the cytotoxic effect of bone marrow cell and is used the marrow stimulant. Can use the marrow stimulant by several paths, comprise intravenous and hypodermic injection. The application concentration of marrow stimulant extensively changes along with many factors, and scope is 0.1-100 μ g/kg body weight usually, and can single dose or a plurality of time points in chemotherapy or radiotherapy scheme use with multi-agent. Yet, usually before or after using chemotherapeutics or radiotherapy, use the marrow stimulant. The service condition that it will be understood by those skilled in the art that the marrow stimulant will be along with concrete marrow stimulant and therapeutic scheme and is changed.

Those skilled in the art will understand, and as mentioned above, MPIF-1 can be used for strengthening the effectiveness of hemopoieticgrowth factor usually. These hemopoieticgrowth factors comprise hematopoietin (stimulating red blood cell to generate) and IL-3 (stimulating more, the multispectral of primordial stem cell is growth factor), increase thus the number of all haemocyte types. Other comprises the hybrid molecule of stem cell factor, GM-CSF and G-CSF and hematopoietin, IL-3 and SCF, GM-CSF and G-CSF.

Marrow containment property pharmaceutical composition of the present invention also can be used for treating the leukaemia that causes bone marrow cell hyper-proliferative state. Thereby, the present invention also provides and has been used for the treatment of leukemic method, comprise the MPIF-1 that to Leukemia Patients uses marrow containment amount, itself or use separately, or unite one or more chemotactic factor (CF)s that are selected from lower group and use: MIP-1 α, MIP-2 α, PF4, IL-8, MCAF and MRP-2.

" containment proliferation of bone marrow cells " refers to that the cell proliferation and/or the increase that reduce bone marrow cell are in the at a slow speed percentage of the bone marrow cell of recurrent state. As mentioned, " individuality " refers to mammal, preferred people. The pre-incubation of marrow containment composition of the present invention and acetonitrile (ACN) can significantly strengthen the specificity of these chemotactic factor (CF) containment myeloid progenitors. Thereby, preferably such as people such as H.E.Broxmeyer, Ann.Hematol., 71 (5): 235-246,1995 and the open WO 94/13321 (complete being collected herein by reference) of PCT described in, before using with marrow containment property composition of the present invention and ACN pre-incubation.

Marrow containment property composition of the present invention can be united multiple chemotherapeutics and be used, and comprises alkylating agent, such as mustargen, aziridine, methyl melamine, alkylsulfonate, nitroso ureas and triazine; Antimetabolite is such as folacin, pyrimidine analogue (particularly fluorouracil and cytarabine) and purine analogue; Natural products is such as vinca alkaloids, epipodophyllotoxin, antibiotic, enzyme and biological answer-reply instrumentality; With mix product, such as platinum coordination complex, amerantrone, substituted ureas (such as hydroxycarbamide), methylhydrazine derivative and AC containment thing.

Can use chemotherapeutics with concentration known by a kind of known technology. Marrow containment property composition of the present invention can be used altogether with chemotherapeutics, and perhaps separate administration was namely used before or after using chemotherapeutics.

Some chemotactic factor (CF), can suppress the marrow of (at least part of blocking-up) marrow containment of the present invention property composition such as MIP-1 β, MIP-2 β and GRO-α and contain effect. Thereby, in another embodiment, the invention provides for the method that suppresses the marrow containment, comprise before being exposed to separately marrow containment agent MPIF-1 or uniting one or more the marrow containment inhibitor that is selected from MIP-1 β, MIP-2 β and GRO-α of administration effective dose that exposes among MIP-1 α, MIP-2 α, PF4, IL-8, MCAF and the MRP-2. Protection for the damage of being induced by cytotoxic agent

Polypeptide of the present invention also can be used for the damage to cell, tissue or organ that reduces or prevent to be induced by cytotoxic agent.

The damage of normal tissue occurs because being exposed to cytotoxic agent. Cytotoxic agent comprises radiation and chemotherapeutics. Radiation can be to be exposed to radiation because of accident, environment, occupation, diagnosis and treatment. The common adverse effect of normal tissue injury or treatment of cancer such as radiotherapy, chemotherapy and radiotherapy and chemotherapy combined therapy. This damage of normal tissue usually is called normal structure " impact ", tissue " toxicity ", " morbidity ", " complication " and organizes " reaction " (acute, subacute or late period).

Thereby, the invention provides composition and the method for the normal tissue injury that is used for the treatment of and prevents to be caused by radiation, radiotherapy, chemotherapy and other cytotoxic agent.

Normal structure toxicity, especially acute toxicity (namely the treatment several days or a few week in occur), can cause pain, and cause other complication, such as infection. In addition, acute toxicity has limited the dosage of cancer therapeutic agent, can damage thus the effect for the treatment of of cancer. In addition, even when toxic effect is subclinical in early days (do not cause morbidity and not dose limitation), they also can cause late period (being also referred to as chronic) impact (i.e. several months or generation in several years after treatment). Late Effect comprise for example sterile, late onset is downright bad and fibrillatable and the cancer of being induced by mitogen.

The invention provides the composition and the method that are used for the treatment of with prophylaxis of acute, subacute and late period normal structure toxicity.

The damage to normal cell and tissue of being induced by cytotoxic agent (such as radiation and chemotherapy) relates to several approach. Damage can be direct or indirect. Coup injury is to be caused by the effect of cytotoxic agent to Cell Component, comprises strand and double-strand break in the chromosome, and the damage of free radical and active oxygen cell membrane and other Cell Component. Indirect injury is that the downstream events by the cytotoxic agent primary action causes. These downstream events comprise that non-viable non-apoptotic cell or tissue discharge free radical, injury of blood vessel, normal immune response and inflammatory response.

The damage to cell, tissue and organ that polypeptide of the present invention and polynucleotides are treated and prevented to be induced by cytotoxic agent by regulating and control at least a above-mentioned direct and indirect approach.

Damage may be to be replied by the loss of (being called the stem cell model) of potential mitotic cell, the injury of blood vessel that causes anoxic and other impact, normal host reparation (to comprise and induce immediate early gene such as Jun and EGR1, induce proinflammatory cytokine such as interleukin and TNF, induce inflammatory cytokine such as TGF β, PDGF, BFGF, and induce the cascade of the secondary cell factor), the impact of inflammatory response, the interaction between the various kinds of cell type (such as inflammatory cell, matrix functioning cell and fibroblast) cause.

Polypeptide of the present invention and polynucleotides can be regulated and control at least a above-mentioned damage cause.

Can one or more mode inducing fibrosis: induce the monocyte and the macrophage that in radiating tissue, exist to generate proinflammatory cytokine, in inflammatory response, raise thus other macrophage; The initial loss of epithelium and stroma cell is induced inflammation; Radiation is by inducing AP-1 to induce the expression of the fibroblast factor.

Polypeptide of the present invention and polynucleotides regulation and control cause Fibrotic at least a approach.

Cell is replied radiation and other cytotoxic agent in several ways: the ceramide that is formed by the film sphingomyelin activates the JNK approach, causes apoptosis, and sign is to form apoptotic body; The apoptosis of being induced by other approach; Mitosis associated death, sign are to form small nut (MN); With the aging by born of the same parents' toxin-induced, wherein cell has metabolic activity, but can not divide.

Polypeptide of the present invention and polynucleotides can be regulated and control at least a above-mentioned cell response for radiation and other cytotoxic agent.

The reagent (such as polypeptide of the present invention) of regulation and control normal structure toxicity is called chemical regulation thing, toxicity protective agent, protective agent, cytoprotection thing and saves agent. These reagent can be used for reducing or preventing the side effect for the treatment of of cancer, and are used for prevention or treat the tissue damage that is caused by radiation exposure.

When being used for this paper, the term cytotoxic agent refers to chemotherapeutics (being also referred to as antitumor agent) and radiation (such as accident radiation, professional radiation, environmental radiation, radiotherapy, radiotherapy comprises for example fractional radiotherapy, non-fractional radiotherapy and the radiotherapy of super section) and radiotherapy and chemotherapy combined therapy. The radiation type also comprises ionization (γ) radiation, corpuscular radiation, low energy emission (LET), high energy emission (HET), ultra violet radiation, infrared emission, visible light and photosensitive radiation. Cytotoxic agent comprises preferential kill tumor cell or destroys the reagent of the cell cycle of fast breeding cell, also is included in the upper reagent that is used for prevention or reduces growth of tumour cell for the treatment of. Chemotherapeutics is also referred to as antineoplastic, and is well known in the art. When being used for this paper, chemotherapy comprises the treatment of using single chemotherapeutics or using agent combination. In the experimenter of needs treatment, chemotherapy can combined surgery be treated or radiotherapy, perhaps unites other antineoplaston form.

Radiation also comprises the ionization radiation, and it is high energy emission (such as the X ray or the gamma-rays that generate ion pair in object), the radiation of high linear energy transfer, low linear energy transfer radiation, alpha ray, β ray, neutron beam, accelerated electron beam and ultraviolet ray. Radiation also comprises photon and fission-spectrum neutron.

Polypeptide of the present invention and polynucleotides can be protected normal cell and organize the impact that avoids cytotoxic agent, such as radiation as herein described and chemotherapeutics.

Exemplary chemotherapeutics is vinca alkaloids, epipodophyllotoxin, anthracycline, antibiotic, actinomycin D, plicamycin, puromycin, Gramicidin D, taxol (Taxol , Bristol Myers Squibb), colchicin, cytochalasin B, ipecine, maytansine and amsacrine (with " mAMSA "). The 1277-1280 page or leaf has been described vinca alkaloids. The example of vinca alkaloids is vincristin, vinblastine and eldisine (desacetyl vinblastine amide) Goodman and Gilman " Pharmacological Basis of Therapeutics " (the materia medica basis of therapeutic agent), the 7th edition, 1985. 1280-1281 has described the epipodophyllotoxin class. The example of epipodophyllotoxin is Etoposide, Etoposide o-quinone and Teniposide. " the Pharmacological Basis of Therapeutics " of Goodman and Gilman (the materia medica basis of therapeutic agent), the 7th edition, 1985, the 1283-1285 pages or leaves have been described the anthracycline antibiotics. The antibiotic example of anthracycline is daunomycin, adriamycin, mitoxantraone and bisanthrene. " the Pharmacological Basis of Therapeutics " of Goodman and Gilman (the materia medica basis of therapeutic agent), the 7th edition, 1985, the 1281-1283 pages or leaves have been described actinomycin D (being also referred to as dactinomycin D). " the Pharmacological Basis of Therapeutics " of Goodman and Gilman (the materia medica basis of therapeutic agent), the 7th edition, 1985, the 1287-1288 pages or leaves have been described plicamycin (being also referred to as mithramycin). Other chemotherapeutics comprises cis-platinum (Platinol , Bristol Myers Squibb), NSC-241240 (Carboplatin) (Paraplatin , Bristol Myers Squibb), mitomycin (Mutamycin , Bristol Myers Squibb), hemel (Hexalen , U.S. Bioscience company), endoxan (Cytoxan , Bristol Myers Squibb), lomustine (lomustine, CCNU) (CeeNU , Bristol Myers Squibb), BCNU (carmustine, BCNU) (BiCNU , Bristol Myers Squibb).

Can also comprise Aclacnomycin A, Aclacinomycin, acronycine, adriamycin, aldesleukin (proleulzin), hemel, aminoglutethimide, aminoglutethimide (Cytadren), aminooimidazole carboxylic acid amides, amsacrine (m-AMSA with co-administered other therapeutic agent of polynucleotides of the present invention and polypeptide; Amsidine), anastrazole (arimidex), ancitabine, anthracycline, peace Anthramycin, asparaginase (elspar), azacitidine, azacitidine (draw and reach mycin), nitrogen ornithine, azaserine, azauridine, 1,1 '; 1 "-phosphinothioylidynetris aziridine, azirino (2 ', 3 ': 3,4) pyrroles (1,2-α) indoles-4,7-diketone, BCG (theracys), BCNU, BCNU vinyl chloride nitroso ureas, benzamide, 4-(two (2-chloroethyl) amino) benzenebutanoic acid, bicalutamide, Dichloroethyl nitroso ureas, bleomycin, bleomycin (blenozane), bleomycins, bromodeoxyribouridine, Broxuridine, busulfan (busulfan), urethanes, NSC-241240, NSC-241240 (paraplatin), Carmustine, Carmustine (BCNU; BiCNU), Chlorambucil (chlorambucil), vinyl chloride nitroso ureas, chorozotocin (DCNU), chromomycin A3, suitable retinoic acid, cisplatin (cis-ddpl; Platinol), cladribine (2-chlorodeoxyadenosine; 2cda; Leustatin) coformycin, cycloleucine, endoxan, Sendoxan, Chlorambucil, cytarabine, cytarabine hydrochloride (cytosar-u), 2-deoxidation-2-(((the methyl nitroso is amino) carbonyl) amino)-D-Glucose, Dacarbazine, dactinomycin D (cosmegen), daunoblastin, hydrochloric acid daunoblastin (daunorubicin hydrochloride), decarbazine, decarbazine (DTIC-dome), demecolcine, dexamethasone, dianydrogalactitol, diazonium oxygen nor-leucine, diethylstilbestrol, docetaxel (taxotere), ADMh (adriamycin), ADMh, Eflornithine, estramustine, estramustine phosphate sodium (emcyt), ethiodized oil, ethoglucid, urethanes, ethylmethane sulfonate, Etoposide (VPl6-213), Suwei A amine, floxuridine, floxuridine (fudr), fludarabine (fludara), fluorouracil (5-FU), Fluoxymesterone (FL), Flutamide, Flutamide (eulexin), fluxuridine, gallium nitrate (granite), gemcitabine (gemzar), genistein, 2-deoxidation-2-(3-methyl-3-nitroso uride)-D-glucopyranose, Goserelin (zoladex), ifosfamide (iflex), the ifosfamide (MAID) that contains mesna, interferon, interferon-' alpha ', Intederon Alpha-2a, α-2b, α-n3, proleulzin, MIBG, the MIBG MIBG, Mexician scammony (camptosar), Accutane (accutane), ketoconazole, 4-(two (2-chloroethyl) amino)-L-Phe, Serine diazonium acetate, lentinan, formyl tetrahydrofolic acid, leuprorelin acetate (p-GLU-HIS-TRP-SER-TYR-D-TRP-LEU-ARG-PRO-GLY-NH2), levamisol (ergamisol), lomustine (CCNU; Cee-NU), mannomustin, maytansine, mechlorethamine, hydrochloric acid mechlorethamine (mustargen), acetic acid xyprogesterone (medroxyprogesterone acetate, depo provera), megestrol acetate (menace), melengestrol acetate, melphalan (melphalan), menogaril, purinethol, purinethol (purinethol), anhydrous purinethol, MESNA, mesna (mesne), methanesulfonic acid, ethyl ester, amethopterin (mtx; Amethopterin), methyl-ccnu, mimosine, Misonidazole, mithramycin, mitoantrone, dibromannitol, mitoguazone, mitolactol, mitomycin (mutamycin), mitomycin C, mitotane (o, p '-DDD; Lysodren), mitoxantrone, mitoxantrone hydrochloride (novantrone), Mopidamol, N, N-two (2-chloroethyl) tetrahydrochysene-2H-1,3,2-oxazaphosphorin-2-amine-2-oxide, N-(1-Methylethyl)-4-((2-methyl hydrazine) methyl) benzamide, N-methyl-two (2-chloroethyl) amine, nicardipine, Nilutamide (nilandron), Nimustine, nitra-amine, nitrogen mustard, nocodazole, nogalamycin, Sandostatin LAR Depot (sandostatin), pacilataxel (taxon), taxol, pactamycin, pegaspargase (PEGx-1), penta Nysfungin, (2 '-deoxycoformycin), NK-631, peptichemio, photophoresis, plicamycin (mithramycin), quinone, pipobroman, plicamycin, podofilox, podophyllotoxin, porphyromycin, prednisone, procarbazine, hydrochloric acid procarbazine (procarbazine), prospidium, puromycin, Puromycin aminonucleoside, (psoralen+ultraviolet a) for PUVA, pyran co-polymer, rapamycin, the s-AzGR, 2,4,6-three (1-aziridinyl)-s-triazine, Semustine, showdomycin, sirolimus, streptozotocin (streptozotocin), suramin, TAMOXIFEN CITRATE (nolvadex), taxon, Tegafur, teniposide (VM-26; Vumon); tenuazonic acid; TEPA; Testolactone; Thiotef; thioguanine; Thiotef (thioplex); the ladder Luolong; topotecan; vitamin A acid (vesanoid); triethyleneiminobenzoquinone; trichodermin; the triethylene glycol diglycidyl ether; Trimetrexate (neutrexin); three (1-aziridinyl) phosphine oxide; three (1-aziridinyl) phosphine sulfide; three (aziridinyls) are to the benzo quinone; three (aziridinyl) phosphine sulfide; uracil mastard; arabinosy ladenosine; vidarabine phosphate; vinblastine; Vinblastine Sulfate (vinblastine sulfate); sulfuric acid vincristin (vincristine); eldisine; Vinorelbine; Vinorelbine tartrate (NA); (1)-mimosine; 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitroso ureas; ((3-amino-2 for (8S-is suitable)-10-; 3-6-three deoxidations-α-L-lysol-pyranoid form hexose-based) oxygen)-7; 8; 9; 10-tetrahydrochysene-6; 8; 11-trihydroxy-8-(glycolyl)-1-methoxyl group-5; 12-aphthacene diketone; the meta-iodine benzyl of 131-guanidine (1-131 MIBG); 5-(3; 3-dimethyl-1-triazenyl)-1H-imidazoles-4-formyl ammonia; 5-(two (2-chloroethyl) amino) 2; 4-(1H; 3H)-hybar X; 2; 4; 6-three (1-aziridinyl)-s-thiazine; 2; 3; 5-three (1-aziridinyl)-2; 5-cyclohexadiene-1; the 4-diketone; 2-chloro-N-(2-chloroethyl)-N-methyl ethyl-amine; N; N-two (2-chloroethyl) tetrahydrochysene-2H-1; 3; 2-oxazaphosphorin-2-amine-2-oxide; 3-denitrification uracil; 3-iodine benzyl guanidine; 5; 12-aphthacene diketone; 5-azacitidine; 5 FU 5 fluorouracil; (1aS; 8S; 8aR; 8bS)-6-amino-8-(((amino carbonyl) oxygen) methyl)-1; 1a; 2; 8; 8a; 8b-six hydrogen-8a-methoxyl group-5-methyl azirino (2 '; 3 ': 3; 4) pyrrolo-(1; 2-a) indoles-4, the 7-diketone; the 6-aza uridine; Ismipur; guanozola; and combination.

Can comprise adriamycin and Doxetaxel, TOPO, taxol, NSC-241240 and taxol, taxol, cisplatin and radiation, 5 FU 5 fluorouracil (5-FU), F-FU and radiation, Toxotere, fludarabine, Ara C, Etoposide, vincristin and vinblastine with co-administered preferred therapeutic agents and the combination of polynucleotides of the present invention and polypeptide.

Exemplary chemotherapeutics also comprises doxetaxel (Toxotere ) and TOPO (Hycamtin ). Other chemotherapeutics and other cytotoxic agent comprise hereinafter described in " pharmaceutical composition ".

Other chemotherapeutics and other cytotoxic agent comprise that hereinafter " epi-position and antibody " and this paper other parts are described, and other reagent well-known in the art.

Polynucleotides of the present invention and polypeptide can prevent and/or treat for radiation insult.

Avoid for Cell protection, tissue and organ low, in or the infringement of high dose radiation and after carrying out preventative or therapeutic administration, polynucleotides of the present invention and polypeptide will protect human or animal's individuality and colony to avoid the damage that is caused by radiation exposure. This damage comprise that gastronintestinal system disorder, weight loss, radiation sickness, radiation burn, endocrine disturbance, goitre, eye illness such as dry eye syndrome, diseases associated with inflammation, psychopathic disorder, respiratory disorders, Genitourinary are disorderly, the circulatory system is disorderly and cancer such as leukaemia and thyroid cancer, and other disorder well-known in the art. The damage that is caused by radiation exposure comprise to the damage of cell and tissue all as indicated above, the destruction of the damage of cell DNA, cellular function is induced (comprise the secondary tumor of being induced by treatment is induced with other cancer induce) such as destroying DNA function, cell death, cancer.

Some regional nuclear proliferation and nuclear test accident in the world are rising. In addition, the utilization of nuclear energy and industrial expansion such as the processing of nuclear fuel and the dismounting of nuclear weapon, have all increased the risk of radiation exposure. For example, in 50 years, 4 key industry radioactive accidents are reported in Kistym (former Soviet Union) and Wind-Scale (Britain), Three Mile Island (U.S.) in 1979 and the Chernobyl (former Soviet Union) in 1986 of nineteen fifty-seven in the past. These radiation discharge the extensive exposure that causes multiple radioactive level. Because the accident of Chernobyl nuclear plant, this area has experienced with interior and in addition resident and animal (such as domestic animal) and has seriously influenced.

Early stage in the 1950's, find the infringement that cysteamine and relevant aminoalkyl mercaptan can protect organism to avoid radiating. Particularly, before being exposed to X ray, give mouse with these materials after, they have reduced the lethal effect of X ray. Studying to find better radioprotector. For example, Amifostine and other aminoalkyl dihydrogen phosphorothioate phosphate (U.S. 3,892,824) have been developed at first as protective agent, especially for for X ray or the nuclear that in military conflict, may meet with radiation. The most promising reagent is the Amifostine (WR 2721, S-2 (3-amino propyl amino)-ethyl-D2EHDTPA) that degradation in vivo becomes amino alkyl hydrosulfide, and its effect is similar to cysteamine. Yet, the poor use that limits WR 2721 of clinical tolerance (Cairnie, Radiation Res., 94:221,1983; The people such as Turrisi, " Radioprotectors and Anticarcinogens " (radioprotector and carcinogenesis are former), Nygaard and Simic compile, Academic publishing house, New York, 681-694 page or leaf, 1983; Blumberg, Int.J.Radiation Oncology Biol.Phys., 8:561,1982).

The invention provides the guard method for the damage of being induced by radiation; it be applicable to the radiation before and/or among and/or use afterwards; and still effective at relatively low non-toxic concn; thereby can be used in mammal (such as the people); and can look like once and repeatedly to use, as will be explained hereinafter.

As mentioned above, polypeptide of the present invention and polynucleotides can be protected, improve and treat cell, tissue and organ and be avoided by the damage of radiating or other cytotoxic agent causes, and increase the survival of the individuality that is exposed to cytotoxic agent (such as radiation). Before exposing, among and/or use afterwards after, the order of severity by radioactive damage will be eliminated or reduce to polypeptide of the present invention and polynucleotides.

Radiation exposure may be the result of accident, intentional, inherent, external, professional, environmental exposure, radon, nuclear pollution for example, and comprises the radiation that is discharged by for example nuclear explosion, nuclear accident or solar flare. This exposure can betide the survivor of for example workman, army personnel, the common people, emergency treatment people, nuclear explosion and the nuclear accident of nuclear power industry, staff, patient and the astronaut of sanitation and health-care field. Polynucleotides of the present invention and polypeptide also can be used for providing treatment or the protection for the radiation in other source, such as emergency treatment people, the common people or army personnel, and perhaps space traveler's radiation that may meet with.

Thereby, can use to preventing and/or treating property the damage that polynucleotides of the present invention and polypeptide are caused by radiation exposure with prevention, reduction or treatment.

The invention provides for the protection of or the individual method that avoids by radioactive damage for the treatment of, comprise polypeptide of the present invention or the polynucleotides of individuality being used effective dose. Polypeptide of the present invention and polynucleotides are for the protective agent by radioactive damage, and can before the radiation exposure, among and/or use afterwards. As above and hereinafter described, protective agent can be used for preventative and curative methods for the treatment of such as polynucleotides of the present invention and polypeptide.

Polypeptide of the present invention and polynucleotides can be used for the individuality that preventative and therapeutic treatment might be exposed to radiation, such as workman, army personnel, the astronaut of nuclear power industry, be engaged in the medical domain staff of the diagnosis that relates to radiation and methods for the treatment of or be exposed to the patient of radiation because of diagnosis or therapeutic purposes. As described in this paper other parts, polypeptide of the present invention and polynucleotides can attach for the cancer radiation method, and wherein polypeptide or polynucleotides are the selective protection normal structure, and so that cancerous tissue suffers radiotherapeutic destruction.

Therefore, can use polypeptide of the present invention or polynucleotides in prevention or treatment to the individuality that the radiation exposure risk is arranged. Possible radiation insult will be prevented or reduce to preventative purposes, and the damage that exists will be treated or reduce to therapeutic use and slow down, reduce or prevent other damage.

Effective dose should be understood to is enough to realize the polypeptide of the present invention of desired effects or the amount of polynucleotides. This effect can be prophylactic effects or therapeutic effect, and perhaps the two is furthermore. Effective dose depends on many factors, comprises type and the quantity of the individual radiation that exposes, and application program etc., this is well known in the art or is described in hereinafter. For example, dosage is described in hereinafter and runs through specification.

Polypeptide of the present invention is presented in the therapeutic process that uses chemotherapeutics and radiation and protects intestines and stomach (seeing embodiment 16-18). In addition, after using after radiation, polypeptide of the present invention shows can reduce the damage (seeing embodiment 16-18) that has radiation to induce in the intestines and stomach.

Thereby polypeptide of the present invention can be used for protective epithelium. " epithelium " refers to the covering of body interior and outer surface, comprises the layer of vascular and other loculus. It is comprised of the cell that links to each other by a small amount of adhesion substance. Shape according to the number of plies, the degree of depth, superficial cell is divided into several types with epithelium. Epithelial cell comprises the epithelial cell of corneal epithelium, barrett's epithelium, capsula glomeruli parietal layer epithelium, cilliated epithelium, columnar epithelium, corneal epithelium, cuboiodal epithelium, epithelium ductus semicircularis, glaze epithelium, false epithelium, germinal epithelium, tooth glaze epithelium, galandular epithelium, glomerulus visceral layer epithelium, stratified epithelium, lens epithelium, mesenchymal epithelium, olfactory epithelium, squamous epithelium, PE, protective epithelium, pseudostratified epithelium, pyramidal epithelium, respiratory epithelium, rod epithelium, seminaferous epithelium, sensory epithelium, simple epithelium, scaly epithelium, stratified epithelium, subcapsular epithelium, epithelium of gingival sulcus, tessellated epithelium, transitional epithelium and eye, tongue, body of gland, oral mucosa, duodenum, ileum, jejunum, caecum, nasal passage, oesophagus, colon, mammary gland and female and male reproductive system.

" body of gland " refers to that specialization is to secrete the cell aggregation of the material that need to have nothing to do with its eubolism. The example that can comprise epithelial body of gland comprises: lymph node, accessory gland, acinous gland, gastric gland, glandula parotis accessoria, adrenal gland, aggregated lymphatic follicles, the A Erwalan gland, circumanal gland, acinous gland, bulbourethral gland, the other body of active gland, glandulae linguales anteriores, apocrine gland, areolar gland, the artery gland, arteriococcygeal gland, the spoon gland, the A Saili gland, avicenna's gland, atribiliary gland, nodi lymphatici axillares, the Ba Tuolin gland, bauhin's gland, the Bao Nujiateng gland, glands of biliary mucosa, blandin and Nuhn gland, incretory, the BoerhaareShi gland, rich promise gland, bowman's gland, brachial lymph nodes, bronchial gland, bruch's glands, brunner gland, buccal glands, bulbourethral gland, cardiac gland, carotid body, abdomen is the gland ALN initiatively, ceruminous gland, cervical gland, choroid plexus, ciaccio's glands, the conjunctiva Moll gland, circumanal gland, the triumphant gland of clo, Cobelli gland, arteriococcygeal gland, sweat gland, compound gland, lymph node, conjuctival gland, examine the amber gland, cutaneous gland, cytogenic gland, hemaden, brunner's gland, the Di Weier inner membrance, von Ebner's gland, sweat gland, egli's glands, incretory, endo-epithelial gland, esophageal gland, excretory gland, exocrine gland, follicular glands of the duct, fundus gland, gastric gland, lymphonodi gastrici, cover her gland, gonad, the gum gland, lattice Lay gland, lymph node, glomerate glands, lingual gland and palatine gland, glands,Guerin's, pharyngeal mucous gland, crypts of Haller, gland of Harder, haversian glands, hedonic glands, haemolymphonodus, haemolymphonodus, the hematopoiesis gland, haemolymphonodus, henry is strangled gland, the bile duct gland, mixed gland, hibernating gland, holocrine gland and incretory.

The further example of body of gland comprises that neck moves the gland bead, the intermediate gland, bonnot's gland, interstitial gland, enteraden, endo-epithelial gland, intramuscular glands of tongue, the jugular vein lymph node, glandulae mucosae conjunctivae, labial gland, lachrymal gland, accessory lacrimal glands, mammary gland, glands of large intestine, apocrine sweat gland, the larynx gland, stomach tongue lenticular nucleus gland, Li Bei bends the grace gland, glandulae linguales anteriores, lingual gland, gland of Littre, Luschka gland, lymph node, extraparotid lymph glands, buccal glands, mammary gland, secondary mammary gland, glandula submandibularis, glands of Manz, the Mel gland, meibomian gland, merocrine gland, lymphonodi mesenterici, mesocolic lymph nodes, mixed gland, glandulae molares, More's film, the cell monolayer gland, gland of Montgomery, glands,Morgagni's, buccal gland, the synovial membrane gland, muccus gland, muccus gland, lingual gland, glands of auditory tube, the duodenum muccus gland, Stuckey difficult to understand pipe difficult to understand muccus gland, polycellular gland, myometrial gland, naboth glands, the special folliculus of Naboo, glandula nasalis, the neck gland, odoriferous glands of the prepuce, sebaceous glands, olfactory gland, gastric gland, arachnoid villi, palatine gland, lymphoglandulae pancreaticolienales, glands,parafrenal, parathyroid gland, woman's paraurethra, the parotid gland, glandula parotis accessoria, pectoral lymph nodes, proper gastric glands, sweat gland, the Pai Er gland, pharyngeal mucous gland, Philips's gland, pineal body and pituitary.

Other example of body of gland comprises the Pu Waliye gland, polyptychic gland, preen gland, pregnancy glands, accessory thyroid glands, preputial gland, prostate, puberty glands, pyloric gland, acinous gland, glandulae linguales posteriores, glandulae molares, the Li Weinusi gland, rosenmuller gland, acinous gland, salivary gland, belly salivary gland, the parotid gland, salivary gland and sublingual gland, the special gland of Saunders, permitted to strangle gland, sebaceous glands, glanduiae sebaceae conjunctivales, sentinel gland, seromucous gland, serous gland, serres' gland, sigmund's glands, glands,Skene's, simple gland, intestinal glands, folliculi lymphatici solitarii intestini crassi, splenoid gland, side's look tal fibre gland, palatine gland, infraauricular lymph nodes, sublingual gland, glandula submandibularis, sweat gland, adrenal gland, accessory adrenal gland, suzanne's gland, sweat gland, the synovial membrane gland, Meibomian gland, theile's glands, thymus gland, thyroid gland, accessory thyroid glands, lingual gland, tracheal gland, follicles in trachoma, tubular gland, acinotubular gland, tympanic gland, preputial gland, simple gland, glandula urethralis, prop up the property glandula urethralis, uropygial gland uterine gland, uterine gland, vaginal gland, haemolymphonodus, greater vestibular gland and glandule, virchow's gland, vitellarium, greater vestibular gland, the Wa Erdaier gland, weber's glands, the Wolflin gland, zeis gland and zuckerkandl gland.

The further example of body of gland comprises gland, Tai Ersong gland, tiedemann gland, acinotubular gland, thachoma glands, greater vestibular gland, the graceful gland of gas, WepferShi gland and WolferShi gland on albuminous glands, gathering gland, glands of auditory tube, secondary sweat gland, bulbourethral gland, pharynx cardiac gland, glands of auditory tube, lymph follicle, galeati's glands, buccal glands, HarverShi gland, inguinal lymph nodes, interrenal gland, KnollShi gland, Luschka gland, the lucky gland of Pierre's skin, marror lymph node, master gland, glandula submandibularis, glands,Mery's, nuhn's gland, Meibomian gland, all glands of tracheae, hair follicle gland, seminal vesicle, glandula submandibularis, sweat gland, the hyoid bone.

Thereby MPIF-1 can be used for protecting the cell in any these cells or these bodies of gland.

MPIF-1 can be used for protection or reduces the damage of being induced by cytotoxic agent in muscle cell (such as cardiac muscle cell, Skeletal Muscle Cell and smooth muscle cell), epithelial cell (such as squamous cell (comprising endothelial cell), cuboidal epithelium and columnar epithelial cell), the nerve fiber cell (such as neuron and neuroglia). MPIF-1 also can be used for protecting or reducing the damage to musculature, nerve fiber, epithelial tissue, endothelial tissue and connective tissue of being induced by cytotoxic agent.

In addition, MPIF-1 can be used for protecting or reducing the damage to cell, terminal differentiation cell, multipotential stem cell, typing CFU-GM and prepattern stem cell in cell, the non-division in the division of being induced by cytotoxic agent.

Thereby MPIF-1 can be used for treating the damage of nervous system cell, such as neuron, comprises cortical neuron, intrerneuron, centre effect neuron, periphery effect neuron and bipolar neuron; And neuroglia, comprise Schwann cell, oligodendroglia, astroglia, microglia and endyma.

In addition, can also use MPIF-1 treatment or protection endocrine and endocrine relevant cell to avoid the infringement of cytotoxic agent, described cell for example comprises: the pituitary gland cell comprises epithelial cell, pituicyte, neuroglia, without particle chromophobe, particle cells chromophil (eosinophil and basophilic granulocyte); Adrenal cells comprises adrenaline secretion cell, non-adrenaline secretion cell, myelocyte, cortical cell (blood vessel is spherical, pencil and desmacyte); Thyroid cell comprises epithelial cell (chief cell and parafollicular cell); Parathyroid cells comprises epithelial cell (chief cell and acidophil); Pancreatic cell comprises islet cells (α, β and delta cell); The pineal body cell comprises parenchyma and Deiter's cells; Thymocyte comprises parafollicular cell; Testicular cell comprises seminiferous tubule cell, interstitial cell (Leydig Schwann Cells), spermatogonium, sperm mother cell (primary and secondary), spermatoblast, sperm, Sertoli Schwann Cells (sertoli cell) and myoid cell; Gonad cell comprises egg cell, oogonium, egg mother cell, granular cell, theca cell (inside and outside), germinal epithelium cell and follicle cell (original, cryptomere, maturation and atretic follicle).

MPIF-1 can be used for treating the damage of being induced by cytotoxic agent among the myocyte, such as muscle fibril, intratusal fiber and extrafusal fibers. MPIF-1 can be used for treating the damage of being induced by cytotoxic agent in the skeletal system cell, such as Gegenbaur's cell, osteocyte, osteoclast and CFU-GM thereof.

Can use MPIF-1 treatment or protection such as following cell to avoid the infringement of cytotoxic agent: heart cell (cardiac muscle cell); Blood and lymphocyte comprise the stem cell to the hematopoietin sensitivity, red blood cell, leucocyte is (such as eosinophil, basophilic granulocyte, and neutrophil cell (granular cell), with lymphocyte and monocyte (without granular cell)), blood platelet, tissue macrophages (histocyte), the organ specificity macrophage is (such as Kupffer cell (Kupffer cell), pulmonary alveolar macrophage, and microglia), bone-marrow-derived lymphocyte, the T lymphocyte is (such as cytotoxic T cell, helper cell, with inhibition T cell), megaloblast, monoblast, myeloblast, lymphoblast, primitive erythroblast, megakaryoblast, premonocyte, premyeloblast, prolymphocyte, early stage normoblast, megacaryocyte, middle normoblast, juvenile cell is (such as the juvenile form juvenile cell, the merogenesis juvenile cell, and polymorphonuclear granulocyte), late period normoblast, desmacyte, and bone marrow cell.

Can also treat respiratory system cell (such as cilium endothelial cell and alveolar cell) with MPIF-1, with the damage that reduces or prevention is induced by cytotoxic agent. Can also treat or protect the urinary system cell, such as the nephron, cilium endothelial cell, granular cell, little endothelial cell and sertoli cell. Can also use MPIF-1 treatment or protection digestive system cell, such as simple columnar epithelium cell, mucomembranous cell, acinar cells, parietal cell, chief cell, zymogenic cell, peptic cells, enterochromaffin cell, calyciform cell, argentaffin and G cell. Can treat sensory cell with prevention or reduce the cytotoxicity damage with MPIF-1, such as: auditory system cell (hair cell); The olfactory system cell comprises olfactory receptor cell and columnar epithelial cell; Balance/vestibular organ cell comprises hairy cell and supportint cell; The vision system cell comprises chromatophore, epithelial cell, light receptor neuron (retinal rod and the cone), gangliocyte, amakrine, Beale's ganglion cells and horizontal cell.

In addition, can treat following cell to reduce or the damage of prevention cytotoxicity with MPIF-1: the endothelial cell of the cell of the cell of mesenchymal cell, stroma cell, hair cell/folliculus, adipocyte, simple epithelium tissue (scaly epithelium, cuboiodal epithelium, columnar epithelium, cilium columnar epithelium and pseudostratified ciliated columnar epithelium), stratified epithelium tissue (stratified squamous epithelium (angling and not angling), stratified cuboidal epithelium and transitional epithelium), calyciform cell, mesenteric mesaraic endothelial cell, small intestine, the endothelial cell of large intestine, tie up the endothelial cell of managing endothelial cell capillaceous, the endothelial cell of little vascular tissue, the endothelial cell of artery, arteriolar endothelial cell, the endothelial cell of vein, venular endothelial cell and bladder etc.

MPIF-1 also protects and treats cytotoxicity damage in the phoirocyte, forms (net form) tissue (comprising corium), feltwork connective tissue, elastic connective tissue, reticular connective tissue and adipose connective tissue such as loose knot. Also can comprise cartilage cell, adipocyte, periosteum cell, bone inner cell, odontoblast, Gegenbaur's cell, osteoclast and osteocyte with the cell of MPIF-1 protection and the connective tissue for the treatment of.

MPIF-1 also protects cell, prostatic cell and the pneumonocyte of endothelial cell, liver cell, horn cell and substrate horn cell, myocyte, maincenter and peripheral neverous system.

MPIF-1 also can protect the epithelial cell in lung, breast, pancreas, stomach, small intestine and the large intestine. But MPIF-1 protective epithelium cell generates calyciform cell and other epithelial cell such as sebaceous cell, hair follicle, liver cell, II type pneumonocyte, mucoprotein, and their CFU-GM that comprises in skin, lung, liver and the intestines and stomach.

MPIF-1 can protect liver cell; MPIF-1 can be used for prevention or weaken acute or chronic hepatitis in prevention or treatment thus, and the burst or the inferior explosive liver failure that are caused by treatment of cancer (such as chemotherapy and/or radiotherapy) and environment or accident radiation exposure.

MPIF-1 also can be used for reducing the enterotoxication side effect that is caused by cytotoxic agent treatment (such as radiation or chemotherapy). MPIF-1 has the cytoprotective effect to mucous membrane of small intestine. MPIF-1 also can be used for prevention or weaken catarrh and for reducing the catarrh (such as oral cavity, esophagus, intestines, colon, rectum and anal ulcer) that is caused by chemotherapy and other cytotoxic agent in prevention or treatment.

IBD (such as CrohnShi disease and ulcerative colitis) is destroyed and the disease that causes by small intestine or colorectal mucosa surface. Thereby MPIF-1 can be used for promoting the reconstruction of mucomembranous surface, thereby helps to heal faster the also development of preventing inflammatory enteropathy. Estimate that the MPIF-1 treatment has remarkable result to whole GI mucus generation, and can be used for protecting intestinal mucosa to avoid the infringement of cytotoxic agent. Thereby the present invention also provides the method that is used for prevention or treatment membrane disease or pathology event (comprising ulcerative colitis, CrohnShi disease and the impaired Other diseases of mucous membrane), comprises the MPIF-1 that uses effective dose. The present invention is similar provides the method for the oral cavity that is used for prevention or treatment and caused by cytotoxic agent (comprising and the impaired relevant odynophagia of pharynx periglottis), esophagus, stomach, intestines, colon and mucous membrane of rectum inflammation.

In addition, MPIF-1 can be used for preventing and reduces plurality of reagents to the damage of lung. MPIF-1 can prevent or treat alveolar and bronchiolar epithelium damage. For example, can effectively treat inhalation injury (namely being caused by smoking) and the radioactive damage that causes bronchiolar epithelium and alveolar necrosis with MPIF-1. Equally, MPIF-1 can be used for protecting II type pneumonocyte.

MPIF-1 can be used for the treatment of or prevent the damage to corium and epidermis, ocular tissue, dental tissue, oral cavity clinically, and the complication relevant with antineoplastic with general or locality radiotherapy. MPIF-1 also can be used for treating the skin forfeiture.

MPIF-1 also can be used for reducing the enterotoxication side effect that is caused by radiation, chemotherapy or the treatment of other cytotoxicity. MPIF-1 has the cytoprotective effect to mucous membrane of small intestine. MPIF-1 also can be used for prevention or weaken catarrh and for reducing the catarrh that is caused by chemotherapy, radiation and other cytotoxic agent (such as oral cavity, esophagus, intestines, colon, rectum and anal ulcer) in prevention or treatment. Thereby the present invention also provides the method for prevention or treatment membrane disease or pathology event (comprising ulcerative colitis, CrohnShi disease and the impaired Other diseases of mucous membrane), comprises the MPIF-1 that uses effective dose. The present invention is similar provides the method that is used for prevention or treatment oral cavity (comprising and the impaired relevant odynophagia of pharynx periglottis), esophagus, stomach, intestines, colon and mucous membrane of rectum inflammation, and no matter cause concrete reagent or the form of this damage.

In addition, MPIF-1 can be used for treating and/or preventing bubble and the burn that is caused by chemicals; The ovary that is caused by the chemotherapeutics treatment damages, for example by radiating or the cystitis that chemotherapy is induced and the damage of intestines of being induced by high dose chemotherapy.

MPIF-1 can be used for preventing or reducing the Poisoning injury of kidney of being induced by chemotherapeutics, radiation or other cytotoxic agent.

The present invention also provide for the protection of individuality avoid radiating, the method for the impact of chemotherapy or the treatment of other cytotoxic agent, comprise the MPIF-1 that uses effective dose. The present invention also provide for reducing or prevention by the method that is exposed to the tissue damage that radiation, chemotherapeutics or other cytotoxic agent cause, comprise the MPIF-1 that uses effective dose. Individuality may be because of many former thereby be exposed to radiation, comprises for therapeutic purposes (for example for the overmedication proliferative diseases), because of the radio isotope accident discharging into environment or in the process of intrusive mood or non-intrusion type medical diagnostic method (such as X ray). In addition, the individuality of a lot of numbers they the workplace or family in be exposed to radioactive radon. The long-term environment exposure that continues has been used for calculating expection and has subtracted longevity estimated value (W.Johnson and K.Kearfott, Health Phys., 73:312-319,1997). Shown in embodiment 17-18, protein of the present invention has strengthened the survival of the animal that is exposed to radiation. Thereby MPIF-1 can be used for increasing the survival rate of the individuality that suffers the damage of being induced by radiation, the individual radiation that avoids sublethal dose of protection, and increase the treatment ratio that disease torments radiation in (such as the hyper-proliferative sexual disorder) treatment.

MPIF-1 also can be used for protecting individuality to avoid the infringement of radiation, chemotherapeutics and other cytotoxicity agent dose that usually can not tolerate. By this way or during use as described herein, can radiotherapy/exposure, chemotherapy or the treatment of other cytotoxic agent/before exposing, afterwards and/or among use MPIF-1. When treatment suffered from disease and torments high developing stage individual of (such as the hyper-proliferative sexual disorder), the radiation of high dose and chemotherapeutics may be useful especially.

On the other hand, the invention provides for prevention or treat the method for following situation, comprise the MPIF-1 that individuality is used effective dose: the hematopoiesis syndrome that the pulmonary fibrosis of inducing such as the oral cavity of being induced by radiation and gastrointestinal damage, catarrh, intestines fibrillatable, rectitis, by radiation, the pneumonia of being induced by radiation, the pleura of being induced by radiation retract, induced by radiation and poisoned by the marrow that radiation is induced.

Thereby, MPIF polynucleotides of the present invention or polypeptide can be used for suppressing Normocellular damage at radiotherapy, chemotherapy and targeted radiotherapy, comprise the damage to myeloid progenitor. The radioimmunotherapy of targeted radiotherapy such as malignant tumor, transfer and associated disorders. Associated disorders such as leukaemia (comprising acute leukemia (such as acute lymphatic leukemia, acute myelocytic leukemia (comprising myeloblast, promyelocyte, myelomonocyte, monocyte and erythroleukemia)) and chronic leukemia (such as chronic myeloid (granulocytic) leukaemia and chronic lymphocytic leukemia)), polycythemia vera, lymthoma (sick such as the sick and non-HodgkinShi of HodgkinShi), Huppert's disease, WaldenstromShi macroglobulinemia, heavy chain disease and solid tumor. Solid tumor includes but not limited to sarcoma and cancer, such as fibrosarcoma, myxosarcoma, the lipid sarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangioendothelial sarcoma, lymphangioendothelial sarcoma, synovialoma, celiothelioma, the EwingShi knurl, leiomyosarcoma, rhabdomyosarcoma, colon cancer, cancer of the stomach, the pancreas cancer, mastocarcinoma, oophoroma, prostate cancer, squamous cell carcinoma, the matrix cells cancer, gland cancer, syringocarcinoma, carcinoma of sebaceous glands, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, cephaloma, bronchus source property cancer, clear-cell carcinoma, hepatoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, WilmShi knurl (nephroblastoma), cervical carcinoma, testicular tumor, lung cancer, ED-SCLC, carcinoma of urinary bladder, epithelioma, glioma, neuroastrocytoma, medulloblastoma, craniopharyngioma, ependymocytoma, pinealoma, hemangioblastoma, acoustic neurinoma, oligodendroglioma, menangioma, melanoma, neuroblastoma, and retinoblastoma. These disorders also comprise metastatic medullary thyroid, shaping neuroastrocytoma, spongioblastoma, follicular lymphoma, colon cancer, cardiac tumor, lung cancer, intestinal cancer, carcinoma of testis, cancer of the stomach, myxoma, myomata, lymthoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi sarcoma and oophoroma. Can use other disorder of polypeptide of the present invention, polypeptide fragment and variant, activator and antagonist (comprising antibody) and other Antybody therapy to be well known in the art, also can be disclosed herein.

MPIF-1 can use separately, perhaps unites one or more other reagent uses of giving for the protective effect of radiation or other reagent. Many cell factors (such as IL-1, TNF, IL-6, IL-12) show gives this protective effect. Consult such as people such as R.Neta J.Exp.Med., 173:1177,1991. In addition, IL-11 is presented at and unites radiation and chemotherapy (people such as X.X.Du, Blood; 83:33,1994), and the thoracic injury (people such as C.A.Redlich who is induced by radiation; J.Immun., 157:1705-1710,1996) rear protection small intestinal cell. Several growth factors also show the protective effect of giving for radiation exposure, and (36:337-340,1997 for the people such as I.Ding, Acta Oncol. such as fibroblast growth factor and TGF-β-3; The people such as C.Potten, Br.J.Cancer, 75:1454-1459,1997).

Can comprise with other radioprotector that polypeptide of the present invention and polynucleotides are used calcium antagonist (WO 93/02670), (US 4 for polyethylene glycol, 676,979), (US 4 for polyvinylpyrrolidone, 676,979), (US 4 for poly glycol monomethyl ether, 676,979), the acid amides of methoxy poly (ethylene glycol) and amine and salt, with chelating agent such as EDTA, DTPA, and EGTA (WO 98/47858), (US 5 with other metallothionein induction thing for manganese, 008,119), (US 5 for WR-2721,424,471), WR-1065, (US 5 for other group thiophosphate, 869,338), (US 5 for polyamide mercaptan, 217,964), (US 5 for SCF/IL-3/GM-CSF combination or single therapy scheme, 620,685), (US 5 for beta carotene and Dunaliella algae preparation, 948,823), (US 5 for alkaloidal phosphorus derivant, 981,512), (US 5 for thymalin and L-Glu-L-Trp, 770,576), cell factor is such as IL-1, TNF, stem cell factor, and IL-12 (Neta, Stem Cell, 15 (supplementary issue 2): 207-210,1997), copper chelate (the people such as Sorenson, Proc.Soc.Exp.Biol.Med., 210:191-204,1995), (US 5 for the D-factor/growth hormone/IL-1/ tumor necrosis factor sub-portfolio or single therapy scheme, 843,422), Actihaemyl (CAS RN No.37239-28-4), Amifostine (WR 2721), 2-amino-single sodium salt (the cystaphos of ethyl mercaptan dihydrogen phosphoric acid (ester), phosphocysteamine), 3-(two (2-chloroethyl) carbamic acid) estradiol (estramustine), 2-amino-ethyl mercaptan (cysteamine), 2,2 '-two sulfo-s two (ethamine) (cystamine), S-2-amino-ethyl thiocarbamide bromide hydrogen bromide (AET), copper chelate such as Cu (II) 2 (3,5-diisopropyl salicylic acid) 4 (Cu (II) 2 (3,5-DIPS) 4), Fe, Mn, the essential metal element chelate (people such as Sorenson with Zn, Proc.Soc.Exp.Biol. Med., 210:191-204,1995), 2-(allyl sulfide generation) pyrazine, alkaloidal phosphorus derivant (austrian patent numbers 377 988 and 354 644; US 5,981, and 512) etc., and combination.

Polynucleotides of the present invention and polypeptide also can be used with the emesis agent, such as 2-(ethylenebis dithiocarbamate)-10-(3-(4-methyl isophthalic acid-piperazinyl) propyl group)-10H-phenthazine (ethyl thioproperazine), 1-(p-chloro-α-phenylbenzyl)-4-(meta-methyl-benzyl)-piperazine (meclozine) etc., and combination. Polynucleotides of the present invention and polypeptide also can and combine with disclosed herein or other therapeutic agent that this area is known and use.

Hemorrhagic cystitis is with some disease condition and is exposed to the relevant syndrome of medicine, virus and toxin. It shows as the diffuse hemorrhage of bladder endodermis. Known treatment comprises in the bladder, general and non-drug therapy (N.J.West, Pharmacotherapy, 17:696-706,1997). Some cytotoxic agent of clinical use has side effect, namely causes the inhibition to normal epithelial propagation in the bladder, causes the collapse of potential life-threatening ulcer and epithelial layer. For example, endoxan is that mainly bio-transformation becomes the cytotoxic agent of active alkanisation metabolin by the functional particles body oxidase system that mixes in liver. The growth mechanism that these metabolins are intervened malignant cell in the fast breeding of susceptibles is considered to relate to crosslinked (" Physician ' s Desk Reference " (desktop reference book of internist), 1997) of DNA of tumor cell.

Endoxan is the example of cytotoxic agent of cystitis of causing bleeding in some patient, and this complication can be serious, is fatal in some case. The fibrillatable of uropoiesis bladder also may with or without cystitis. This damage is considered to that endoxan metabolin in the urine causes by being excreted to. There are several days usually in the blood urine that is caused by endoxan, but also may continue. In serious case, need internal medicine or surgical intervention. Serious hemorrhagic cystitis situation causes treated with cyclophosphamide pulse discontinuous. In addition, uropoiesis bladder malignant tumour betides treated with cyclophosphamide pulse in two years usually, and betides among the patient who before suffers from hemorrhagic cystitis (consulting cyclophosphamide packing insert). Endoxan has poisonous impact to prostate and male reproductive system. Treated with cyclophosphamide pulse can cause sterile, and causes orchiatrophy to a certain degree.

Those of ordinary skill will understand, the MPIF-1 polypeptide effective dose (comprising with or be not effective to marrow containment agent or marrow containment property inhibitor the amount of the MPIF-1 polypeptide of marrow containment) that can rule of thumb be identified for treating the individuality that needs rising MPIF-1 activity level for indicating every kind of situation using MPIF-1. Can unite one or more pharmaceutics in pharmaceutical composition can accept excipient and use the polypeptide with MPIF-1 activity.

MPIF-1 also can be by apoptosis-induced leukaemia and the abnormality proliferation cell (such as tumour cell) of being used for the treatment of. MPIF-1 is apoptosis-induced in the HPC group.

MPIF-1 can be used for amplification prematurity HPC by temporarily preventing differentiation, such as granulocyte, macrophage or monocyte. These bone marrow cells can be in vitro culture. Thereby for the purpose of bone-marrow transplantation and/or gene therapy, MPIF-1 also can be at external instrumentality as candidate stem cell. Because stem cell is rare, and it is the most useful to carry out gene therapy for quiding gene, so MPIF can be used for the population of stem cells of separation and concentration. Cultured cell comes stem cell enrichedly in the situation of cytotoxin (such as 5-FU) that can be by killing cell in the quick division in existence, and wherein stem cell will be subject to the protective effect of MPIF-1. Can send these stem cells back to Patients Following Bone Marrowtransplantation, perhaps then be used for transfection expectation gene to carry out gene therapy. In addition, MPIF-1 can be expelled in the individual body, cause individual marrow to discharge stem cell in peripheral blood. Can separate these stem cells, to be used for the operation of autologous bone marrow transplantation or gene therapy. After the patient finishes chemotherapy or radiotherapy, the stem cell that separates can be returned the patient.

In addition, because MPIF-1 is influential to T lymphocyte and macrophage, so MPIF-1 might enhancement antigen be the ability that delivery cell (APC) absorbs virus, bacterium or other foreign substance, processing and presents to the lymphocyte of being responsible for immune response. MPIF-1 also can regulate and control the interaction of APC and T lymphocyte and bone-marrow-derived lymphocyte. MPIF-1 can provide costimulatory signal in Antigen presentation, instruct responsive cell survival, propagation, differentiation, secrete other cell factor or solubility medium or selectively eliminate responsive cell by apoptosis-induced or other cell death mechanism. MPIF-1 because showing, APC helps HIV is transferred to the CD4+T lymphocyte, so also can affect this ability and prevent the lymphocyte infected by HIV or by other virus of APC mediation. APC, T lymphocyte or other cell type also are like this to the initial infection of HIV, EBV or any other this viroid.

In addition, prove recently that MIP-1 α acceptor is taken on and help HIV to enter person monocytic cell and the lymphocytic confactor of T that this has proposed interesting possibility: namely MPIF-1 and variant thereof may be intervened the process (seeing embodiment 11) that HIV enters cell. Thereby MPIF-1 can be used as for invasion and is subject to virus and the retroviral antivirotic that MIP-1 α acceptor helps.

By stimulating the T lymphocyte to resist the intrinsic activity of bacterium and virus infections and other foreign body, MPIF-1 can take on immune enhancer. These activity can be used for the normal response (infecting such as allergia) to exotic antigen, and to the immune response of tumour or optimum growth (comprising solid tumor and leukaemia).

Owing to these reasons, the present invention can be used for the panimmunity System Dependent disorder of diagnosis or treatment mammal (preferred people). These disorders comprise tumour, cancer and the imbalance of any immune cell function, include but not limited to autoimmunity, arthritis, leukaemia, lymthoma, immune containment, pyemia, wound healing, acute and chronic infection, the immunity by cell-mediated, humoral immunity, IBD, marrow containment, etc.

Therefore, MPIF-1 can be used for promoting wound healing by control target immunocyte infiltration impingement. Similarly, polypeptide of the present invention can be by attracting and activate the microbicidel leucocyte to strengthen the host for the defence of chronic infection (such as mycobacterium).

The another kind of purposes of this polypeptide is to come suppressor T cell propagation by the biosynthesis that suppresses IL-2, for example in autoimmune disease and lymphocytic leukemia.

MPIF-1 also can be used for suppressing keratinization of epidermis cell proliferation, and this is useful in psoriasis (keratinocyte hyper-proliferative), because find the Langerhans Hemapoiesis MIP-1 α in the skin.

MPIF-1 can be by raising the removing fragment and promoting the inflammatory cell of connective tissue and be used for scabbing of prevent injuries agglutination by control by the beta mediated fibrillatable of excessive TGF. In addition, this peptide species can be used for treating apoplexy, piastrenemia, pulmonary embolism and bone marrow proliferation sexual disorder, because MPIF-1 can increase vasopermeability. Pharmaceutical composition

The MPIF-1 polypeptide drug composition comprises the separation MPIF-1 polypeptide of the present invention of effective dose, particularly can effectively increase the MPIF-1 mature form of MPIF-1 activity in this individuality. Consider the other factors that each patient's clinical condition (using especially separately the side effect of MPIF-1 treatment), the delivery site of MPIF-1 peptide composition, the method for using, the plan of using and practitioner know, can the mode consistent with good medical practice prepare and take these compositions. Thereby, can be identified for by these considerations " effective dose " of the MPIF-1 polypeptide of this paper purpose.

Polypeptide of the present invention, antagonist or activator and suitable pharmaceutics carrier can be united use. These compositions comprise this polypeptide for the treatment of effective dose, and pharmaceutics acceptable carrier or excipient. This carrier includes but not limited to salt solution, BS, dextrose, water, glycerine, ethanol and combination thereof. The pattern that preparation should be fit to use.

" pharmaceutics acceptable carrier " refers to the preparation adminicle of nontoxic solid, semisolid or liquid filling agent, diluent, lapping or any type. When being used for this paper, term " stomach and intestine are outer " refers to comprise in intravenous, intramuscular, the peritonaeum, breastbone is interior, subcutaneous and the mode of administration of intra-arterial injection and infusion.

By slow-released system also can be suitable use the MPIF-1 polypeptide. The suitable example of slow releasing composition comprises the semipermeable polymers matrix of formative substance form (such as film or micro-capsule). Sustained-release matrix comprises polyactide class (U.S. Patent number 3,773,919 and EP 58,481), the copolymer of Pidolidone and the γ-ethyl-Pidolidone (people such as U.Sidman, Biopolymers, 22:547-556,1983), poly-(HEMA) (people such as R.Langer, J.Biomed. Mater.Res., 15:167-277,1981 and R.Langer, Chem.Tech., 12:98-105,1982), the ethene vinyl-acetic ester (people such as R.Langer, the same) or poly-D-(-)-3-hydroxybutyrate (EP 133,988). The MPIF-1 peptide composition of slowly-releasing also comprises the MPIF-1 polypeptide of liposome. Can prepare the liposome that comprises the MPIF-1 polypeptide: DE 3,218,121 by known method itself; The people such as Epstein, Proc.Natl.Acad.Sci. USA, 82:3688-3692,1985; The people such as Hwang, Proc.Natl.Acad.Sci. USA, 77:4030-4034,1980; EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese patent application 83-118008; U.S. Patent number 4,485,045 and 4,544,545; With EP 102,324. Usually, liposome is little (approximately 200-800 dust) single-layer type, and wherein lipid content can be regulated selected ratio to reach best MPIF-1 polypeptide therapy greater than about 30 molar percentage cholesterol.

For stomach and intestine are used outward, in one embodiment, usually by can being accepted carrier (be that described carrier is nontoxic at used dosage and concentration to acceptor, and compatible with other composition in the preparation) with the injectable forms (solution, suspension or emulsion) of UD and pharmaceutics, the MPIF-1 of required purity mixes to prepare the MPIF-1 polypeptide. For example, preparation preferably contains no oxidizing agent and known other compound harmful to polypeptide.

Usually by contacting to prepare preparation with solid carrier liquid-carrier or fine dispersion or both with the MPIF-1 polypeptide is even and close. Then, if necessary, product can be made the preparation shape of expectation. Preferred vector is the outer carriers of stomach and intestine, the solution that more preferably oozes with acceptor blood etc. The example of this carrier comprises water, salt solution, RingerShi liquid and glucose solution. Non-aqueous carrier (such as fixed oil and ethyl oleate) and liposome also are useful in this article.

Carrier can suitably comprise the additive of minute quantity, such as the material that strengthens isotonicity and chemical stability. These materials are nontoxic at used dosage and concentration to acceptor, comprise buffer solution, such as phosphate, citrate, succinate, acetic acid and other organic acid or its salt; Antioxidant is such as ascorbic acid; Low-molecular-weight (being less than about 10 residues) polypeptide is such as pR60 or tripeptides; Protein is such as serum albumin, gelatin or immunoglobulin (Ig); Hydrophilic polymer is such as polyvinylpyrrolidone; Amino acid is such as glycine, glutamic acid, aspartic acid or arginine; Monose, disaccharides and other carbohydrate comprise cellulose or derivatives thereof, glucose, mannose or dextrin; Chelating agent is such as EDTA; Sugar alcohol is such as sweet mellow wine or sorbierite; Equilibrium ion is such as sodium; And/or nonionic surface active agent, such as polysorbate (polysorbate), poloxalkol (poloxamers) or PEG.

Usually under the condition of the about 3-8 of pH, in these carriers, prepare the MPIF-1 polypeptide with the concentration of about 0.1mg/ml-100mg/ml, preferred 1mg/ml-10mg/ml. The use that should be appreciated that above-mentioned some excipient, carrier or stabilizing agent can cause the formation of MPIF-1 polypeptide salt.

When MPIF-1 and/or its variant were used for the treatment of people's hyper-proliferative sexual disorder as marrow protectant and as the part of chemotherapy regimen, being used for the appropriate dose scope that intravenous uses was 0.01 μ g/kg body weight-10 μ g/kg body weight. In addition, dosage intravenous that can 0.1,1.0,10 and 100 μ g/kg body weight is used MPIF-1. Indicating the MPIF-1 dosage of the marrow protection that is applicable to the people from the Data Extrapolation of zooscopy is 0.016 μ g/kg body weight.

Using MPIF-1 and/or its variant as to the treatment of cell, tissue and organ cell's toxic damages the time, can after being exposed to cytotoxic agent, it be applied to the people. As to the prevention of the cytotoxicity of cell, tissue and organ damage the time, can before exposure, use MPIF-1 and/or its variant, perhaps can before being exposed to cytotoxic agent and afterwards, all use.

In addition, MPIF-1 and/or its variant can be used and once reach given number of days (such as 3 days) every day. In addition, when being used for chemotherapy regimen, can before using chemotherapeutics, MPIF-1 be applied to the people. For example, can and use MPIF-1 before using two days of chemotherapeutics, before one day the same day.

When in order to treat the bone marrow proliferation sexual disorder MPIF-1 and/or its variant being applied to the people, application dosage is can be with MPIF-1 identical during as marrow protectant. When being applied to the people in order to treat the bone marrow proliferation sexual disorder, can subcutaneous administration MPIF-1.

The MPIF-1 that being used for the treatment of property is used must be aseptic. By filtering and easily to realize degerming through aseptic filter membrane (such as 0.2 μ m film). Usually therapeutic MPIF-1 peptide composition is placed the container with aseptic gateway, for example intravenous solution bag or have the bottle of the transparent stopper of hypodermic needle.

Usually with the MPIF-1 polypeptide as aqueous solution or be used for the container that heavy molten lyophilized formulations is stored in UD or multiple dose, for example in the airtight ampoule bottle or bottle. The example of lyophilized formulations is that 5ml is poured into the bottle of 10ml through 1% (w/v) of aseptic filtration MPIF-1 aqueous solution, and with the freeze-drying of gained mixture. Can prepare infusion solution by the MPIF-1 polypeptide that uses the heavy molten freeze-drying of antibacterial water for injection.

The present invention also provides pharmaceutics packing or the kit of the one or more containers that comprise one or more compositions that pharmaceutical composition of the present invention is housed. Can sign and issue with preparation, the application of management medicine or biological products or the government organs of selling the points for attention of form with these containers, its reflection obtained people's medication preparation, use or the license of marketing organization. In addition, polypeptide of the present invention and other therapeutic compound can be united use.

The present invention also provides the method that treats and/or prevents disease or disorder (such as any or multiple disease disclosed herein or disorder) by the therapeutic agent of the experimenter being used effective dose. Therapeutic agent refers to polynucleotides or polypeptide (comprising fragment and variant), their activator or antagonist and/or their antibody, and the associating pharmaceutics can be accepted bearer type (such as sterile carrier).

Consider each patient clinical condition (use especially separately MPIF-1 treatment side effect), deliver site, application process, other factors that the plan of using and practitioner know, prepare and take MPIF-1 in the mode consistent with good medical practice. Thereby, can be identified for by these considerations " effective dose " of this paper purpose.

Although as mentioned above, but in treatment consideration, but every dose of stomach and intestine of general recommendations to use the scope of total pharmaceutics effective dose of MPIF-1 outward be about 1 μ g/kg weight in patients/sky-10mg/kg weight in patients/sky. More preferably, this dosage is 0.01mg/kg weight in patients/sky at least, to people's about 0.01-1mg/kg body weight/day most preferably. If successive administration, usually with about 1 μ g/kg body weight/hour-about 50 μ g/kg body weight/hour dose rates, by injection every day 1-4 time, perhaps use MPIF-1 by for example continuous h inf with Micropump. Also can adopt the packed solution of intravenous. Observe and change the time interval that reacts after required treatment time and the treatment and as if change with desired effects.

Can followingly use MPIF-1: oral, rectum, stomach and intestine are outer, in the pond, in the vagina, in the peritonaeum, local (by powder, ointment, gel, drops or through the skin patch), cheek or mouth or nose spray delivery. " pharmaceutics can be accepted carrier " refers to the preparation adminicle of nontoxic solid, semisolid or liquid filling agent, diluent, lapping or any type. When being used for this paper, term " stomach and intestine are outer " refers to comprise in intravenous, intramuscular, the peritonaeum, breastbone is interior, subcutaneous and the mode of administration of intra-arterial injection and infusion.

By slow-released system also can be suitable use MPIF-1. The example of slowly-releasing MPIF-1 is that oral, rectum, stomach and intestine are outer, in the pond, in the vagina, in the peritonaeum, local (by powder, ointment, gel, drops or through the skin patch), cheek or mouth or nose spray delivery. " pharmaceutics can be accepted carrier " refers to the preparation adminicle of nontoxic solid, semisolid or liquid filling agent, diluent, lapping or any type. When being used for this paper, term " stomach and intestine are outer " refers to comprise in intravenous, intramuscular, the peritonaeum, breastbone is interior, subcutaneous and the mode of administration of intra-arterial injection and infusion.

By slow-released system also can be suitable use the MPIF-1 polypeptide. The example of slowly-releasing MPIF-1 comprises suitable polymeric material (such as the semipermeable polymers matrix of formative substance form, such as film or micro-capsule), suitable hydrophobic material (for example can accept the emulsion in the oil) or ion exchange resin and a small amount of soluble derivative (such as a small amount of soluble-salt).

Sustained-release matrix comprises polyactide class (U.S. Patent number 3,773,919 and EP 58,481), the copolymer of Pidolidone and the γ-ethyl-Pidolidone (people such as Sidman, Biopolymers, 22:547-556,1983), poly-(HEMA) (people such as Langer, J. Biomed.Mater.Res., 15:167-277,1981 and Langer, Chem.Tech., 12:98-105,1982), the ethene vinyl-acetic ester (people such as Langer, the same) or poly-D-(-)-3-hydroxybutyrate (EP 133,988).

The MPIF-1 of slowly-releasing comprises that also the MPIF-1 of liposome (consults Langer, Science, 249:1527-1533,1990 usually; The people such as Treat, " Liposomes in the Therapy of Infectious Disease and Cancer " (liposome in infectious disease and the cancer therapy), Lopez-Berestein and Fidler compile, Liss, New York, 327 pages of 317-and 353-365 page or leaf, 1989). Can prepare the liposome that comprises MPIF-1: DE 3,218,121 by known method itself; The people such as Epstein, Proc.Natl.Acad. Sci.USA, 82:3688-3692,1985; The people such as Hwang, Proc.Natl.Acad. Sci.USA, 77:4030-4034,1980; EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese patent application 83-118008; U.S. Patent number 4,485,045 and 4,544,545; With EP 102,324. Usually, liposome is little (approximately 200-800 dust) single-layer type, and wherein lipid content can be regulated selected ratio to reach best MPIF-1 polypeptide therapy greater than about 30 molar percentage cholesterol.

In also having an embodiment, MPIF-1 (consults Langer, sees above by what the mode of pump was used; Sefton, CRC Crit.Ref.Biomed.Eng., 14:201,1987; The people such as Buchwald, Surgery, 88:507,1980; The people such as Saudek, N.Engl. J.Med., 321:574,1989).

Langer, Science, 249:1527-1533 discloses other controlled release system in 1990 the summary.

For stomach and intestine are used outward, in one embodiment, usually can accept carrier with the injectable forms (solution, suspension or emulsion) of UD with pharmaceutics by the MPIF-1 with required purity mixes to prepare the MPIF-1 polypeptide, described carrier is nontoxic at used dosage and concentration to acceptor, and compatible with other composition in the preparation. For example, preparation preferably contains no oxidizing agent and known other compound harmful to MPIF-1.

Usually by with the MPIF-1 polypeptide even and close contact to prepare preparation with liquid-carrier or the meticulous solid carrier that separates or both. Then, if necessary, product can be made the preparation shape of expectation. Preferred vector is the outer carriers of stomach and intestine, the solution that more preferably oozes with acceptor blood etc. The example of this carrier comprises water, salt solution, RingerShi liquid and glucose solution. Non-aqueous carrier (such as fixed oil and ethyl oleate) and liposome also are useful in this article.

Carrier can suitably comprise the additive of minute quantity, such as the material that can strengthen isotonicity and chemical stability. These materials are nontoxic at used dosage and concentration to acceptor, comprise buffer solution, such as phosphate, citrate, succinate, acetic acid and other organic acid or its salt; Antioxidant is such as ascorbic acid; Low-molecular-weight (being less than about 10 residues) polypeptide is such as pR60 or tripeptides; Protein is such as serum albumin, gelatin or immunoglobulin (Ig); Hydrophilic polymer is such as polyvinylpyrrolidone; Amino acid is such as glycine, glutamic acid, aspartic acid or arginine; Monose, disaccharides and other carbohydrate comprise cellulose or derivatives thereof, glucose, mannose or dextrin; Chelating agent is such as EDTA; Sugar alcohol is such as sweet mellow wine or sorbierite; Equilibrium ion is such as sodium; And/or nonionic surface active agent, such as polysorbate, poloxalkol or PEG.

Usually under the condition of the about 3-8 of pH, in these carriers, prepare MPIF-1 with the concentration of about 0.1mg/ml-100mg/ml, preferred 1mg/ml-10mg/ml. The use that should be appreciated that above-mentioned some excipient, carrier or stabilizing agent can cause the formation of MPIF-1 polypeptide salt.

Any medicine that being used for the treatment of property is used must be aseptic. Filter and to be easy to realize degerming by passing aseptic filter membrane (such as 0.2 μ m film). Usually MPIF-1 is placed the container with aseptic gateway, for example intravenous solution bag or have the bottle of the transparent stopper of hypodermic needle.

Usually with MPIF-1 as aqueous solution or be used for the container that heavy molten lyophilized formulations is stored in UD or multiple dose, for example in the airtight ampoule bottle or bottle. The example of lyophilized formulations is that 5ml is poured into the bottle of 10ml through 1% (w/v) of aseptic filtration MPIF-1 aqueous solution, and with the freeze-drying of gained mixture. Can prepare infusion solution by the MPIF-1 polypeptide that uses the heavy molten freeze-drying of antibacterial water for injection.

The present invention also provides pharmaceutics packing or the kit of the one or more containers that comprise one or more compositions that pharmaceutical composition of the present invention is housed. Can sign and issue with preparation, the application of management medicine or biological products or the government organs of selling the points for attention of form with these containers, its reflection has obtained the license of preparation, application or the marketing organization of people's medication. In addition, MPIF-1 can unite use with other therapeutic compound.

MPIF-1 can use separately, perhaps unites adjuvant and uses. Can include but not limited to that with the adjuvant that MPIF-1 uses alum, alum add deoxycholate (ImmunoAg), MTP-PE (Biocine company), QS-21 (Genentech company), BCG and MPL. In a specific embodiments, MPIF-1 and alum are co-administered. In another embodiment, MPIF-1 and QS-21 are co-administered. Can include but not limited to other adjuvant that MPIF-1 uses monophosphoryl lipid matter immunomodifier, AdjuVax 100a, QS-21, QS-18, CRL1005, aluminium salt, MF-59 and virion adjuvant technology. Can include but not limited to the vaccine that MPIF-1 uses the vaccine for MMR (measles,mumps,rubella), polio, varicella, lockjaw/diptheria, hepatitis A, hepatitis B, haemophilus influenzae B, pertussis, pneumonia, influenza, LymeShi disease, rotavirus, cholera, yellow fever, encephalitis B, polio, rabies, typhoid fever and pertussal protective effect. (as mixture, separating but the while) or sequential application combinations thereof can accompany. This comprises the situation of using together agent combination as the therapeutic mixture, and separately but use simultaneously the flow process (as entering same individual by the intravenous route that separates) of agent combination. " associating " used and also comprised and at first give a kind of composition or reagent gives alternative separate administration subsequently.

MPIF-1 can use separately, perhaps unites other therapeutic agent. Can include but not limited to the co-administered therapeutic agent of MPIF-1 other member, cytotoxic agent, chemotherapeutics, radiation, radiation sensitizer, targeted radiotherapy, antibiotic, antivirotic, steroid and non-steroidal anti-inflammatory agents, immunotherapeutic agent, radioimmunoassay detection agent, cell factor and/or the growth factor of TNF family. (as mixture, separating but the while) or sequential application combinations thereof can accompany. This comprises the displaying of using together agent combination as the therapeutic mixture, also has the flow process (as entering same individual by the intravenous route that separates) of separating but using simultaneously agent combination. " associating " used and also comprised and at first give a kind of composition or reagent gives alternative separate administration subsequently.

In one embodiment, other member of composition of the present invention and TNF family is co-administered. TNF that can be co-administered with MPIF-1, TNF molecule relevant or the TNF sample includes but not limited to TNF-α, Lymphotoxin-α (LT-α is also referred to as TNF-β), LT-β (being found in compound heterotrimer LT-α 2-β), OPGL, FasL, CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L, TNF-γ (international publication number WO 96/14328), AIM-I (international publication number WO 97/33899), endocrine factor-α (endokine-alpha) (international publication number WO 98/07880), TR6 (international publication number WO 98/30694), OPG, soluble form with middle sex factor-α (neutrokine-α) (international publication number WO 98/18921), OX40, and nerve growth factor (NGF), and Fas, CD30, CD27, CD40, soluble form with 4-IBB, TR2 (international publication number WO 96/34095), DR3 (international publication number WO 97/33904), DR4 (international publication number WO 98/32856), TR5 (international publication number WO 98/30693), TR6 (international publication number WO 98/30694), TR7 (international publication number WO 98/41629), TRANK, TR9 (international publication number WO 98/56892), TR10 (international publication number WO 98/54202), 312C2 (international publication number WO 98/06842), and TR12, and CD154, CD70, the soluble form of CD153.

In certain embodiments, MPIF-1 and antiretroviral agent, NRTI, non-nucleoside reverse transcriptase inhibitor and/or protease inhibitors are co-administered. Can include but not limited to RETROVIR with the co-administered NRTI of MPIF-1^TM(retrovir/AZT), VIDEX^TM(Didanosine/ddl), HIVID^TM(Zha Xi is guest/ddC), ZERI also^TM(stavudine/d4T)、EPIVIR ^TM(lamivudine/3TC) and COMBIVIR^TM(retrovir/lamivudine). Can include but not limited to VIRAMUNE with the co-administered non-nucleoside reverse transcriptase inhibitor of MPIF-1^TM(neyirapine)、RESCRIPTOR ^TM(delavirdine) and SUSTIVA^TM(efayirenz). Can include but not limited to CRIXIVAN with the co-administered protease inhibitors of MPIF-1^TM(indinayir)、NORVIR ^TM (ritonavir)、INVIRASE ^TM(saquinavir) and VIRACEPT^TM(nelfinavir). In specific embodiment, antiretroviral agent, NRTI, non-nucleoside reverse transcriptase inhibitor and/or protease inhibitors can be united use with any with MPIF-1, infect with treatment AIDS and/or prevention or treatment HIV.

In other embodiments, MPIF-1 can be co-administered with anti-opportunistic infect agent. Can include but not limited to TRIMETHOPRIM-SULFAMETHOXAZOLE with the co-administered anti-opportunistic infect agent of MPIF-1^TM、DAPSONE ^TM、PENTAMIDINE ^TM、 ATOVAQUONE ^TM、ISONIAZID ^TM、RIFAMPIN ^TM、PYRAZINAMIDE ^TM、ETHAMBUTOL ^TM、 RIFABUTIN ^TM、CLARITHROMYCIN ^TM、AZITHROMYCIN ^TM、GANCICLOVIR ^TM、 FOSCARNET ^TM、CIDOFOVIR ^TM、FLUCONAZOLE ^TM、ITRACONAZOLE ^TM、 KETOCONAZOLE ^TM、ACYCLOVIR ^TM、FAMCICOLVIR ^TM、PYRIMETHAMINE ^TM、 LEUCOVORINE ^TM、NEUPOGEN ^TM(filgrastim/G-CSF) and LEUKINE^TM(sargramostim/GM-CSF). In a specific embodiment, with MPIF-1 and TRIMETHOPRIM-SULFAMETHOXAZOLE^TM、DAPSONE ^TM、PENTAMIDINE ^TM, and/or ATOVAQUONE^TMUnite use with any, with prophylactic treatment or prevention opportunistic Pneumocystis carinii (Pneumocysits carinii) pneumonia. In another specific embodiment, with MPIF-1 and ISONIAZID^TM、RIFAMPIN ^TM、PYRAZINAMIDE ^TM, and/or ETHAMBUTOL^TMUnite use with any, with prophylactic treatment or prevention opportunistic mycobacterium avium (Mycobacterium avium) MOI. In another specific embodiment, with MPIF-1 and RIFABUTIN^TM、CLARITHROMYCIN ^TM, and/or AZITHROMYCIN^TMUnite use with any, infect with prophylactic treatment or prevention opportunistic Much's bacillus (Mycobacterium tuberculosis). In another specific embodiment, with MPIF-1 and GANCICLOVIR^TM、FOSCARNET ^TM, and/or CIDOFOVIR^TMUnite use with any, with prophylactic treatment or the cytomegalovirus infection of prevention opportunistic. In another specific embodiment, with MPIF-1 and FLUCONAZOLE^TM、ITRACONAZOLE ^TM, and/or KETOCONAZOLE^TMUnite use with any, with prophylactic treatment or prevention opportunistic fungal infection. In another specific embodiment, with MPIF-1 and ACYCLOVIR^TMAnd/or FAMCICOLVIR^TMUnite use with any, infect with prophylactic treatment or prevention opportunistic I type and/or II herpes simplex virus type. In another specific embodiment, with MPIF-1 and PYRIMETHAMINE^TMAnd/or LEUCOVORIN^TMUnite use with any, draw body (Toxoplasma gondii) with prophylactic treatment or prevention opportunistic mouse and infect. In another specific embodiment, with MPIF1 and LEUCOVORIN^TMAnd/or NEUPOGEN^TMUnite use with any, infect with prophylactic treatment or prevention opportunistic bacterium.

In another embodiment, MPIF-1 and antivirotic are co-administered. Can include but not limited to acyclovir, virazole, amantadine and remantidine with the co-administered antivirotic of MPIF-1.

In another embodiment, MPIF-1 and Antibiotic combination are used. Can include but not limited to Amoxicillin, beta-lactamase, aminoglycoside, beta-lactam (glycopeptide), beta-lactamase, clindamycin, chloramphenicol, cynnematin, Ciprofloxacin, Ciprofloxacin, erythromycin, fluoquinolone, macrolide, metronidazole (MET), penicillin, quinolone, rifampin, streptomysin, sulfanilamide (SN), tetracycline, trimethoprim, trimethoprim-Sulfamethoxazole and vancomycin with the co-administered antibiotic of MPIf-1.

Can include but not limited to the nospecific immunity containment agent of the co-administered routine of MPIF-1 steroids, cyclosporin, the similar thing of cyclosporin, endoxan methyl prednisone, prednisone, imuran, FK-506,15-deoxyspergualin and other immunity containment agent that can play a role by the function of containment responsiveness T cell.

In specific embodiment, MPIF-1 and immunodepressant are co-administered. Can include but not limited to ORTHOCLONE with the co-administered immunosupress agent formulation of MPIF-1^TM(OKT3)、 SANDIMMUNE ^TM/NEORAL ^TM/SANGDYA ^TM(cyclosporin), PROGRAF^TM (tacrolimus)、CELLCEPT ^TM(mycophenolic acid), imuran, glucocorticoid and RAPAMUNE^TM(siroliumus). In specific embodiment, immunodepressant can be used for preventing the repulsion to organ or bone-marrow transplantation.

In another embodiment, use separately MPIF-1, perhaps unite one or more Intravenous immunoglobuin preparations and use. Can include but not limited to GAMMAR with the Intravenous immunoglobuin that MPIF-1 uses^TM、IVEEGAM ^TM、SANDOGLOBULIN ^TM、GAMMAGARD S/D ^TM, and GAMIMUNE^TM In a specific embodiment, MPIF-1 and Intravenous immunoglobuin preparation are co-administered in transplantation therapy (such as the marrow value of moving).

In another embodiment, use separately MPIF-1, perhaps unite antiinflammatory and use. Can include but not limited to the antiinflammatory that MPIF-1 uses glucocorticoid and on-steroidal antiinflammatory, aminoaryl carboxylic acid derivates, Arylacetic acids derivative, arylbutyric acid derivatives, aryl carboxylic acid, aryl propionic acid derivatives, pyrazoles, pyrazolone, salicyclic acid derivatives, thiazine carboxylic acid amides, e-acetylamino caproic acid, S-adenosylmethionine, 3-amino-4-hydroxybutyric acid, amixetrine, Bendazac, benzydamine, BCP, Difenpiramide, ageroplas, Emorfazone, guaiazulene, Nabumetone, aulin, orgotein, Oxaceprol, paranyline, perisoxal, pifoxime, proquazone, proxazole and tenidap.

In another embodiment, MPIF-1 composition and chemotherapeutics are co-administered. Can include but not limited to the chemotherapeutics that MPIF-1 uses: antibiotic derivatives is (such as adriamycin (Adriamycin^TM), bleomycin, daunorubicin and dactinomycin D); Antiestrogenic (such as tamosifen); Antimetabolite (such as 5 FU 5 fluorouracil (5-FU), methotrexate, floxuridine, Interferon Alpha-2b, glutamic acid, plicamycin, purinethol and 6-thioguanine); Cytotoxic agent (such as Carmustine, BCNU, lomustine, CCNU, cytarabine, endoxan, estramustine, hydroxycarbamide, procarbazine, mitomycin, busulfan, cisplatin and vincristin sulfate); Hormone (such as Medroxyprogesterone, sodium phosphate estramustine, ethinyloestradiol, estradiol, megestrol acetate acetate, methyltestosterone, diethylstilbestrol diphosphonic acid, Chlorotrianisene and testosterone); Nitrogen mustard derivatives (replacing group such as mephalen, chorambucil, mechlorethamine (mustargen) and thiophene); Steroids and combination thereof (such as bethamethasone sodium phospharate); With other chemotherapeutics (such as dicarbazine, asparaginase, mitotane, vincristin sulfate, vinblastine sulfate and Etoposide).

In a specific embodiment, any combinatorial association of MPIF-1 and CHOP (endoxan, adriamycin, vincristin and prednisone) or CHOP composition is used. In another embodiment, any combining of MPIF-1 and Rituxmab and CHOP or Rituxmab and CHOP composition used.

In another embodiment, MPIF-1 and cell factor are co-administered. Can include but not limited to the cell factor that MPIF-1 uses: IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, anti-CD40, CD40L, IFN-γ and TNF-α. In another embodiment, MPIF-1 can use with any interleukin, includes but not limited to IL-1 α, IL-1 β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20 and IL-21.

In another embodiment, MPIF-1 and angiogenic proteins are co-administered. Can include but not limited to the blood vessel generation albumen that MPIF-1 uses the growth factor (GDGF) of being derived by glioma, as disclosed among the European patent numbering EP 399,816; By platelet-derived growth factor-A (PDGF-A), as disclosed among the European patent numbering EP 682,110; By platelet-derived growth factor-B (PDGF-B), as disclosed among the European patent numbering EP 282,317; Placenta growth factor (PIGF) is as disclosed among the international publication number WO 92/06194; Placenta growth factor-2 (PIGF-2), such as people such as Hauser, Growth Facotrs, 4:259-268, disclosed in 1993; VEGF (VEGF) is as disclosed among the international publication number WO 90/13649; VEGF-A (VEGF-A) is as disclosed among the European patent numbering EP 506,477; VEGF-2 (VEGF-2) is as disclosed among the international publication number WO 96/39515; Vascular endothelial growth factor B (VEGF-B); Vascular endothelial growth factor B-186 (VEGF-B186) is as disclosed among the international publication number WO 96/26736; VEGF-D (VEGF-D) is as disclosed among the international publication number WO 98/02543; VEGF-D (VEGF-D) is as disclosed among the international publication number WO 98/07832; And VEGF-E (VEGF-E), as disclosed among the Deutsche Bundespatent numbering DE19639601. Above-mentioned document is collected herein by reference.

In another embodiment, MPIF-1 and hemopoieticgrowth factor are co-administered. Can include but not limited to LEUKINE with the hemopoieticgrowth factor that MPIF-1 uses^TM (SARGRAMOSTIM ^TM) and NEUPOGEN^TM(FLIGRASTIM ^TM)。

In another embodiment, MPIF-1 and fibroblast growth factor are co-administered. Can include but not limited to the fibroblast growth factor that MPIF-1 uses FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14 and FGF-15.

In other embodiments, MPIF-1 and other therapeutic or Prevention scheme are co-administered, such as radiotherapy.

In other embodiments, also can use polynucleotides of the present invention, polypeptide, activator and/or antagonist with anti-angiogenesis. The representative examples of anti-angiogenesis comprises: the various forms of the inhibitor-1 of the tissue depressant of the anti-intrusion factor, retinoic acid and derivative thereof, taxol, suramin, metalloproteinases-1, the tissue depressant of metalloproteinases-2, plasminogen activator, the inhibitor-2 of plasminogen activator and light " d family " transition metal.

Light " d family " transition metal comprises for example vanadium, molybdenum, tungsten, titanium, niobium and tantalum. These transition metal can form transition metal composite. The suitable complexes of above-mentioned transition metal comprises oxo transition metal composite.

The representative examples of vanadium compound comprises vanadyl complexes, such as vanadic acid and vanadyl compound. Suitable vanadic acid compound comprises metavanadate and orthovanadate compound, such as ammonium metavanadate, sodium metavanadate and sodium orthovanadate. Suitable vanadyl compound comprises for example vanadium oxygen ethylacetoacetone compound and vanadic sulfate, comprises the vanadic sulfate hydrate, such as vanadic sulfate monohydrate and trihydrate.

The representative examples of tungsten and molybdenum compound also comprises oxo compound. Suitable oxo tungsten compound comprises wolframic acid and tungsten oxide compound. Suitable wolframic acid compound comprises ammonium tungstate, artificial schellite, sodium tungstate dihydrate and wolframic acid. Suitable tungsten oxide comprises tungsten (IV) oxide and tungsten (VI) oxide. Suitable oxo molybdenum compound comprises molybdic acid, molybdenum oxide and molybdenyl compound. Suitable molybdic acid compound comprises ammonium molybdate and hydrate, sodium molybdate and hydrate thereof and potassium molybdate and hydrate thereof. Suitable molybdenum oxide comprises molybdenum (VI) oxide, molybdenum (VI) oxide and molybdic acid. Suitable molybdenyl compound comprises for example molybdenyl acetylacetonate. The tungsten that other is suitable and molybdenum compound comprise the hydroxy derivatives of being derived by for example glycerine, tartaric acid and sugar.

Other anti-angiogenesis also can be used for content of the present invention widely. Representative examples comprises platelet factor 4; Protamine sulfate; Sulfuric acid chitin derivative (by queen crab shell preparation) people such as (, Cancer Res., 51:22-26,1991) Murata; Sulfated polysaccharide peptide glycan compound (SP-PG) (function of this compound can be enhanced by the existence of steroids (such as estrogen) and tamosifen citrate); Staurosporin; The instrumentality of matrix metabolism comprises for example proline analogs, cis hydroxyproline, d, L-3,4-dehydroproline, sulphur proline, α, α-connection pyridine, aminopropionitrile fumarate; 4-propyl group-5-(4-pyridine)-2-(3-H)-oxazolone; Amethopterin; Mitoxantrone; Heparin; Interferon; 2 macroglobulin-serum; ChIMP-3 (people such as Pavloff, J.Bio.Chem., 267:17321-17326,1992); Chymostatin (286:475-480,1992 for the people such as Tomkinson, Biochem.J.); Myristyl sulfuric acid cyclodextrin; Eponemycin; Camptothecine; Fumidil (people such as Ingber, Nature, 348:555-557,1990); Disodium aurothiomalate (" GST "; Matsubara and Ziff, J.Clin.Invest., 79:1440-1446,1987); Anticollagenase-serum; α 2-antiplasmin (people such as Holmes, J.Biol.Chem., 262 (4): 1659-1664,1987); Bisantrene (national cancer association, National Cancer Institute); Lobenzarit Disodium (N-(2)-carboxyl phenyl-4-chlorine chrloroanthracene ketone acid disodium or " CCA "; The people such as Takeuchi, Agents Actions, 36:312-316,1992); Se Lidemi; The tubulation steroids; AGM-1470; The carboxyamino imidazoles; And metal protease inhibitors, such as BB94. Mode of administration

Can understand, can reduce the situation that causes by using standard or the normal level that MPIF-1 albumen treats in the individuality by the MPIF-1 activity. Thereby, the present invention also provides treatment to need the method for the individuality of raising MPIF-1 activity level, comprise the MPIF-1 that this individuality is used the pharmaceutical composition that the present invention who comprises effective dose separates the MPIF-1 polypeptide, particularly mature form, to improve the MPIF-1 level in this individuality.

The quantity and the dosage that are applied to experimenter's MPIF-1 will depend on many factors, such as the essence of the pattern of using, situation to be treated and evolution doctor's judgement. Use this pharmaceutical composition with the amount that is enough to treat and/or prevent specific indication. Generally speaking, consider use path, symptom, etc., will use polypeptide with the amount of about at least 10 μ g/kg body weight, and, in most of the cases, will use polypeptide with the amount that is no more than about 10mg/kg body weight/day, preferred dosage is about 10 μ g/kg body weight/day.

Although as mentioned above, but in treatment consideration, but every dose of stomach and intestine of general recommendations to use the scope of total pharmaceutics effective dose of MPIF-1 polypeptide outward be about 1 μ g/kg weight in patients/sky-10mg/kg weight in patients/sky. Even more preferably, this dosage is 0.01mg/kg body weight/day at least, to people's about 0.01-1mg/kg body weight/day most preferably. If successive administration, usually with about 1 μ g/kg body weight/hour-about 50 μ g/kg body weight/hour dose rates, by injection every day 1-4 time, perhaps use the MPIF-1 polypeptide by for example continuous h inf with Micropump. Also can adopt the packed solution of intravenous. Observe change required treatment time length and treatment after length blanking time that reacts as if change with desired effects.

Can followingly use the pharmaceutical composition that comprises MPIF-1 of the present invention: oral, rectum, stomach and intestine are outer, in the pond, in the vagina, in the peritonaeum, local (by powder, ointment, gel, drops or through the skin patch), cheek or mouth or nose spray delivery. Gene therapy

According to the present invention, chemotactic factor (CF) and can be used by these polypeptide of expression in vivo as activator or the antagonist of polypeptide, Here it is " gene therapy " often said.

Thereby for example, the polynucleotides of available code polypeptide (DNA or RNA) exsomatize to be transformed the cell from the patient, and then improved cell being offered need to be with the patient of polypeptide treatment. These methods are well known in the art. For example, the flow process that can know by this area is come engineered cells by the retroviral particle with the RNA that comprises code book invention polypeptide.

Similarly, the method that can know by for example this area, engineered cells is to express in vivo polypeptide in vivo. As road known in the art, can use to the patient producer's cell of the retroviral particle that can produce the RNA that contains code book invention polypeptide, with engineered cells in vivo and express in vivo polypeptide. In view of instruction of the present invention, should be apparent for those skilled in the art for these and other method of using by this method polypeptide of the present invention. For example, the expression vector that is used for engineered cells not only can be retrovirus, also can be adenovirus, and described adenovirus can be used in vivo engineered cells after uniting with suitable delivery vector.

Can be by the retrovirus retroviral plasmid vector of deriving, described virus includes but not limited to Moloney murine sarcoma virus, Moloney murine leukemia virus, spleen necrosis virus, Rous sarcoma virus and Harvey sarcoma virus.

In a preferred embodiment, retrovirus expression vector is pMV-7, its flank is the LTR (LTR) of Moloney murine sarcoma virus, and comprises the drug resistance gene the selected neo that is subject to herpes simplex virus (HSV) thymidine kinase (tk) promoter regulation. Coded sequence (people such as P.T.Kirschmeier, DNA, 7:219-225,1988) is convenient to import in unique EcoRI and HindIII site.

Carrier comprises one or more suitable promoters, includes but not limited to retroviral LTR; The SV40 promoter; Human cytomegalovirus (CMV) promoter (people such as Miller, Biotechniques, 7 (9): 980-990,1989); Or any other promoter (for example the cell promoter such as the eukaryotic promoter, includes but not limited to histone, polIII and beta-actin promoter). In view of the teachings contained herein, the selection of suitable promoter is apparent to those skilled in the art.

The nucleotide sequence of code book invention polypeptide is subject to the control of suitable promoter, includes but not limited to viral thymidine kinase promoter, such as the herpes simplex virus thymidine kinase promoter; The natural promoter of the gene of retrovirus LTR, beta-actin promoter and this polypeptide of control coding.

Adopt retroviral plasmid vector transduction package cell line to form producer's clone. But the example of the incasing cells of transfection includes but not limited to PE501, PA317, GP+am12. Carrier can be by any known method transduction incasing cells of this area. These methods include but not limited to use and the CaPO of electroporation, liposome₄Precipitation.

Producer's clone generates the infectious retroviral vector particle of the nucleotide sequence that comprises coded polypeptide. Then can use these retroviral vector particles eukaryotic of in external or body, transduceing. To express the nucleotide sequence of coded polypeptide through the eukaryotic of transduction. Transducible eukaryotic includes but not limited to fibroblast and endothelial cell.

Another aspect of the present invention is the gene therapy that is used for the treatment of disorder, disease and situation. Gene therapy relates to nucleic acid (DNA, RNA and antisense DNA or RNA) sequence importing animal, to realize the expression of MPIF-1 polypeptide of the present invention. The method requires the polynucleotides of coding MPIF-1 polypeptide to be operatively connected promoter and to express necessary any other genetic elements of described polypeptide by target tissue. This gene therapy and delivery technology are known in this area, consult for example WO 90/11092 (being collected herein by reference).

Thereby for example then the available stripped cell of transforming from the patient of polynucleotides (DNA or RNA) that comprises the promoter that is operatively connected the MPIF-1 polynucleotides offers improved cell the patient with the polypeptide treatment. These methods are well known in the art. Such as consulting the people such as A.Belldegrun, J.Natl.Cancer Inst., 85:207-216,1993; The people such as M.Ferrantini, Cancer Research, 53:1107-1112,1993; The people such as M.Ferrantini, J.Immunology, 153:4604-4615,1994; The people such as T.Kaido, Int.J.Cancer, 60:221-229,1995; The people such as H.Ogura, Cancer Research, 50:5102-5106,1990; The people such as L.Santodonato, Human Gene Therapy, 7:1-10,1996; The people such as L.Santodonato, Gene Therapy, 4:1246-1255,1997; With the people such as J.-F.Zhang, Cancer Gene Therapy 3:31-38,1996 (being collected herein by reference). In one embodiment, the cell of transformation is arterial cell. Can be by directly being injected to artery, artery surrounding tissue or by the conduit injection arterial cell being imported the patient again.

As detailed below, can deliver MPIF-1 polynucleotides construction by any method that injectable materials is delivered to zooblast, such as being expelled in tissue (heart, muscle, skin, lung, the liver etc.) gap. Can in the acceptable liquid of pharmaceutics or aqueous carrier, deliver MPIF-1 polynucleotides construction.

In one embodiment, deliver the MPIF-1 polynucleotides with the form of exposed polynucleotides. Term " exposed " polynucleotides (DNA or RNA) refer to not contain the sequence of any delivery carrier, wherein deliver carrier and play effect auxiliary, that promote or be convenient to enter cell, comprise virus sequence, virion, Liposomal formulation, lipofectin reagent or precipitating reagent etc. Yet, also can in the well-known preparation of Liposomal formulation, lipofectin reagent and those skilled in the art, deliver the MPIF-1 polynucleotides. These methods are described in for example U.S. Patent number 5,593,972,5,589,466 and 5,580,859 (being collected herein by reference).

The MPIF-1 polynucleotide carrier construction that uses in the gene therapy preferably neither is incorporated in the host genome, does not also comprise the construction that allows the sequence that copies. Suitable carrier comprises can be by pWLNEO, pSV2CAT, pOG44, pXT1 and the pSG of Stratagene acquisition; Can be by pSVK3, pBPV, pMSG and the pSVL of Pharmacia acquisition; With pEF1/V5, pcDNA3.1 and the pRc/CMV2 that can be obtained by Invitrogen. Other suitable carrier will be apparent to those skilled in the art.

Any strong promoter that those skilled in the art will know that all can be used for driving the expression of MPIF-1 polynucleotide sequence. Suitable promoter comprises adenovirus promoter, such as adenovirus major late promoter; Or allogeneic promoter, such as cytomegalovirus (CMV) promoter; Respiratory Syncytial Virus(RSV) (RSV) promoter; But inducible promoter is such as MMT promoter, metallothionein promoter; The heat shock promoter; The albumin promoter; The ApoAI promoter; People's globin promoter; Viral thymidine kinase promoter is such as the herpes simplex virus thymidine kinase promoter; Retrovirus LTR; The beta-actin promoter; With the human growth hormone (HGH) promoter. Promoter also can be the natural promoter of MPIF-1.

Different from other gene therapy technology, the major advantage that exposed nucleotide sequence is imported target cell is the synthetic instantaneous person's character of polynucleotides in the cell. Studies show that can be with non-replicating dna sequence dna transfered cell, so that the generation of the expectation polypeptide that reaches 6 months to be provided.

MPIF-1 polynucleotides construction can be delivered to the tissue space of animal, comprise muscle, skin, brain, lung, liver, spleen, marrow, thymus gland, the heart, lymph, blood, bone, cartilage, pancreas, kidney, gall-bladder, stomach, intestines, testis, ovary, uterus, rectum, nervous system, eye, body of gland and connective tissue. Tissue space comprises the mucopolysaccharide matrix that flows of the iuntercellular between the collagenous fibres of reticular fibre, vascular wall or room wall elastomer at organ-tissue, fibr tissue, perhaps wraps up in the connective tissue of muscle cell or the same matrix in the lacuna osseous. The blood plasma of circulation also is similar with the occupied gap of vasculolymphatic lymph liquid. Because following reason preferably is delivered to the musculature gap. Can deliver easily them by being injected to the tissue that comprises these cells. Preferably they are delivered to noble cells lasting, non-division and in wherein expressing, although can in the cell of differentiation or not exclusively differentiation, realize delivering and expressing, such as blood stem cell or SF. The ability of muscle cell picked-up and expression polynucleotides is strong especially in the body.

For the injection of exposed nucleotide sequence, the effective dose of DNA or RNA will be about 0.05mg/kg body weight-about 50mg/kg body weight. Preferred dose is the about 20mg/kg of about 0.005mg/kg-, more preferably about about 5mg/kg of 0.05mg/kg-. Certainly, the technical staff of ordinary skill will understand, and this dosage will change according to the tissue site of injection. Those of ordinary skills are easy to measure the suitable and effective dose of nucleotide sequence, and this dosage depends on situation to be treated and the path of using.

Preferably using the path is the stomach and intestine outer pathway that is injected to tissue space. But also can use other stomach and intestine outer pathway, such as the suction of aerosol, especially for the delivery to lung or bronchial tissue, throat or schneiderian membrane. In addition, exposed MPIF-1 DNA construction can be delivered to artery by used conduit in the operation in the angioplasty process.

Deliver exposed polynucleotides by any method that this area is known, include but not limited to deliver direct needle injection, intravenous injection, local application, conduit infusion and the so-called particle gun in site ". These delivering methods are known in this area.

Also can deliver construction, such as virus sequence, virion, Liposomal formulation, lipofectin reagent, precipitation reagent etc. with delivering carrier. These delivering methods are known in this area.

In certain embodiments, MPIF-1 polynucleotides construction is compound in the Liposomal formulation. Be used for Liposomal formulation of the present invention and comprise cation (positively charged), anion (electronegative) and neutral preparation. Yet cationic-liposome particularly preferably is because can form closely charge recombination body between cationic-liposome and the polyanionic nucleic acid. Cationic-liposome shows and can mediate DNA (84:7413-7416 1987, is collected herein by reference for the people such as Felgner, Proc.Natl.Acad.Sci.USA); MRNA (people such as Malone, Proc. Natl.Acad.Sci.USA, 86:6077-6081,1989, be collected herein by reference); With deliver in the born of the same parents of the transcription factor of purifying (265:10189-10192 1990, is collected herein by reference for the people such as Deb s, J.Biol.Chem.).

Cationic-liposome is easy to obtain. For example; N-[1-(2,3-dioleoyl oxygen) propyl group]-N, N; N-triethylamine (DOTMA) liposome is particularly useful; and can obtain (to consult such as people such as Felgner by Lipofectin trade mark (GIBCO BRL, Grand Island, New York); Proc.Natl.Acad.Sci.USA; 84:7413-7416,1987, be collected herein by reference). Other liposome that can buy comprises transfectace (DDAB/DOPE) and DOTAP/DOPE (Boehringer).

Use technology well-known in the art to prepare other cationic-liposome by the material that is easy to obtain. Consult the synthetic of DOTAP (1,2-two (oleoyl oxygen)-3-(trimethylamine groups) propane) liposome that PCT publication number WO 90/11092 (being collected herein by reference) for example describes. Explained the preparation of DOTMA liposome in the document, consulted such as people such as P.Felgner, Proc.Natl. Acad.Sci.USA, 84:7413-7417 is collected herein by reference. Available similar approach prepares liposome by other cationic lipid material.

Anionic liposome and neutral fats plastid are easy to obtain equally, such as being obtained by Avanti Polar Lipids (Birmingham, Ala.), perhaps can be easy to the material preparation that is easy to obtain. These materials comprise phosphatidyl, choline, cholesterol, phosphatidyl-ethanolamine, DOPC (DOPC), DOPG (DOPG), DOPE (DOPE) etc. These materials can mix with proper ratio with DOTMA and DOTAP parent material. The method for preparing liposome with these materials is well known in the art.

For example, commercially available DOPC (DOPC), DOPG (DOPG) and DOPE (DOPE) can be used for multiple combination, add or do not add cholesterol, the liposome that preparation is conventional. Like this, for example can be by passing into nitrogen drying 50mg DOPG at ultrasonic vial and 50mg DOPC prepares the DOPG/DOPC vesica. Sample placed under the vavuum pump spend the night, and second day makes its hydration with deionized water. Then in covering vial, use the Heat Systems 350 type Ultrasound Instrument with reversing cup (water-bath type) probe, arrange down in maximum, water-bath was with the 15EC circulation time, to sample ultrasonic 2 hours. In addition, can be ultrasonic and prepare electronegative vesica, to produce MLV, perhaps by being pressed through nucleopore membranes, to produce the monolayer vesicle of discontinuous size. Other method is known and available to those skilled in the art.

Liposome comprises MLV (MLV), little monolayer vesicle (SUV) or large monolayer vesicle (LUV), wherein preferred SUV. Use method well-known in the art to prepare multiple liposome-nucleic acid complex. Consult such as people such as Straubinger, Methods of Immunology, 101:512-517,1983, be collected herein by reference. For example, be prepared as follows the MLV that contains nucleic acid: phospholipid membrane is deposited on the glass tube walls, and the solution with material to be wrapped carries out aquation subsequently. Prepare SUV by abundant ultrasonic MLV, to produce the equal a group of unilamellar liposome. Material to be wrapped is added to ready-made MLV suspension, then ultrasonic. When use contains the liposome of cation lipid, the lipid film of drying is resuspended in suitable solution such as sterilized water or isotonic buffer solution such as 10mM Tris/NaCl, ultrasonic, then ready-made liposome is directly mixed with DNA. Because the combination of positively charged liposome and electronegative DNA, so liposome and DNA form very stable compound. Find that SUV can be used for little nucleic acid fragment. Prepare LUV by many methods well-known in the art. Common method comprises Ca²⁺-EDTA chelating (people such as Papahadjopoulos, Biochem.Biophys.Acta, 394:483,1975; The people such as Wilson, Cell, 17:77,1979); Ether injection (D.Deamer and A.Bangham, Biochim.Biophys., 443:629,1976; The people such as Ostro, Biochem.Biophys.Res.Commun., 76:836,1977; The people such as Fraley, Proc.Natl.Acad.Sci.USA, 76:3348,1979); Detergent dialysis (H.Enoch and P.Strittmatter, Proc.Natl. Acad.Sci.USA, 76:145,1979); With anti-phase evaporation (REV) (people such as Fraley, J.Biol.Chem., 255:10431,1980; F.Szoka and D.Papahadjopoulos, Proc.Natl.Acad.Sci.USA, 75:145,1978; The people such as Schaefer-Ridder, Science, 215:166,1982) (being collected herein by reference).

Usually, the ratio of DNA and liposome is about 10: 1 to about 1: 10. Preferred ratio is about 5: 1 to about 1: 5, and preferred ratio is about 3: 1 to about 1: 3. Still preferred ratio is about 1: 1.

U.S. Patent number 5,676,954 (being collected herein by reference) have been reported and will be expelled in the Mice Body with the compound genetic stocks of cationic-liposome carrier. U.S. Patent number 4,897,355,4,946,787,5,049,386,5,459,127,5,589,466,5,693,622,5,580,859,5,703,055 and international publication number WO 94/29469 (being collected herein by reference) cation lipid that is used for DNA is transfected into cell and mammal is provided. U.S. Patent number 5,589,466,5,693,622,5,580,859,5,703,055 and international publication number WO 94/29469 (being collected herein by reference) provide DNA-cation lipid complex be delivered to mammiferous method.

In certain embodiments, use the retroviral particle that comprises RNA to exsomatize or the interior engineered cells of body, described RNA comprises the sequence of the MPIF-1 that encodes. The retrovirus of retroviral plasmid vector of can deriving includes but not limited to Moloney murine leukemia virus, spleen necrosis virus, Rous sarcoma virus, Harvey sarcoma virus, avian leukosis viruses, gibbon ape leukemia virus, human immunodeficiency virus, bone marrow proliferative sarcoma virus and mammary tumor virus.

Use retroviral plasmid vector transduction package cell line, to form producer's clone. But the example of the package cell line of transfection includes but not limited to PE501, PA317, R-2, R-AM, PA12, T19-14X, VT-19-17-H2, RCRE, RCRIP, GP+E-86, GP+envAm12 and DAN clone (Miller, Human Gene Therapy, 1:5-14,1990, complete being collected herein by reference). Any method carrier transduction incasing cells that can know by this area. These methods include but not limited to use and the CaPO of electroporation, liposome₄Precipitation. In a candidate scheme, retroviral plasmid vector can be wrapped in the liposome, or the coupling lipid, then be applied to the host.

Producer's clone can produce the infectious retroviral vector particle of the polynucleotides that comprise the MPIF-1 that encodes. Then can adopt these retroviral vector particles eukaryotic of in external or body, transduceing. Eukaryotic through transduction will be expressed MPIF-1.

In some other embodiment, exsomatize or the interior engineered cells of body with the MPIF-1 polynucleotides that are contained in the adenovirus vector. Can operate adenovirus, make its coding and express MPIF-1, simultaneously deactivation it in the normal replication capacity of lytic virus in life cycle. Can in the situation of viral DNA unconformity in the host cell chromosome, realize the expression of adenovirus, alleviate thus inserting the worry of mutagenesis. In addition, adenovirus is as the intestines vaccine of living for many years, has fabulous security people such as (, Am.Rev.Respir.Dis., 109:233-238,1974) A.R.Schwartz. At last, many examples have been set forth by adenovirus mediated transgenosis, comprise the lung (people such as M.A.Rosenfeld, Science, 252:431-434,1991 that alpha1 Anti-trypsin and CFTR are transferred to cotton mouse; The people such as Rosenfeld, Cell, 68:143-155,1992). In addition, attempt setting up adenovirus as the further investigation of human cancer causative factor all negative people such as (, Proc.Natl.Acad.Sci.USA, 76:6606,1979) M.Green.

Can be used for suitable adenovirus vector of the present invention and for example be described in Kozarsky and Wilson, Curr.Opin.Genet.Devel., 3:499-503,1993; The people such as Rosenfeld, Cell, 68:143-155,1992; The people such as Engellhardt, Human Genet.Ther., 4:759-769,1993; The people such as Yang, Nature Genet., 7:362-369,1994; The people such as Wilson, Nature, 365:691-692,1993; With U.S. Patent number 5,652,224 (being collected herein by reference). For example, adenovirus vector Ad2 is useful, and it can be grown in 293 cells. These cells comprise the E1 district of adenovirus, and constitutive expression E1a and E1b, and they are by providing complementary with defective adenoviral by the product of the gene that lacks on the carrier. Except Ad2, other adenovirus kind (such as Ad3, Ad5 and Ad7) also can be used for the present invention.

Preferably, being used for adenovirus of the present invention is replication defective. The adenovirus require helper virus of replication defective and/or the help of package cell line are to form infectious particles. The virus that obtains can infection cell, and can express the polynucleotide of interest that is operatively connected with promoter, but in most cells reproducible not. The adenovirus of replication defective can be lacked one or more following genes all or part of: E1a, E1b, E3, E4, E2a, or, L1 to L5.

In some other embodiment, exsomatize or the interior engineered cells of body with adeno-associated virus (AAV). AAV is the defective virus of natural generation, and it needs helper virus to produce infectious particles (N.Muzyczka, Curr.Topics in Microbiol.Immunol., 158:97,1992). It also is its DNA to be incorporated into one of minority virus in the Unseparated Cell. The few carrier to 300 base-pairs that contains AAV gets final product packaged and integration, but the spatial constraints of foreign DNA is at about 4.5kb. Producing and using the method for this AAV is known in this area. Consult for example U.S. Patent number 5,139,941,5,173,414,5,354,678,5,436,146,5,474,935,5,478,745 and 5,589,377.

For example, be used for suitable AAV carrier of the present invention and will comprise that dna replication dna, capsidation and host cell integrate necessary all sequences. The Application standard cloning process inserts the AAV carrier with MPIF-1 polynucleotides construction, can be people such as Sambrook such as those, " Molecular Cloning:A Laboratory Manual " (molecular cloning: laboratory manual), cold spring port publishing house, New York, the method that finds in 1989. Then use any standard technique, comprise lipofection, electroporation, calcium phosphate precipitation etc., restructuring AAV carrier is transfected in the incasing cells that has infected helper virus. Suitable helper virus comprises adenovirus, cytomegalovirus, vaccinia virus or herpesviral. In case incasing cells is transfected and infection, they comprise generation the infectious AAV virion of MPIF-1 polynucleotides construction. Then these virions are used for the eukaryotic of transduceing in stripped or the body. Cell through transduction will comprise the MPIF-1 polynucleotide constructs that is incorporated in its genome, and will express MPIF-1.

The another kind of method of gene therapy comprises that being operatively connected allos control zone and endogenous polynucleotide sequence (such as coding MPIF-1) by homologous recombination (consults that for example U.S. Patent number is open on June 24th, 5,641,670,1997; International publication number WO is open on September 26th, 96/29411,1996; International publication number WO is open on August 4th, 94/12650,1994; The people such as Koller, Proc.Natl.Acad.Sci.USA, 86:8932-8935,1989; With the people such as Zijlstra, Nature, 342:435-438,1989). This method comprises activating and is present in the target cell but the gene of usually not expressing or expressing to be lower than aspiration level in cell.

The standard technique preparation of using this area to know comprises the polynucleotides construction of the target sequence of promoter and promoter flank. Suitable promoter is described herein. Target sequence and endogenous sequence are fully complementary, to allow between promoter-target sequence and endogenous sequence homologous recombination occuring. The target sequence will fully be expected 5 ' end of endogenous polynucleotide sequence near MPIF-1, promoter is operatively connected with endogenous sequence after homologous recombination.

Useful PCR increase promoter and target sequence. Preferably, the promoter of amplification comprises different restriction enzyme sites 5 ' with 3 ' end. Preferably, 3 ' end of first target sequence comprises promoter 5 ' the terminal identical restriction enzyme sites with amplification, and 5 ' end of second target sequence comprises the restriction enzyme sites identical with the promoter 3 ' end of amplification. The promoter of digest amplification and target sequence, and connect together.

Or to expose the form of polynucleotides, or with the form of transfection promoter (such as the liposome that above describes in detail, virus sequence, virion, totivirus, lipofection thing, precipitating reagent etc.) associating, promoter-target sequence construct thing is delivered to cell. Promoter-target sequence be can deliver by any method, direct pin injection, intravenous injection, local application, conduit infusion, particle accelerator etc. comprised. Hereinafter describe these methods in detail.

Cell is with uptake promoter-target sequence construct thing. Between construction and endogenous sequence homologous recombination occurs, endogenous MPIF-1 sequence is placed under the control of this promoter. Then the expression of the endogenous MPIF-1 sequence of promoters driven.

The polynucleotides of coding MPIF-1 can together be used with other polynucleotides of coding angiogenic proteins. The example of angiogenic proteins includes but not limited to acid and basic fibroblast growth factor, VEGF-1, VEGF-2, VEGF-3, EGF α and β, by platelet-derived ECGF, by platelet-derived growth factor, tumor necrosis factor α, HGF, IGF, colony stimulating factor, macrophage colony stimulatory factor, granulocyte/macrophage colony stimulatory factor and nitric oxide synthase.

Preferably, the polynucleotides of coding MPIF-1 comprise the secretory signal sequence that promotes the protein secretion. Burst is usually located in the polynucleotide encoding to be expressed district, towards or be positioned at 5 ' end of code area. Burst can be homology or allos for polynucleotide of interest, and for the cell for the treatment of transfection, can be homology or allos. The method chemical synthesis burst that in addition, can use this area to know.

Can use any mode of administration to use any above-mentioned polynucleotides construction, as long as this pattern causes one or more molecules to provide the scale of result for the treatment of to reach with q.s. This comprises implantation or topical application in direct pin injection, general injection, conduit infusion, trajectory emission injection, particle accelerator (i.e. " particle gun "), the long-acting medicament of gelatin foam, other commercially available long-acting drug agent material, osmotic pumps (for example Alza micropump), oral or suppository solid (sheet or ball) pharmaceutics preparation and the surgical procedures. For example, the exposed plasmid of calcium phosphate precipitation is injected directly in rats'liver and the rat spleen, or the plasmid of protein parcel is injected directly in the portal vein, expression (the people such as Kaneda that can cause foreign gene in the rats'liver, Science, 243:375,1989).

The method for optimizing of local application is by direct injection. Preferably, will be applied in the artery district by compound recombinant molecule of the present invention with delivering carrier by direct injection or local application. Composition is locally applied in the artery district to refer to composition is expelled in the artery in several centimetres and preferred several millimeters.

The another kind of method of local application is with around polynucleotides construction of the present invention contact surgical wound or its. For example, the patient can experience operation, and the polynucleotides construction is coated in tissue surface in the wound, or construct is expelled in the tissue district in the wound.

Can be used for the therapeutic composition that general uses and comprise of the present invention recombinant molecule compound with targeting Delivery carrier of the present invention. Being used for suitable delivery carrier that general uses comprises and contains the liposome that is useful on the part of carrier target specific site.

The method for optimizing that general is used comprises intravenous injection, aerosol, oral and deliver through skin (part). Can use the standard method of this area to carry out intravenous injection. Also can use the standard method of this area to carry out aerosol and deliver (consult such as people such as Stribling, Proc. Natl.Acad.Sci.USA, 189:11277-11281 1992, is collected herein by reference). Can be by the compound carrier that can bear the degraded of animal intestinal digestive ferment of polynucleotides construction of the present invention be carried out oral delivery. The example of these carriers comprises plastic capsule or tablet, as road known in the art. Can by with polynucleotides construction of the present invention with can transdermal lipophilic reagent (for example DMSO) mix to carry out the part and deliver.

Treat definite responsible many factors of delivering the material effective dose, comprise the age of the chemical constitution of this material for example and BA, animal and accurate situation and the order of severity thereof that body weight, needs are treated and use the path. Therapeutic frequency relies on many factors, such as the amount of every dose of polynucleotides construction of using, and experimenter's health and case. Accurately will be determined by the physician who cures mainly or animal doctor amount, dosage number and medicine time.

Therapeutic composition of the present invention can be applied to any animal, preferred mammal and birds. Preferred mammal comprises people, dog, cat, mouse, rat, rabbit, sheep, ox, horse and pig, and wherein the people is particularly preferred. Antisense and ribozyme (antagonist)

In specific embodiments, be nucleic acid or its complementary strand of cloning contained nucleotide sequence in 75676 corresponding to contained sequence and/or preservation in SEQ ID NO:1 or 6 according to antagonist of the present invention. In one embodiment, antisense sequences is that section produces in vivo. In another embodiment, antisense sequences is that separate administration (is consulted for example O ' Connor, J. Neurochem., 56:560,1991; " Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression " (oligodeoxynucleotide is as the antisense inhibitor of gene expression), CRC publishing house, Boca Raton, FL, l988). Antisense technology perhaps forms by triple helix by antisense DNA or RNA, can be used for controlling gene and expresses. Antisense technology is described in for example Okano, J.Neurochem., 56:560,1991; " Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression " (oligodeoxynucleotide is as the antisense inhibitor of gene expression), CRC publishing house, Boca Raton, FL, 1988. Triple helix forms and is described in such as people such as Lee Nucleic Acids Research, 6:3073,1979; The people such as Cooney, Science, 241:456,1988; With the people such as Dervan, Science, 251:1300,1991. These methods based on be the combination of polynucleotides and complementary DNA or RNA.

For example, the purposes that c-myc and c-myb antisense RNA construction be used for to suppress non-lymphocytic leukemia clone HL-60 and other cell line growth (people such as Wickstrom, 1988 had before been described; The people such as Anfossi, 1989). These experiments are undertaken by cell is incubated with oligoribonucleotide external. WO 91/15580 has described the similar flow process that is used for purposes in the body. In brief, a pair of oligonucleotides of the given antisense RNA of following generation: add EcoRI site (5 ' end) and HindIII site (3 ' end) at the sequence flank with front 15 base complementrities of opening code-reading frame. Then, with this to oligonucleotides in 90 ℃ of heating 1 minute, then connect buffer solution (20mM Tris pH7.5,10mM MgCl at 2x₂, 10mM dithiothreitol (DTT) (DTT) and 0.2mM ATP) in annealing, and connect the EcoRI/HindIII site of retroviral vector pMV7 (WO 91/15580).

For example, 5 ' coded portion of the polynucleotides of code book invention mature polypeptide can be used for the antisense rna oligonucleotide of the about 10-40 base-pair of design length. The DNA oligonucleotides of a regional complementarity of the gene that design and participation are transcribed prevents transcribing and producing of acceptor thus. The antisense RNA oligonucleotides can be hybridized in vivo with mRNA, and blocking-up mRNA molecule is translated into receptor polypeptides.

In one embodiment, in cell, generate MPIF-1 antisensenucleic acids of the present invention by transcribing of exogenous array. For example, transcription vector or its part generate antisensenucleic acids of the present invention (RNA). This class carrier should comprise the sequence of coding MPIF-1 antisensenucleic acids. This class carrier can be kept outside chromosome or be incorporated in the chromosome, generates the antisense RNA of expectation as long as it can be transcribed. This class carrier can make up by the recombinant DNA technology method standard of this area. Carrier can be other material of knowing for plasmid, virus or this area of copying at vertebrate cells and express. The sequence of coding MPIF-1 or the expression of its fragment can be undertaken by any promoter in the effect of the preferred people's cells play of vertebrate that this area is known. These promoters include but not limited to SV40 early promoter district (Bernoist and Chambon, Nature, 29:304-310,1981), the promoter (Yamamoto etc. that comprise in the Rous sarcoma virus 3 ' LTR, Cell 22:787-797,1980); Herpesvirus thymine deoxyriboside kinase promoter (people such as Wagner, Proc.Natl.Acad.Sci.USA, 78:1441-1445,1981), the regulating and controlling sequence of the metallothionein gene (people such as Brinster, Nature, 296:39-42,1982), etc.

Anti sense nucleotide sequence of the present invention comprises and this sequence of at least a portion complementation of the rna transcription of MPIF-1 gene. Yet, and do not require absolute complementation (although preferred). The sequence of " complementary with at least a portion of RNA " refers to have enough complementarity in this article, therefore can hybridize with RNA, forms the sequence of stable duplex; In the situation of double-stranded MPIF-1 antisensenucleic acids, can detect thus the strand of duplex DNA, maybe can measure triplex and form. The hybridization ability will depend on complementary degree and the length of antisensenucleic acids. Generally speaking, hybrid nucleic acid is larger, can comprise with MPIF-1 RNA mispairing just manyly, but still forms stable duplex (or being triplex in some cases). Those skilled in the art use the standard method of determining the hybridization complex fusing point can determine the tolerance degree of mispairing.

With 5 ' end of information for example 5 ' non-translated sequence until and the oligonucleotides that comprises the complementation of AUG initiation codon the most effective in suppressing translation. Yet, be presented at the sequence of mRNA 3 ' non-translated sequence complementation and suppress also effective in the mRNA translation. Usually consult R.Wagner, Nature, 372:333-335,1994. Thereby, can be used for the antisense method with 5 ' or the 3 ' untranslated of MPIF-1 shown in Figure 20, the oligonucleotides of noncoding region complementation, to suppress the translation of endogenous MPIF-1 mRNA. Should comprise the complement of AUG initiation codon with the oligonucleotides of 5 ' the non-translational region complementation of mRNA. With the ASON of mRNA code area complementation be not so effectively to translate mortifier, but can use according to the present invention. Whether no matter be designed to hybridize with 5 ', 3 ' or the code area of MPIF-1 mRNA, the length of antisensenucleic acids should be at least 6 nucleotides, and preferred length is the antisensenucleic acids of 6-about 50 nucleotides. Aspect specific, oligonucleotides is 10 nucleotides at least, at least 17 nucleotides, at least 25 nucleotides, or at least 50 nucleotides.

Polynucleotides of the present invention can be strand or double-stranded DNA or RNA or its chimeric mixture or derivative or modified version. Oligonucleotides can partly be modified in base portion, sugar moieties or phosphate backbone, so as such as the stability of improving nucleic acid, hybridization, etc. Oligonucleotides can comprise other additional group, the factor such as peptide (as for target host cell receptor in the body) or the transhipment of promotion cross-cell membrane (is consulted such as people such as Lestinger, Proc.Natl. Acad.Sci.USA, 86:6553-6556,1989; The people such as Kemaitre, Proc.Natl. Acad.Sci.USA, 84:648-652,1987; PCT publication number WO 88/09810, be disclosed on December 15th, 1988) or promote the factor of crossing over blood-brain barrier (to consult for example PCT publication number WO 89/10134, be disclosed on April 25th, 1988), the cutting agent that triggers of hybridization (consults such as people such as Kro1, BioTechniques, 6:958-976,1988) or insert agent and (consult for example Zon, Pharm.Res., 5:539-549,1988). For this purpose, oligonucleotides can be puted together another kind of molecule, the cutting agent that crosslinking agent, transport agents, the hybridization that triggers such as peptide, hybridization triggers, etc.

ASON can comprise at least a modified base portion, be selected from and include but not limited to 5 FU 5 fluorouracil, 5-bromouracil, the 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, the 4-acetylcytosine, 5-(carboxyl hydroxymethyl) uracil, 5-carboxymethyl aminomethyl-2-sulphur uridine, 5-carboxymethyl aminomethyl uracil, dihydrouracil, β-D-galactosylation queosine, inosine, the N-6-isopentenyl gland purine, the 1-methyl guanine, M1I, 2, the 2-dimethylguanine, the 2-methyl adenine, the 2-methyl guanine, the 3-methylcystein, 5-methylcytosine, the N-6-adenine, the 7-methyl guanine, 5-methyl aminomethyl uracil, 5-methoxyl group aminomethyl-2-thiouracil, β-D-MANNOSE base queosine, 5 '-methoxyl group carboxymethyl uracil, the 5-methoxyuracil, 2-methyl sulfo--N-6-isopentennyladenine, uracil-5-fluoroacetic acid (V), wybutoxosine, pseudouracil, queosine, 2-sulfo-cytimidine, 5-methyl-2-paper substrate, the 2-paper substrate, the 4-paper substrate, the 5-methyluracil, uracil-5-fluoroacetic acid methyl esters, uracil-5-fluoroacetic acid (V), 5-methyl-2-paper substrate, 3-(3-amino-3-N-2-carboxylic propyl group) uracil, (acp3) w, with 2,6-diaminopurine.

ASON can also comprise at least a modified sugar moieties, and it is selected from the group that includes but not limited to arabinose, 2-fluorine arabinose, xylulose and hexose.

In also having an embodiment, ASON comprises at least a modified phosphate backbone, be selected from and include but not limited to following group: thiophosphate, phosphorodithioate, amino thiophosphate, phosphoramidate, phosphorodiamidite, methyl phosphonate, alkyl phosphotriester and formacetal, or its analog.

In also having another embodiment, ASON is a kind of a end group isomery oligonucleotides. A kind of a end group isomery oligonucleotides and complementary RNA form special double-stranded heterozygote, and be wherein different from common b unit, two chains be parallel to each other (people such as Gautier, Nucl.Acids Res., 15:6625-6641,1987). Oligonucleotides is 2 '-O-methyl nucleotide (people such as Inoue, Nucl.Acids Res., 15:6131-6148,1987) or chimeric RNA-DNA analog (people such as Inoue, FEBS Lett., 215:327-330,1987).

The synthetic polynucleotides of the present invention of the standard method that can know by this area, as use the automation dna synthesizer (such as available from Biosearch, Applied Biosystems, etc.). For example, can be by people such as Stein, Nucl.Acids Res., 16:3209,1988 method is synthesized the thiophosphate oligonucleotides, can use controlled pore size glass, polymer holder to prepare the methyl phosphonate oligonucleotides (people such as Sarin, Proc.Natl.Acad.Sci.USA, 85:7448-7451,1988), etc.

Although can use the GEM 132 with the complementation of MPIF-1 coding region sequence, most preferably those and the GEM 132 of the non-translational region complementation of transcribing.

Also comprise catalytic RNA according to potential antagonist of the present invention, or ribozyme (is consulted for example PCT publication number WO 90/11364, is disclosed in October 4 nineteen ninety; The people such as Sarver, Science, 247:1222-1225,1990). Although can use the mRNA that destroys MPIF-1 at the ribozyme of specific recognition sequence place, site cutting mRNA, the preferred hammerhead ribozyme that uses. Hammerhead ribozyme is by the position of flanking region appointment cutting mRNA, described flanking region and said target mrna formation complementary base pair. Unique requirement is the sequence that said target mrna has two following bases: 5 '-UG-3 '. Structure and the generation of hammerhead ribozyme are well known in the art, and fully are described in Haseloff and Gerlach, Nature, 334:585-591,1988. At the nucleotides sequence column memory of MPIF-1 at many potential hammerhead ribozyme cleavage sites (Figure 1A-1B). Preferably, engineered ribozyme so that the cutting recognition site be positioned at MPIF-1 mRNA 5 ' end near; Namely increase efficient and the intracellular accumulation that reduces non-functional mRNA transcript.

In the antisense method, ribozyme of the present invention can be formed by modified oligonucleotides (such as for improvement of stability, targeting, etc.), and should be delivered in the cell of expression in vivo MPIF-1. Can be with mentioned above about importing the identical mode of antisense code dna with the DNA construction transfered cell of encoding ribozyme. The method for optimizing of delivering comprises that use " coding " is subject to the DNA construction of the ribozyme of strong constitutive promoter (such as pol III or pol II promoter) control, also suppresses translation so that will generate the ribozyme of q.s through transfectional cell to destroy endogenous MPIF-1 information. Because ribozyme is different from antisense molecule, it is catalytic, therefore only needs lower intracellular concentration can bring into play effect.

Antagonist/agonist compound can be used for suppressing polypeptide of the present invention to Growth of Cells and the proliferation function (stimulation that namely tumor vessel is generated) of tumour cell and tissue, block thus or prevent unusual Growth of Cells and propagation, for example tumour form or growth in.

Antagonist/activator also can be used for preventing hypervascular disease, and prevents the propagation of cataract extraction operation epithelium posterius lens cell. Avoid the mitogenic activity of polypeptide of the present invention also expecting in some cases, such as the ISR behind the balloon angioplasty.

Antagonist/activator also can be used for treating disease as herein described.

Thereby, the invention provides by the patient being used (a) for the antisensenucleic acids of polynucleotides of the present invention and/or (b) treating the method for disorder or disease for the ribozyme of polynucleotides of the present invention, include but not limited to listed, relevant with the overexpression of polynucleotides of the present invention disorder or disease in the entire chapter application. Epi-position and antibody

The present invention contain the epi-position that comprises following polypeptide or consisting of polypeptide: the polypeptide with amino acid sequence of SEQ ID NO:2, number the polypeptide of 75676 contained polynucleotide sequence codings by the ATCC preserved material, or by in rigorous hybridization conditions mentioned above or the complementary series of numbering 75676 contained sequences with sequence or the ATCC preserved material of SEQ ID NO:1 or 6 under than the low stringency hybridization condition polypeptide of the polynucleotide encoding of hybridization occurs. The present invention also contain the epi-position of code book invention peptide sequence polynucleotide sequence (such as disclosed sequence in SEQ ID NO:1 or 6), code book invention peptide sequence epi-position polynucleotide sequence complementary strand polynucleotide sequence and the polynucleotide sequence of hybridization occurs with complementary strand under rigorous hybridization conditions mentioned above or low rigorous condition condition.

When being used for this paper, term " epi-position " refers to have antigenicity or immunogenic part in the polypeptide in animal (preferred mammal, optimum is chosen). In a preferred embodiment, the polypeptide that comprises epi-position is contained in the present invention, and the polynucleotides of this peptide species of encoding. When being used for this paper, " immunogenicity epi-position " is defined as the part that causes antibody response in the protein in animal, can measure by any known method in this area, (consult such as people such as Geysen such as the method that hereinafter described is used for generation antibody, Proc.Natl.Acad.Sci.USA, 81:3998-4002,1983). When being used for this paper, term " antigenic epitopes " but be defined as that the antibody immunologic opsonin can be by any method mensuration well-known in the art, for example immunoassay as herein described in conjunction with the part of its antigen in the protein. Immunologic opsonin still needn't be got rid of the cross reaction with other antigen in conjunction with not comprising non-specific binding. Antigenic epitopes is not necessarily immunogenic.

Can generate by any conventional method the fragment (consult for example R. A.Houghten, Proc.Natl.Acad.Sci.USA, 82:5131-5135 1985, is further described in U.S. Patent number 4,631,211) of performance epi-position function.

In the present invention, antigenic epitopes preferably comprises at least 4, at least 5, at least 6, at least 7, more preferably at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, most preferably about 15-about 30 amino acid whose sequences. The length that comprises the preferred polypeptide of immunogenicity or antigenic epitopes is at least 10,15,20,25,30,35,40,45,50,55,60,65,70,75,80,85,90,95 or 100 amino acid residues. Other non-exclusionism preferred antigens epi-position comprises antigenic epitopes disclosed herein and part thereof. But antigenic epitopes can be used for for example generating the antibody of this epi-position of specific binding, comprises monoclonal antibody. Preferred antigenic epitopes comprises antigenic epitopes disclosed herein, and any combination of two, three, four, five or more these antigenic epitopes. The target molecule that antigenic epitopes can be used as in the immunoassay (is consulted such as people such as Wilson Cell, 37:767-778,1984; The people such as Sutcliffe, Science, 219:660-666,1983).

Similarly, can according to method well-known in the art, the immunogenicity epi-position be used for such as inducing antibody (to consult such as people such as Sutcliffe, see above; The people such as Wilson see above; The people such as Chow, Proc.Natl.Acad.Sci.USA, 82:910-914; With the people such as Bittle, J.Gen.Virol., 66:2347-2354,1985). Preferred immunogenicity epi-position comprises immunogenicity epi-position disclosed herein, and any combination of two, three, four, five or more these immunogenicity epi-positions. Can with carrier protein (such as albumin) to animal system (such as rabbit or mouse) present comprise one or more immunogenicity epi-positions or consisting of polypeptide to cause antibody response, perhaps, if this polypeptide long enough (about at least 25 amino acid) can be presented polypeptide so and need not carrier. Yet, comprise and few show that to 8-10 amino acid whose immunogenicity epi-positions be enough to cause at least can be in conjunction with the antibody of the linear epitope in the sex change polypeptide (as in the Western trace).

According to method well-known in the art, include but not limited to immunity in the body, external immunity and phage display method, epi-position of the present invention is carried polypeptide and be can be used for inducing antibody. Consult such as people such as Sutcliffe, see above; The people such as Wilson see above; And Bittle, J.Gen. Virol., 66:2347-2354,1985. If immunity in the use body, available free peptide immune animal; Yet, can be by peptide coupling macromolecular carrier (such as keyhole  hemocyanin (KLH) or tetanus toxoid) be strengthened tiring of anti-peptide antibody. For example, can use the peptide that will contain cysteine residues such as maleimide benzoyl-joints such as N-hydroxy-succinamide ester (MBS) to be coupled on the carrier, and use bridging agent (such as glutaraldehyde) more commonly used other peptide can be coupled on the carrier. Can be for example by in the peritonaeum and/or intracutaneous injection contain the emulsion of any other adjuvant that about 100 μ g peptides or carrier protein and Freunds adjuvant or known immune stimulatory reply, with the peptide immune animal of free or coupling carrier, such as rabbit, rat and mouse. May need several times booster shots, about two weeks of interval for example, providing with the anti-peptide antibody of tiring, its free peptide that can be for example be adsorbed onto solid phase surface by use detects through the ELISA determination method. Can improve by the selection of anti-peptide antibody from the tiring of the anti-peptide antibody of immune serum, for example according to method well-known in the art is adsorbed onto on the peptide on the solid support and wash-out is selected antibody.

Those skilled in the art will understand, and as mentioned above, the polypeptide of the present invention that comprises immunogenicity or antigenic epitopes can merge other peptide sequence. For example, polypeptide of the present invention can merge constant domain or its part (CH of immunoglobulin (Ig) (IgA, IgE, IgG, IgM)₁、CH ₂、 CH ₃, their any combination or part) produce chimeric polyeptides. These fusions can promote purifying and can increase half life in the body. For example, the chimeric protein that is comprised of each domain of the first two domain of people CD4 polypeptide and mammalian immune globulin heavy chain or constant region of light chain namely shows this phenomenon. Consult for example EPA 394,827; The people such as Traunecker, Nature, 331:84-86,1988. For the antigen (such as insulin) of puting together FcRn binding partners (such as IgG or Fc fragment), proved that antigen passes the epithelium barrier and enters immune send be enhanced (consulting for example PCT publication number WO 96/22024 and WO 99/04813). The fusion that has the dimeric structure that is connected by disulfide bond because of IgG part disulfide bond aspect combination and other molecule that neutralizes than the MPIF-1 albumen of independent monomeric form or fragment is more effective (consults such as people such as Fountoulakis, J.Biochem., 270:3958-3964,1995). The nucleic acid of the above-mentioned epi-position of encoding also can be recombinated with epitope tag (such as hemagglutinin (HA) label or flag label), with detection and the purifying that helps expressed polypeptide. For example, the system of being described by people such as Janknecht can make things convenient for to the non-sex change fusion of expressing purifying people such as (, Proc.Natl.Acad.Sci.USA, 88:8972-897,1991) Janknecht in Human cell line. In this system, genes of interest is subcloned in the vaccinia virus recombinant plasmid, so that the amino terminal label that the fusion of the opening code-reading frame of this gene translation property is comprised of six histidine residues. This label is taken on the matrix binding structural domain of fusion. In the future self-infection the extract loading of cell of vaccinia virus recombinant to Ni²⁺Nitrilo-acetic acid-agarose column, and with the protein that contains imidazole buffer selective elution histidine mark.

Can be by gene shuffling, primitive reorganization, extron reorganization and/or codon reorganization (being referred to as " DNA reorganization ") technology one-tenth in next life other fusion of the present invention. The activity that DNA reorganization can be used for regulating polypeptide of the present invention, these methods can be used for generating the polypeptide with change activity, and the activator of polypeptide and antagonist. Usually consult U.S. Patent number 5,605,793,5,811,238,5,830,721,5,834,252 and 5,837,458, and the people such as Patten, Curr.Opinion Biotechnol., 8:724-733,1997; Harayama, Trends biotechnol., 16 (2): 76-82,1998; The people such as Hansson, J.Mol.Biol., 287:265-276,1999; And M.M.Lorenzo and R.Blasco, Biotechniques, 24 (2): 308-313,1998 (with these patents and deliver that thing is complete to be collected herein by reference). In one embodiment, can reorganize to realize corresponding to the polynucleotides of SEQ ID NO:1 or 6 with by the change of the polypeptide of these polynucleotide encodings by DNA. In another embodiment, can reorganize to realize to the polynucleotides of polypeptide shown in the coding SEQ ID NO:2 with by the change of the polypeptide of these polynucleotide encodings by DNA. DNA reorganization comprises by homology or site-specific recombinates to assemble two or more DNA sections, produces variation in polynucleotide sequence. In another embodiment, can be before restructuring, the random mutagenesis by fallibility PCR, random nucleotide insertion or other method changes polynucleotides of the present invention or coded polypeptide. In another embodiment, can be with one or more elements of the polynucleotides of code book invention polypeptide, motif, section, partly, one or more elements of domain, fragment etc. and one or more heterologous molecule, motif, section, partly, the restructuring such as domain, fragment. Antibody

But other polypeptide of the present invention relates to immunologic opsonin in conjunction with polypeptide, polypeptide fragment or the variant of SEQ ID NO:2 of the present invention and/or antibody and the T cell antigen receptor (TCR) (measuring by the immunoassay for measuring specific antibody-antigen combination well-known in the art) of epi-position. Fragment, antiidiotype (anti-Id) antibody (comprising such as the anti-Id antibody for antibody of the present invention) and any above-mentioned epi-position that antibody of the present invention includes but not limited to polyclone, monoclonal, multiple specific, people, humanization or chimeric antibody, single-chain antibody, Fab fragment, F (ab ') fragment, produced by the Fab expression library are carried fragment. When being used for this paper, term " antibody " refers to the immunologic competence part of immunoglobulin molecules and immunoglobulin molecules, but namely comprises the molecule of the antigen binding site of immunologic opsonin conjugated antigen. Immunoglobulin molecules of the present invention can be that any classification (for example IgG, IgE, IgM, IgD, IgA and IgY), subclass of immunoglobulin molecules is (such as IgG₁、IgG ₂、IgG ₃、IgG ₄、IgA ₁And IgA₂) or somatotype.

Most preferably antibody is human antigen's binding antibody fragment of the present invention, includes but not limited to Fab, Fab ', F (ab ')₂, the Fv (sdFv) that connects of Fd, scFv (scFv), single-chain antibody, disulfide bond and comprise V_LOr V_HThe fragment of domain. Antigen binding antibody fragment (comprising single-chain antibody) can only comprise the variable region or also have following domain complete or part: hinge area, CH₁、CH ₂, and CH₃Domain. The present invention also comprises and also comprises variable region and hinge area, CH₁、CH ₂, and CH₃The Fab of any combination of domain. Antibody of the present invention can from any animal origin, comprise birds and mammal. Preferably, antibody is people, mouse (such as Mouse and rat), donkey, sheep, rabbit, goat, cavy, camel, horse or chicken. When being used for this paper, " people " antibody comprises the antibody of the amino acid sequence with human immunoglobulin(HIg), and comprise the antibody that is separated by human immunoglobulin(HIg) library or transgenic animals, described transgenic animals have imported one or more human immunoglobulin(HIg)s and have not expressed endogenous immunoglobulin (Ig), as hereinafter with such as the U.S. Patent number 5 of authorizing the people such as Kucherlapati, described in 939,598.

Antibody of the present invention can be monospecific, bispecific, tri-specific or more much higher heavy specific. Multiple specific antibody can have specificity for the different epi-positions of polypeptide of the present invention, or can all have specificity for polypeptide of the present invention and for allos epi-position (such as heterologous polypeptide or solid support material). Consult for example PCT publication number WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; The people such as Tutt, J.Immunol., 147:60-69,1991; U.S. Patent number 4,474,893,4,714,681,4,925,648,5,573,920,5,601,819; The people such as Kostelny, J.Immunol., 148:1547-1553,1992.

Antibody of the present invention can be identified or epi-position or the part of the polypeptide of the present invention of specific binding are described or illustrated by it. Epi-position or polypeptide portion can as described hereinly be illustrated, for example and C terminal position terminal by N, the size by the adjacent amino acid residue or as show and figure in listed. But the antibody of any epi-position of specific binding the present invention or polypeptide also can foreclose. Therefore, but the present invention includes the antibody of specific binding polypeptide of the present invention, and allow it is foreclosed.

Antibody of the present invention also can be described or illustrate by their cross reactivity. Comprise not the antibody in conjunction with any other analog, orthoevolution homologue or the homologue of polypeptide of the present invention. The present invention also comprises can be in conjunction with having the antibody of the polypeptide of at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55% and at least 50% homogeneity (use this area know calculate with method described herein) with polypeptide of the present invention. In specific embodiment, the mouse of antibody of the present invention and human protein and corresponding epi-position thereof, rat and/or rabbit homologue generation cross reaction. The present invention also comprises not with having with polypeptide of the present invention and is lower than 95%, is lower than 90%, is lower than 85%, is lower than 80%, is lower than 75%, is lower than 70%, is lower than 65%, is lower than 60%, is lower than 55% and be lower than the antibody that the polypeptide of 50% homogeneity (use this area know calculate with method described herein) combines. In specific embodiment, cross reaction mentioned above is about any monospecific antigenicity disclosed herein or immunogenic polypeptide, or the combination of 2,3,4,5 or more specific antigenic and/or immunogenic polypeptide. The present invention also comprises can be in conjunction with the antibody by the coded polypeptide of the polynucleotides that hybridization occur at rigorous hybridization conditions (as described herein) polynucleotides lower and of the present invention. Antibody of the present invention also can be described or illustrate by the binding affinity of they and polypeptide of the present invention. Preferred binding affinity comprises that those dissociation constants or Kd are lower than 5 * 10^-2M、10 ^-2M、5×10 ^-3M、10 ^-3M、5×10 ^-4M、10 ^-4M、5×10 ^-5M、10 ^-5M、 5×10 ^-6M、10 ^-6M、5×10 ^-7M、10 ^-7M、5×10 ^-8M、10 ^-8M、5×10 ^-9M、10 ^-9M、5×10 ^-10M、 10 ^-10M、5×10 ^-11M、10 ^-11M、5×10 ^-12M、10 ^-12M、5×10 ^-13M、10 ^-13M、5×10 ^-14M、 10 ^-14M、5×10 ^-15M or 10^-15The affinity of M.

The antibody that the present invention also provides contestable to suppress the combination of antibody and epi-position of the present invention is such as any method mensuration that is used for determining competitive binding of knowing by this area, for example immunoassay as herein described. In preferred embodiments, the combination of antibody competition inhibition and epi-position reaches at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60% or at least 50%.

The present invention also provides contestable to suppress the antibody of the combination of monoclonal antibody and polypeptide of the present invention (polypeptide of preferred SEQ ID NO:2). Can measure competitive the inhibition by any technology that this area is known, for example use competitive binding assay method described herein. In preferred embodiments, the combination of the polypeptide of antibody competition inhibition monoclonal of the present invention and SEQ ID NO:2 reaches at least 90%, at least 80%, at least 70%, at least 60% or at least 50%.

Antibody of the present invention can be taken on activator or the antagonist of polypeptide of the present invention. For example, the present invention includes the interactional antibody of part or all of destruction receptor/ligand and polypeptide of the present invention. Preferably, antibody of the present invention can be in conjunction with antigenic epitopes disclosed herein or its part. The present invention also comprises receptor specific antibody and ligand specificity's antibody. The present invention also comprises the receptor specific antibody that does not stop ligand binding but stop receptor activation. Can measure receptor activation (being signal transduction) by other technology that technology described herein or this area are known. For example, can following mensuration receptor activation: by immunoprecipitation subsequently Western engram analysis (for example mentioned above) detect the phosphorylation (such as tyrosine or serine/threonine) of acceptor or its substrate. In specific embodiment, provide and to have suppressed ligand activity or receptor active reach at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60% or at least 50% antibody of (activity when lacking antibody).

The present invention also comprises the receptor specific antibody that not only stops ligand binding but also stop receptor activation, and the antibody of identification receptor-ligand complex and the preferred not unconjugated acceptor of specific recognition or unconjugated part. Similarly, but the present invention includes binding partner and stop the neutralizing antibody of ligand binding acceptor, but and binding partner stop thus receptor activation but do not stop the antibody of ligand binding acceptor. The present invention also comprises the antibody of activated receptor. These antibody can be taken on receptor stimulating agent, i.e. reinforcement or activation all or part part mediate the biologically active of receptor activation effect, for example by inducing the dimerization of acceptor. Antibody can be elaborated into activator, antagonist or the inverse agonist of biologically active (particular biological that comprises peptide of the present invention disclosed herein is active). The method that can use this area to know prepares above-mentioned antibody agonist. Consult for example PCT publication number WO 96/40281; U.S. Patent number 5,811,097; The people such as Deng, Blood, 92 (6): 1981-1988,1998; The people such as Chen, Cancer Res., 58 (16): 3668-3678,1998; The people such as Harrop, J.Immunol., 161 (4): 1786-1794,1998; The people such as Zhu, Cancer Res., 58 (15): 3209-3214,1998; The people such as Yoon, J. Immunol., 160 (7): 3170-3179,1998; The people such as Prat, J.Cell.Sci., 111 (Pt2): 237-247,1998; The people such as Pitard, J.Immunol.Methods, 205 (2): 177-190,1997; The people such as Liautard, Cytokine, 9 (4): 233-241,1997; The people such as Carlson, J.Biol.Chem., 272 (17): 11295-11301,1997; The people such as Taryman, Neuron, 14 (4): 755-762,1995; The people such as Muller, Structure, 6 (9): 1153-1167,1998; The people such as Bartunek, Cytokine, 8 (1): 14-20,1996 (being collected herein by reference all documents are complete).

Antibody of the present invention can be used for such as but not limited to purifying, detection and target polypeptide of the present invention, comprises diagnosis and methods for the treatment of in external and the body. For example, antibody can be used for the level that quantitative and qualitative analysis is measured biological sample polypeptide of the present invention in immunoassay. Consult such as people such as Harlow, and " Antibodies:A Laboratory Manual " (antibody: lab guide), publishing house of cold spring harbor laboratory, the 2nd edition, 1988 (complete being collected herein by reference).

As will be detailed later, antibody of the present invention can use separately or unite use with other composition. Can further the recombinate N that is blended in heterologous polypeptide or C of antibody is terminal, or chemically conjugated on polypeptide or other component (comprise covalency with non-covalent puting together). For example, molecule and effector molecules molecule that antibody of the present invention can be recombinated and be merged or put together label in can be used as the detection assay method are such as heterologous polypeptide, medicine, radionuclide or toxin. Consult for example PCT publication number WO 92/08495, WO 91/14438, WO 89/12624, U.S. Patent number 5,314,995 and EP 396,387.

Antibody of the present invention comprises modified derivative, namely is covalently attached to antibody by the molecule with any type, does not reply so that covalent attachment does not stop antibody to produce antiidiotype. For example; antibody derivatives comprises modified antibody; such as by the cutting of the derivatization of glycosylation, acetylation, PEGization, phosphorylation, amidatioon, known protection/blocking groups, proteolysis, with being connected of cell ligand or other oroteins etc., but be not limited only to this. Can carry out any of many chemical modifications by known technology, include but not limited to that the metabolic of specificity chemical cleavage, acetylation, formylated, tunicamycin is synthetic, etc. In addition, derivative can comprise one or more nonclassical amino acids.

Any appropriate method that can know by this area becomes antibody of the present invention next life. Can generate by the several different methods that this area is known the polyclonal antibody for purpose antigen. For example, polypeptide of the present invention multiple host animal be can be applied to, rabbit, mouse, rat etc. be included but not limited to, to induce the generation of the serum that contains the antigentic specificity polyclonal antibody.

Multiple adjuvant can be used for increasing immunological response, this depends on host species, include but not limited to Fu Shi (fully with incomplete) adjuvant, mineral coagulant (such as aluminium hydroxide), surface reactive material (such as lysolecithin), poly polyalcohol, polyanion, peptide, oil emu, keyhole  hemocyanin, dinitrophenol and the potential people's of can be used for adjuvant (such as BCG vaccine (BCG, Bacille Calmette-Gu é rin) and Corynebacterium). These adjuvants are well known in the art.

Can prepare monoclonal antibody with a variety of technology that this area is known, comprise and use hybridoma, recombinant and display technique of bacteriophage or its associating. For example, can use hybridoma technology to generate monoclonal antibody, comprise that those this areas are known and such as people such as Harlow, " Antibodies:A Laboratory Manual " (antibody: lab guide), publishing house of cold spring harbor laboratory, the 2nd edition, 1988; The people such as Hammerling, " Monoclonal Antibodies and T Cell Hybridomas " (monoclonal antibody and T quadroma), 563-681 page or leaf, Elsevier, New York, the technology of instruction in 1981 (being collected herein by reference described document is complete). When being used for this paper, term " monoclonal antibody " is not limited to the antibody by the hybridoma technology generation. Term " monoclonal antibody " refers to derived from the monospecific polyclonal antibody of (comprising any eucaryon, protokaryon or phage clone), rather than generates its method.

Prepare and the method for screening specific antibody is conventional and well-known in this area with hybridoma technology, be specified in embodiment (such as embodiment 22 and 30). In a non-limiting example, with polypeptide of the present invention or express the cellular immunity mouse of this polypeptide. In case detect immune response, as in mice serum, detecting antigen-specific antibodies, then gather in the crops mouse spleen, and separating Morr. cell. Then by well-known technology splenocyte is merged any suitable myeloma cell, for example from the cell of clone SP20 (can be obtained by ATCC). Select and the clone hybridization knurl by limiting dilution assay. Then the technology of knowing by this area can be in conjunction with the cell of the antibody of polypeptide of the present invention to the secretion of hybridoma colony assay. Can be by generating the ascites that usually contains high-level antibody with positive hybridoma clone immune mouse.

Therefore, the invention provides the method that generates monoclonal antibody, and the antibody that generates by this method, comprise: the hybridoma of cultivating secretion antibody of the present invention, wherein preferably hybridoma is that splenocyte and the myeloma cell of the mouse by merging the antigen immune of the present invention of use by oneself produces, and then the hybridoma screening that is obtained by fusion is secreted and can be cloned in conjunction with the hybridoma of the antibody of polypeptide of the present invention.

Can generate by known technology the antibody fragment of identification defined epitope. For example, use such as papain (producing the Fab fragment) or pepsin (produce F (ab ')₂Fragment), can generate Fab of the present invention and F (ab ') by proteolysis cutting immunoglobulin molecules₂Fragment. F (ab ')₂Fragment comprises variable region, constant region of light chain and heavy chain CH₁Domain.

For example, the multiple phage display method that can use also that this area knows becomes antibody of the present invention next life. In the phage display method, the functional antibodies domain is illustrated in the surface of the phage particle that carries their polynucleotide sequence of coding. In a specific embodiment, this bacteriophage can be used for showing the antigen binding structural domain of being expressed by library or combinatorial antibody library (such as people or mouse). But the bacteriophage of the antigen binding structural domain of expressing binding purpose antigen is selected or identified to available antigen, as using through the antigen of mark or in conjunction with (or capturing) antigen on solid phase surface or the pearl. The bacteriophage that is used for these methods is filobactivirus normally, comprise fd and M13, wherein merged by the binding structural domain of the phage expression with the stable Fv antibody structure territory of Fab, Fv or disulfide bond and phage gene III or the restructuring of gene VIII protein. The example that can be used for generating the phage display method of antibody of the present invention comprises those people such as Brinkman, J.Immunol.Methods, 182:41-50,1995; The people such as Ames, J.Immunol. Methods, 184:177-186,1995; The people such as Kettleborough, Eur.J. Immunol., 24:952-958,1994; The people such as Persic, Gene, 187:9-18,1997; The people such as Burton, Advances in Immunology, 57:191-280,1994; Please number PCT/GB91/01134 among the PCT; PCT publication number WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; With U.S. Patent number 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,82 1,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727,5,733,743 and 5,969,108 (complete being collected herein by reference).

Described in above-mentioned document, after bacteriophage is selected, can be by bacteriophage separation antibody code area, and for the Fab that generates complete antibody (comprising human antibodies) or any other expectation, and in host's (comprising mammalian cell, insect cell, plant cell, yeast and bacterium) of any expectation, express, just as will be detailed later. For example, also can adopt for restructuring and generate Fab, Fab ' and F (ab ')₂The technology of fragment, the method for using this area to know is such as those PCT publication numbers WO 92/22324; The people such as Mullinax, BioTechniques, 12 (6): 864-869,1992; The people such as Sawai, AJRI, 34:26-34,1995; With the people such as Better, Science, 240:1041-1043, disclosed method in 1988 (being collected herein by reference described document is complete).

The example that can be used for generating the technology of scFv and antibody comprises those U.S. Patent numbers 4,946,778 and 5,258,498; The people such as Huston, Methods in Enzymology, 203:46-88,1991; The people such as Shu, PNAS, 90:7995-7999,1993; With the people such as Skerra, Science, 240:1038-1040, the technology of describing in 1988. For some purposes, comprise and using in the human body of antibody and the application of vitro detection determination method, preferably use chimeric, humanization or people's antibody. Chimeric antibody is a kind of like this molecule, and wherein the different piece of antibody is derived from different animal species, such as having derived from the variable region of mouse monoclonal antibody and the antibody of human immunoglobulin(HIg) constant region. The method that is used for the generation chimeric antibody is known in this area. Consult such as people such as Morrison Science, 229:1202,1985; The people such as Oi, BioTechniques, 4:214,1986; The people such as Gillies, J.Immunol.Methods, 125:191-202,1989; U.S. Patent number 5,807,715; 4,816,567; With 4,816,397 (complete being collected herein by reference). Humanized antibody is from can be in conjunction with the antibody molecule of inhuman species antibody of expectation antigen, and it has from one or more complementary determining regions (CDR) of inhuman species with from one or more framework regions of human immunoglobulin(HIg) molecule. Usually, will use from the framework residue in the alternative people's framework region of the corresponding residue of CDR donor, to change the combination of (advantageous embodiment) antigen. These frameworks substitute and can identify by method well-known in the art, as by CDR and the interactional modeling of framework residue, to identify antigen in conjunction with important framework residue, and by sequence relatively, (consult such as people such as Queen with the non-common framework residue of identifying ad-hoc location, U.S. Patent number 5,585,089; The people such as Riechmann, Nature, 332:323,1988, be collected herein by reference). The multiple technologies that can use this area to know make the antibody humanization, and for example (EP 239,400 for the CDR grafting; PCT publication number WO 91/09967; U.S. Patent number 5,225,539; 5,530,101; With 5,585,089), inlay or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology, 28 (4/5): 489-498,1991; The people such as Studnicka, Protein Engineering, 7 (6): 805-814,1994; The people such as Roguska, PNAS, 91:969-973,1994) and chain reorganization (U.S. Patent number 5,565,332).

Complete people's antibody is desirable especially for the therapeutic treatment of human patients. Can generate people's antibody by the several different methods that this area is known, comprise that above-described use is from the phage display method of the antibody library of human immunoglobulin(HIg) sequence. Also can consult U.S. Patent number 4,444,887 and 4,716,111; With PCT publication number WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735 and WO 91/10741 (complete being collected herein by reference).

Also can prepare people's antibody with transgenic mice, described mouse can not the endogenous immunoglobulin (Ig) of expressive function, but can express the human immunoglobulin gene. For example, can be at random or by homologous recombination people's heavy chain and light chain immunoglobulin gene compound are imported mouse embryo stem cell. Perhaps, except people's heavy chain and light chain gene, people variable region, constant region and variable region can be imported mouse embryo stem cell. When importing human immunoglobulin gene's seat by homologous recombination, can separate or make simultaneously murine heavy chain and light chain immunoglobulin gene to lose function. Particularly, the homozygosity in JH district disappearance has prevented that endogenous antibody from generating. Embryonic stem cell after amplification is modified and microinjection in the blastocyst to produce gomphosis mouse. Then cultivate gomphosis mouse to produce the offspring of isozygotying who expresses people's antibody. With usual way with selected antigen (such as all or part polypeptide of the present invention) immune transgenic mouse. Can use conventional hybridization knurl technology by the monoclonal antibody that obtains in the transgenic mice of immunity for antigen. The human immunoglobulin(HIg) transgenosis that transgenic mice carries is reset in the B cell differentiation procedure, experiences subsequently type conversion and somatic mutation. Thereby, use this technology, might generate upper useful IgG, IgA, IgM and the IgE antibody for the treatment of. About the summary of this technology of preparation people antibody, consult Lonberg and Huszar, Int.Rev.Immunol., 13:65-93,1995. Consult for example PCT publication number WO 98/24893 for this technology that generates people's antibody and human monoclonal antibodies and the discussing in detail of scheme that generate these antibody; WO 92/01047; WO 96/34096; WO 96/33735; European patent number 0 598 877; U.S. Patent number 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; With 5,939,598, complete being collected herein by reference. In addition, companies such as Abgenix (Freemont, CA) and GenPharm (San Jose, CA) can carry out to use with similar method mentioned above provides work for people's antibody of selected antigen.

Can use the technology that is called " directiveness is selected " to generate the fully human antibodies of the selected epi-position of identification. In this method, selected non-human monoclonal antibodies example (such as mouse antibodies) is used for instructing selection people such as (, Bio/technology, 12:899-903,1988) Jespers of the fully human antibodies of the identical epi-position of identification.

In addition, use the well-known technology of those skilled in the art, can be used for generating conversely the anti-idiotype of " simulation " polypeptide of the present invention for the antibody of polypeptide of the present invention, (consult for example Greenspan and Bona, FASEB J., 7 (5): 437-444,1989; And Nissinoff, J.Immunol., 147 (8): 2429-2438,1991). For example, can in conjunction with and the competitive polypeptide multimerization that suppresses polypeptide of the present invention and/or can be used for generating " simulation " polypeptide polymerization and/or binding structural domain with the antibody of the combination of part, thereby can in conjunction with and in the antiidiotype of polypeptide and/or its part. These neutrality antiidiotypes or its Fab fragment can be used for therapeutic scheme with in and polypeptide ligand. For example, these anti-idiotypes can be used for blocking thus its BA in conjunction with polypeptide of the present invention and/or in conjunction with its ligand/receptor.

Term " combination of polypeptide of the present invention and part " includes but not limited to the combination of ligand polypeptide of the present invention and acceptor; The combination of receptor polypeptides of the present invention and part; The combination of antibody of the present invention and antigen or epi-position; The combination of antigen of the present invention or epi-position and antibody; The combination of antibody of the present invention and anti-idiotype; The combination of anti-idiotype of the present invention and part; The combination of anti-idiotype of the present invention and acceptor; The combination of anti-anti-idiotype antibody of the present invention and part, acceptor or antibody; Deng.

As another example, can in conjunction with and the competitive antibody that activates polypeptide of the present invention or its part can be used for generating mimic peptide binding structural domain and/or activation structure territory, thereby can in conjunction with and the anti-idiotype of activating polypeptide and/or its part. These activity anti-idiotypes or its Fab fragment can be used for therapeutic scheme, with the activating polypeptide part. For example, these anti-idiotypes can be used for activating thus its BA in conjunction with polypeptide of the present invention, and/or activate thus its BA in conjunction with the ligand/receptor of polypeptide of the present invention. The polynucleotides of encoding antibody

The present invention also provide the nucleotide sequence that comprises code book invention antibody or its fragment or consisting of polynucleotides. The present invention also is encompassed in rigorous or the polynucleotides of hybridization occurs than the polynucleotides of (as hereinbefore defined) under the low stringency hybridization condition and encoding antibody (but the antibody of preferred specific binding polypeptide of the present invention, preferably can be in conjunction with the antibody of the polypeptide of the amino acid sequence with SEQ ID NO:2).

Can obtain polynucleotides by any method that this area is known and measure the nucleotide sequence of polynucleotides. For example, if the nucleotide sequence of antibody is known, can be assembled the polynucleotides of encoding antibody by the oligonucleotides of chemical synthesis (such as people such as Kutmeier so, BioTechniques, 17:242 is described in 1994), in brief, comprise the synthetic overlapping oligonucleotides that comprises the Sequence of encoding antibody, anneal and connect these oligonucleotides, the oligonucleotides that then connects by pcr amplification.

Perhaps, can be generated by the nucleic acid in suitable source the polynucleotides of encoding antibody. If can not obtain comprising the clone of nucleic acid of specific antibodies of encoding, but the sequence of antibody molecule is known, but so chemical synthesis or by appropriate sources (such as the antibody cDNA library, perhaps by any tissue of expressing this antibody or cell (such as the hybridoma of selecting to be used for the expressing antibody of the present invention) cDNA library that makes up or the nucleic acid of separation (preferred polyA+RNA)) obtain the nucleic acid of this immunoglobulin (Ig) of coding, comprise in the rear approach use can with the pcr amplification of the synthetic primer of antibody coding sequence 3 ' and 5 ' terminal hybridization, perhaps use for the special oligonucleotide probe of specific gene sequence, identified the cDNA clone of this antibody of for example encoding through the clone by cDNA library. Then can use any method well-known in the art to be cloned in the reproducible cloning vector by the amplification of nucleic acid that PCR produces.

In case determined nucleotide sequence and the corresponding amino acid sequence of antibody, just can use the method for the nucleotide sequence operation well-known in the art to operate the nucleotide sequence of antibody, (consult such as people such as Sambrook such as recombinant DNA technology, direct mutagenesis, PCR etc., " Molecular Cloning, A Laboratory Manual " (molecular cloning, lab guide), the 2nd edition, publishing house of cold spring harbor laboratory, cold spring port, NY, 1990 and the people such as Ausubel compile, " Current Protocols in Molecular Biology " (molecular biology general scheme), John Wiley﹠Sons, NY, 1998, complete being collected herein by reference), with the antibody that generation has the different aminoacids sequence, for example produce amino acid replacement, disappearance and/or insertion.

In a specific embodiment, can be by method well-known in the art, such as the known amino acid sequence of relatively other heavy chain and variable region of light chain determining the sequence hypervariable region, thereby the amino acid sequence that checks heavy chain and/or variable region of light chain domain is to identify the sequence of complementarity-determining region (CDR). Use conventional recombinant DNA technology, one or more CDR can be inserted in the framework region, as inserting people's framework region so that non-human antibody's humanization is as above aforementioned. Framework region can be natural generation or total framework region, preferred people's framework region (consulting such as people such as Chothia J.Mol.Biol., 278:457-479, people's framework region tabulation of 1998). Preferably, but by the antibody of the polynucleotide encoding specific binding polypeptide of the present invention of framework region and CDR combination results. Preferably, as mentioned above, can in framework region, carry out one or more amino acid replacements, and preferably, amino acid replacement has improved the combination of antibody and its antigen. In addition, these methods can be used for the variable region cysteine of one or more participation intrachain disulfide bonds is substituted or lacks, to produce the antibody molecule that lacks one or more intrachain disulfide bonds. Other polynucleotides change also to be contained within the scope of the invention and belongs in the art technology scope.

In addition, can use exploitation be used for to generate technology (people such as Morrison, Proc.Natl.Acad.Sci.USA, 81:851-855,1984 of " chimeric antibody "; The people such as Neuberger, Nature, 312:604-608,1984; The people such as Takeda, Nature, 314:452-454,1985), be about to arrive with the gene splicing from the human antibody molecules with suitable BA from the gene with the specific mouse antibodies molecule of suitable antigen. As mentioned above, chimeric antibody is that wherein different piece is derived from the molecule of different animals species, has derived from the variable region of mouse mAb and the molecule of human immunoglobulin(HIg) constant region, such as humanized antibody such as those.

Perhaps, can adopt technology (U.S. Patent number 4,946,778 of describing for the preparation of single-chain antibody; Bird, Science, 242:423-42,1988; The people such as Houston, Proc.Natl.Acad.Sci.USA, 85:5879-5883,1988; With the people such as Ward, Nature, 334:544-54,1989) generate single-chain antibody. Single-chain antibody is to form by single chain polypeptide is received together, produced to the heavy chain in FV district and light chain segments through the amino acid bridging. Also can use the technology (people such as Skerra, Science, 242:1038-1041,1988) of assembling function Fv fragment in Escherichia coli. Generate the method for antibody

Can prepare antibody of the present invention, particularly chemical synthesis by any known method for the synthesis of antibody that this area is known, or preferred recombination and expression techniques.

The recombinant expressed expression vector that needs to make up the polynucleotides that comprise encoding antibody of antibody of the present invention or its fragment, derivative or analog (such as heavy chain or light chain or the single-chain antibody of the present invention of antibody of the present invention). In case obtain code book invention antibody molecule or the heavy chain of antibody or the polynucleotides of light chain or its part (preferably comprising heavy chain or light chain variable domain), just can use method well-known in the art to make up the carrier that is used for generating antibody molecule of the present invention by recombinant DNA technology. Thereby, this paper describes that the polynucleotides that comprise the antibody coding nucleotide sequence by expression prepare method of protein. Method well-known in the art can be used for making up and comprises antibody coding sequence and the suitable expression vector of transcribing and translate control signal. These methods comprise for example interior Genetic Recombination of extracorporeal recombinant DNA technology, synthetic technology and body. The present invention provides the replicable vector of the nucleotide sequence that comprises the code book invention antibody molecule that is operatively connected with promoter or its heavy chain or light chain or heavy chain or light chain variable domain thus. These carriers can comprise the nucleotide sequence of encoding antibody molecule constant region and (consult for example PCT publication number WO 86/05807; PCT publication number WO 89/01036; With U.S. Patent number 5,122,464), and the variable domains of antibody can be cloned into carrier for the expressed heavy chain or light chain.

Can expression vector be transferred in the host cell by routine techniques, then cultivate transfectional cell to generate antibody of the present invention by routine techniques. Thereby, the present invention includes the host cell of the polynucleotides that contain the code book invention antibody that is operatively connected with allogeneic promoter or its heavy chain or light chain or single-chain antibody of the present invention. In expressing a preferred embodiment of double-chain antibody, can be in host cell the carrier of coexpression encoding heavy chain and light chain, with the immunoglobulin molecules of the expressed, as detailed below.

Multiple host-expression vector system can be used for expressing antibody molecule of the present invention. These host-expression systems have represented the carrier that can be used to produce purpose coded sequence and subsequent purificn, and transform with suitable nucleotide coding sequence or transfection after original position express the cell of antibody molecule of the present invention. These include but not limited to following microorganism, such as the bacterium (such as Escherichia coli, bacillus subtilis) that transforms with the recombinant phage dna that comprises antibody coding sequence, DNA or cosmid DNA expression vector; Yeast (such as saccharomyces, pichia) with the recombinant yeast expression vector conversion that comprises antibody coding sequence; Insect cell system with the recombinant virus expression vector that comprises antibody coding sequence (such as baculoviral) infection; With the recombinant virus expression vector that comprises antibody coding sequence (such as cauliflower mosaic virus, CaMV; Tobacco mosaic virus (TMV) TMV) infects or with the plant cell system of the recombinant plasmid expression vector that comprises antibody coding sequence (such as Ti-plasmids) conversion; Perhaps carry and comprise derived from the genomic promoter of mammalian cell (such as metallothionein promoter) or derived from the mammalian cell system (such as COS, CHO, BHK, 293,3T3 cell) of the recombinant expression construct thing of the promoter of mammalian virus (such as gland virus stage starting, vaccinia virus 7.5K promoter). Preferred bacterium cell (such as Escherichia coli), more preferably the eukaryotic expression of whole recombinant antibody molecule (especially for) is used for the expressing recombinant antibody molecule. For example, uniting of mammalian cell (such as Chinese hamster ovary cell (CHO)) and carrier (such as the main immediate early gene promoter element from human cytomegalovirus) is (people such as Foecking of effective expression system of antibody, Gene, 45:101,1986; The people such as Cockett, Bio/technology, 8:2,1990).

In bacterial system, can be according to the intended purpose of the antibody molecule to be expressed favourable multiple expression vector of selection. For example, when needs generate a large amount of this albumen for the production of the pharmaceutical composition of antibody molecule, may wish that carrier instructs the high level expression of the fusion protein product that is easy to purifying. These carriers include but not limited to the coli expression carrier pUR278 (people such as Ruther, EMBO J., 2:1791,1983), wherein can be connected to separately antibody coding sequence in the carrier and meet the reading frame of lacZ coded sequence, thereby generate fusion; PIN carrier (Inouye and Inouye, Nuclei Acids Res., 13:3101-3109,1985; Van Heeke and Schuster, J.Biol.Chem., 24:5503-5509,1989); Deng. The pGEX carrier can be used for the formal representation polypeptide with the fusion that forms with glutathione S-transferase (GST). Usually, these fusions are solubilities, and be easy to by cell lysis by absorption and be attached on matrix glutathione-sepharose 4B, obtain purifying there being wash-out in the situation of free glutathione subsequently. Can design the pGEX carrier to comprise fibrin ferment or factor Xa protease cutting site, the target gene product of DCRP can partly be released by GST.

In the insect system, with the carrier of Autographa californica nucleopolyhedrosis virus (AcNPV) as the expressing heterologous gene. Virus is grown in fall army worm (Spodoptera frugiperda) cell. Antibody coding sequence can be cloned into separately in the viral nonessential region (for example polyhedron gene), and place under the control of AcNPV promoter (for example polyhedrin promoter).

In mammalian host cell, can utilize multiple expression system based on virus. Using in the situation of adenovirus as expression vector, a purpose antibody coding minute sub-connection adenovirus can transcribed/translated the control complex, such as late promoter and three component targeting sequencings. Then can this mosaic gene be inserted the adenoviral gene group by restructuring in external or the body. Insertion at viral genome nonessential region (such as E1 or E3 zone) has generation vigor and can (consult for example Logan and Shenk by the recombinant virus of expression antibody molecule in the host who infects, Proc.Natl.Acad. Sci.USA, 81:355-359,1984). Effective translation of the antibody coding sequence that inserts may also need specific initial signal. These signals comprise ATG initiation codon and the sequence of adjoining. In addition, initiation codon must with the expectation coded sequence the reading frame homophase, to guarantee the translation of whole insetion sequence. These external sources translation control signals and initiation codon can be multiple sources, natural or synthetic. Can improve by comprising suitable transcriptional enhancer element, transcription terminator etc. the efficient (consulting such as people such as Bittner Enzymology method, 153:51-544,1987) of expression.

In addition, can select to regulate the expression of insetion sequence or the host cell strain of modification and processed gene product in the certain desired mode. These modifications (such as glycosylation) of protein and processing (such as cutting) may be important to the function of protein. Process after the translation of different hosts cell for protein and gene outcome and modification has characteristic and specific mechanism. Can select suitable clone or host system correctly to be modified and process to guarantee expressed foreign protein. For this purpose, can use the processing with correct primary transcribe, the glycosylation of gene outcome and the cyto-architectural eukaryotic host cell of phosphorylation. These mammalian host cells include but not limited to CHO, VERA, BHK, Hela, COS, MDCK, 293,3T3, WI38, breast cancer cell line particularly, such as BT483, Hs578T, HTB2, BT20 and T47D, and MCF-10, such as CRL7030 and Hs578Bst.

For generation recombinant protein long-term, high yield, preferred stably express. For example, can transform the clone of stably express antibody molecule. Be not to use the expression vector comprise the virus replication starting point, but with being subject to suitably expressing control element (such as promoter, enhancer sequence, transcription terminator, polyadenylation site etc.) but control, comprise selected marker but do not contain the DNA transformed host cell of functional or intact virus origin of replication. After importing foreign DNA, improved cell was grown in rich medium 1-2 days, then change selective medium into. Selected marker in the recombinant plasmid is given the resistance of selecting, allow cytotostatic with plasmid integration in their chromosome, and growth forms the plaque that can clone and be extended to clone. This method can be favourable the clone of be used for to transform expressing antibody molecule. These improved clones are for screening and assessment is direct or indirect and the interactional compound of antibody molecule may be particularly useful.

Can use the multiple choices system, include but not limited to herpes simplex virus thymidine kinase (people such as Wigler, Cell, 11:223,1977), hypoxanthine-guanine phosphoribosyl transferase (Szybalska and Szybalski, Proc.Natl.Acad.Sci.USA, 48:202,1992) and the adenine phosphoribosyl transferase (people such as Lowy, Cell, 22:817,1980), they can be respectively applied to tk-, hgprt-or aprt-cell. The antimetabolite resistance can be used as the basis of the following gene of screening: dhfr equally, gives methotrexate resistance (people such as Wigler, Proc.Natl.Acad.Sci.USA, 77:357,1980; O ' Hare et al, Proc.Natl.Acad.Sci.USA, 78:1527,1981); Gpt gives mycophenolic acid resistance (Mulligan and Berg, Proc.Natl.Acad.Sci.USA, 78:2072,1981); Neo gives the resistance to aminoglycoside G-418 (people such as Goldspiel, Clinical Pharmacy, 12:488-505,1993; Wu and Wu, Biotherapy, 3:87-95,1991; Tolstoshev, Ann.Rev.Pharmacol.Toxicol., 32:573-596,1993; Mulligan, Science, 260:926-932,1993; Morgan and Anderson, Ann.Rev.Biochem., 62:191-217,1993; May, TIB TECH, 11 (5): 155-215,1993); And hygro, give hygromycin resistance (people such as Santerre, Gene, 30:147,1984). The method that the recombinant DNA field is generally known can conventionally be used for the recombinant clone that selection is expected, and these methods are described in such as people such as Ausubel and compile, " Current Protocols in Molecular Biology " (molecular biology general scheme), John Wiley ﹠ Sons, New York, 1993; Kriegler, " Gene Transfer and Expression, A Laboratory Manual " (transgenosis and expression, lab guide), Stockton publishing house, New York, 1990; The people such as Dracopoli compile, " Current Protocols in Human Genetics " (human genetics general scheme), the 12nd and 13 chapters, John Wiley ﹠ Sons, New York, 1994; With the people such as Colberre-Garapin, J.Mol.Biol., 150:1,1981 (being collected herein by reference).

(summary is consulted Bebbington and Hentschel can to increase to increase the expression of antibody molecule by carrier, " DNA cloning " (dna clone) the 3rd volume, " The use of vectors based on gene amplification for the expression of cloned genes in mammaliab cells " (purposes that in mammalian cell, is used for the gene of expression cloning based on the carrier of gene magnification), Academic publishing house, New York, 1987). When the mark in the carrier system of expressing antibody can increase, then the raising of inhibitor level will increase the copy number of marker gene in the host cell culture. Because the zone of amplification is relevant with antibody gene, so the generation of antibody also will increase people such as (, Mol.Cell. Biol., 3:257,1983) Crouse.

Available two kinds of expression vector cotransfection host cells of the present invention, the first vector encoded polypeptide of being derived by heavy chain wherein, the second vector encoded is by the polypeptide of derived light chain. These two kinds of carriers can comprise identical selected marker, so that heavy chain and light chain polypeptide equivalent are expressed. Perhaps, can use coding and can express the two single carrier of heavy chain and light chain polypeptide. In this case, before light chain should place heavy chain, to avoid excessive poisonous free heavy chain (Proudfoot, Nature, 322:52,1986; Kohler, Proc.Natl.Acad.Sci.USA, 77:2197,1980). The coded sequence of heavy chain and light chain can comprise cDNA or genomic DNA.

In case by animal generation, chemical synthesis or recombinant expressed antibody molecule of the present invention, just can carry out purifying by any method that is used for the purifying immunoglobulin molecules that this area is known, for example chromatography (such as ion-exchange, affine (particularly for the specific antigen of albumin A affine) and size exclusion chromatography), centrifugal, difference dissolving or be used for any other standard technique of protein purification. In addition, antibody of the present invention or its fragment can merge with allogeneic polypeptide sequence described herein or that this area is known, so that purifying.

The present invention is also contained restructuring and is merged or chemically conjugated (comprising covalency and non-covalent puting together) polypeptide of the present invention (or its part, at least 10,20,30,40,50,60,70,80,90 or 100 amino acid of preferred polypeptide), to produce fusion. Fusion needs not to be directly, also can occur by joint sequence. Antibody can have for the specificity except polypeptide of the present invention (or its part, 10,20,30,40,50,60,70,80,90 or 100 amino acid of preferred polypeptide) antigen in addition at least. For example, can by polypeptide of the present invention is merged or put together specific antibody for the specific cells surface receptor utilize antibody with polypeptide target of the present invention to specific cell type, can be in external or the body. The method that can use this area to know utilize to merge or the antibody of puting together polypeptide of the present invention carries out in vitroimmunoassay method and purification process. Consult such as people such as Harbor, see above; With PCT publication number WO 93/231232; EP 439,095; The people such as Naramura, Immunol.Lett., 39:91-99,1994; U.S. Patent number 5,474,981; The people such as Gillies, PNAS, 89:1428-1432,1992; The people such as Fell, J.Immunol., 146:2446-2452,1991 (complete being collected herein by reference). Conjugate and chelate

The present invention also comprises the composition that comprises the fusion or puted together the polypeptide of the present invention in the antibody structure territory except the variable region. For example, polypeptide of the present invention can merge or put together antibody Fc zone or its part. The antibody moiety that is blended in polypeptide of the present invention can comprise constant region, hinge area, CH₁Domain, CH₂Domain and CH₃Domain, or any combination of total territory or its part. Polypeptide also can merge or put together the aforementioned polypeptides part to form polymer. For example, the Fc part that is blended in polypeptide of the present invention can form dimer by the disulfide bond between the Fc part. Can prepare by the part of fused polypeptide and IgA and IgM more much higher aggressiveness form. The method that be used for to merge or put together polypeptide of the present invention and antibody moiety is known in this area. Consult for example U.S. Patent number 5,336; 603,5; 622,929; 5,359,046; 5,349,053; 5,447,851; 5,112,946; EP 307,434; EP 367,166; PCT publication number WO 96/04388; WO 91/06570; The people such as Ashkenazi, Proc.Natl.Acad.Sci.USA, 88:10535-10539,1991; The people such as Zheng, J.Immunol., 154:5590-5600,1995; With the people such as Vil, Proc.Natl.Acad.Sci.USA, 89:11337-11341,1992 (complete being collected herein by reference).

As mentioned above, the method that can use this area to know will merge corresponding to the polypeptide of polypeptide, polypeptide fragment or the variant of SEQ ID NO:2 or put together above-mentioned antibody moiety, with half life in the body that increases polypeptide, or be used for immunoassay. In addition, can merge or put together above-mentioned antibody moiety corresponding to the polypeptide of SEQ ID NO:2, so that purifying. The example of a report has described that (EP 394,827 by the first two domain of people CD4 polypeptide and the heavy chain of mammalian immune globulin or the chimeric protein that each domain of constant region of light chain forms; The people such as Traunecker, Nature, 331:84-86,1988). The polypeptide of the present invention that merges with the antibody with dimeric structure (owing to IgG) that disulfide bond is connected or put together may be at combination and neutralization other minute period of the day from 11 p.m. to 1 a.m than the higher (people such as Fountoulakis of the efficient of monomeric secreted protein matter or protein fragments self, J.Biochem., 270:3958-3964,1995). In many cases, the Fc part in the fusion is useful in treatment and diagnosis, thereby can cause for example improvement of pharmacokinetic properties (EP A 232,262). Perhaps, disappearance Fc partly expects behind expression, detection and purified fusion protein. For example, the Fc part may hinder treatment and diagnosis (if with fusion antigen as immunity inoculation). In drug development, for example with human protein (such as hIL-5) and Fc partial fusion, identify that to be used for the high flux screening determination method antagonist of hIL-5 (is consulted the people such as Bennett, J.Molecular Recoginition, 8:52-58,1995; The people such as Johanson, J.Biol.Chem., 270:9459-9471,1995).

In addition, antibody of the present invention or its fragment can merge flag sequence, such as the peptide of being convenient to purifying. In a preferred embodiment, marker amino acid sequence is six histidine peptides, such as in pQE carrier (QIAGEN company, 9259 Eton Avenue, Chatsworth, CA, 91311) label that provides in, and other, many can the acquisition by commercial sources in them. Such as people such as Gentz, Proc.Natl.Acad.Sci.USA, 86:821-824, described in 1989, for example six histidines can be convenient to the purifying of fusion. Other peptide tag that can be used for purifying includes but not limited to " HA " label, and it is corresponding to the epi-position of being derived by influenza hemagglutinin protein matter (people such as Wilson, Cell, 37:767,1984) and " flag " label.

As mentioned above, but the present invention includes the antibody of at least a MPIF-1 epi-position of specific binding, but also may get rid of the antibody of specific binding any epi-position of the present invention or polypeptide. Thereby antibody can have the specificity for MPIF-1, perhaps can have for the polypeptide beyond the MPIF-1 and the specificity of epi-position.

Antibody or its fragment of having puted together diagnosticum or therapeutic agent also contained in the present invention. Antibody can be used for diagnosis, for example is used for growth or the process of monitoring tumour as a part of clinical trial flow process, as determining the effect of given therapeutic scheme. Can be by the antibody coupling detectable substance being convenient to detection. The example of detectable substance comprises positron emission material and the on-radiation paramagnetic metal ion of plurality of enzymes, prothetic group, fluorescent material, luminescent material, bioluminescent material, radioactive material, the multiple positron emission computerized tomography method of use. The technology that can use this area to know, with detectable substance directly or indirectly (by intermediate, the joint of knowing such as this area) coupling be conjugated to antibody or its fragment on. Consult for example U.S. Patent number 4,741,900, about puting together in antibody to be used as the metal ion according to diagnosticum of the present invention. The example of suitable enzyme comprises horseradish peroxidase, alkaline phosphatase, beta galactosidase or acetylcholinesterase; The example of suitable prothetic group compound comprises Streptavidin/biotin and avidin/biotin; The example of suitable fluorescent material comprises umbelliferone, fluorescein, fluorescein isothiocynate, rhodamine, dichlorotriazine base amine fluorescein, dansyl Cl or phycoerythrin; The example of luminescent material comprises luminol; The example of bioluminescent material comprises luciferase, luciferin and aequorin; The example of suitable radioactive material comprises¹²⁵I、 ¹³¹I、 ¹¹¹In or⁹⁹Tc。

In addition, antibody or its fragment can be puted together the therapeutic part, such as cytotoxin, as cytotoxicity, suppress cell, cytocidal reagent, therapeutic agent or radioactive metal ion, such as alpha emitter, such as²¹³Bi. Cytotoxin or cytotoxic agent comprise any reagent harmful to cell. Example comprises taxol, Cytochalasin B, Gramicidin D, ethidium bromide, emetine, mitomycin, Etoposide, tenoposide, vincristin, vinblastine, colchicine, adriamycin, daunorubicin, dihydroxy anthracin diketone, mitoxantrone, mithramycin, actinomycin D, 1-dehydrogenation testosterone, glucocorticoid, procaine, totokaine, lidocaine, Propranolol and puromycin and analog or homologue. Therapeutic agent includes but not limited to antimetabolite (such as methotrexate, Ismipur, 6-thioguanine, cytarabine, 5 FU 5 fluorouracil decarbazene), alkylating agent (such as mechlorethamine, thiophene for sending (DDP) cis-platinum of Chlorambucil, melphalan, BCNU (BSNU) and lomustine (CCNU), endoxan, busulfan, dibromannitol, streptozotocin, mitomycin C, suitable-dichloro diamines platinum (II)), anthracycline (such as daunorubicin (being called in the past daunomycin) and adriamycin), antibiotic (such as D actinomycin D, bleomycin, mithramycin, Anthramycin (AMC)) and antimitotic agent (such as vincristin and vinblastine).

Conjugate of the present invention can be used for modifying specific biological answer-reply, and therapeutic agent or drug moiety should not be construed as and be limited to classical chemotherapeutant. For example, drug moiety can be protein or the polypeptide with expectation BA. These protein can comprise for example toxin, all coin abrins, ricin A, PE or diphtheria toxin; Protein, such as TNF, alpha-interferon, beta-interferon, nerve growth factor, by platelet-derived growth factor, tissue plasminogen activator, apoptosis agent, such as TNF-α, TNF-β, AIM I (consulting international publication number WO 97/33899), AIM II (consulting international publication number WO 97/34911), the FasL (people such as Takahashi, Int.Immunol., 6:1567-1574,1994), VEGI (consulting international publication number WO 99/23105), thrombolytics or anti-angiogenic agent, such as blood vessel element processed or Endothelin processed; Or biological answer-reply regulator, such as lymphokine, il-1 (IL-1), proleulzin (IL-2), interleukin-6 (IL-6), granulocyte-macrophage colony stimutaing factor (GM-CSF), granulocyte colony stimulating factor (G-CSF) or other growth factor.

Also antibody can be attached on the solid support, this is particularly useful in the purifying of immunoassay or target antigen. These solid supports include but not limited to glass, cellulose, polyacrylamide, nylon, polypropylene, polyvinyl chloride or polystyrene.

In addition, MPIF-1 polypeptide or its variant, fragment, activator and antagonist can be puted together diagnosticum or therapeutic agent, such as those above with reagent as herein described or well-known in the art. The MPIF conjugate can be used for diagnosis as herein described and well-known in the art or therapeutic purposes. For example, MPIF can put together radio isotope and be used for diagnosis or treatment.

Be well known in the art for the technology of these therapeutic partly being puted together antibody, consult such as people such as Arnon, " Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy " (monoclonal antibody that is used for medicine immunity target in the treatment of cancer), in " Monoclonal Antibodies And Cancer Therapy " (monoclonal antibody and treatment of cancer) that the people such as Reisfeld compile, the 243-56 page or leaf, Alan R.Liss company, 1985; The people such as Hellstrom, " Antibodies For Drug Delivery " (antibody that is used for drug delivery), in " Controlled Drug Delivery " (controlled drug delivery) that the people such as Robinson compile, the 2nd edition, the 623-53 page or leaf, Marcel Dekker company, 1987; Thorpe, " Antibody Carriers Of Cytotoxic Agents In Cancer Therapy:A Review " (antibody carrier of the cytotoxic agent in the treatment of cancer: look back), in " Monoclonal Antibodies ' 84:Biological And Chinical Applications " (monoclonal antibody ' 84: biology and clinical practice) that the people such as Pinchera compile, the 475-506 page or leaf, 1985; " Analysis; Results; And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy " (to analysis, result and prospect of the therapeutic use of radiolabelled antibody in treatment of cancer), in " the Monoclonal Antibodies For Cancer Detection And Therapy " of the people such as Baldwin volume (monoclonal antibody that is used for cancer detection and treatment), the 303-16 page or leaf, Academic publishing house, 1985; With the people such as Thorpe, Immunol.Rev., 62:119-158,1982.

Antibody, protein (comprising polypeptide of the present invention) and little molecule can be used as target and pre-targeted molecular. Can pass through the well-known method radioactive label of those of ordinary skills these molecules of the present invention, include but not limited to that the radioactive label chelating in antibody and antibody phage library is used for the agent of target RIT. Consult such as people such as DeNardo Clin.Cancer Res., 5 (10S): 3213s-3218s, 1999; The people such as Quadri, Q.J.Nucl.Med., 42:250-261,1998 (complete being collected herein by reference).

For chelating, different chemical bonds can be inserted between antibody and the radiolabeled chelate. Have the reactive radioactive label monoclonal antibody of target antigen and can in vivo cytotoxicity or diagnostic Isotopic selectivity be delivered malignant cell. Useful diversity and plastic antibody gene provide the structure of pre-targeted molecular. By selecting the unprimed phage antibody library of people, can obtain to have the reactive single chain antibody fragments of target antigen (being scFv) diversity array. Also can by hybridoma Direct Cloning scFv, be convenient to operate subsequently the phage library of (modifying such as affinity maturation and specificity) with structure. The scFv that is selected by these sources has proved that for the affinity of its specific antigen target wide spectrum is in conjunction with feature. Can will be cloned in the diabody molecule as box from heavy chain of antibody (VH) and light chain (VL) gene of selected scFv. Hereinafter, come specificity to destroy the method for cell by use polypeptide of the present invention with toxin or cytotoxic drug precursor, this application further has been discussed.

Perhaps, antibody can be puted together the second antibody to form the different conjugate of antibody, such as Segal at U.S. Patent number 4,676, (complete being collected herein by reference) described in 980.

No matter whether antibody put together the therapeutic part, uses separately or associational cells virulence factor and/or cell factor, all useful as therapeutics.

Radiolabeled antibody, protein and little molecule are cytotoxic agents, and can be used for targeted radiotherapy (such as radioimmunotherapy) or radio-immunity detection (such as radioimmunodiagnosis and immune imaging). In order to protect or treat normal structure, cell and organ, can or use afterwards MPIF-1 before using radiolabeled antibody, protein or little molecule, in the process.

As mentioned above, but the present invention includes the antibody of at least a MPIF-1 epi-position of specific binding, but also can get rid of the antibody of specific binding any epi-position of the present invention or polypeptide. Thereby antibody can have the specificity for MPIF-1, perhaps can have for the polypeptide beyond the MPIF-1 and the specificity of epi-position. In one embodiment, in the process of the radioimmunotherapy of cancer or another kind of disease, with the co-administered radiolabeled antibody of MPIF-1 polypeptide, protein or little molecule. Before using the RIT agent, in the process or when using afterwards, normal cell be protected and/or be treated to the MPIF polypeptide can, and such as myeloid progenitor, and other normal cell, tissue and organ avoid damage.

In another preferred embodiment, composition of the present invention and following co-administered: Rituximab (anti-CD-20 monoclonal antibody; Rituxan^TM), Ibritumomab tiuxetan (puted together⁹⁰The anti-CD-20 monoclonal antibody of Y; Zevalin^TM), Tositumomab (puted together¹³¹I；Bexxar ^TM)、Rituximab(Rituxan ^TM)、Trastuzumab(Herceptin ^TM), OvaRex MoAb (for the monoclonal antibody of the CA 125 in the oophoroma), Denileukin difitox (diphtheria toxin conjugate; Ontak ), AA98 (can be in conjunction with microvessel structure but not in conjunction with the monoclonal antibody of normal structure; The people such as Xiyun, summary 82,1999), Adrecolomab (anti-general gland cancer antigen), anti-CEA antibody (puted together Clin.Cancer Res., 5 (supplementary issues):¹³¹I; The people such as Behr, Amer.Soc.of Clin.Oncol.35th Annual Meeting, Atlanta (the 35th annual meeting of Atlanta U.S. Clinical Oncology association): make a summary 1025,1999), anti-TAG72 glycoprotein antibody, anti-PMSA antibody, anti-HLA-DR10 β antibody, anti-VEGF antibody, anti-CXCR4 antibody, CH225 (anti-EGFR chimeric antibody; The people such as Mendelsohn, Amer. Soc.of Clin.Oncol.35th Annual Meeting, Atlanta (the 35th annual meeting of Atlanta U.S. Clinical Oncology association): summary 1502,1999), CP-358,774 (the people such as Karp, Amer.Soc.of Clin.Oncol.35th Annual Meeting, Atlanta, GA (the 35th annual meeting of Georgia Atlanta U.S. Clinical Oncology association): summary 1499,1999) and the ZD 1839 (people such as Karp, Amer.Soc.of Clin.Oncol. 35th Annual Meeting, Atlanta, GA (the 35th annual meeting of Georgia Atlanta U.S. Clinical Oncology association): the summary 1500,1999) (the two all is the micromolecular inhibitor of EGFR), IDEC-Y2B8 (and puted together can in conjunction with⁹⁰The anti-CD 20 antibodies of the molecule of Y; The people such as Witzi, " Phase I/II trial of IDEC-Y2B8 radioimmunotherapy for treatment of relapsed or refractory CD20-positive B-cell non-Hodgkin ' s lymphoma " (the I/II phase that is used for the treatment of the IDEC-Y2B8 radioimmunotherapy of the non-Hodgkin lymphomas of the positive B cell of CD2-recurrence or refractory tests), J. Clin.Oncol., 1999, in the printing; The people such as Bastion, Blood, 86:3257-3263,1995),¹³¹I-MH-14F(ab) ₂Anticancer embryonal antigen monoclonal antibody (people such as Juweid, J.Nucl. Med., 41 (1): 93-103, in January, 2000; Comment is seen J.Nucl.Med., 41 (1): 104-106, in January, 2000),⁶⁷(Lym is the lymphocytic mouse monoclonal antibody of preferential targeted malignant to Cu-2IT-BAT-Lym-1; Chelating agent is combination⁶⁷The Isosorbide-5-Nitrae of Cu, 7,11-tetraazacyclododecane tetradecane-N, N ', N ", N -four acetic acid; The people such as O ' Donnel, J.Nucl.Med., 40 (12): 2014-2020, in December, 1999),¹⁸⁸Anti-NCA antigen-antibody BW250/183 (the anti-granulocyte of rhenium mark; Use the direct Radiolabelling method of three-(2-carboxyethyl) phosphine (TCEP); The people such as Seitz, Eur.J.Nucl.Med., 26 (10): 1265-1273, in October, 1999), IDEC-Y2B8 (⁹⁰Y ibritumomab tiuxetan; Covalent bond chelating radio isotope⁹⁰The mouse IgG of the MX-DTPA of yttrium (tiuxetan)₁The κ anti-CD-20 monoclonal antibody; Witzig, J. Clin.Oncol., 17 (12): 3793-3803, in December, 1999),²¹³Bi-HuM195 (anti-CD 33; The people such as Sgouros, J.Nucl.Med., 40 (11): 1935-1946, in December, 1999),⁶⁷Copper-2-imido sulfane-6-[p-(acetyl bromide is amino) benzyl]-TETA-Lym-1; The people such as O ' Donnel, Clin.Cancer Res., 5 (10 supplementary issue): 3330s-3336s, in October, 1999),⁹⁰Y-BC-4 (the mouse monoclonal antibody of identification tenascin; Can use the p-isothiocyanic acid benzyl derivative (ITC-Bz-DTPA) of chelating agent two inferior second triamido pentaacetic acids to prepare stable⁹⁰The monoclonal antibody conjugate of Y mark; The people such as Riva, Clin.Cancer Res., 5 (10 supplementary issue): 3275s-3280s, in October, 1999),¹³¹The cG250 chimeric mAb G250 (cG250) of the I mark (people such as Steffens, Clin.Cancer Res., 5 (10 supplementary issue): 3268s-3274s, in October, 1999), the anticancer embryonal antigen of bispecific/anti-two inferior second triamido pentaacetic acid (DTPA) antibody and¹³¹Other target of I Di-DTPA haptens (people such as Vuillez, Clin.Cancer Res., 5 (10 supplementary issue): 3259s-3267s, in October, 1999) and radioimmunotherapy comprises HLA-DR, CD19 and CD22.

Method that be used for to analyze the protective effect of RIT process present composition normal tissue is well known in the art, and comprises using seeking bone and thereby irradiation marrow composition⁸⁹The strontium infusion is studied the body inner model of bone marrow injury. Following survey article also provides the guide that uses antibody, radiotherapy and immune radiotherapy: " Physical and biological targeting of radiotherapy " (physics of radiotherapy and biological targets to), Acta Oncol., 38 (supplementary issues 13): 75-83,1999; " Radioimmunodiagnosis and therapy " (radioimmunodiagnosis and treatment), Cancer Treat.Rev, 26 (1): 3-10, in February, 2000; " Antibodies in the therapy of colon caneer " (antibody in the treatment of colon cancer), Semin.Oncol., 26 (6): 683-690, in December, 1999; " Overview of the clinical development of rituximab:first monoclonal antibody anpproved for the treatment of lymphoma " (review of the clinical development of rituximab: approval is used for the treatment of lymphadenomatous the first monoclonal antibody), Semin.Oncol., 26 (5 supplementary issue 14): 66-73, in October, 1999; " Radiolabeled antibody therapy of B-cell lymphomas " (the radiolabelled antibody therapy of B cell lymphoma), Semin.Oncol., 26 (5 supplementary issues 1): in October, 58-65,1999; " Strategies for developing effective radioimmunotherapy for solid tumors " (being used for the strategy that exploitation is used for effective radioimmunotherapy of solid tumor), Clin.Cancer Res., 5 (10 supplementary issue 1): 3219s-3223s, in October, 1999; The technological progress of the radiotherapy of " Technical advances in radiotherapy of head and neck tumors " head and tumor colli), Hematol. Oncol.Clin.North Am., 13 (4): 811-823, in August, 1999; " Use of monoclonal antibodies for the diagnosis and treatment of bladder cancer " (monoclonal antibody is for the diagnosis and treatment of the purposes of carcinoma of urinary bladder), Hybridoma, 18 (3): 219-224, in January, 1999.

In another embodiment, the invention provides the method that the composition that will comprise polypeptide of the present invention (as comprise the MPIF polypeptide of having united heterologous polypeptide, heterologous nucleic acids, toxin or prodrug or the composition of anti-MPIF antibody) is delivered to target cell (such as B or T cell, monocyte, macrophage and the neutrophil cell of expressing MPIF). MPIF polypeptide of the present invention or anti-MPIF antibody can be through hydrophobic, hydrophilic, ion and/or covalent interactions and are united heterologous polypeptide, heterologous nucleic acids, toxin or prodrug.

In one embodiment, the invention provides the method that present composition specificity is delivered to cell by using the polypeptide of the present invention (such as MPIF polypeptide or anti-MPIF antibody) of having united heterologous polypeptide or nucleic acid. In one embodiment, the invention provides for the method that therapeutic protein is delivered to target cell. In another embodiment, the invention provides for single-chain nucleic acid (such as antisense or ribozyme) or double-strandednucleic acid (as being incorporated into cellular genome or copying and transcribed DNA as episome) are delivered to the method in the target cell.

In another embodiment, the invention provides the method that by using the polypeptide of the present invention (such as MPIF polypeptide or anti-MPIF antibody) of having united toxin or cytotoxic drug precursor specificity is destroyed cell.

For example, can use and put together radioisotopic MPIF and destroy the leukaemia, treat thus leukaemia.

In a specific embodiment, the invention provides the method that by using the MPIF polypeptide of having united toxin or cytotoxic drug precursor specificity is destroyed T or the cytophyletic cell of B leukaemia or the lymthoma of T or B cell (as relate to).

In another specific embodiment, the invention provides the method that by using the anti-MPIF antibody of having united toxin or cytotoxic drug precursor specificity is destroyed the cell (such as monocytic leukemia or lymthoma) of monocyte pedigree.

In another embodiment, the invention provides by using the polypeptide of the present invention (such as MPIF polypeptide or anti-MPIF antibody) of having united toxin or cytotoxic drug precursor specificity and destroy the cell method of (as destroying the cell amboceptor of inflammation). The cell amboceptor of inflammation comprises for example T cell, monocyte, dendritic cells, astroglia, messangial cell, macrophage, neutrophil cell and relates to the cell of graft rejection (acute or chronic).

In another embodiment, use the non-MPIF molecule of having united toxin or cytotoxic drug precursor with MPIF-1, so that MPIF-1 can prevent or treat normal cell, tissue or organ damage, perhaps protect myeloid progenitor.

" toxin " refer to can in conjunction with and activate the compound of endogenous cell poisonous effect system system, comprise catalytic subunit, other cytotoxic agent of radio isotope, holotoxin, modification toxin, toxin or do not exist in normal cell or on its surface and at any molecule or the enzyme of determining to cause under the condition cell death. Can include but not limited to the radio isotope that this area is known according to the toxin that method of the present invention is used, compound is such as can be in conjunction with antibody (or it comprises complementary fixing part), thymidine kinase, endonuclease, RNA enzyme, alpha toxin, ricin (WA), abrin, ETA, diphtheria toxin, saporin, momordin, gelonin, PAP, α-sarcin and the cholera toxin of the endogenous cell poisonous effect system inherence or that induce system. " toxin " also comprise cytotoxicity, suppress cell or cytocidal reagent, therapeutic agent or radioactive metal ion, such as alpha emitter, such as²¹³Bi or other radio isotope, such as¹⁰³Pd、 ¹³³Xe、 ¹³¹I、 ⁶⁸Ge、 ⁵⁷Co、 ⁶⁵Zn、 ⁸⁵Sr、 ³²P、 ³⁵S、 ⁹⁰Y、 ¹⁵³Sm、 ¹⁵³Gd、 ¹⁶⁹Yb、 ⁵¹Cr、 ⁵⁴Mn、 ⁷⁵Se、 ¹¹³Sn、 ⁹⁰Yttrium,¹¹⁷Tin,¹⁸⁶Rhenium,¹⁶⁶Holmium and¹⁸⁸Rhenium; Luminous marker is such as luminol; And fluorescent marker, such as fluorescein and rhodamine, and biotin.

Can use the technology of knowing this area and come mark antibody of the present invention. These technology include but not limited to that difunctional use of puting together agent (consults for example U.S. Patent number 5,756,065; 5,714,631; 5,696,239; 5,652,361; 5,505,931; 5,489,425; 5,435,990; 5,428,139; 5,342,604; 5,274,119; 4,994,560; With 5,808,003; Complete being collected herein by reference). Cytotoxin or cytotoxic agent comprise any reagent harmful to cell. Example comprises taxol, Cytochalasin B, Gramicidin D, ethidium bromide, emetine, mitomycin, Etoposide, tenoposide, vincristin, vinblastine, colchicine, adriamycin, daunorubicin, dihydroxy anthracin diketone, mitoxantrone, mithramycin, actinomycin D, 1-dehydrogenation testosterone, glucocorticoid, procaine, totokaine, lidocaine, Propranolol and puromycin and analog or homologue. Therapeutic agent includes but not limited to antimetabolite (such as methotrexate, Ismipur, 6-thioguanine, cytarabine, 5 FU 5 fluorouracil decarbazene), alkylating agent (such as mechlorethamine, thiophene for sending (DDP) cis-platinum of Chlorambucil, melphalan, BCNU (BSNU) and lomustine (CCNU), endoxan, busulfan, dibromannitol, streptozotocin, mitomycin C, suitable-dichloro diamines platinum (II)), anthracycline (such as daunorubicin (being called in the past daunomycin) and adriamycin), antibiotic (such as D actinomycin D, bleomycin, mithramycin, Anthramycin (AMC)) and antimitotic agent (such as vincristin and vinblastine).

" prodrug of toxin cell " refers to be become by the enzymic transformation that usually exists the non-toxic compound of cytotoxic compound in cell. Can include but not limited to according to the cytotoxic drug precursor that method of the present invention is used the phenoxy-acetamide derivative of phosphoric acid derivatives, cytarabine, daunorubicin and daunorubicin of glutamyl derivative, Etoposide or the mitomycin C of benzoic acid mustard seed alkylating agent.

Other preferred embodiment of the present invention includes but not limited to MPIF polypeptide, MPIF polynucleotides and function activator thereof the purposes in using hereinafter.

In a specific embodiment, use MPIF polypeptide of the present invention or polynucleotides or its activator or antagonist (such as anti-MPIF antibody) with treatment, prevention, detect and/or diagnosing chronic myelogenous leukemia, acute myeloid leukaemia, leukaemia, histiocytic leukemia, monocytic leukemia (such as acute monocytic leukemia), leukaemia eeticulosis, Shilling type monocytic leukemia and/or by the cell of monocyte and/or monokaryon and/or other leukaemia of tissue derived.

Thereby, use MPIF polypeptide of the present invention or polynucleotides or its activator or antagonist (such as anti-MPIF antibody) with treatment, prevention, detection and/or diagnosis leukaemia, such as acute lymphocytic (lymphatic) leukaemia (ALL), it can comprise and not break up B cellular type, common B cellular type, pre B cell type and B cellular type; Acute myeloid (marrow or bone marrow cell) leukaemia (AML), it can comprise the bone marrow cell type that is not divided into, be divided into bone marrow cell type, progranulocyte type (APL), become myelomonocyte type, monoblast type, erythroleukemia type and megakaryoblast type; Chronic lymphocytic (lymph) leukaemia (CLL), it can comprise B cellular type, T cellular type, prolymphocyte type, S é zary syndrome (the leukaemia stage of CTCL), pilocytic and lymphoma leukemia (leukaemia of namely seeing in advanced stage in malignant lymphoma changes); With chronic bone marrow cell (marrow, marrow or granulocytic) leukaemia (CML), for example wherein tumour clone is red blood cell, megacaryocyte, monocyte, T cell or B cell.

In another specific embodiment, use MPIF polypeptide of the present invention or polynucleotides or its activator or antagonist with treatment, prevention, diagnosis and/or improve monocytic leukemia sample reaction, as what in tuberculosis for example, see.

In another specific embodiment, use MPIF polypeptide of the present invention or polynucleotides or its activator or antagonist with treatment, prevention, diagnosis and/or improve monocarpotic cellularity leukocytosis, monocarpotic cellularity leukopenia, monocytopenia and/or monocytosis,mononucleosis.

In another specific embodiment, use MPIF polypeptide of the present invention or polynucleotides or its activator or antagonist with treatment, prevention, diagnosis and/or improve graft to versus-host disease or graft rejection.

In another specific embodiment, use MPIF polypeptide of the present invention or polynucleotides or its activator or antagonist with treatment, prevention, diagnosis and/or improve anaemia.

In another specific embodiment, use MPIF polypeptide of the present invention or polynucleotides or its activator or antagonist with treatment, prevention, diagnosis and/or improve B cell malignant tumor, transform sick such as ALL, Hodgkins disease, non-Hodgkins lymthoma, chronic lymphocytic leukemia, plasmacytoma, Huppert's disease, Burkitt lymphomas and EBV.

In another embodiment, use MPIF polypeptide of the present invention or polynucleotides or its activator or antagonist with treatment, prevention and/or diagnosis of fibrosis and the situation relevant with fibrillatable, such as, but not limited to fibrillatable and SymmersShi clay main line fiber type under fibrillatable, main line fiber type, replacement fibrosis, the adventitia around fibrillatable, the muscle around fibrillatable, the center under lung fibrosis, cystic fibrosis (comprising such as cystic fibrosis of the pancreas, Clarke-Hadfield syndrome, pancreas fibrocystic disease, mucoviscidosis and viscidosis), myocardium internal membrane of heart fibrillatable, idiopathic retroperitoneal fibrosis, pia mater fibrillatable, fibrosis of mediastinum, the nodositas epidermis.

In highly preferred embodiment, use MPIF polypeptide of the present invention or polynucleotides or its activator or antagonist with treatment, prevention and/or diagnosis catarrh, especially relevant with chemotherapy or radiotherapy catarrh.

MPIF polypeptide of the present invention or polynucleotides and/or its activator and/or antagonist also can be used for treatment, prevention and/or diagnose organ rejection or graft to versus-host disease (GVHD) and/or the situation relevant with it. The organ rejection destroys transplanted tissue by the host immune cell by immune response and occurs. Similar, also relate to immune response among the GVHD, but in this case, the immune cell destruction host cell of external transplanting. But using Immunosuppression, to reply the MPIF polynucleotides of the present invention of (the particularly propagation of T cell, monocyte or other mediator of inflammation, differentiation or chemotaxis) or polypeptide and/or its activator and/or antagonist may be effective therapy of prevention organ rejection or GVHD. In addition, proved in the mouse and can cause in conjunction with the disappearance of the CCR1 acceptor of MPIF-1 the survival of allograft to prolong the (people such as Gao, " Targeting of the chemokine receptor CCR1 suppresses development of acute and chronic cardiac allograft rejection " (target of chemokine receptors CCR1 is contained the development of acute and chronic allograft rejection), J.Clin.Invest., 105 (1): 35-44, in January, 2000). Thereby, will be useful by for example interaction of MPIF-1 antagonist blocking-up MPIF-1 or other CCR1 part and its CCR1 acceptor for prophylaxis of acute and chronic transplanting rejection.

Similar, MPIF polypeptide of the present invention or polynucleotides and/or its activator and/or antagonist also can be used for regulating and control inflammation. For example, but propagation and the differentiation of the cell that MPIF polynucleotides of the present invention or polypeptide and/or its activator and/or antagonist inflammation-inhibiting relate in replying. These molecules can be used for treatment, prevention and/or diagnosis inflammatory conditions, chronic and acute situation all can, comprise that chronic prostatitis, granulomatous prostatitis and malakoplakia, the pulmonary lesion, inflammatory bowel disease, the CrohnShi that induce with the infection (replying syndrome (SIRS) such as septic shock, pyemia or systemic inflammatory) of inflammation-related, ischemic-weight perfusion injury, endotoxin mortality, arthritis, the hyperacute rejection by complement-mediated, ephritis, by cell factor or chemotactic factor (CF) are sick or by the excessive disease that causes of production of cell factor (such as TNF or IL-1).

In a specific embodiment, anti-MPIF antibody of the present invention is used for the treatment of, prevents, regulates and control, detects and/or diagnose inflammation.

In a specific embodiment, anti-MPIF antibody of the present invention is used for the treatment of, prevents, regulates and control, detects and/or diagnose inflammatory disorderly.

In a preferred embodiment, with composition of the present invention and following co-administered: the CD40L of CD40L (CD40L), soluble form is (such as AVREND^TM), biological active fragment, variant, derivative, anti-CD40L antibodies (such as activator or antagonist antibody) and/or the anti-CD 40 antibodies (such as activator or antagonist antibody) of CD40L.

This area will recognize, the conjugate that is applied to the MPIF polypeptide and chelate that the conjugate of above describing with relevant antibody herein and chelate can be equal to. Immunophenotyping

Antibody of the present invention can be used for the immunophenotyping of clone and biological sample. The translation thing of gene of the present invention can be used as cell specific marker or more specifically as the cell marking at the differentiation of particular cell types and/or ripe various stage differential expressions. Monoclonal antibody for specificity epitope or epi-position combination can be used to screen the cell mass of expressing this mark. Can utilize multiple technologies to use the cell mass of monoclonal antibody screening presentation markup, comprise the magnetic separating that uses the antibody sandwich magnetic bead, (consult for example U.S. Patent number 5 with " elutriation " and the flow cytometry of the antibody that is attached to solid matrix (i.e. flat board), 985,660; With the people such as Morrison, Cell, 96:737-49,1999).

These technology can be screened specific cell mass, such as " non-own " cell that can be found in hematology malignant tumor (being the remaining disease (MRD) of Minimum Residual in the acute leukemic patient) and the transplanting, to prevent graft to versus-host disease (GVHD). Perhaps, these technology can be used to screen candidate stem cell and the CFU-GM that can experience propagation and/or differentiation, such as the cell in the human cord blood. The antibody binding assay

Can measure by any method that this area is known the immunologic opsonin combination of antibody of the present invention. Spendable immunoassay includes but not limited to competitive and noncompetitive is measured system, utilizes technology such as Western blotting, radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), " sandwich " immunoassay, immunoprecipitation assay, precipitin reaction, GDP reaction, immune diffusion measurement method, aggreation determination method, complement fixation determination method, immunoradiometric assay method, fluorescence immunoassay, albumin A immunoassay. These determination methods are conventional and well-knownly (to consult " Current Protocols in Molecular Biology " (the molecular biology general scheme) compiled such as people such as Ausubel in this area, the 1st volume, John Wiley ﹠ Sons company, New York, 1994, complete being collected herein by reference). Hereinafter sketched exemplary immunoassay (but being not limited to this).

The immunoprecipitation scheme generally includes: cell lysis group in such as lysis buffers such as the RIPA buffer solutions that adds protein phosphatase and/or protease inhibitors (such as EDTA, PMSF, AKOLINE, sodium vanadate) (1%NP-40 or Triton X-100,1% NaTDC, 0.1%SDS, 0.15M NaCl, 0.01M crack acid sodium pH7.2,1%Trasylol), in cell lysate, add purpose antibody, in 4 ℃ of insulation a period of times (such as 1-4 hour), in cell lysate, add albumin A and/or Protein G sepharose 4B, in 4 ℃ of insulations about 1 hour or more of a specified duration, in lysis buffer, clean pearl, and pearl is resuspended in the SDS/ sample loading buffer. Can come by for example western blot analysis the ability of purpose of appraisals antibody mediated immunity precipitation specific antigen. It will be appreciated by those skilled in the art that modifiable parameter with the combination of increase antibody synantigen and reduce background (as using sepharose 4B precleaning cell lysate). Consult " Current Protocols in Molecular Biology " (the molecular biology general scheme) compiled such as people such as Ausubel about the further discussion of immunoprecipitation scheme, the 1st volume, John Wiley ﹠ Sons company, New York, 1994,10.16.1.

Western blot analysis generally includes: the preparation protein example, with protein example in polyacrylamide gel electrophoresis (such as 8%-20%SDS-PAGE, molecular weight according to antigen), protein example is transferred to film (such as celluloid, PVDF or nylon) from polyacrylamide gel, closing membrane in the confining liquid PBS of 3%BSA or defatted milk (as contain), in cleaning buffer solution (such as the PBS-polysorbas20), clean film, being used in the primary antibodie (purpose antibody) of diluting in the sealing buffer solution contacts/detect/insulation film, in cleaning buffer solution, clean film, be used in that sealing dilutes in the buffer solution puted together enzymatic substrate (such as horseradish peroxidase or alkaline phosphatase) or Geigers (as³²P or¹²⁵I) two anti-(it identifies primary antibodie, such as anti-human antibody) contact/detect/insulation film, clean film in cleaning buffer solution, and detect whether there is antigen. It will be appreciated by those skilled in the art that and to change parameter to increase detection signal and to reduce background noise. Consult " Current Protocols in Molecular Biology " (the molecular biology general scheme) compiled such as people such as Ausubel about the further discussion of Western blotting scheme, the 1st volume, John Wiley ﹠ Sons company, New York, 1994,10.8.1.

ELISA comprises: preparation antigen, hole with antigen coated 96 hole microtiter plates, Xiang Kongzhong adds the purpose antibody of having puted together detectable compounds such as enzymatic substrate (such as horseradish peroxidase or alkaline phosphatase), insulation a period of time, and detect whether there is antigen. In ELISA, purpose antibody needn't be puted together detectable compounds; On the contrary, but Xiang Kongzhong adds two anti-(its identifying purpose antibody) puted together detectable compounds. In addition, can not be to use antigen coated hole, but with antibody sandwich to the hole. In this case, in the hole after coated, add purpose antigen after, can add and put together the two anti-of detectable compounds. It will be appreciated by those skilled in the art that to change parameter with the increase detection signal, and other ELISA that this area is known makes a variation. Consult " Current Protocols in Molecular Biology " (the molecular biology general scheme) compiled such as people such as Ausubel, the 1st volume, John Wiley Sons company, New York, 1994,11.2.1 about the further discussion of ELISA.

Can measure the binding affinity of antibody and antigen and the dissociation rate of antibody-AI by the competitive binding assay method. An example of competitive binding assay method is radioimmunoassay, comprising: in the situation of the unlabelled antigen that has cumulative amount, will through labelled antigen (as³H or¹²⁵I) be incubated with purpose antibody, and detect the antibody that is attached on labelled antigen. Can draw to analyze by the data obtained by Scatchard and measure purpose antibody for the affinity of specific antigen with in conjunction with dissociation rate. Also can measure and two anti-competitions with radioimmunoassay. In this case, in the unmarked two anti-situations that have cumulative amount, with antigen with puted together labeled compound (as³H or¹²⁵I) purpose antibody is incubated together. Treatment-antibody

The invention still further relates to the therapy based on antibody, comprise antibody of the present invention is applied to animal (preferred mammal, optimum is chosen) patient, to treat one or more disclosed diseases, disorder or situation. Therapeutic compound of the present invention includes but not limited to the nucleic acid (comprising fragment disclosed herein, analog and derivative and anti-idiotype) of antibody of the present invention (comprising fragment disclosed herein, analog and derivative) and code book invention antibody. Antibody of the present invention can be used for treating, suppress or unconventionality expression and/or active relevant disease, disorder and the situation of prevention and polypeptide of the present invention, includes but not limited to any or various diseases, disorder and situation as herein described. Include but not limited to alleviate the symptom relevant with these diseases, disorder or situation with unconventionality expression and/or active relevant the treating and/or preventing of disease, disorder and situation of polypeptide of the present invention. Pharmaceutics that can know in this area or as herein described can accept to provide in the composition antibody of the present invention.

The approach that the therapeutic of antibody of the present invention is used be included in generally part in the body or general in conjunction with polynucleotides of the present invention or polypeptide or the direct cytotoxicity (as by complement (CDC) or effector cell (ADCC) mediation) by antibody. In these methods some hereinafter have been described in detail in detail. Lattice is according to instruction provided herein, and those skilled in the art need not too much experiment and namely know how antibody of the present invention is used for diagnosis, monitoring or therapeutic purposes.

Antibody of the present invention can be favourable other monoclonal of associating or chimeric antibody or lymphokine or hemopoieticgrowth factor (such as IL-2, IL-3, IL-7) use, for example for increasing with the interactional effector cell's of antibody number or activity.

Antibody of the present invention can be used separately or unite the treatment (such as radiotherapy, chemotherapy, hormonotherapy, immunotherapy and antitumor agent) of other type and be used. Usually, preferably use the product of the source of species identical with patient's species or species reactive (for antibody). Thereby, in a preferred embodiment, in order to treat or to prevent and people's antibody, fragment, derivative, analog or nucleic acid are applied to people patient.

For immunoassay and the therapy for the disorder relevant with polynucleotides of the present invention or polypeptide (comprising its fragment), preferably use high-affinity and/or the interior inhibition of strong body and/or the neutrality antibody in polypeptide of the present invention or polynucleotides or its fragment or zone. These antibody, fragment or zone preferably have the affinity for polynucleotides of the present invention or polypeptide (comprising its fragment). Preferred binding affinity comprises that those dissociation constants or Kd are lower than 5 * 10^-2M、10 ^-2M、5×10 ^-3M、 10 ^-3M、5×10 ^-4M、10 ^-4M、5×10 ^-5M、10 ^-5M、5×10 ^-6M、10 ^-6M、5×10 ^-7M、10 ^-7M、 5×10 ^-8M、10 ^-8M、5×10 ^-9M、10 ^-9M、5×10 ^-10M、10 ^-10M、5×10 ^-11M、10 ^-11M、5×10 ^-12M、 10 ^-12M、5×10 ^-13M、10 ^-13M、5×10 ^-14M、10 ^-14M、5×10 ^-15M or 10^-15The affinity of M. The antibody gene therapy

In a specific embodiment, by the mode of gene therapy, use the nucleic acid of the coded sequence that comprises antibody or its functional derivatives, with treatment, inhibition or prevention unconventionality expression and/or active relevant disease or the disorder with polypeptide of the present invention. Gene therapy refers to treat by the experimenter being used nucleic acid expression or effable. In this embodiment of the present invention, nucleic acid generates the protein by they codings, and the latter can mediate result for the treatment of.

This area can all can be according to the present invention for any gene therapy method that utilizes. Exemplary methods has hereinafter been described.

The general summary of gene therapy is consulted the people such as Goldspiel, Clinical Pharmacy, 12:488-505,1993; Wu and Wu, Biotherapy, 3:87-95,1991; Tolstoshev, Ann.Rev.Pharmacol.Toxicol., 32:573-596,1993; Mulligan, Science, 260:926-932,1993; Morgan and Anderson, Ann. Rev.Biochem., 62:191-217,1993; And May, TIBTECH, 11 (5): 155-215,1993. The method of the spendable recombinant DNA technology that this area is generally known is described in " Current Protocols in Molecular Biology " (the molecular biology general scheme) that the people such as Ausubel compiles, John Wiley ﹠ Sons company, New York, 1993; And Kriegler, " Gene Transfer and Expression, A Laboratory Manual " (transgenosis and expression, lab guide), Stockton publishing house, New York, 1990.

One preferred aspect, compound comprises the nucleotide sequence of encoding antibody, described nucleotide sequence is the part of expression vector, this expression vector is expressed antibody or its fragment or chimeric protein or heavy chain or light chain in suitable host. Particularly, in these nucleotide sequences, promoter and antibody coding region are operatively connected, but described promoter can be induction type or composing type, optional tissue is specific. In another specific embodiment, use such nucleic acid molecules, wherein antibody coding sequence and any other expectation sequence flank are the zones that can promote the homologous recombination in expectation site in the genome, intrachromosomal expression (Koller and the Smithies of antibody encoding nucleic acid are provided thus, Proc.Natl.Acad.Sci.USA, 86:8932-8935,1989; The people such as Zijlstra, Nature, 342:435-438,1989). In specific embodiment, the antibody molecule of expression is single-chain antibody; Perhaps, this nucleotide sequence comprises the heavy chain of encoding antibody and the sequence of light chain or its fragment.

Nucleic acid directly or indirectly can be delivered to the patient, in the situation of directly delivering, the patient directly is exposed to nucleic acid or carries the carrier of nucleic acid, in the situation of indirectly delivering, at first at external use nucleic acid transformant, then transplants cells in the patient body. These two kinds of methods are called in the body or stripped gene therapy.

In a specific embodiment, direct administration of nucleic acid sequence in vivo, thus express in vivo to generate coded product. This can realize by the several different methods that any this area is known, for example by they being configured to the part of suitable nucleic acid expression vector, and following using: for example (consult U.S. Patent number numbers 4 by familiar lacunas type or attenuation type retrovirus or other viral vectors, 980,286) infection, or the direct injection by naked DNA, or the use by microparticle bombardment is (such as particle gun; Biolistic, Dupont), or coated with lipid or cell surface receptor or transfection agents, be wrapped in liposome, particulate or the micro-capsule, or by being connected with the known peptide that enters nuclear, by with can carry out part by receptor-mediated endocytosis and be connected and (consult for example Wu and Wu, J.Biol.Chem., 262:4429-4432,1987) (this can be used for the cell type of targeting specific expressed receptor) etc., make them become type in the born of the same parents. In another embodiment, can form nucleic acid-part complex, wherein part comprises the fusion viral peptide that destroys endosome, makes nucleic acid avoid lysosomal degraded. In also having an embodiment, by the targeting specific acceptor, targeted cells specificity in the nucleic acid body can be taken in and expressed and (consult for example PCT publication number WO 92/06180; WO 92/22635; WO 92/20316; WO 93/14188; WO 93/20221). Perhaps, nucleic acid can be imported in the born of the same parents, and mix host cell DNA by homologous recombination and express (Koller and Smithies, Proc.Natl.Acad. Sci.USA, 86:8932-8935,1989; The people such as Zijlstra, Natue, 342:435-438,1989).

In a specific embodiment, used the viral vectors of the nucleotide sequence that comprises code book invention antibody. For example, can use retroviral vector (consulting the people such as Miller, Meth. Enzymol., 217:581-599,1993). These retroviral vectors comprise virus genomic correct packing and are incorporated into necessary composition in the host cell DNA. To be cloned into one or more carriers for the nucleotide sequence of the encoding antibody of gene therapy, described carrier is convenient to gene delivery to the patient. More details about retroviral vector are consulted the people such as Boesen, Biotherapy, 6:291-302,1994, it described the use retroviral vector with the mdr1 gene delivery to candidate stem cell, make stem cell more can tolerate chemotherapy. Other document of setting forth the purposes of retroviral vector in gene therapy is: the people such as Clowes, J.Clin. Invest., 93:644-651,1994; The people such as Kiem, Blood, 83:1467-1473,1994; Salmons and Gunzberg, Human Gene Therapy, 4:129-141,1993; With Grossman and Wilson, Curr.Opin.in Genetics and Devel., 3:110-114,1993.

Adenovirus is the another kind of viral vectors that can be used for gene therapy. Adenovirus is for being attractive especially carrier with gene delivery to airway epithelial. Adenovirus natural infection airway epithelial causes slight disease at there. Other target based on the delivery system of adenovirus is liver, central nervous system, endothelial cell and muscle. Adenovirus has the advantage that can infect Unseparated Cell. Kozarsky and Wilson, Curr.Opin.in Genetics and Devel., 3:499-503,1993 provide the summary of relevant gene therapy based on adenovirus. The people such as Bout, Human Gene Therapy, 5:3-10,1994 have set forth with adenovirus vector the airway epithelial of transgenosis to rhesus macaque. Adenovirus is used for other example of gene therapy and consults the people such as Rosenfeld, Science, 252:431-434,1991; The people such as Rosenfeld, Cell, 68:143-155,1992; The people such as Masrangeli, J.Clin.Invest., 91:225-234,1993; PCT publication number WO 94/12649; With the people such as Wang, Gene Therapy, 2:775-783,1995. In a preferred embodiment, use adenovirus vector.

Adeno-associated virus (AAV) also is proposed to be used in gene therapy (people such as Walsh, Proc.Soc. Exp.Biol.Med., 204:289-300,1993; U.S. Patent number 5,436,146).

The another kind of method of gene therapy relates to by such as electroporation, fat transfection, by the transfection of calcium phosphate mediation, or the cell of the method such as virus infections during transgenosis to tissue is cultivated. Transfer method generally includes selected marker is transferred to cell. Then cell is placed under the selection pressure, absorbed and expressed the cell that is transferred gene to separate those. Then these cells are delivered to the patient.

In this embodiment, first with the nucleic acid transfered cell, then the recombinant cell that obtains is carried out using in the body. This importing can be undertaken by any method that this area is known, include but not limited to transfection, electroporation, microinjection, with the virus or the phage vector that comprise nucleotide sequence infect, Fusion of Cells, by the transgenosis of Chromosome-encoded, by the transgenosis of Microcell-mediated, protoplast fusion etc. This area is known for many technology of foreign gene transfered cell (are consulted for example Loeffler and Behr, Meth.Enzymol., 217:599-618,1993; The people such as Cohen, Meth.Enzymol., 217:618-644,1993; Cline, Pharmac.Ther., 29:69-92m, 1985, and can use according to the present invention, only otherwise destroying essential growth and the physiologic function of recipient cell gets final product. This technology should be transferred to cell with nucleic acid stability, so that the cell expressible nucleic acid, and preferred, can be by cell filial generation Inheritance and expression.

Can the recombinant cell that obtain be delivered to the patient by the several different methods that this area is known. The preferred intravenous of restructuring haemocyte (such as candidate stem cell or CFU-GM) is used. Estimate that the cell concentration that uses depends on the effect of expectation, patient's situation etc., and can be determined by those skilled in the art.

The cell that can import nucleic acid for the purpose of gene therapy is contained cell type any expectation, obtainable, includes but not limited to epithelial cell, endothelial cell, horn cell, fibroblast, muscle cell, liver cell; Haemocyte is such as T lymphocyte, bone-marrow-derived lymphocyte, monocyte, macrophage, neutrophil cell, eosinophil, megacaryocyte, granulocyte; Multiple stem cell or CFU-GM are particularly as deriving from candidate stem cell or the CFU-GM of marrow, Cord blood, peripheral blood, tire liver etc.

In a preferred embodiment, the cell for gene therapy is that the patient is from body.

In the embodiment of recombinant cell for gene therapy, with the nucleotide sequence transfered cell of encoding antibody, make cell or its filial generation can express them, then recombinant cell is carried out using in the body, with the performance result for the treatment of. In a specific embodiment, stem cell or CFU-GM have been used. Can in-vitro separation and any stem cell of keeping and/or CFU-GM all can be potential use according to this embodiment of the present invention and (consult for example PCT publication number WO 94/08589; Stemple and Anderson, Cell, 71:973-985,1992; Rheinwald, Meth. Cell Bio., 21A:229,1980; With Pittelkow and Scott, Mayo Clinic Proc., 61:771,1986).

In a specific embodiment, but the nucleic acid that imports for the purpose of gene therapy comprises the inducible promoter that is operatively connected with the code area, so that can whether control by controlling suitable existence of transcribing derivant the expression of nucleic acid.

Confirm treatment or prophylactic activity

Preferably before compound of the present invention or pharmaceutical composition are used for the people, treatment or the prophylactic activity of expectation are carried out testing in vitro, then carry out the body build-in test. For example, be used for the treatment of proof compound or pharmaceutical composition or the external test method of prevention effectiveness and comprise that compound is to the effect of clone or patient tissue sample. Can measure compound or composition to the effect of clone and/or tissue samples with the technology that those skilled in the art will know that, include but not limited to that rosette forms determination method and lysis determination method. According to the present invention, can be used for determining whether that the external test method that can use specific compound comprises cell in vitro cultivation determination method, wherein cultivate the patient tissue sample and be exposed to or by the alternate manner administered compound, and observe this compound to the effect of tissue samples. Therapeutic/preventative using and composition-antibody

The invention provides the method that the compounds of this invention by the experimenter being used effective dose or pharmaceutical composition (preferred antibody of the present invention) are treated, suppressed and prevent. One preferred aspect, compound is basically pure (as being substantially free of the material that limits its effect or produce unexpected side effect). The preferred animal of experimenter includes but not limited to ox, pig, horse, chicken, cat, dog etc., preferred mammal, and optimum is chosen.

Spendable prescription and application process are as mentioned above when compound comprises nucleic acid or immunoglobulin (Ig); Other suitable prescription and use the path and can be selected from hereinafter described. Known have a multiple delivery system, and can be used for using compound of the present invention, as be wrapped in liposome, particulate or the micro-capsule, can express compound recombinant cell, (consult for example Wu and Wu by receptor-mediated endocytosis, J.Biol.Chem., 262:4429-4432,1987), with nucleic acid construct be the part etc. of retrovirus or other carrier. Introduction method includes but not limited in the corium, in the muscle, in the peritonaeum, in the intravenous, subcutaneous, nose, dura mater is outer and oral path. Can come administered compound or composition by any convenient path, for example inject, absorb by epithelium or mucocutaneous layer (such as oral mucosa, rectum and intestinal mucosa) by infusion or bolus, and can use with other BA agent. Use can be whole body or local. In addition, may wish by any suitable path pharmaceutics compound of the present invention or composition to be imported central nervous system, comprise in the ventricles of the brain and intrathecal injection; Can be convenient to intraventricular injection by the intraventricular catheter that for example is attached to storage (such as the Ommaya storage). Also can be by for example carrying out pulmonary administration with inhalator or sprayer and the preparation that contains aerosol.

In a specific embodiment, may wish pharmaceutics compound of the present invention or composition are locally applied to the zone that needs are treated; This can by the local infusion in (but not being limited to) surgical procedures for example, topical application (such as the wound dressing after the combined surgery), by injection, by conduit, by suppository or by implant (as described in implant be porous, non-porous or gel-like material, comprise film, such as sialastic film or fiber) realize. Preferably, use protein of the present invention when (comprising antibody), must be noted that to use protein not adsorb material on it.

In another embodiment, can in carrier, deliver compound or composition, particularly liposome and (consult Langer, Science, 249:1527-1533,1990; The people such as Treat, in " Liposomes in the Therapy of Infectious Disease and Cancer " (liposome in communicable disease and the treatment of cancer) book of Lopez-Berestein and Fidler volume, Liss, New York, the 353-365 page or leaf, 1989; Lopez-Berestein, the same, the 317-327 page or leaf; Usually consult simultaneously).

In also having an embodiment, can in controlled release system, deliver compound or composition. In one embodiment, can use pump (to consult Langer, see above; Sefton, CRC Crit.Ref.Biomed.Eng, 14:201,1987; The people such as Buchwald, Surgery, 88:507,1980; The people such as Saudek, N.Engl.J.Med., 321:574,1989). In another embodiment, (consult " Medical Applications of Controlled Release " (medical application of controlled release), Langer and Wise compile can to use polymeric material, CRC publishing house, Boca Raton, Florida, 1974; " Controlled Drug Bioavailability, Drug Product Design and Performance " (bioavilability of controlled drug, the design of drug products and performance), Smolen and Ball compile, Wiley, New York, 1984; Ranger and Peppas, J.Macromol.Sci.Rev. Macromol. Chem., 23:61,1983; The people such as Levy, Science, 228:190,1985; The people such as During, Ann.Neurol., 25:351,1989; The people such as Howard, J.Neurosurg., 71:105,1989). In also having an embodiment, controlled release system can place the treatment target (being brain) near, thereby only need the sub-fraction of whole-body dose (to consult for example Goodson, " Medical Applications of Controlled Release " (medical application of controlled release), see above, the 2nd volume, 115-138 page or leaf, 1984).

Langer, Science, 249:1527-1533 has discussed other restricted delivery system in 1990 the review.

In the specific embodiments of nucleic acid of coded protein at the compounds of this invention, but administration of nucleic acid is to promote the protein expression of its coding in the body, namely by being the part of suitable nucleic acid expression vector with nucleic acid construct, and (consult U.S. Patent number 4 by for example retroviral vector, 980,286) or by direct injection or by microparticle bombardment (such as particle gun; Biolistic, Dupont) or with lipid or cell surface receptor or transfection agents is coated or (consult such as people such as Joliot by connecting the known homeobox sample peptide that enters nuclear, Proc. Natl.Acad.Sci.USA, 88:1864-1868,1991) etc. use, it is entered in the born of the same parents. Perhaps, nucleic acid can be imported in the born of the same parents, and mix host cell DNA by homologous recombination and express.

The present invention also provides pharmaceutical composition. These compositions comprise compound and the pharmaceutics for the treatment of effective dose can accept carrier. In a specific embodiment, term " pharmaceutics is acceptable " refers to through the approval of the management organization of federation or state government or lists in the animal that can be used in American Pharmacopeia or other the generally acknowledged pharmacopeia, and clearer and more definite can be used for the people. Term " carrier " refers to diluent, adjuvant, excipient or the carrier used with therapeutic agent. These pharmaceutics carriers can be sterile liquids, such as water and oil, comprise derive from oil, animal, plant or, synthetic oil, for example peanut oil, soybean oil, mineral oil, sesame wet goods. When intravenous drug administration composition, water is preferred carrier. Salting liquid and dextrose hydrate and glycerite also can be used as liquid-carrier, and be especially true for Injectable solution. Suitable pharmaceutics excipient comprises starch, glucose, lactose, sucrose, gelatin, Fructus Hordei Germinatus, rice, flour, chalk, silica gel, odium stearate, glycerol monostearate, talcum, sodium chloride, skimmed milk power, glycerine, propane diols, ethylene glycol, water, ethanol etc. If necessary, composition also can comprise a small amount of wetting agent or emulsifying agent, or the pH buffer. These compositions can be the forms of solution, suspension, emulsion, tablet, pill, capsule, pulvis, sustained release preparation etc. Composition can be mixed with suppository with traditional bond and carrier (such as triglycerides). Oral formulations can comprise standard vector, such as the sweet mellow wine of pharmaceutical grade, lactose, starch, dolomol, saccharin sodium, magnesium carbonate etc. E.W.Martin has described the example of suitable pharmaceutics carrier in " Remington ' s Pharmaceutical Sciences " (Remington's Pharmaceutical Science). These compositions will comprise the compound for the treatment of effective dose, be preferably purified form, and an amount of carrier, so that the form that is suitable for being applied to the patient to be provided. Prescription should be fit to administering mode.

In a preferred embodiment, according to old process composition is mixed with and is suitable for the pharmaceutical composition that intravenous is applied to the people. Usually, the composition of using for intravenous is the solution of sterile isotonic water-containing buffering liquid. In case of necessity, composition also can comprise solubilizer and local anesthetic (such as lidocaine) to alleviate the pain of injection site. Usually, with unit dosage form separately or mix various compositions are provided, for example provide as the freeze-dried powder in the airtight container (such as ampoule bottle or sachette) that indicates the amount of activating agent or without the form of aqueous concentrate. When using composition by infusion, the available infusion bottle of aseptic pharmaceutical grade water or salt solution that is equipped with is with its packing. When using composition by injection, ampoule bottle sterilized water or a salt solution that can be provided for injecting mixes various compositions before using.

But compound ingredients of the present invention becomes neutrality or salt form. The acceptable salt of pharmaceutics comprises the salt that those and anion form, such as those salt derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid etc., the salt that forms with those and cation is such as those salt derived from sodium, potassium, ammonium, calcium, iron hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histamine, procaine etc.

Can be determined at treatment by standard clinical techniques, suppress and the unconventionality expression of prevention and polypeptide of the present invention and/or active relevant disease or disorder in the amount of effective the compounds of this invention. In addition, can choose the dosage range that adopts the external test method to help identify the best wantonly. The exact dose that uses in preparation also will depend on uses path and disease or the disorderly order of severity, and should decide according to practitioner's judgement and each patient's situation. Can be by the dosage in external or animal model test macro source-response curve extrapolation effective dose.

As for antibody, the dosage that is applied to the patient is 0.1mg/kg weight in patients-100mg/kg weight in patients normally. Preferably, the dosage that is applied to the patient is 0.1mg/kg weight in patients-20mg/kg weight in patients, more preferably 1mg/kg weight in patients-10mg/kg weight in patients. Usually people's antibody has long half life with comparing from the antibody of other species, and this will give the credit to the immune response to allogenic polypeptide. Thereby, usually be possible than people's antibody and the less frequency of administration of low dosage. In addition, can strengthen antibody picked-up and tissue penetration (as entering brain) by modifying (such as esterified), thereby reduce application dosage and the frequency of antibody of the present invention.

The present invention also provides and has contained one or more drug packages or kits that the container of one or more compositions of pharmaceutical composition of the present invention is housed. The optional points for attention of signing and issuing with the government organs of preparation, application or the sale of management medicine or biological products with container, these points for attention reflect preparation, the application of relevant people's medication or sell the license that has obtained this mechanism. Diagnosis and imaging-antibody

But the specific binding desired polypeptides can be used for diagnostic purpose through labelled antibody and derivative thereof and analog, with detect, unconventionality expression and/or relevant disease and/or the disorder of activity of diagnosis or monitoring and polypeptide of the present invention. The invention provides the detection to the unconventionality expression of desired polypeptides, comprise that (a) measures the expression of desired polypeptides in individual cell or the body fluid with one or more to the special antibody of desired polypeptides; (b) gene expression dose and standard gene expression level are compared, measure thus the polypeptide gene expression than the increase of standard expression or reduce the indication unconventionality expression.

The invention provides for the disorderly diagnostic method of diagnosis, comprise that (a) measures the expression of desired polypeptides in individual cell or the body fluid with one or more to the special antibody of desired polypeptides; (b) gene expression dose and standard gene expression level are compared, the polypeptide gene expression of measuring thus is than increase or the reduction indication particular disorder of standard expression. For cancer, the neurological susceptibility that exists the transcript of a large amount relatively may indicate disease to form in the biopsy from individuality perhaps may be provided at the means that disease occurs detecting before the actual clinical symptom. Such more definite diagnosis can allow healthy professional person to adopt preventive measure or early stage interventional therapy, thus the formation of pre-anti-cancer or further develop.

Can use the classical immunohistology method that those skilled in the art will know that, utilize the protein level in the TPPA biological sample of the present invention (to consult such as people such as Jalkanen J. Cell.Biol., 101:976-985,1985; The people such as Jalkanen, J.Cell.Biol., 105:3087-3096,1987). Can be used for detecting other method based on antibody that protein gene expresses and comprise immunoassay, such as enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA). Suitable assay for antibodies label is known in this area, comprises the enzyme labeling thing, such as glucose oxidase; Radio isotope, such as iodine (¹²⁵I、 ¹²¹I), carbon (¹⁴C), sulphur (³⁵S), tritium (³H), indium (¹¹²In) and technetium (⁹⁹Tc); Luminous marker is such as luminol; And fluorescence labeling, such as fluorescein and rhodamine, and biotin.

One aspect of the present invention is disease or disorderly diagnosis and detection relevant with the unconventionality expression of polypeptide of the present invention in the animal (preferred mammal, optimum is chosen). In one embodiment, diagnosis comprises: (a) to the experimenter use (for example in parenteral, the subcutaneous or peritonaeum) but the specific binding desired polypeptides for the treatment of effective dose through labelled molecule; (b) after using, wait for a period of time, so that preferentially assemble (with unconjugated labelled molecule is removed to background level) at the patient position of this expression of polypeptides through labelled molecule; (c) measure background level; (d) detect among the experimenter through labelled molecule, thus, detect and be higher than background level through labelled molecule and indicate this experimenter to have specified disease or the disorder relevant with the unconventionality expression of desired polypeptides. Can measure background level by several different methods, comprise that the amount of the labelled molecule that will detect and the standard value of determining for particular system in advance compare.

This area will be understood, and experimenter's size and used imaging system will determine to generate the amount of the needed imaging moiety of diagnostic image. In the situation of radio isotope part, for people experimenter, radioactive injection volume scope is about 5-20 millicurie normally⁹⁹Tc. Antibody or antibody fragment through mark will preferentially be assembled at the cell position of containing specified protein. The in-vivo tumour imaging is described in the people such as S.W.Bruchiel, " Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments " (the immune pharmacokinetics of radiolabelled antibody and fragment thereof), in " the Tumor Imaging:The Radiochemical Detection of Cancer " of S.W.Bruchiel and B.A.Rhodes volume (tumor imaging: the radiochemistry of cancer detects) book, the 13rd chapter, Masson publishing company, 1982.

Depend on multiple variable, the type and the mode of administration that comprise used label, make after using labelled molecule preferentially be gathered in the experimenter site and with unconjugated labelled molecule remove to the time interval of background level be 6-48 hour or 6-24 hour or 6-12 hour. In another embodiment, the time interval after using is 5-20 days or 5-10 days.

In one embodiment, disease or disorderly monitoring are by repeating disease or disorderly diagnostic method carries out, and for example, diagnosing rear 1 month first, diagnose first rear 6 months, are diagnosing first rear 1 year etc. and carry out the method.

Whether the method that is used for the body interscan of using this area to know can detect the patient and exist through labelled molecule. These methods depend on the type of used label. Those skilled in the art can be identified for detecting the proper method of particular marker. The method and apparatus that can be used for diagnostic method of the present invention includes but not limited to computed tomography (CT), body scan such as positron emission tomography (position emission tomography, PET), magnetic resonance imaging and ultrasonography.

In a specific embodiment, use the labelled with radioisotope molecule, and use radiation to reply the type operating theater instruments and in the patient, detect people such as (, U.S. Patent number 5,441,050) Thurston. In another embodiment, use the fluorescent chemicals labelled molecule, use fluorescence to reply the type scanning means and in the patient, detect. In also having an embodiment, use the positron emitting metal labelled molecule, use positron emission tomography in the patient, to detect. In also having an embodiment, use the paramagnetic zond labelled molecule, use magnetic resonance imaging (MRI) in the patient, to detect. Antibody and other kit

The invention provides the kit that can be used for said method. In one embodiment, kit comprises antibody of the present invention in one or more containers, the antibody of preferred purifying. In a specific embodiment, kit of the present invention comprises basically the polypeptide that separates, described polypeptide contain with kit in contained antibody the epi-position of specific immune response can occur. Preferably, kit of the present invention also comprises not the control antibodies that reacts with desired polypeptides. In another specific embodiment, kit of the present invention comprises for detection of the means of the combination of antibody and desired polypeptides (but can put together detection substrate such as antibody, such as fluorescent chemicals, zymolyte, radioactive compound or luminophor, but the second antibody that maybe can identify the first antibody can be puted together detection substrate).

In another specific embodiments of the present invention, kit is to comprise diagnostic kit for the serum of the specific antibody of proliferative and/or carcinous polynucleotides and polypeptide for screening. This kit can comprise not the control antibodies that reacts with desired polypeptides. This kit can comprise the polypeptide antigen that basically separates, and described polypeptide antigen contains the epi-position that specific immune response can occur with at least a anti-polypeptide antigen antibody. In addition, this kit comprises for detection of the means of the combination of this antibody and antigen (can put together such as antibody can be by the fluorescent chemicals of Flow cytometry, such as fluorescein or rhodamine). In a specific embodiment, kit can comprise restructuring polypeptide antigen that generate or chemical synthesis. The polypeptide antigen of kit also can be attached on the solid support.

In a more particular embodiment, the detection means of kit mentioned above comprises that polypeptide antigen adheres to the solid support on it. This kit also can comprise the anti-human antibody that does not adhere to, use the report molecular labeling. The combination of antibody that in this embodiment, can be by described report molecular labeling detects the combination of antibody and polypeptide antigen.

In another embodiment, the present invention includes the diagnostic kit that comprises the serum of polypeptide antigen of the present invention for screening. Diagnostic kit comprise can with the antibody that basically separates of polypeptide or polynucleotides antigen generation specific immune response, with for detection of the means of the combination of polynucleotides or polypeptide antigen and antibody. In one embodiment, antibody is attached on the solid support. In a specific embodiment, antibody can be monoclonal antibody. The detection means of kit can comprise the second through the monoclonal antibody of mark. Perhaps, detection means can additionally comprise the competitive antigen through mark.

In a kind of diagnostic configuration, test serum reacts with the solid-phase reagent with surface conjunction antigen (obtaining by method of the present invention). After being bonded to reagent and removing unconjugated serum composition by cleaning with specific antigen-antibody, this reagent and the anti-human antibody of report molecular labeling are reacted, thus with solid support on the amount of anti-antigen-antibody of combination the report molecule is attached on the reagent. Cleaning reagent is unconjugated through labelled antibody to remove again, and measures the amount of the report molecule that links to each other with this reagent. Usually, the report molecule is detectable enzyme by insulation solid phase in the situation that has suitable fluorescence, luminous or colorimetric substrates (Sigma, St.Louis, MO).

Can be by prepare the solid phase surface reagent of said determination method for the known technology that protein material is attached to solid support (such as polymeric beads, test strips, 96 orifice plates or filtering material). These adherence methods generally include protein to the non-specific adsorption of holder or protein (usually the passing through free amine group) covalent attachment to the chemical active radical on the solid support (such as activated carboxyl, hydroxyl or aldehyde radical). Perhaps, can will be combined with biotinylated antigen with the coated plate of Streptavidin.

Thereby, the invention provides be used to the mensuration system or the kit that carry out this diagnostic method. Kit usually comprise with the holder of the recombinant antigen of surface conjunction and for detection of the anti-antigen-antibody of surface conjunction, with the anti-human antibody of report molecular labeling.

For the ease of understanding the following example, through describing some frequent method and/or term that occurs.

The name of " plasmid ": at first be the p of small letter and/or then be the capitalization the letter and/or digital. The initial plasmid of this paper can obtain by commercial sources, and the public can unrestrictedly obtain or can be according to disclosed flow process by obtainable plasmid construction. In addition, knowing in this area with the plasmid that plasmid described herein is equal to, is apparent to those skilled in the art.

" digestion " of DNA refers to the restriction enzyme catalyze cleavage DNA that only acts on some sequence among the DNA. Multiple restriction enzyme used herein can obtain by commercial sources, and the road uses their reaction condition, confactor and other requirement as known to persons of ordinary skill in the art. In order to analyze, usually in the buffer solution of about 20 μ l, use the enzyme of 1 μ g plasmid or dna fragmentation and about 2 units. For the DNA isolation fragment to make up plasmid, usually in larger volume with the enzymic digestion 5-50 μ g DNA of 20-250 unit. Being used for the suitable buffer of specific limited enzyme and the amount of substrate can be specified by manufacturer. Usually in about 1 hour of 37 ℃ of insulations, still can change temperature retention time according to supplier's indication. After the digestion, directly the electrophoresis product is expected fragment to separate on polyacrylamide gel.

Use the people such as D.Goeddel, Nucleic Acids Res., 8:4057,1,980 8% polyacrylamide gels of describing carry out the size separation of cutting fragment.

" oligonucleotides " but refer to a poly deoxynucleosides chain of chemical synthesis or the poly deoxynucleosides chain of two complementations. These synthetic oligonucleotides do not have 5 ' phosphoric acid, thereby can not connect another oligonucleotides, unless add phosphoric acid with ATP existing in the kinase whose situation. Synthetic oligonucleotides will connect not dephosphorylized fragment.

" connection " refers between two double stranded nucleic acid fragments to form the process (people such as T.Maniatis, the same, the 146th page) of phosphodiester bond. Unless stated otherwise, use known buffer solution and condition, the per 0.5 μ g roughly dna fragmentation to be connected of equimolar amounts realizes connecting with the T4 dna ligase of 10 units.

Unless stated otherwise, such as F.Graham and A.Van der Eb, Virology transforms described in the method for 52:456-457,1973.

Above the present invention is carried out general description, will be more readily understood the present invention by reference the following example. Embodiment provides with way of example, is not intended to limit the present invention.

The bacterial expression of EXAMPLE Example 1:MPIF-1 and purifying

At first use the dna sequence dna ATCC#75676 of PCR Oligonucleolide primers amplification coding MPIF-1, described primer is corresponding to 5 ' sequence of the rear MPIF-1 albumen (having lacked signal peptide sequence) of processing and the carrier sequence of MPIF-1 gene 3 '. Add respectively extra nucleotides corresponding to BamHI and XbaI in 5 ' and 3 ' of sequence. 5 ' Oligonucleolide primers has sequence 5 '-TCAGGATCCGTCACAAAAGATGCAGA-3 ' (SEQ ID NO:8), this sequence comprises the BamHI restriction enzyme sites, is 18 nucleotides that stopped the MPIF-1 coded sequence of amino acid code beginning by the supposition of protein after the processing subsequently. 3 ' sequence 5 '-CGCTCTAGAGTAAAACGACGGCCAGT-3 ' (SEQ ID NO:9) comprises the complementary series in XbaI site.

These restriction enzyme sites are corresponding to the restriction enzyme sites on the bacterial expression vector pQE-9 (Qiagen company, Chatsworth, CA). PQE-9 coding antibiotic resistance (Amp^r), bacterium origin of replication (ori), IPTG regulation type promoter operon (P/O), ribosome bind site (RBS), 6-His label and restriction enzyme sites. Then with BamI and XbaI digestion pQE-9. The sequence of amplification is connected among the pQE-9, and inserts with the form of the coded sequence reading frame that meets histidine-tagged and RBS. Then transform coli strain M15/rep4 (Qiagen company) with connecting mixture. M15/rep4 comprises the plasmid pREP4 of multicopy, and it is expressed the lacI repressor and gives kalamycin resistance (Kan^r). Identify transformant by the energy for growth on the LB flat board, and select ampicillin/kalamycin resistance bacterium colony. Isolated plasmid dna is also confirmed by restriction analysis. The overnight incubation (O/N) in the LB fluid nutrient medium that adds Amp (100 μ g/ml) and Kan (25 μ g/ml) that is cloned in of construction will be comprised. With 1: 100-1: 250 ratio, inoculate a large amount of culture mediums with the O/N culture. Make Growth of Cells to optical density 600 (O.D.600) be 0.4-0.6. Then add IPTG (isopropyl-B-D-thiogalactoside) to final concentration 1mM. IPTG induces P/O to remove by deactivation lacI repressor, causes the increase of gene expression. Cell was cultivated 3-4 hour again. Then pass through centrifugal cell harvesting. Cell precipitation is dissolved in chaotropic agent 6M guanidine hydrochloride. After the clarification, under the condition that the protein that allows to contain the 6-His label is combined closely, by the Ni chelate column by the MPIF-1 of this solution purification dissolving people such as (, J. Chromatography, 411:177-184,1984) E.Hochuli. By wash-out MPIF-1 (95% is pure) on the post, for renaturation, be adjusted to 3M guanidine hydrochloride, 100mM sodium phosphate, 10mM glutathione (reduced form) and 2mM glutathione (oxidized form) with 6M guanidine hydrochloride pH5.0. Insulation was dialysed protein after 12 hours to the 10mM sodium phosphate in this solution.

Perhaps, use following without Tag primer with Gene cloning in plasmid pQE70: 5 ' primer: 5 ' CCCGCA TGC GGG TCA CAA AAG ATG CAG 3′(SEQ ID NO：10)

SphI 3 ' primer: 5 ' AAAGGA TCC TCA ATT CTT CCT GGT CTT 3′(SEQ ID NO：11)

BamHI terminator Escherichia coli are optimized the structure of MPIF-1

In order to increase the expression of MPIF-1 in the escherichia expression system, gene codon is optimized to the Escherichia coli preference codon. Optimization district for synthetic MPIF-1 has generated a series of 4 kinds of oligonucleotides: mpif-1 oligomer numbering 1-4 (listing in hereinafter). These overlapping oligomers are used for 7 of following condition take turns the PCR reaction:

95 ℃ of 20sec of sex change

58 ℃ of 20sec anneal

Extend 72 ℃ of 60sec

7 take turns synthetic after, add 5 ' primer (the ACA T in this zone in the PCR reaction of the initial reaction that contains 6 kinds of oligonucleotides of 1 μ lGC ATG CGU GUU ACC AAA GAC GCU GAA ACC GAA UUC AUG AUG UCC (SEQ ID NO:12)) and 3 ' primer (the GCC C in this whole zoneAA GCT TTC AGT TTT TAC GGG TTT TGA TAC GGG (SEQ ID NO:13)). Use following condition, this product amplification 30 taken turns:

95 ℃ of 20sec of sex change

55 ℃ of 20sec anneal

Extend 72 ℃ of 60sec

Pass through the product that this end reaction generates with SphI and HindIII restrictive diges-tion; and be cloned among the same pQE70 with SphI and HindIII cutting. Express these clones, discovery has higher expression and does not have said mutation. mpif1： 5′GCA TGC GUG UUA CCA AAG ACG CUG AAA CCG AAU UCA UGA UGU CCA AAC UGC CGC UGG AAA ACC CGG UUC UGC UGG ACC GUU UCC ACG C3′ ( SEQ ID NO：14 ) mpif2： 5′GCU GGA AUC CUA CUU CGA AAC CAA CUC CGA AUG CUC CAA ACC GGG UGU UAU CUU CCU GAC CAA AAA AGG UCG UCG UUU CUG CGC UAA CCC GUC CGA CAA ACA GG3′ ( SEQ ID NO：15 ) mpif3： 5′AAG CTT TCA GTT TTT ACG GGT TTT GAT ACG GGT GTC CAG TTT CAG CAT ACG CAT ACA AAC CTG AAC CTG TTT GTC GGA CGG GTT AGC GC3′ ( SEQ ID NO：16 ) mpif4： 5′GGT TTC GAA GTA GGA TTC CAG CAG GGA GCA CGG GAT GGA ACG CGG GGT GTA GGA GAT GCA GCA GTC AGC GGA GGT AGC GTG GAA ACG GTC CAG C 3′ ( SEQ ID NO：17 ) MPIF-1

Use above listed Escherichia coli is optimized the MPIF-1 construction by 5 ' the terminal deletion mutant that makes up of MPIF-1 gene. Being used for making up the 5 ' primer that lacks lists in hereinafter. Optimize the listed pcr amplification that carries out of MPIF-1 construction about Escherichia coli as mentioned. With 5 ' site of NcoI restrictive diges-tion Δ 17-A qe6, Δ 23, Δ 28 products, with HindIII restrictive diges-tion 3 ' site, and be cloned among the plasmid pQE60 that digests with NcoI and HindIII. With 5 ' site of all other products of SphI restrictive diges-tion, with its 3 ' site of HindIII restrictive diges-tion, and be cloned among the plasmid pQE70 that digests with SphI and HindIII.

Used 5 ' primer is as follows: Δ 17-A qe6 (pQE60) 5 ' NcoI gc gca g ccatgThe amino acid sequence of g aa aac ccg gtt ctg ctg gac 3 ' (SEQ ID NO:18) these deletion mutants: MENPVLLDRFHATSADCCISYTPRSIPCSLLESYFETNSECSKPGVIFLTK KGRRFCANPSDKQVQVCMRMLKLDTRIKTRKN (SEQ ID NO:19) Δ 16-A qe7 (pQE70) 5 ' SphI gc cat g gcatgThe amino acid sequence of this deletion mutant of c tg gaa aac ccg gtt ctg ctg gac (SEQ ID NO:20): MLENPVLLDRFHATSADCCISYTPRSIPCSLLESYFETNSECSKPGVIFLT KKGRRFCANPSDKQVQVCMRMLKLDTRIKTRKN (SEQ ID NO:21) Δ 23 (pQE60) 5 ' NcoI gc gca g ccatgThe amino acid sequence of this deletion mutant of g ac cgt ttc cac gct acc tcc (SEQ ID NO:22): MDRFHATSADCCISYTPRSIPCSLLESYFETNSECSKPGVIFLTKKGRRF CANPSDKQVQVCMRMLKLDTRIKTRKN (SEQ ID NO:23) Δ 24 (pQE70) 5 ' SphI gcc atg gcatgThe amino acid sequence of this deletion mutant of c gtt tcc acg cta cct cc (SEQ ID NO:24): MRFHATSADCCISYTPRSIPCSLLESYFETNSECSKPGVIFLTKKGRRFC ANPSDKQVQVCMRMLKLDTRIKTRKN (SEQ ID NO:4) Δ 28 (pQE60) 5 ' NcoI gcg cag ccatgThe amino acid sequence of this deletion mutant of g cta cct ccg ctg act gct gc (SEQ ID NO:25): MATSADCCISYTPRSIPCSLLESYFETNSECSKPGVIFLTKKGRRFCANPS DKQVQVCMRMLKLDTRIKTRKN (SEQ ID NO:26) S70A mutant (the 70th Ser sudden change adult Ala) (pQE70) antisense ttc gaa gta ggc ttc cag cag (SEQ ID NO:27) has adopted ctg ctg gaa gcc tac ttc gaa (SEQ ID NO:28) 5 ' SphI total length gcc atg gcatgThe amino acid sequence of this deletion mutant of c gtg tta cca aag acg ctg aaa cc (SEQ ID NO:29): MRVTKDAETEFMMSKLPLENPVLLDRFHATSADCCISYTPRSIPCSLLE aYFETNSECSKPGVIFLTKKGRRFCANPSDKQVQVCMRMLKLDTRIKT RKN (SEQ ID NO:30)

Be used for the 3 ' primer that all constructions use: 3 ' Hind III gcc c aagctttca gt ttt tac ggg ttt tga tac ggg(SEQ ID NO：31)

" maturation " MPIF-1 (mutant 1 of Figure 19) that is used for Bacillus coli expression: MRVTKDAETEFMMSKLPLENPVLLDRFHATSADCCISYTPRSIPCSLLE SYFETNSECSKPGVIFLTKKGRRFCANPSDKQVQVCMRMLKLDTRIKT RKN (SEQ ID NO:3) embodiment 2

Be used for to carry the SV40 origin of replication at most of carriers of mammalian cell transient expression MPIF-1 gene order. This allows carrier to be copied to high copy number in the cell (such as the COS cell) of expressing T antigen, and T antigen is to start viral DNA to synthesize needed. Any other mammal cell line also can be used for this purpose.

Typical mammalian expression vector comprises promoter element, protein coding sequence, the termination of mediation mRNA transcription initiation and transcribes the signal required with the transcript polyadenylation. Other element comprises that enhancer, Kozak sequence and flank are to be used for the donor of RNA montage and the intervening sequence of acceptor site. With early stage and late promoter, the LTR (LTR) of retrovirus (such as RSV, HTLVI, HIVI) and the early promoter of cytomegalovirus (CMV) of SV40, can realize efficiently transcribing. Yet, also can use cell signal (such as the human actin promoter). Being used for putting into practice suitable expression vector of the present invention comprises such as pSVL and pMSG (Pharmacia, Uppsala, Sweden), the carrier such as pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146) and pBC12MI (ATCC 67109). Operable mammalian host cell comprises Hela cell, 283 cells, H9 cell and Jurkart cell, NIH 3 T 3 cells in vitro and C127 cell, Cos1 cell, Cos7 cell and CV1 cell, cercopithecus aethiops cell, quail QC1-3 cell, mouse Lcell and Chinese hamster ovary cell.

Perhaps, can in comprising the stable cell lines that is incorporated into the gene in the chromosome, express this gene. Can identify and separate transfectional cell with the cotransfection of selected markers such as dhfr, gpt, neomycin, hygromycin.

Also can increase rotaring redyeing gene with the coded protein of great expression. Dihyrofolate reductase (DHFR) is a kind of useful mark, the clone that can be used to cultivate the genes of interest that carries hundreds if not thousands of copies. Another kind of useful selected marker is glutamine synthase (GS) (people such as Murphy, Biochem.J., 227:277-279,1991; The people such as Bebbington, Bio/Technology, 10:169-175,1992). Use these marks, can in selecting culture medium, cultivate the cell that mammalian cell and selection have maximum resistance. These clones comprise the amplification gene that is incorporated in the chromosome. Chinese hamster ovary (CHO) cell is usually used in producing protein.

Expression vector pC1 and pC4 comprise LTR (LTR) strong promoter (people such as Cullen of Rous sarcoma virus, Molecular and Cellular Biology, in March, 1985: 438-447) and the cmv enhancer fragment (people such as Boshart, Cell, 41:521-530,1985). MCS such as restriction enzyme cleavage site BamHI, XbaI and Asp718, is convenient to clone genes of interest. Carrier also comprises 3 ' introne, polyadenylation and the termination signal of rat proinsulin protogene. A. at COS cells restructuring MPIF-1

Expression plasmid CMV-MPIF-1-HA is derived from carrier pcDNAI/Amp (Invitrogen), it comprises: (1) SV40 origin of replication, (2) ampicillin resistance gene, (3) Escherichia coli origin of replication, (4) CMV promoter is followed by polylinker district, SV40 introne and polyadenylation site. Be cloned in the polylinker district of carrier at the HA label of its 3 ' end with the dna fragmentation of the complete MPIF-1 precursor of coding and with identical reading frame fusion, therefore, expression of recombinant proteins is subject to the guidance of CMV promoter. As mentioned above, the HA label is corresponding to the epi-position (people such as I.Wilson, Cell, 37:767,1991) derived from influenza hemagglutinin protein. The fusion of HA label and our target protein makes it possible to be easy to detect recombinant protein with the antibody of identification HA epi-position.

The plasmid construction strategy has hereinafter been described:

Using following two kinds of primers to comprise the HindIII site by dna sequence dna ATCC#75676:5 ' primer 5 '-GGAAAGCTTATGAAGGTCTCCGTGGCT-3 ' (SEQ ID NO:32) that PCR makes up coding MPIF-1 on clone's original EST, is 18 nucleotides of the MPIF-1 coded sequence that begun by initiation codon subsequently; 3 ' sequence 5 '-CGCTCTAGATCAAGCGTAGTCTGGGACGTCGTATGGGTAATTCTTCCTGGTCTTGA TC C-3 ' (SEQ ID NO:33) comprises last 20 nucleotides (not comprising terminator codon) of complementary series, translation stop codon, HA label and the MPIF-1 coded sequence in XbaI site. Therefore, the PCR product comprises HindIII site, MPIF-1 coded sequence, is the HA label that merges with identical reading frame, translation stop codon and the XbaI site that follows the HA label closely subsequently. Be connected the dna fragmentation and the carrier pcDNAI/Amp connection of being connected of pcr amplification with the XbaI restriction enzyme with HindIII. To connect mixture and be transformed among the coli strain SURE (Stratagene Cloning Systems, La Jolla, CA), will transform culture and be coated on the ampicillin medium flat board and selection. By the transformant isolated plasmid dna, whether there is correct fragment by restriction analysis with check. In order to express restructuring MPIF-1, by DEAE-DEXTRAN method (J.Sambrook, E.Fritsch, T.Maniatis, " Molecular Cloning:A Laboratory Manual " (molecular cloning: lab guide), publishing house of cold spring harbor laboratory, 1989), use the expression vector rotaring redyeing COS cell. By radioactive label and immuno-precipitation (E.Harlow and D.Lane, " Antibodies:A Laboratory Manual " (antibody: lab guide), publishing house of cold spring harbor laboratory, 1988), detect the MPIF-1-HA protein expression. After the transfection 2 days, use³⁵S-cysteine labeled cell 8 hours. Then gather in the crops culture, and with detergent (RIPA buffer solution: 150mM NaCl, 1% NP-40,0.1% SDS, 0.5% DOC, 50mM Tris, pH7.5) (people such as I.Wilson, the same, 37:767,1984) cell lysis. Cell lysate and culture medium all use the HA monoclonal antibody specific to precipitate. The protein of precipitation is analyzed at the 15%SDS-PAGE gel. B. clone and expression in Chinese hamster ovary celI

Carrier pC1 is used for the MPIF-1 protein expression. Plasmid pC1 is the derivative of plasmid pSV2-dhfr (ATCC numbering 37146). These two kinds of plasmids are included in the mouse DHFR gene under the control of SV40 early promoter. Can be by select Chinese hamster ovary cell or other cell with the shortage dihydrofoilic acid activity of these plasmid transfections at the middle cultured cell of the selective medium (α-MEM, Life Technologies) that adds the chemotherapeutics methotrexate. The existing many records of amplification DHFR gene (are consulted for example F.W.Alt, R.M.Kellems, J.R.Bertino and R.T.Schimke, J.Biol.Chem., 253:1357-1379,1978 in methotrexate (MTX) resisting cell; J.L.Hamlin and C.Ma, Biochem.et Biophys.Acta, 1097:107-143,1990; M.J.Page and M.A.Sydenham, Biotechnology, 9:64-68). The cell of growing in the cumulative MTX of concentration has developed drug resistance by the excessive generation of target enzyme DHFR, and the excessive generation of DHFR is the result of DHFR gene magnification. If the second gene is linked to each other with the DHFR gene, the then common coamplification of this gene and overexpression. This is the mode that this area is used for cultivating the clone that comprises the above gene of 1000 copies. Subsequently, after removing methotrexate, clone comprises the amplification gene that is incorporated in the chromosome.

In order to express genes of interest, plasmid pC1 comprises the strong promoter (people such as Cullen of the LTR (LTR) of Rous sarcoma virus, Molecular and Cellular Biology, in March, 1985: 438-447) with the fragment of being separated by human cytomegalovirus (CMV) immediate early gene enhancer (people such as Boshart, Cell, 41:521-530,1985). The downstream of promoter is the Single restriction enzyme cleavage site that can be used for integrator gene: BamHI, follows 3 ' introne and polyadenylation site by rat proinsulin protogene. Other efficient promoter also can be used for expressing, such as people's beta-actin promoter, SV40 is early stage or the LTR of late promoter or other retrovirus (such as HIV and HTLVI). For the polyadenylation of mRNA, also can use such as other signal from human growth hormone (HGH) or globin gene.

Also can after using selected marker (such as gpt, G418 or hygromycin) cotransfection, select to carry the stable cell lines that is incorporated into the gene in the chromosome. Preferably use during beginning to surpass a kind of selected marker, add methotrexate such as G418.

With restriction enzyme BamHI digested plasmid pC1, the flow process of then knowing by this area Roll phosphatase dephosphorylation. Again then by 1% Ago-Gel carrier of separating.

Use the dna sequence dna (ATCC numbering 75676) corresponding to the PCR Oligonucleolide primers amplification coding MPIF-1 of gene 5 ' and 3 ' sequence.

5 ' primer has sequence:

5′AAA GGA TCC GCC ACC ATG AAG GTC TCC GTG GTC 3′

BamHI KOZAK (SEQ ID NO:34), the MPIF-1 albumen coded sequence that comprises BamHI restriction enzyme sites (underscore) and Fig. 1 (SEQ ID NO:1) is a part of. As described below, in being inserted into expression vector after, 5 ' end of the amplified fragments of encoding human MPIF-1 provides effective signal peptide. Such as M.Kozak, J.Mol.Biol., 196:947-950,1987 is described, be used for to start among the suitable carrier part that places construction of the useful signal of eukaryotic translation.

3 ' primer has sequence: 5 ' AAAGGA TCC TCA ATT CTT CCA GGT CTT 3′

BamHI Stop (SEQ ID NO:35) comprises Asp718 restriction site and a part of with the nucleotides of Fig. 1 (SEQ ID NO:1) listed MPIF-1 coded sequence complementation, comprises terminator codon.

As mentioned above, the fragment of separating amplification by 1% Ago-Gel, with endonuclease BamHI and Asp718 digestion, and then on 1% Ago-Gel purifying.

Connect fragment and the dephosphorylized carrier that separates with the T4 dna ligase. Then transform the Escherichia coli HB101 cell, and use restriction enzyme BamHI to identify the bacterium that comprises the plasmid pC1 that inserts with correct orientation. Confirm to insert the sequence of gene by dna sequencing. Transfection CHO-DHFR cell

The Chinese hamster ovary cell that lacks active DHFR enzyme is used for transfection. Use lipofection people such as (, the same) Felgner cotransfection 5 μ g expression plasmid C1 and 0.5 μ g plasmid pSV-neo. Plasmid pSV2-neo comprises dominant selectable marker neo gene, and this neo gene is from Tn5, and the enzyme of coding is given the resistance to one group of antibiotic (comprising G418). Inoculating cell in the α-MEM that adds 1mg/ml G418. After 2 days, use trypsin digestion and cell, be seeded to hybridoma clone dull and stereotyped (Greiner, Germany) and cultivated 10-14 days. After this, with the single clone of Trypsin Induced, and be seeded to the 6 hole culture dishes that contain variable concentrations methotrexate (25nM, 50nM, 100nM, 200nM, 400nM). The clone that then will grow in the maximum concentration methotrexate transfers in the 6 new orifice plates that contain higher concentration methotrexate (500nM, 1 μ M, 2 μ M, 5 μ M). Repeat identical flow process, grow until be cloned in the concentration of 100 μ M.

By Western engram analysis and SDS-PAGE, analyze the expression of required gene outcome. The expression pattern of embodiment 3:MPIF-1 in people's tissue

Carry out the Northern engram analysis with the expression of MPIF-1 in identifier's tissue. Use RNAzol^TMThe B system (Biotecx Laboratories company, 6023 South Loop East, the Houston, TX 77033) the total cell RNA sample of separation. To organize the total RNA of about 10 μ g of separation to separate at 1% Ago-Gel by every kind of nominator, and transfer to (Sambrook, Fritsch and Maniatis on the nylon leaching film, " Molecular Cloning:A Laboratory Manual " (molecular cloning: lab guide), cold spring port publishing house, 1989). According to Stratagene Prime-It kit, the 50ng dna fragmentation is carried out labeled reactant. With Select-G-50 post (Boulder, CO 80303 for 5 Prime-3 Prime companies, 5603 Arapahoe Road) purifying through marker DNA. Then with filter membrane and radiolabeled total length MPIF-1 gene in 1,000,000cpm/ml at 0.5M NaPO₄Spend the night in 65 ℃ of hybridization among pH7.4 and the 7%SDS. With 0.5 * SSC, 0.1%SDS, clean twice in room temperature, again in 60 ℃ of cleanings twice, then with the sensitization screen filter membrane exposure is spent the night in-70 ℃. Embodiment 4: use baculovirus expression system to express and purifying chemotactic factor (CF) MPIF-1

Be used for expressing the recombinate shape virus infection SF9 cell of MPIF-1 cDNA with design. With MOI=2 infection cells, and in 28 ℃ of cultivations 72-96 hour. Remove cell fragment by low-speed centrifugal by infecting culture. In supernatant, add protease inhibitor cocktail to final concentration 20 μ g/ml Pefabloc SC, 1 μ g/ml leupeptin, 1 μ g/ml E-64 and 1mM EDTA. By 20-30 μ l supernatant application of sample being monitored the MPIF-1 level in the supernatant to the 15%SDS-PAGE gel. Detecting MPIF-1 is visible 9kDa band, corresponding to the expression of several mg/L. Be further purified MPIF-1 by three step purifying flow processs: the Heparin-binding affinity chromatography. The supernatant of baculoviral culture is mixed with the buffer solution of 1/3 volume (100mM HEPES/MES/NaOAc pH6), and with 0.22 μ m membrane filtration. Then sample is splined on Heparin-binding post (HEI poros 20, Bi-Perceptive System company). In the linear gradient of the 50-500mM NaCl that is dissolved in 50mM HEPES/MES/NaOAc pH6, in about 300mM NaCl wash-out MPIF-1; Cation-exchange chromatography. To be carried out by the MPIF-1 of heparin chromatography enrichment 5 times of dilutions with the buffer solution that contains 50mM HEPES/MES/NaOAc pH6. Then the mixture that obtains is applied to cation exchange column (S/M poros 20, Bio-Perceptive System company). In the linear gradient of the 25-300mM NaCl that is dissolved in 50mM HEPES/MES/NaOAc pH6, in 250mM NaCl wash-out MPIF-1; Size exclusion chromatography. After cation-exchange chromatography, (1.4 * 45cm) are further purified MPIF-1 for HW50, TOSO HAAS by being applied to size-exclusion column. The MPIF-1 fraction is positioned near the 13.7kDa molecular weight standard (RNA enzyme), corresponding to the dimer of this protein.

After three step purifying mentioned above, judge that by the Coomassie blue stain of SDS-PAGE gel the MPIF-1 that obtains surpasses 90% pure (Fig. 3).

Also the MPIF-1 of purifying tested endotoxin/LPS pollution. According to LAL determination method (Bio Whittaker), LPS content is lower than 0.1ng/ml. Embodiment 5: the impact that the fresh separated bone marrow cell colony that the M-CIF of baculovirus expression and MPIF-1 stimulate M-CSF and SCF forms

With low-density bone marrow cells in mice group organizing in the culture dish of processing in 37 ℃ of insulations 1 hour to remove other cell on monocyte, macrophage and adsorption plastic surface. Then non-adherent cell group is coated with 10,000 cell/dishes and contain in the agar medium, have or lack the factor shown in Figure 8 in the described culture medium. Culture is incubated 10 days (88%N in 37 ℃₂、 5％CO ₂, and 7%O₂), and under inverted microscope, colony is marked. Data are stated average colony number as, and derive from parallel 3 mensuration of carrying out. The Lin that embodiment 6:MPIF-1 and M-CIF stimulate IL-3 and SCF^-The impact of bone marrow cell group propagation and differentiation

Use the negative flow process of selecting to obtain the bone marrow cells in mice group of being rich in original HPC, wherein use one group of monoclonal antibody (anti-cdllb, CD4, CD8, CD45R and Gr-1 antigen) and magnetic bead to remove the committed cell of most of pedigrees. With the cell mass (pedigree exhausts cell) that obtains with 5 * 10⁴Individual cell/ml coats and adds in IL-3 (5ng/ml) and stem cell factor (SCF) growth medium (100ng/ml), exists in the described culture medium or lack to specify chemotactic factor (CF) (50ng/ml). At moist incubator (7%O₂、5％CO ₂, and 88%N₂) in 37 ℃ of insulations after 7 days, harvesting, and measure HPP-CFC and prematurity CFU-GM. In addition, by the expression of FACScan to some differentiation antigen of cell analysis. The colony data state as average colony number (+/-SD), and the every group of cells of must the doing for oneself mensuration (Fig. 9) of in 6 dishes, carrying out. Embodiment 7:MPIF-1 suppresses to reply the colony formation of IL-3, M-CSF and GM-CSF

Gather bone marrow cells in mice by femur and shin bone, separate in the Fei Keer density gradient, and remove monocyte by plastics absorption. With the cell mass that obtains add IL-3 (5ng/ml), GM-CSF (5ng/ml), M-CSF (10ng/ml) and G-CSF (10ng/ml) based on the culture medium of MEM in incubated overnight. With these cells with 1,000 cell/dish is coated in the CFA method based on agar, wherein there are IL-3 (5ng/ml), GM-CSF (5ng/ml) or M-CSF (5ng/ml), and comprise or do not contain MPIF-1 (50ng/ml). Data are stated the percentage that colony forms the colony number that forms when only containing specificity factor as. Two experiments have shown the data of describing with the mean value of two parallel discs, and error bar is indicated the standard deviation (Figure 11) of each experiment. Embodiment 8: through the expression of gene therapy

Obtain fibroblast by skin histology biopsy method by the experimenter. The tissue that obtains is placed tissue culture medium (TCM) and is divided into small pieces. The fritter tissue is placed on the wetted surface of tissue culture flasks, place about 10 in each bottle. Bottle is put upside down, tightened, and at room temperature place and spend the night. After at room temperature 24 hours, blake bottle is put upside down, at the bottom of tissue block still is fixed on bottle, added fresh culture (for example HamShi F12 culture medium contains 10%FBS, penicillin and streptomysin). Then it is incubated about 1 weeks in 37 ℃. At this moment, add fresh culture, change once every a couple of days subsequently. After cultivating for 2 weeks, the individual layer fibroblast has appearred again. With the Trypsin Induced individual layer and scrape in the larger blake bottle.

Be pMV-7 people such as (, DNA, 7:219-25,1988) P.T.Kirschmeier of Moloney murine sarcoma virus LTR with EcoRI and HindIII digestion flank, process with the Roll phosphatase subsequently. With linear carrier at Ago-Gel separately and use the bead purifying.

CDNA with the PCR primer amplification code book invention polypeptide that corresponds respectively to 5 ' and 3 ' terminal sequence. 5 ' primer comprises the EcoRI site, and 3 ' primer comprises the HindIII site. Under the condition that has T4 DNA ligase, that equivalent Moloney murine sarcoma virus linear backbone and EcoRI-HindIII fragment is added together. Keep the mixture that obtains being suitable for connecting under the condition of two fragments. With connecting mixture transform bacteria HB101, then coat on the agar that contains kanamycins, comprise the genes of interest of correct insertion to confirm carrier.

In the Eagles culture medium (DMEM) that the DulbeccoShi that contains 10% cow's serum (CS), penicillin and streptomysin revises, facultative pA317 or GP+am12 incasing cells are grown in tissue is cultivated converge density. Then in culture medium, add the MSV carrier that comprises described gene, use the carrier transduction incasing cells. Now, incasing cells generates the infectious viral particle (be called producer cell with incasing cells this moment) that comprises this gene.

To in producer's cell of transduction, adding fresh culture, subsequently by the dull and stereotyped culture medium of gathering in the crops of the 10cm of the producer's cell that converges. The culture medium that has consumed that will contain infectious viral particle to remove producer's cell of desorption, then infects fibroblast with this culture medium by the millopore membrane filtration. Converge flat board by fibroblastic time and remove culture medium, change to rapidly the culture medium from producer's cell. Remove this culture medium, change to fresh culture. If virus titer is high, will infects so in fact all fibroblasts, and need not to select. Very low if tire, must use the retroviral vector with selected marker (such as neo or his) so.

Then, improved fibroblast is expelled among the host separately, or the cytodex3 microcarrier bead grow to converge after the injection. Fibroblast generates protein now. Embodiment 9: external marrow protection

As what above prove, MPIF-1 is the establishment agent of low multiplication potentiality colony forming cell (LPP-CFC) (generating the myeloid progenitor of granulocyte and monocyte pedigree). In order to prove that MPIF-1 can protect LPP-CFC to avoid the cytotoxicity of cell cycle effect chemotherapeutics, separates pedigree by mouse bone marrow cells and exhausts cell mass (Lin^-Cell) and having cytokine profiles and comprise or do not contain in the situation of MPIF-1 be incubated. After 48 hours, one group of every kind of culture is accepted 5-FU, then continues insulation 24 hours, measures the LPP-CFC number of survival this moment by the former determination method of clone. Shown in Figure 21 A, exist in the situation of MPIF-1, about 40% LPP-CFC is protected and the cytotoxicity that avoids being induced by 5-FU; Lacking MPIF-1 but exist in the situation of irrelevant protein, observing the protection of LPP-CFC seldom (＜5%). Under identical condition of culture, high proliferation potentiality colony forming cell (HPP-CFC) is not subject to the protection of MPIF-1, proves that the protective effect of MPIF-1 has specificity.

Use chemotherapeutics Ara-C to replace 5-FU to carry out similar experiment. Shown in Fig. 1 5B, LPP-CFC has been subject to the remarkable protection from wild type MPIF-1 and saltant MPIF-1 (being mutant-1, about the description of this mutant embodiment 11 that sees below). Thereby MPIF-1 can protect LPP-CFC to avoid the cytotoxicity of being induced by chemotherapeutics 5-FU and Ara-C. Embodiment 10: marrow protection in the body

External marrow protection presentation of results; if the key cells type in the mechanism of chemotherapeutics in the marrow can access the protection of MPIF-1; the bone marrow toxicity that is caused by cytotoxic drug so; the serious side effects of namely observing in the cancer patient of experience chemotherapy just can improve. In order to prove marrow protection in the body, two kinds of experiments in mouse, have been carried out. In a kind of experiment, to one group of mouse (the 4th group) injection every day (I.P.) 1.0mg/kg MPIF-1, each minor tick 24 hours reaches three days, and at the 3rd day these mouse is also injected (I.P.) 150mg/kg 5-FU. Pump pickle (the 1st group), only with MPIF-1 (the 2nd group) or the animal of only using 5-FU (the 3rd group) in contrast. Then, after using 5-FU, put to death 4 animals from every group on the 3rd, 6 and 10 day, to measure white blood cell (WBC) counting in the peripheral blood. As shown in figure 16, it is very little on the impact of WBC counting only to inject MPIF-1. Just as expected, 5-FU processes that the WBC counting significantly reduces in the circulation that causes behind the 5-FU the 6th day. Importantly, the animal display ratio of processing with MPIF-1 before using 5-FU is only counted with WBC in the blood of the high approximately twice of the animal of 5-FU processing. Thereby, before 5-FU, process mouse with MPIF-1 and cause adding quick-recovery by neutrophil cell minimizing disease.

Candidate stem cell in the marrow and multipotency CFU-GM are responsible for recovering all hematopoietic lineages after chemotherapy. In normal individual, the division of these cells is more not frequent, thereby escapes the single dose chemotherapeutics. Yet, if after first dose, used second dose with interior in three days, will kill these cells so, because during this period fast division of the crucial CFU-GM type in the marrow.

In order to prove that MPIF-1 can protect these cell types in the marrow, has carried out following experiment. Use 3 groups of mouse (every group of 6 animals) of following processing to test: the 1st group, at the 1st, 2 and 3 day pump pickle; The 2nd group, at the 0th and 3 day injection 5-FU; The 3rd group, at the 0th and 3 day injection 5-FU, at the 1st, 2 and 3 day injection MPIF-1 (seeing Figure 17). At the 6th and 9 day results marrow, use the former determination method of the well-known clone of those skilled in the art to measure HPP-CFC and LPP-CFC frequency. The result proves, than the animal of only processing with 5-FU, uses HPP-CFC and the fast quick-recovery of LPP-CFC frequency that MPIF-1 caused the 9th day before second dose of 5-FU. Embodiment 11: about the research of MPIF-1 mutant

Many MPIF-1 variants of N end brachymemma have been identified and have characterized. Figure 19 has showed the amino terminal sequence of these variants of measuring by the Edman edman degradation Edman. For example, mutant-2 ,-3, the-7 and the-the 8th, spontaneous appearance in the purge process of MPIF-1 mature form, said preparation is called Formulation K 0871. Similar, mutant-2 ,-3, the-4 and the-the 5th is found in another batch purifying MPIF-1 preparation (preparation HG00300-B7). Since can not with these variants each other purifying separately, Formulation K 0871 and HG00300-B7 are used in experiment therefore as mentioned below. Except the terminal methionine of N, the mutant-the 6th that its amino terminal sequence is identical with mutant-3 generates by mutagenesis in vitro. The mutant-1 identical with wild type also generates by mutagenesis except the terminal methionine of N. In addition, found the another kind of splicing form (mutant-9) of MPIF-1, the protein (Figure 19) of 137 amino acid of this cDNA clones coding (Figure 20 A). Mutant-9 relatively discloses with the amino acid sequence of MPIF-1, between MPIF-1 sequence the 45th and 46 residues 18 amino acid whose insertions is arranged, the 46th arginic forfeiture of MPIF-1 (Figure 20 B). Hereinafter summarized the BA of these MPIF-1 mutant proteins. The cellular calcium migration

In the above-described embodiments, MPIF-1 albumen shows the calcium migration that can make in the monocyte. In the person monocytic cell wild type and saltant MPIF-1 have been tested them and induced the ability of cellular calcium migration, end user MIP-1 α is as positive control. Test following carrying out: separate the person monocytic cell by elutriation, by with 1 * 10⁶Individual cell is containing 1mM CaCl₂、2mM MgSO ₄, 5mM glucose and 10mM HEPES pH7.4 and add among the 1ml HBSS of 2.5mM Indo-1/ methyl acetate and loaded the Indo-1/ methyl acetate in 30 minutes in 37 ℃ of insulations. Then with HBSS cleaning cell and with 5 * 10⁵Individual cell/ml is resuspended in same buffer, stimulates in 37 ℃ with the appointment protein of various concentration. 330nm by monitoring Indo-1 on Hatchi F-2000 sepectrophotofluorometer excites and 405nm and 485nm launch to measure and reply cellular calcium ((Ca²⁺) i) change and the fluorescence signal of inducing.

The result proves that the specific activity wild type of Formulation K 0871, HG00300-B7 and mutant-9 is high 10 times, and mutant-6 can not make a distinction with wild type, the specific activity wild type of mutant-1 high about 2 times (Figure 21). Because MIP-1 α and MPIF-1 are 45% same aspect the one-level amino acid sequence, therefore interestingly measure them and whether interact with same receptor. In order to explore this possibility, studied MPIF-1 and made the calcium of being induced by MIP-1 α move the ability of desensitization. Figure 22 A and 22B show that MIP-1 α and MPIF-1 wild-type protein can make the other side mobilize the ability of calcium to desensitize mutually in monocyte, but MCP-4 (another kind of beta-chemokine) is not all right.

In similar experiment, Formulation K 0871, HG00300-B7 and mutant-1 ,-6 and-9 can be blocked the calcium migration of being induced by MIP-1 α. This experiment is following carrying out: described about disclosed experiment among Figure 21 as mentioned, and measure the person monocytic cell and specify the calcium migration of protein to reply to 100ng/ml. For desensitization research, at first monocyte is exposed to a kind of factor, after replying of the first processing got back to baseline, in same cell, add the second factor. (-) signal designation is not replied the second factor, and (+) signal designation has excitant to reply (Figure 23) to the first factor.

As if thereby MPIF-1 and saltant variant thereof interact with MIP-1 α, perhaps share the composition of cell surface receptor. Nearest proof MIP-1 α acceptor is taken on promotion HIV and is entered person monocytic cell and the lymphocytic confactor of T, and this has proposed interesting possibility: namely MPIF-1 and variant thereof may be intervened the process that HIV enters cell. Chemotaxis

The chemotaxis that the human peripheral blood mononuclear cell (PBMC) that mensuration is replied for MPIF-1 and the variant thereof of various concentration in 96 hole NeuroProbe chemotaxis grooves partly (mainly is comprised of lymphocyte and monocyte). Experiment is following carrying out: cell cleaned 3 times with the HBSS (HBSS/BSA) that contains 0.1%BSA, and with 2 * 10⁶/ ml is resuspended for mark. Add calcein (calcein)-AM (Molecular Probes) to final concentration 1mM, and cell is incubated 30 minutes in 37 ℃. After the insulation, cell is cleaned 3 times with HBSS/BSA. Then will be resuspended to 8 * 10 through labeled cell⁶/ ml, and with 25ml suspension (2 * 10⁵Individual cell) is assigned on each of 96 hole chemotactic flat boards in groove. Chemoattractant is assigned in the lower groove in each hole with various concentration. With polycarbonate leaching film (3-5mm aperture; Do not contain PVP; NeuroProbe company) separates up and down groove. Allow cell migration 45-90 minute, then use Cytofluor 11 fluorescence plate readers (PerSeptive Biosystems) that migrating cell (being attached to filter membrane bottom surface and lower groove) number is carried out quantitatively. The concentration of active peak value is observed in numeric representation, and the numeric representation in the bracket is with respect to the multiple of inducing of background.

Result shown in Figure 24 proves that Formulation K 0871 and HG00300-B7 are the chemotaxis derivants stronger than wild type, and mutant-1 and mutant-6 can not make a distinction with wild type. Impact on the formation of LPP-CFC colony

In order to measure the MPIF-1 variant to the impact that the LPP-CFC colony forms, a limited number of bone marrow cells in mice is coated in the soft agar that contains culture medium, added cytokine profiles in the culture medium, comprise or do not contain the MPIF-1 variant of multiple concentration. Experiment is following carrying out: with low-density bone marrow cells in mice group coating (1,500 cell/3.5cm diameter culture dishes) in the agar that contains culture medium, comprise or do not contain the appointment MPIF-1 variant of various concentration in the culture medium, but have following restructuring mouse cell factor IL-3 (5ng/ml), SCF (100ng/ml), IL-1 α (10ng/ml) and M-CSF (5ng/ml). Then culture dish is incubated 14 days in incubator for tissue culture, mark to the LPP-CFC colony this moment under inverted microscope. Data shown in Figure 25 are merged by several different experiments, and wherein every kind of condition is measured double.

Result's proof reaches the maximum desired valid density of 50 % that suppresses than the low 20-100 of wild type doubly in the situation of Formulation K 0871 and HG00300-B7; Mutant-6 is than 10 times of low 2-of wild type (Figure 25). Thereby the N terminal amino acid disappearance of MPIF-1 albumen causes the effectiveness of molecule to increase. Embodiment 12: stem cell migration in the body of being induced by MPIF-1

In order to prove that MPIF-1 stimulates stem cell migration, the following experiment in vivo. 6 mouse are used for each processed group (C57Black6/J, female, about 6 ages in week). Give every injected in mice (I.P.) salt solution (vehicle Control) or MPIF-15 μ g. After 30 minutes, by the mouse blood sampling, and by Coulter counter analysis WBC. Then, merge the blood from every group of all 6 animals, and analyze Gr.1+ cell and the dual positive cell of CD34.Sca-1+ by FACScan. The WBC counting is stated mean value ± S.D. as, and the FACScan data are stated the percentage with respect to total cell as. Owing to the expection characteristic of thinking that the dual positive cell of CD34.Sca-1+ is showed candidate stem cell, the MPIF-1 of presentation of results shown in Figure 26 can be used as the stem cell mobilization agent. Carrying out MPIF-1 in the embodiment 13:5-FU processing procedure processes and to cause blood platelet and granulocytic recovery faster

Two kinds of major complications that caused by chemotherapy are that neutrophil cell reduces disease (blood neutrophil cell counting reduces) and thrombopenia (platelet count reduction). Alleviate neutrophil cell at clinical middle use granulocyte colony stimulating factor (G-CSF) at present and reduce disease. Known G-CSF forms at the colony of stimulated in vitro colony forming unit-granulocyte (CFU-G), generates at animal model moderate stimulation granulocyte. TPO (Tpo) is carrying out be used to the clinical testing that alleviates thrombopenia. Known Tpo forms at the colony of stimulated in vitro colony forming unit-megacaryocyte (CFU-Meg), the thrombopenia moderate stimulation thrombocytopoiesis that experiment is induced in animal. The major limitation of G-CSF in clinical is that it is invalid in alleviating the neutrophil cell minimizing disease of carrying out many circulations patients undergoing chemotherapy. This might be owing to exhausted CFU-G, i.e. the target cell of G-CSF effect in the marrow. Tpo also may meet with mutually and share a common fate, shown in initial clinical test results. Can prevent that any reagent that exhausts G-CSF and Tpo target cell in chemotherapy process all will have great clinical value. The MPIF-1 of data declaration hereinafter can satisfy this clinical needs.

Among the embodiment formerly, the colony that MPIF-1 is presented at external inhibition dual intensity granulocyte/monocyte myeloid progenitor forms. Particularly, the digital proof that

embodiment

9 and 10 provides, MPIF-1 can protect original multipotency myeloid progenitor to avoid the cytotoxicity of being induced by 5-FU in vitro and in vivo. Estimate that these multipotency CFU-GMs can produce the further typing CFU-GM of all marrow pedigrees, comprise CFU-G and CFU-Meg. Carrying out following experiment carries out MPIF-1 with proof and processes and can cause blood platelet and granulocytic recovery faster in the 5-FU of 2 or 3 circulations processing procedure. Materials and methods

Use C57BL6 female mice (7-10 age in week), average weight 19.4g (± 1.1S.D., n=150). In whole experimentation, under standard recipe and raising condition (illumination/dark cycle and temperature), raise all mouse. MPIF-1 preparation (HG00304-E6) generates in Escherichia coli, and representative lacks the truncated-type MPIF-1 (being the MPIF-1 mutant-3 interpolation N terminal M et among Figure 19) of 23 N terminal amino acids of maturation protein. Clinical grade G-CSF (Neupogen ) is available from Shady Grove Pharmacy, Rockville, MD 20850 (Neupogen  is made by Amgen company, Amgen Center, and Thousand Oaks, CA 91320). 5 FU 5 fluorouracil (5-FU) is dissolved in common salt solution and fresh preparation available from Sigma Chemicals before facing use. Similarly, with the buffer solution dilution G-CSF that contains 10mM sodium acetate, 5% (wt/v) sweet mellow wine, 0.004% (v/v) Tween 80 pH4.0. Suitable puted together fluorogen, for the rat monoclonal antibody of mouse CD41a, Gra.1 and Mac.1 antigen available from Pharmingen.

5 groups of mouse of following processing (30 every group):

The 1st group at the-2 ,-1,0,6, the 7 and 8 days common salt solution of I.P. injection 0.1ml, as normal control;

The 2nd group at the 0th and 8 day I.P. injection 0.2ml 5-FU solution (100mg/kg body weight);

The 3rd group such as the 2nd group of injection 5-FU, in addition at the-2 ,-1,0,6,7 and 8 days I.P. injection 0.1ml MPIF-1 solution (1.0mg/kg body weight).

The 4th group such as the 2nd group of injection 5-FU, in addition at the 1st, 2,3,9,10 and 11 day I.P. injection 0.1ml G-CSF solution (0.5mg/kg body weight).

The 5th group as the 2nd group the injection 5-FU, as the 3rd group the injection MPIF-1, and as the 4th group the injection G-CSF.

Then 6 animals of scheduled date analysis from every group, with blood platelet and the granulocyte recovery of monitoring peripheral blood and marrow level. Should be noted that after the 5-FU administration first time mouse of analyzing in 6 and 8 days do not accept the second time of MPIF-1 or 5-FU and process.

In the coated pipe of EDTA, collect the peripheral blood that is gathered by rear eye socket, and analyze to measure blood platelet (CD41a positive events) and granulocyte (the dual positive cell of Gra.1 and Mac.1) counting by FACS Vangage immediately. Should be noted that analytical method used herein and animal species do not allow to obtain absolute counting. On the contrary, granulocyte is stated as with respect to total leukocytic percentage, and blood platelet is evaluated as the CD41a positive events in per 15 seconds on the sorter. Then put to death mouse, the Application standard method obtains bone marrow cell. Same by the percentage of facs analysis bone marrow cell with Gra.1 and the dual positive cell group of Mac.1 in the monitoring marrow. Owing in the stage that these antigens not fully aware of begin to express, therefore estimate that Gra.1 and the dual positive cell of Mac.1 in the marrow are heterogeneous with regard to stage of development and ripe potentiality in the granulocyte pedigree.

Also use the former determination method of body outer clone to analyze marrow to measure the frequency of the former CFU-GM of clone. In brief, in double-deck agar culture systems, carry out high proliferation potentiality colony forming cell (HPP-CFC) and low multiplication potentiality colony forming cell (LPP-CFC) determination method. In 3.5cm diameter culture dish, the MEM that adds 20% FBS, 0.5% Difco agar, 7.5ng/ml mIL-3,75ng/ml mSCF, 7.5ng/ml hM-CSF and 15ng/ml mIL-1 α with 1ml prepares bottom. Then cover this layer with 0.5ml mouse bone marrow cell suspension, thereby in the MEM that contains 20% FBS and 0.3% agar, have 2,000 cells/culture dish. Allow top agar solidify about 15 minutes in room temperature. Then with culture dish incubator for tissue culture (37 ℃, 88%N₂、5 ％CO ₂, and 7%O₂) in insulation 14 days, and under inverted microscope, colony is marked. In this experiment, reported total colony counting.

The FACS data are to obtain by analyzing the material that is obtained by every group of 3 animal of each time point, and cloning former determination method is to use the cell that is obtained by every group of 6 animal of each time point to carry out. At last, the representative of the data point of the 1st group of experiment is by the numerical value of common mouse (the 1st group) acquisition of pump pickle. The result

In order to monitor hematoblastic recovery situation in the peripheral blood, measure the steady-state level of CD41a positive cell by FACS Vantage. As shown in Figure 27, the processing of the MPIF-1 before the 5-FU (the 3rd group) causes hematoblastic recovery more faster, much better than than what observe in the mouse (the 2nd group) with the 5-FU+ saline treatment. As what estimate, G-CSF process in the mouse (the 4th group) platelet recovery dynamics can not with making a distinction of in 5-FU+ saline treatment mouse, observing. Equally, with comparing of in the mouse (the 3rd group) of only processing with MPIF-1, observing, 5-FU is processed mouse use G-CSF to add MPIF-1 (the 5th group) very little on hematoblastic whole steady-state level impact. Thereby, before 5-FU processes, mouse is carried out the MPIF preliminary treatment and cause hematoblastic fast quick-recovery in the peripheral blood.

Recover by the granulocyte of the steady-state level of Gra.1 in the blood and the dual positive cell of Mac.1 quantitatively being monitored in the peripheral blood. As shown in Figure 28, to mouse carry out 5-FU process cause for the first time and for the second time 5-FU process that the steady-state level of Gra.1 and the dual positive cell of Mac.1 sharply reduces in rear 6 days blood. The MPIF-1 preliminary treatment has two kinds of beneficial effects: with comparing of observing in the mouse (the 2nd group) with the 5-FU+ saline treatment, the degree (extent of deterioration of Gra.1 and the dual positive cell of Mac.1) of neutrophil cell minimizing disease is light, and regeneration rate is faster. As what estimate, after processing, uses 5-FU the fast quick-recovery that G-CSF (the 4th group) causes Gra.1 and the dual positive cell of Mac.1 in the blood. Yet the recovery extent that neutrophil cell reduces disease in the mouse of processing with G-CSF significantly is lower than to be observed in the mouse (the 3rd group) of processing with MPIF-1 on the 8th day. Using MPIF-1, to add G-CSF (the 5th group) quite remarkable to the effect of granulocyte loss and recovery, and namely the steady-state level of Gra.1 and the dual positive cell of Mac.1 is more much higher than what observe in the mouse of only processing with MPIF-1 or G-CSF in the blood showed of these mouse. Thereby as if as shown in figure 28, MPIF-1 and G-CSF can bring into play the addition effect when using altogether.

As mentioned above, monitor the recovery of marrow level by FACS Vantage method and the former determination method of clone. The result who obtains with FACS is illustrated in Figure 29. As what estimate, the level of Gra.1 and the dual positive cell group of Mac.1 significantly reduced at 6-14 days in the marrow (the 2nd group) of processing with 5-FU, yet returned to normal level at the 16th day. Before 5-FU, process mouse (the 3rd group) with MPIF-1, can eliminate fully by the Gra.1 of 5-FU mediation and this impact of the dual positive cell loss of Mac.1. Surprisingly, G-CSF (the 4th group) can prevent that Gra.1 and the dual positive cell of Mac.1 from replying exhausting that 5-FU for the first time takes medicine, but can not prevent for the second time. This may be because the availability of G-CSF target cell and the time that G-CSF uses. In add the mouse (the 5th group) that G-CSF processes with MPFI-1, observe similar replying, although for the first time behind the 5-FU the 8th day recovery extent more much higher than what in the mouse of only processing with MPIF-1 or G-CSF, observe.

From clone former determination method data display in Figure 30. CFU-GM frequency in the marrow is replied 5-FU and is kept reducing in whole 14 days of experimental session, the sign of recovery was arranged on the 16th day. Being reduced in the front mouse with the MPIF-1 processing of 5-FU of this CFU-GM frequency is eliminated. On the contrary, with G-CSF process mouse maintain normal or MPIF-1 process aspect the CFU-GM frequency of finding in the marrow invalid. Using G-CSF adds MPIF-1 the impact of CFU-GM frequency in the marrow is seemed complicated. General introduction property preclinical pharmacology form

Following form (table 2,3 and 4) has been summarized external and the interior primary and secondary pharmaceutical research of body. Mention in the table 2,3 and 4 batch key

With MPIF-1 batch of many compositions codings name of the form (such as maturation, total length or variant) of the indication organism of marking protein and expression product. The carrier (being the B=baculoviral, C=Chinese hamster ovary celI, E=Escherichia coli) that the letter of name hyphen back, end is indicated the organism of marking protein or is used for expressing. Form or the variant of last 3 expressed protein of numeral indication of hyphen front (are 300=total length MPIF-1,301=MPIF-1 Δ 17 variants, the amino terminal of the ripe MPIF-1 of 302=and mature amino acid sequence has added methionine residues, 304=MPIF-1 Δ 23 variants, 311=total length MPIF-1). Thereby whether the form of batch coded MPIF-1 albumen of name indication, host cell be with the form (if secretion) of secretory protein and secretory protein. For example, the total length MPIF-1 albumen of baculovirus vector expression is used in the HG00300-B5 indication. In addition, because the MPIF-1 of this system expression of use is subject to the processing of insect host cell, so the secreted form of this protein is ripe MPIF-1.

An exception of above-mentioned nomenclature is a batch HG00300-B7. This batch comprises the mixture of four kinds of different MPIF-1 polypeptide. The inventor thinks that the generation of these polypeptide is results of the proteolysis cutting MPIF-1 that occurs in the purge process. Embodiment 11 has discussed the MPIF-1 variant that exists among the HG00300-B7.

Table 2: elementary pharmacology-external

Experimental design	Cell type	MPIF-1 Δ 23 dosage	Chemical reagent	The result
Experimental design	Cell type	MPIF-1 Δ 23 dosage	Chemical reagent	The result	The impact (use mouse bone marrow cells) that MPIF-1 or MPIF-1 Δ 23 form colony	HPP-CFC LPP-CFC	0.01-100ng/ml	NA	MPIF-1 and MPIF-1 Δ 23 cause that all the dose dependent of LPP-CFC frequency reduces. MPIF-1 Δ 23 is all remarkable more effective than MPIF-1 in all test concentrations. Two kinds of isotypes all have no significant effect the frequency of HPP-CFC.
MPIF-1 Δ 23 is on the impact of artificial blood progenitor cell proliferation	CD34+, people's spinal cord blood	1-1000ng/ml	NA	MPIF-1 Δ 23 is processed and is caused cell survival to be subject to the inhibition of 20%-40%. Presentation of results MPIF-1 Δ 23 is myeloid progenitor inhibitory factors.		HPP-CFC LPP-CFC	0.01-100ng/ml	NA
	CD34+, people's spinal cord blood	1-1000ng/ml	NA		Mensuration is by the specific CFU-GM type of MPIF-1 Δ 23 targets	CD34+, the people	50ng/ml	NA	MPIF-1 Δ 23 suppresses the formation of (50%-64%) CFU-GM and CFU-Mix. The formation of BFU-E, CFU-G, CFU-M and CFU-Meg is not suppressed. The result is defined as MPIF-1 Δ 23 in the inhibitor of human granular leukocyte/monocyte precursory cell.
Characterize the inhibition effect of 23 pairs of mouse bone marrow cells of MPIF-1 Δ	Mouse bone marrow cells	50ng/ml	NA	MPIF-1 Δ 23 is down to 30% of contrast with the frequency of bone marrow cfu-gm colony. The frequency of LPP-CFC colony is down to 24% of contrast. MPIF-1 Δ 23 does not suppress the formation of CFU-E, BFU-E and HPP-CFC colony.		CD34+, the people	50ng/ml	NA
	Mouse bone marrow cells	50ng/ml	NA		Measure MPIF-1 Δ 23 protection pedigrees and exhaust the ability that the bone marrow cell group avoids the cytotoxic effect of 5-FU	Mouse bone marrow cells	NA	5-FU	MPIF-1 Δ 23 protects the LPP-CFC of 40%-50% to avoid the cytotoxicity of being induced by 5-FU. MPIF-1 Δ 23 is not protected HPP-CFC.

Table 3: in elementary pharmacology-body

Experimental design	Species	MPIF-1 batch	MPIF-1 dosage, scheme, path	Chemical reagent	Dosage, scheme, path	Terminal point
Experimental design	Species	MPIF-1 batch	MPIF-1 dosage, scheme, path	Chemical reagent	Dosage, scheme, path	Terminal point	Effect in the body of HPP-CFC and LPP-CFC frequency in MPIF-1 or MPIF-1 Δ 23 human peripheral bloods and the marrow	Mouse	HG00300-B5 HG00304-E2	0.5mg/kg, inject twice every day, interval 8 hours, totally 2 days, i.p.	NA	NA	MPIF-1 Δ 23 significantly reduces the LPP-CFC frequency in the marrow. MPIF-1 Δ 23 changes the impact of LPP-CFC frequency in the blood. MPIF-1 Δ 23 is on the not impact of HPP-CFC frequency.
Measure to be used for providing for the best MPIF-1 Δ 23 of the protective effect of the 5-FU cytotoxic effect scheme of taking medicine	Mouse	HG00304-E2	1mg/kg, variable between the-3 and 0 days, i.p.	5-FU	150mg/k g, the 0th day, i.p.	The-2 ,-1 and 0 day giving MPIF-1 Δ 23 avoids in the cytotoxic effect of 5-FU the most effective at protection marrow.		Mouse	HG00300-B5 HG00304-E2		NA	NA
	Mouse	HG00304-E2	1mg/kg, variable between the-3 and 0 days, i.p.	5-FU	150mg/k g, the 0th day, i.p.		MPIF-1 Δ 23 dose dependents that marrow recovers behind the mensuration 5-FU	Mouse	HG00304-E6	0.01-10mg/kg, the-2 ,-1,0 days, i.p.	5-FU	150mg/k g，i.p.	Observed the dosage dependence on the 4th day and reply, optimal recovery betides the lowest dose level (0.01mg/kg) of test. Do not observe dose response on the 6th day. Obtained bell dosage-response curve, and observed optimum activity at 0.1mg/kg in the 8th day.
Measuring MPIF-1 Δ 23 protects myeloid progenitor to avoid the ability of the infringement of cytotoxicity therapy in vivo	Mouse	HG00304-E6	1mg/kg, the-2 ,-1,0 days, i.p.	5-FU	150mg/k g, the 0th day, i.p.	The colony of the mouse bone marrow cells that the MPIF-1 Δ 23 of using by oneself is processed be formed on 5-FU process got back in rear 7 days normal. Form from the marrow colony of the mouse of only processing with 5-FU and not show recovery this moment.		Mouse	HG00304-E6	0.01-10mg/kg, the-2 ,-1,0 days, i.p.	5-FU	150mg/k g，i.p.
	Mouse	HG00304-E6	1mg/kg, the-2 ,-1,0 days, i.p.	5-FU	150mg/k g, the 0th day, i.p.		Measure MPIF-1 Δ 23 for the protectiveness effect of many circulations chemotherapy	Mouse	HG00304-E6	1mg/kg, the-2 ,-1,0,6,7,8 days	5-FU	100mg/k g, the 0th and 8 day, i.p.	MPIF-1 Δ 23 is protected CFU-GM behind the 5-FU of 2 circulations. Behind the 5-FU of the 2nd circulation, see the most remarkable protection. By the CD45 that derives of hematopoiesis in the blood⁺Cell number is increased in the chemotactic protection effect of showing MPIF-1 Δ 23 in the peripheral blood.
Measure MPIF-1 Δ 23 accelerates the ability mensuration MPIF-1 Δ 23 associating G-CSF of marrow colony, neutrophil cell and platelet recovery after many circulations chemotherapy activity	Mouse	HG00304-E6	1mg/kg, the-2 ,-1,0,6,7,8 days, i.p.	5-FU G-CSF	100mg/k g, the 0th and 8 day, i.p. 0.5mg/k g, the 1st, 2,3,9,10 and 11 day	According to the measurement of Gr-1 and the dual positive cell loss of Mac-1, to compare with the mouse of only processing with 5-FU, the degree that reduces disease with neutrophil cell in the mouse of MPIF-1 and 5-FU processing is lower, and regeneration rate is very fast. Cause the fast quick-recovery of dual positive cell in the blood with the G-CSF processing behind the 5-FU. The 8th day, G-CSF processed recovery extent in the mouse and significantly is lower than to process at MPIF-1 and observes in the mouse. Compare the positive cell that has the higher steady state level in the blood with the mouse of MPIF-1 and G-CSF processing with the mouse of only processing with MPIF-1 or G-CSF. The colony of processing mouse bone marrow cells from 5-FU forms remarkable reduction. MPIF-1 before the 5-FU processes and has eliminated the impact that 5-FU forms colony. With comparing of observing in the mouse of only processing with 5-FU, the platelet recovery of the mouse that usefulness MPIF-1 and 5-FU process is faster, stronger. Add not further impact of G-CSF.		Mouse	HG00304-E6	1mg/kg, the-2 ,-1,0,6,7,8 days	5-FU	100mg/k g, the 0th and 8 day, i.p.

Table 4: secondary pharmacology-external

Experimental design	Cell type	MPIF-1 batch	The MPIF-1 dosage range	The result
Experimental design	Cell type	MPIF-1 batch	The MPIF-1 dosage range	The result	The calcium migration that mensuration is caused by MPIF-1 or MPIF-1 Δ 23	T cell, B cell, monocyte, neutrophil cell, basophilic granulocyte, dendritic cells, NK cell, THP-1 cell	HG00300-B7 HG00302-E2 HG00302-E3 HG00304-E2 HG00304-E3 HG00304-E6 HG00304-E7 HG00301-C1 HG00311-C1	1-1000ng/ml	In monocyte and dendritic cells, observe detectable replying at 100ng/ml. Monocytic series THP-1 replys MPIF-1 Δ 23, has the greatest impact at 100ng/ml.
Measure the chemotactic activity of MPIF-1 Δ 23	T cell, monocyte, neutrophil cell, lymphocyte, eosinophil, basophilic granulocyte, NK cell, blood platelet	HG00300-B5 HG00300-B7 HG00302-E1 HG00302-E2 HG00303-E1 HG00304-E2 HG00304-E6 HG00304-E7	0.1-1000ng/ml	MPIF-1 Δ 23 stimulates the chemotaxis of tranquillization T cell, replys maximum at 10ng/ml. The monocyte of 23 pairs of fresh separated of MPIF-1 Δ has chemotaxis, has the greatest impact at 100ng/ml. In neutrophil cell, observe weak chemotactic response. In other cell of test, do not reply.				1-1000ng/ml
Measure the chemotactic activity of MPIF-1 Δ 23			0.1-1000ng/ml		MPIF-1 or MPIF-1 Δ 23 are on monocytic impact	Monocyte	HG00300-B7 HG00302-E1 HG00302-E2 HG00302-E3 HG00304-E3 HG00304-E6 HG00301-C1 HG00311-C1	0.5-1000ng/ml	MPIF-1 Δ 23 induces the low-level and variable lysosome N-acetyl-β of the monocyte of fresh separated-D-Glucose glycosides enzyme to discharge. MPIF-1 Δ 23 is on the not impact of release of lysosomal enzyme elastoser, Glucuronidase and myeloperoxidase. MPIF-1 Δ 23 is not induced monocyte secretion IL-1 β, TNF-α, IL-10 or IL-12. MPIF-1 Δ 23 is on oxidisability outburst or the not impact of cytotoxicity of the macrophage of activation.
MPIF-1 or MPIF-1 Δ 23 are on the impact of histamine release	Basophilic granulocyte, the people	HG00300-B5 HG00300-B7	1-1000ng/ml	MPIF-1 and MPIF-1 Δ 23 do not induce basophilic granulocyte to discharge histamine.	MPIF-1 or MPIF-1 Δ 23 are on monocytic impact	Monocyte		0.5-1000ng/ml

		HG0030-E2 HG00304-E6
		HG0030-E2 HG00304-E6			MPIF-1 or MPIF-1 Δ 23 are on by the cell-mediated impact that kills and wounds of NK	The NK cell, the people	HG00302-E1 HG00300-B7	1-100ng/ml	MPIF-1 and MPIF-1 Δ 23 be not on being affected by the cell-mediated K562 cell killing of NK that stimulated by IL-2.
MPIF-1 or MPIF-1 Δ 23 are on the impact of platelet aggregation	Blood platelet, the people	HG00302-E1 HG00300-B7	0.1-100ng/ml	MPIF-1 Δ 23 is not induced or is regulated and control platelet aggregation.		The NK cell, the people	HG00302-E1 HG00300-B7	1-100ng/ml
	Blood platelet, the people	HG00302-E1 HG00300-B7	0.1-100ng/ml		MPIF-1 Δ 23 is on the impact of non-transformed human cell growth	Fibroblast, astroglia, Schwann cell, smooth muscle cell, epithelial cell, vein and CMEC, marrow, B cell, T cell, monocyte, neutrophil cell, horn cell	HG00300-B7 HG00300-B5 HG00302-E1	0.1-1000ng/ml	The propagation of the listed cell of research is not induced, strengthens or suppressed to MPIF-1 Δ 23.
MPIF-1 or MPIF-1 Δ 23 are on the impact of IL-6 and prostaglandin release	People's endothelial cell of former generation, lung fibroblast and aortic smooth muscle cell	HG00300-B5 HG00300-B7 HG00302 E1 HG00300-E2 HG00304-E2 HG00301-C1	0.1-1000ng/ml	MPIF-1 and MPIF-1 Δ 23 discharge not impact to IL-6 or prostaglandin.			HG00300-B7 HG00300-B5 HG00302-E1	0.1-1000ng/ml
			0.1-1000ng/ml		The impact that MPIF-1 Δ 23 forms capillary	Former generation CMEC	HG00304-E2	0.1-100ng/ml	MPIF-1 Δ 23 is not induced formation capillaceous external.
MPIF-1 is on the impact of the ability of tumor cell invasion confluent monolayer endothelial cell	Former generation endothelial cell	HG00300-B7	0.1-10ng/ml	Not impact	The impact that MPIF-1 Δ 23 forms capillary	Former generation CMEC	HG00304-E2	0.1-100ng/ml	MPIF-1 Δ 23 is not induced formation capillaceous external.
	Former generation endothelial cell	HG00300-B7	0.1-10ng/ml	Not impact	MPIF-1 or MPIF-1 Δ 23 human peripheral blood monocytes or GA are by the impact of the endothelium of IL-1 activation	Former generation endothelial cell	HG00300-B5 HG00300-B5 HG00304-E6 HG00304-E7 HG00301-C1	0.1-100ng/ml	Not impact

Embodiment 14: use the pHE4-5 expression vector to generate, reclaim and purifying MPIF-1 Δ 23

MPIF-1 is novel people's beta-chemokine. The mature form of MPIF-1 is as 99 amino acid whose peptide secretions, molecular weight 11.2kDa. Identified also that in the initial expression study of MPIF-1 length is 76 amino acid whose truncated-types (MPIF-1 Δs 23). In baculovirus expression system, separate subsequently and subclone MPIF-1 Δ 23. Biological assay indication truncated-type more has activity than total length homologue. Clone and expression

MPIF-1 Δ 23 genes that separated by the aorta inner skin cDNA library at first are subcloned into Single restriction enzyme cleavage site NdeI and Asp718 place (Figure 31) of expression vector pHE4, and be transformed into the coli strain SG 13009 that is derived by K12 and (can be obtained by Susan Gottesman, National Health Association, National Institutes of Health, Bethesda, MD) in. Other coli strain that uses pHE4 to carry out the suitable host of protein expression be can take on and bacterial strain DH5 α and W3110 (ATCC numbering 27325) comprised. The pHE4 carrier comprises strong synthetic promoter and two lac operators. The expression of this promoter is subject to the regulation and control of the existence of lac containment thing, and can use isopropyl ss-D-thiogalactoside (IPTG) or lactose-induced. This plasmid also comprises the synthetic transcription terminator in effective ribosome bind site and MPIF-1 Δ 23 gene downstreams. This carrier also comprises the duplicate field of pUC plasmid and causes transform bacteria to have the neomycin phosphotransferase gene of kalamycin resistance. Manufacture method

The general introduction of sweat. The sweat of MPIF-1 Δ 23 outlines into following phases and is illustrated in Figure 32.

The essentialspecies word bank. The colibacillary main cell storehouse (MCB) of the Plasmid Transformation of expressing MPIF-1 Δ 23 is used in preparation under general Good Manufacturing Practices condition. In glycerinated culture medium, prepare the storehouse as the refrigeration thing, frozen in-80 ℃. After the preparation, MCB is to guarantee not exist the pollution of bacteriophage or other microorganism in test.

The first sub. Preparation the first sub culture in inoculum is housed prepares the band flask with indentation of culture medium. Inoculate shaking flask with 1: 2000 dilution factor with the seed original seed that thaws, and place shaking table, kept 12 hours in 37 ℃ with 225rpm.

Production phase. Be equipped with DO₂, pH, temperature and nutrient fodder control 100 liters of feed supplements-batch fermentation tank in preparation production phase culture. With the first sub culture inoculation productive culture base (37 ℃), so that 600nm optical density (OD) reaches 0.20U/ml. Work as OD₆₀₀When reaching 10 ± 2U/ml, add IPTG (final concentration 20mM) and express with induced protein. Induce rear 4 hours harvestings.

The cell harvesting stage. Use the continuous flow type centrifuge by the centrifugal recovery of 18,000g bacterium. The cell mass that obtains is frozen in-80 ℃. The recovery of MPIF-1 Δ 23

The recovery of MPIF-1 Δ 23 is outlined in Figure 33.

Cell lysis. Bacillus coli cells group is thawed and be resuspended in the resuspended buffer solution of 10 times of volumes. Then through (twice) the homogenizer smudge cells by 7000psi.

Clean inclusion body. In cell lysate, add NaCl to final concentration 0.5M, then use 0.45 μ m filter membrane by concentrated 2 times of grossflow filtration. With cleaning-2 buffer solution (100mM Tris-HCl, 500mM NaCl and the 25mM EDTA-Na of residue retentate at 3 times of volumes₂) in dialysis, subsequently at cleaning-1 buffer solution (100mM Tris-HCl, the 25mM EDTA-Na of 1 times of volume₂) middle dialysis. With cleaning-1 buffer solution retentate is diluted 2 times, and collect insoluble fraction by continuous centrifugal. Perhaps, can pass through the eccentric cleaning inclusion body.

The dissolving inclusion body. The precipitation that obtains after centrifugal is suspended in dissolving buffer solution (100mM Tris-HCl, 1.75M guanidine hydrochloride and the 25mM EDTA-Na that is equal to 9 times of compacting inclusion body volumes₂). At first suspension was stirred 2-4 hour in room temperature, then stirred 12-18 hour in 2 ℃-10 ℃.

Again folding. Suspension is centrifugal, and collect supernatant, with refolding buffer solution (100mM sodium acetate, 125mM NaCl and the 2mM EDTA-Na of 9 times of volumes₂) mix. Diluted material is placed about 2 hours (2 ℃-10 ℃) make the precipitation sedimentation. Filtering material, then can process immediately or frozen reach 72 hours then processing. Purifying

The HS-50 cation-exchange chromatography. The purifying of MPIF-1 Δ 23 is outlined in Figure 34. The filter liquor application of sample is extremely used the POROS HS-50 post of 50mM NaOAc, 150mM NaCl pH5.8-6.2 balance. To use the substep mode elute protein of NaCl (300-1500nM). Merge the fraction with 500mM NaCl wash-out, and 2 times of dilute with waters are used for injection.

HQ-50/CM-20 Anionic/Cationic displacement chromatography. With the merging fraction application of sample that obtains behind the HS-50 chromatography to the columns in series with CM-1 buffer solution balance (before this HQ-50 post, then be the CM-20 post). With NaCl (100-900mM) by CM-20 wash-out MPIF-1 Δ 23. Analyze the fraction of wash-out by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and RPHPLC (HPLC), merge the fraction that those contain MPIF-1 Δ 23, and by ultrafiltration or flow through the HS-50 post extra and concentrate.

Size exclusion chromatography. CM-20 eluent application of sample is extremely used the Sephacryl-100 HR of S-100 buffer solution balance. Collect fraction and analyze by SDS-PAGE and reversed-phase HPLC. Merge the fraction that contains MPIF-1 Δ 23, use 0.2 μ m membrane filtration degerming, and be stored in 2 ℃-10 ℃. The specification of bulk materials

Determined the specification that table 5 is listed for MPIF-1 Δ 23 in bulk

Describe	Specification
Describe	Specification	Outward appearance	Clarification, colourless solution
pH	5.8±0.2	Outward appearance	Clarification, colourless solution
pH	5.8±0.2	Protein concentration (BCA)	1-5mg/ml
The non-reduced condition of purity * reversed-phase HPLC size exclusion HPLC SDS-PAGE (Coomassie blue stain) reducing condition	≥90％ ≥90％ ≥90％ ≥90％	Protein concentration (BCA)	1-5mg/ml
	≥90％ ≥90％ ≥90％ ≥90％	Residual DNA	≤ 100pg/mg protein
Endotoxin king crab ameboid cell lysate gel grumeleuse	≤ 10EU/mg protein	Residual DNA	≤ 100pg/mg protein
Endotoxin king crab ameboid cell lysate gel grumeleuse	≤ 10EU/mg protein	Bioassary method (is passed through Ca²⁺Migration assay is assessed)	Report the result

* the purity of MPIF-1 Δ 23 preparations will compare with marker, and its specification defines at present. The specification of drug products

The drug products of finishing reaches in the table 5 about all specifications of bulk materials, also tests aseptic (12CRF610.12). Embodiment 15: inhibition and MPIF-1 that colony is formed by 23 mediations of MPIF-1 Δ mobilize Ca in the born of the same parents in monocyte²⁺Ability relevant

MPIF-1 Δ 23 suppresses the LPP-CFC colony and forms in external soft agar determination method, and induces the cellular calcium migration in monocyte (comprising the THP-1 cell, i.e. people's myelomonocyte system). Two kinds of determination methods are all for the BA at purifying and stability study assessment MPIF-1 Δ 23. In the LPP-CFC determination method, the mouse bone marrow cell of fresh separated is coated in the soft agar that has cytokine profiles (5ng/ml IL-3,50ng/ml SCF, 5ng/ml M-CSF and 10ng/ml IL-1 α). With culture insulation 14 days, then use inverted microscope that colony is marked.

The calcium migration assay is used the person monocytic cell of fresh separated or the THP-1 cell that loads with Fura-2 (0.2nM/ 1,000,000). After with MPIF-1 Δ 23 irritation cells, by fluorescence photometer assessment Ca²⁺Migration. Ca²⁺Migration assay provides the quick indicator (table 6) of MPIF-1 Δ 23 preparation activity. Table 6: inhibition and MPIF-1 that colony is formed by 23 mediations of MPIF-1 Δ mobilize Ca in the born of the same parents in monocyte²⁺Ability relevant

The MPIF-1 construction/batch/situation	Ca ²⁺Migration (ng/ml) *	LPP-CFC inhibition (ng/ml)+
The MPIF-1 construction/batch/situation	Ca ²⁺Migration (ng/ml) *	LPP-CFC inhibition (ng/ml)+	MPIF-1/HG00300-B5	1000	20-40
MPIF-1 Δ 23/HG00304-E2 preserved 3 months in 4 ℃	100	5-10	MPIF-1/HG00300-B5	1000	20-40
MPIF-1 Δ 23/HG00304-E2 preserved 3 months in 4 ℃	100	5-10	MPIF-1 Δ 23/HG00304 preserved for 1 week	100	5-10
MPIF-1 Δ 23/HG00304 preserved for 4 weeks	100	5-10	MPIF-1 Δ 23/HG00304 preserved for 1 week	100	5-10
MPIF-1 Δ 23/HG00304 preserved for 4 weeks	100	5-10	MPIF-1/HG00302-E2 preserved 3 months in 4 ℃	1000	＞100
MPIF-1 Δ 23/HG00304-E3, first peak of CM post	100	5-10	MPIF-1/HG00302-E2 preserved 3 months in 4 ℃	1000	＞100
MPIF-1 Δ 23/HG00304-E3, first peak of CM post	100	5-10	MPIF-1 Δ 23/HG00304-E4, second peak of CM post	100	5-10
MPIF-1 Δ 23/HG00304-E3, the 3rd peak of CM post	＞1000	＞1000	MPIF-1 Δ 23/HG00304-E4, second peak of CM post	100	5-10

* mobilize the desired least concentration of calcium in person monocytic cell and/or the THP-1 cell. + compared with the control the LPP-CFC colony is formed the concentration that produces 50% inhibition. Preparation and preservation

Aseptic manufacturing MPIF-1 Δ 23 in bulk, and liquid formulations is aseptic, nonrecoverable product. With 50mM sodium acetate, 125mM NaCl pH5.8 buffering protein matter, and install in the 5ml Wheaton I type vial, in 2 ℃ of-8 ℃ of preservations. Stability

Working concentration 1.0mg/ml, with sodium acetate pH5,6 and 7 bufferings, temperature-80 ℃, 2 ℃-8 ℃, 20 ℃-25 ℃ and 2 ℃-8 ℃ protein carry out stability study. Find, in the solution of 10mM sodium acetate, 125mM NaCl pH5-7 in or be lower than 2 ℃-8 ℃ when preserving, MPIF-1 Δ 23 is stable at least 6 months. In the current research of carrying out, will be to sample determination outward appearance, protein concentration, purity (SDS-PAGE (reduction and non-reduced); Anti-phase and size exclusion HPLC) and active (Ca²⁺The migration bioassary method) to reach the specification of previous outline.

At first the MPIF-1 Δ 23 batches (HG00304-E10) that is used for clinical front Study of cytotoxicity is carried out stability study. To be mixed with protein concentration 4.0mg/ml at 50mM NaOAc, 125mM NaCl pH5.9 for 23 batches of the MPIF-1 Δs of these researchs. Preservation condition is-80 ℃, 2 ℃-8 ℃, 25 ℃ and 37 ℃ and relative humidity 60%, and 45 ℃ and relative humidity 75%. The duration of stability study is 12 months in the temperature of height to 25 ℃, and 37 ℃ is 6 months, and 45 ℃ is 1 month. To measure outward appearance, pH, protein concentration, purity (SDS-PAGE (reduction and non-reduced) to stability; Anti-phase and size exclusion HPLC) and active (Ca²⁺The migration bioassary method). To carry out endotoxin measurement method and biological load test at some seclected time. Embodiment 16:MPIF-1 protection intestines and stomach avoid the cytotoxicity by taxol induced

0th, use simultaneously 0.3mg/kg MPIF-1 Δ 23 (subcutaneous) and 10.5mg/kg taxol (in the peritonaeum) in 1 and 2 day, can protect rat to avoid the weight loss relevant with intestines toxicity. Kept body weight at the 0th, 5 day with the rat that 0.3mg/kg MPIF-1 Δ 23 is processed, and the in fact weightening finish at the 9th day. The rat of accepting 0.1mg/kg MPIF-1 Δ 23 or not accepting MPIF-1 Δ 23 was lost about 45g body weight at 0-5 days.

As above, the 0th, 1 and 2 day administered with paclitaxel (Taxol ) and MPIF-1 Δ 23 the two. Each rat body weight is about 185g in the-2 days (namely for the first time administered with paclitaxel and MPIF-1 front 2 days) all groups. By the 5th day, the whose body weight in two test groups (accept taxol and do not accept MPIF-1 Δ 23 or accept 0.1mg/kg MPIF-1 Δ 23) was about 155g. On the contrary, the whose body weight of accepting 0.3mg/kg MPIF-1 Δ 23 is about 175g. 5 days bodies of individuality to the in the control group (neither accepting the rat that taxol is not accepted MPIF-1 Δ 23 yet) about 195g that weighs. The watch for animals ability of the Acute Toxicity that avoids taxol of MPIF-1 has proved that it has the ability that the many cell types of protection (except hematopoietic stem cells, also having such as GI cell) avoid the infringement of cytotoxic agent. Embodiment 17: the deadly model in the Asia of stomach and intestine protection

The 0th day, C57B1/6 female mice (12 age in week) is exposed to total 9Gy137 caesium sublethal exposure (dose rates: 25.54cGy/min), deliver interval 4 hours with twice equal dose 4.5Gy. One group of mouse continuous 4 days (the-2 to+1 days) to called after " MPIF-1 (front) " gives MPIF-1 (1mg/kg/BID i.p.). One group continuous 7 days (by irradiation beginning in namely the 1st day in rear 1 day) to called after " MPIF-1 (afterwards) " gives MPIF-1 (1mg/kg/BID i.p.). Control group is only accepted diluent (the common mice serum of HBSS+0.1%). Drink acidifying water in 7 days all mouse at pre-irradiation. Pre-irradiation 3 days, in acidifying water, add Amoxicillin (0.5mg/ml), continue until shone rear 7 days. Survival, situation and the changes of weight of monitoring mouse. According to the scheduled date each mouse body weight with respect to the percentage of when beginning experiment body weight, data are shown as every group changes of weight percentage (Figure 37-38). The watch for animals ability of the Acute Toxicity that avoids shining of MPIF-1 has proved that it has the ability that the many cell types of protection (except hematopoietic stem cells, also having such as gastrointestinal tract cell) avoid the infringement of cytotoxic agent. Embodiment 18: the deadly model of stomach and intestine protection

The 0th day, C57B1/6 female mice (12 age in week) is exposed to total 11Gy137 caesium lethal exposure (dose rates: 25.54cGy/min), deliver interval 4 hours with twice equal dose 5.5Gy. One group of mouse continuous 4 days (the-2 to+1 days) to called after " MPIF-1 (front) " gives MPIF-1 (1mg/kg/BID i.p.). One group continuous 7 days (by irradiation beginning in namely the 1st day in rear 1 day) to called after " MPIF-1 (afterwards) " gives MPIF-1 (1mg/kg/BID i.p.). Control group is only accepted diluent (the common mice serum of HBSS+0.1%). Drink acidifying water in 7 days all mouse at pre-irradiation. Pre-irradiation 3 days, in acidifying water, add Amoxicillin (0.5mg/ml), continue until shone rear 7 days. Survival, situation and the changes of weight of monitoring mouse. According to the scheduled date each mouse body weight with respect to the percentage of when beginning experiment body weight, data are shown as every group changes of weight percentage (Figure 39-40).

All control groups (not accepting MPIF-1) are to shining rear the 20th day all in the dust. On the contrary, the MPIF-1 of 25 % (afterwards) group and 57% MPIF-1 (front) group were still survived after irradiation on the 38th day. The watch for animals ability of the infringement that avoids lethal exposure of MPIF-1 has proved that it has the ability that the many cell types of protection (such as gastrointestinal tract cell) avoid the infringement of cytotoxic agent. Embodiment 19: the method for measuring cytoprotection

Be used for measuring MPIF-1 is assessment dosage-reduction factor or dosage-modifying factor (DMF) (Weiss, Environ. Health Perspectives, 105:1473-1478,1997 to a kind of method of the relative protective capability of specific cells toxic agents; The people such as Brown, Pharmacol. Ther., 39:157-168,1988; The people such as Yuhas, Int.J.Radiat.Biol., 15:233-237,1973; The people such as Weiss, in " Radiation and the Intestinal Tract " (radiation and enteron aisle) book, the people such as Dubois compile, Boca Raton, FL, CRC publishing house, 183-199 page or leaf, 1995).

For example, with the dose irradiation mouse of certain limit, and use or do not use MPIF-1. DMF by survival in 6-7 days after the full-body exposure of high dose relatively measures MPIF-1 to GI protection. The DMF of survival in 30 days measures MPIF-1 to the protection of hemopoietic system. Embodiment 20: make up N end and/or C end deletion mutant

Following conventional method can be used for cloning N end or C end MPIF-1 deletion mutant. Generally speaking, by the derive Oligonucleolide primers of two kinds of about 15-25 nucleotides of the expectation 5 ' of the polynucleotides of SEQ ID NO:1 or 6 and 3 ' position. 5 ' and 3 ' position of primer is definite according to expecting the MPIF-1 polynucleotide passage. If necessary, the initial sum terminator codon can be added respectively, to express the MPIF-1 polypeptide fragment by the polynucleotide passage coding in 5 ' and 3 ' primer. Preferred MPIF-1 polynucleotide passage be above in " MPIF-1 polypeptide " specification part those disclosed coding N end and C hold the polynucleotide passage of deletion mutant.

Can also in 5 ' and 3 ' primer sequence, add the extra nucleotides that comprises restriction site so that in the expectation carrier, clone the MPIF-1 polynucleotide passage. Use condition suitable PCR Oligonucleolide primers and this paper discussion or that this area is known, by genomic DNA or by preservation cDNA clonal expansion MPIF-1 polynucleotide passage. The MPIF-1 polypeptide fragment that general fashion that can be identical with full-length polypeptide is expressed and purifying is encoded by MPIF-1 polynucleotide passage of the present invention, although because the chemistry between specific fragment and the full-length polypeptide and the difference of physical characteristic, revise in the path may be essential.

Inventive example expressivity and nonrestrictive a kind of method is the polynucleotides of following amplification and clones coding MPIF-1 polypeptide fragment R46-N120: generate 5 ' primer, it comprises restriction enzyme sites, is that the polynucleotide sequence of the polypeptide fragment N end parts that begins with coding R46 is in the initiation codon in the identical reading frame subsequently. Generate 3 ' complementary primer, it comprises restriction enzyme sites, is that the polynucleotide sequence of the MPIF-1 polypeptide fragment C end parts that finishes with N120 with coding is in the terminator codon in the identical reading frame subsequently.

Polynucleotide passage and expression vector with the restriction enzyme digest amplification of identifying the contained site of primer. Then will connect together through the polynucleotides of digestion. With the expression vector after the MPIF-1 polynucleotide passage insertion restrictive diges-tion, preferably MPIF-1 polypeptide fragment code area is placed the downstream of promoter. The Application standard flow process also described in this paper embodiment, will connect mixture and be transformed in the Bacillus coli cells. Also confirmed the identity of cloned DNA by restriction analysis, PCR and dna sequencing by resistance bacterium colony isolated plasmid dna. The protein of embodiment 21:MPIF-1 merges

Preferably the MPIF-1 polypeptide is merged other oroteins. These fusions can be used for multiple application. For example, the fusion of MPIF-1 polypeptide and His label, HA label, albumin A, IgG domain and maltose-binding protein helps purifying (to consult embodiment 5; Also can consult EP A 394,827; The people such as Traunecker, Nature, 331:84-86,1988). Similar, can increase half life in the body with IgG-1, IgG-3 and albuminised fusion. The nuclear localization signal that merges with the MPIF-1 polypeptide can be with the specific subcellular location of protein target, and covalency heterodimer or homodimer can increase or reduce the activity of fusion. Fusion also can produce has the chimeric molecule that surpasses a kind of function. At last, compare with non-fusion, fusion can increase its dissolubility and/or stability. Scheme generates all types of fusion mentioned above described in following scheme that can be by revise outlining polypeptide and the fusion of IgG molecule or the embodiment 2.

In brief, can use 5 ' and 3 ' the terminal primer of crossing over sequence hereinafter described by the people Fc part of pcr amplification IgG molecule. These primers also should have easily restriction enzyme sites, so that be cloned in the expression vector preferred mammal expression vector.

For example, if use pC4 (numbering 209646), people Fc partly can be connected in the BamHI cloning site so. Attention should destroy 3 ' BamHI site. Then, with BamHI again restrictive diges-tion contain the carrier of people Fc part, with the carrier linearisation, and will be connected in this BamHI site by the MPIF-1 polynucleotides that PCR scheme described in the embodiment 1 is separated. The polynucleotides of noting the clone do not contain terminator codon, otherwise will can not generate fusion.

If use the burst of natural generation to become secretory protein next life; PC4 does not need second signal peptide so. Perhaps; if do not use the burst of natural generation, those can modify carrier to comprise allos burst ( consulting for example WO 96/34891 ) . IgG Fc： GGGATCCGGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGCCC AGCACCTGAATTCGAGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCC AAGGACACCCTCATGATCTCCCGGACTCCTGAGGTCACATGCGTGGTGGTGG ACGTAAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGT GGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACG TACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCA AGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAACCCCCATCGAGAA AACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTG CCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGG TCAAAGGCTTCTATCCAAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCA GCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCC TTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGA ACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCA GAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGTGCGACGGCCGCGACTCTAG AGGAT ( SEQ ID NO：42 )

In addition, can be with one or more elements of MPIF-1, primitive, section, partly, one or more elements of domain, fragment etc. and one or more heterologous molecule, primitive, section, partly, the restructuring such as domain, fragment. In preferred embodiments, heterologous molecule is the chemotactic factor (CF) family member. In another preferred embodiment, heterologous molecule is growth factor, such as by platelet-derived growth factor (PDGF), IGF (IGFI), TGF (TGF)-α, EGF (EGF), fibroblast growth factor (FGF), TGF-β, bone morphogenetic protein (BMP)-2, BMP-4, BMP-5, BMP-6, BMP-7, activin A and B, decapentaplegic (dpp), 60A, OP-2, dorsalin, growth and differentiation factor (GDF), tubercle, MIS, inhibin-α, TGF-β 1, TGF-β 2, TGF-β 3, TGF-β 5 and the neurotrophic factor (GDNF) of being derived by neuroglia. Embodiment 22: the generation of antibody

Can prepare antibody of the present invention (consulting " Current Protocols " (general scheme), the 2nd chapter) by several different methods. As an example of these methods, can use the cell of expressing MPIF-1 to animal, to induce the generation of the serum that contains polyclonal antibody. In a preferred method, preparation and purifying MPIF-1 protein formulation make it to be substantially free of natural pollutant. Then this preparation is imported animal, to generate the more polyclonal antiserum of high specific acitivity of tool.

In most preferred method, antibody of the present invention is monoclonal antibody (or its protein bound fragment). Can use hybridoma technology to prepare these monoclonal antibodies (people such as K hler, Nature, 256:495,1975; The people such as K hler, Eur.J.Immunol., 6:511,1976; The people such as K hler, Eur.J.Immunol., 6:292,1976; The people such as Hammerling, in " Monoclonal Antibodies and T-Cell Hybridomas " (monoclonal antibody and T quadroma) book, Elsevier, New York, 563-681 page or leaf, 1981). Generally speaking, these flow processs comprise uses the MPIF-1 polypeptide, more preferably uses the cellular immunity animal (preferred mouse) of expression-secretion type MPIF-1 polypeptide. Can in any suitable culture medium for tissue culture, cultivate these cells; Yet, preferably adding 10% hyclone (in about 56 ℃ of deactivations) and adding about 10g/L nonessential amino acid, about 1, the EarleShi of 000U/ml penicillin and about 100 μ g/ml streptomysins revises cultured cell in the EagleShi culture medium.

Extract the splenocyte of these mouse and merge suitable myeloma cell line. Can adopt any suitable myeloma cell line according to the present invention; Yet preferably adopt parent myeloma cell lines (SP20) (can be obtained by ATCC). After the fusion, in the HAT culture medium, selectively keep the hybridoma of generation, then such as people such as Wands, Gastroenterology, 80:225-232,1981 describedly clone by limiting dilution. Then the hybridoma that this selection of upchecking obtains can be in conjunction with the clone of the antibody of MPIF-1 polypeptide to identify secretion.

Perhaps, can use anti-idiotype, generating in two step flow processs can be in conjunction with other antibody of MPIF-1 polypeptide. It also is antigen thereby might obtain can be in conjunction with the fact of the antibody of the second antibody that this method is utilized antibody self. According to this method, protein-specific antibody is used for immune animal, preferred mouse. Then the splenocyte of this animal is used for generating hybridoma, and the screening hybridoma with identify the antibody that the generated clone that can be blocked by MPIF-1 in conjunction with the ability of MPIF-1 protein specific antibody. These antibody comprise the anti-idiotype for the MPIF-1 protein specific antibody, and can be used for immune animal to induce the formation of other MPIF-1 protein specific antibody.

Can understand, can use according to method disclosed herein Fab and (Fab ') of antibody of the present invention₂With other fragment. The common use of these fragments such as papain (generation Fab fragment) or trypsase (generation F (ab ')₂Fragment) generates by proteolysis cutting. Perhaps, can be by using recombinant DNA technology or becoming secreting type MPIF-1 protein combination fragment next life by the synthetic chemistry method.

For purposes in the body of antibody in the people, may preferably use " humanization " chimeric mAb. Can generate these antibody with the genetic constructs of being derived by the hybridoma that generates monoclonal antibody mentioned above. The method that be used for to generate chimeric antibody in this area be know (summary is consulted Morrison, Science, 229:1202,1985; The people such as Oi, BioTechniques, 4:214,1986; The people such as Cabilly, U.S. Patent number 4,816,567; The people such as Taniguchi, EP 171,496; The people such as Morrison, EP 173,494; The people such as Neuberger, WO 86/01533; The people such as Robinson, WO 87/02671; The people such as Boulianne, Nature, 312:643,1984; The people such as Neuberger, Nature, 314:268,1985). By the antibody fragment of scFv library Separated pin to MPIF-1

The large-scale library of the gene constructed one-tenth antibody fragment of natural generation V that will be separated by people PBL, described antibody fragment comprises the reactivity of the MPIF-1 that may or fail to expose for donor and (consults for example U.S. Patent number 5,885,793, complete being collected herein by reference).

The redemption in library. Described in WO 92/01047, by the RNA structure scFv storehouse of people PBL. In order to save the bacteriophage of showing antibody fragment, use about 10⁹The individual Escherichia coli inoculation 50ml that comprises phasmid contains the 2xYT (2xYT-AMP-GLU) of 1% glucose and 100 μ g/ml ampicillins and shakes and is cultured to O.D. and reaches 0.8. With this culture inoculation of 5ml 50ml2xYT-AMP-GLU, add 2 * 10⁸The helper phage of TU missing gene 3 (M13 Δ gene III consults WO 92/01047), and with culture in 37 ℃ do not shake the insulation 45 minutes, then in 37 ℃ shake the insulation 45 minutes. With culture with 4,000rpm centrifugal 10 minutes, and precipitation is resuspended in 2 liters of 2xYT that contain 100 μ g/ml ampicillins and 50 μ g/ml kanamycins, overnight incubation. Described in WO 92/01047, prepare bacteriophage.

Therefore be prepared as follows not encoding gene III albumen of M13 Δ gene III:M13 Δ gene III helper phage, show that the affinity of bacteriophage (phasmid) conjugated antigen of antibody fragment is larger. Generate infectious M13 Δ gene III particle by cultivate helper phage in the cell that comprises the pUC19 derivative, described pUC19 derivative provides wild type gene III albumen in morphology of phages generating process. With culture in 37 ℃ do not shake the insulation 1 hour, then shake in 37 ℃ and be incubated again 1 hour. Centrifugal sedimentation cell (IEC-Centra 8,4000rev/min, 10min) is resuspended in the 2xYT meat soup (2xYT-AMP-KAN) that 300ml contains 100 μ g/ml ampicillins and 25 μ g/ml kanamycins, and shakes overnight incubation in 37 ℃. By culture medium purifying and concentrated phage particle, be resuspended in 2ml PBS by twice PEG precipitation people such as (, 1990) Sambrook, and with 0.45 μ m filter membrane (Minisart NML; Sartorius) filter, the final concentration that obtains is about 10¹³Transduced unit/ml (amicillin resistance clone).

The elutriation in library. In PBS, spend the night with 4ml 100 μ g/ml or the coated immunity pipe of 10 μ g/ml polypeptide of the present invention (Nunc). Effective 2% Marvel-PBS in 37 ℃ of sealings 2 hours, is then cleaned 3 times with PBS. With about 10¹³The TU bacteriophage is added in the pipe, and in room temperature insulation 30 minutes, over and under rotating disk, then uprightly placed 1.5 hours. Effective PBS/0.1% polysorbas20 is cleaned 10 times, clean 10 times with PBS again. Add 1ml 100mM triethylamine, rotation is 15 minutes under rotating disk, then immediately with 0.5ml 1.0M Tris-HCl pH7.4 neutralization, thus wash-out bacteriophage. Then arise from 37 ℃ of insulations 30 minutes by bacteriophage and bacterium one with wash-out, and bacteriophage is used for infecting 10ml mid-log phase e. coli tg1. Then Escherichia coli are coated on the TYE flat board that contains 1% glucose and 100 μ g/ml ampicillins. Then save the bacterium library that produces with Δ gene 3 helper phages as mentioned above, with for the preparation of the bacteriophage of screening wheel subsequently. Then this process is repeated altogether 4 to take turns affinity purification, take turns the 3rd and 4 and use the wash number of PBS, 0.1 % polysorbas20 to be increased to 20 times, use the wash number of PBS to be increased to 20 times.

The sign of bond. To take turns the bacteriophages of selecting wash-out by the 3rd and 4 and be used for ehec infection HB 2151, and generate soluble scFv people such as (, 1991) Marks by single bacterium colony and be used for measuring. Carry out ELISA with microtiter plate, described plate is coated with the 10pg/ml polypeptide of the present invention that is dissolved in 50mM bicarbonate pH9.6. Then check order by PCR fingerprint technique (consulting WO 92/01047) and further to characterize the clone of the ELISA positive. Embodiment 23: the method that treatment MPIF-1 level reduces

The present invention relates to be used for the treatment of the method that needs the individuality of polypeptide level of the present invention in the rising body, comprise described individuality is used the composition that comprises the activator of the present invention (comprising polypeptide of the present invention) for the treatment of effective dose. In addition, can understand, can treat the situation that is caused by MPIF-1 standard or the reduction of normal expression level in the individual body by using MPIF-1 (preferred secreted form). Thereby the present invention also provides treatment to need the method for the individuality of rising MPIF-1 polypeptide level, comprise described individuality used the therapeutic agent that comprises a certain amount of MPIF-1, thereby in described individual body the activity level of rising MPIF-1.

For example, the patient that MPIF-1 polypeptide level reduces can accept every daily dose 0.1-100 μ g polypeptide/kg body weight, reaches continuous 6 days. Preferably, polypeptide is secreted form. Provide in this paper " pharmaceutical composition " part based on the fine detail of using with the scheme of taking medicine of preparation. Embodiment 24: the method that treatment MPIF-1 level raises

The invention still further relates to the method that treatment need to reduce the individuality of polypeptide level of the present invention in the body, comprise described individuality is used the composition that comprises the antagonist of the present invention (comprising polypeptide of the present invention and antibody) for the treatment of effective dose.

In one embodiment, suppress the generation of MPIF-1 with antisense technology. This technology is to reduce an example of the method for MPIF-1 polypeptide (preferred secreted form) level because of Different types of etiopathogenises.

For example, with 0.5,1.0,1.5,2.0 with diagnosis was suffered from patient's intravenous that the MPIF-1 horizontal abnormality raises in 3.0mg/kg/ days and use antisense polynucleotides, reach 21 days. If the tolerance for the treatment of is fine, repeat this treatment after 7 day rest period so. This paper " pharmaceutical composition " part provides the preparation of antisense polynucleotides. Embodiment 25: the methods for the treatment of of using the gene therapy that exsomatizes

A kind of method of gene therapy is that the Allogenic Cultured Dermal Fibroblasts Transplantation that can express the MPIF-1 polypeptide arrives with it the patient. Generally speaking, fibroblast is obtained by the experimenter by Skin biopsy. The tissue that obtains is placed culture medium for tissue culture and is divided into fritter. The fritter tissue is placed on the wetted surface of tissue culture flasks, place about 10 in each bottle. Bottle is put upside down, tightened, and at room temperature place and spend the night. After at room temperature 24 hours, blake bottle is put upside down, at the bottom of tissue block still is fixed on bottle, added fresh culture (such as HamShi F12 culture medium, containing 10%FBS, penicillin and streptomysin). Then it is incubated about 1 weeks in 37 ℃. At this moment, add fresh culture, per a couple of days is changed once subsequently. After cultivating for 2 weeks, the individual layer fibroblast has appearred again. With the Trypsin Induced individual layer and scrape in the larger blake bottle.

Can use the listed cDNA that corresponds respectively to the PCR primer amplification coding MPIF-1 of 5 ' and 3 ' terminal sequence among the embodiment 1. Preferably, 5 ' primer comprises the EcoRI site, and 3 ' primer comprises the HindIII site. Under the condition that has the T4 dna ligase, that the EcoRI-HindIII fragment of equivalent Moloney murine sarcoma virus linear backbone and amplification is added together. Keep the mixture that obtains being suitable for connecting under the condition of two fragments. With connecting mixture transform bacteria HB101, then coat on the agar that contains kanamycins, comprise the MPIF-1 of correct insertion to confirm carrier.

In the Eagles culture medium (DMEM) that the DulbeccoShi that contains 10% cow's serum (CS), penicillin and streptomysin revises, facultative pA317 or GP+aml2 incasing cells are grown in tissue is cultivated converge density. Then in culture medium, add the MSV carrier that comprises the MPIF-1 gene, use the carrier transduction incasing cells. Now, incasing cells generates the infectious viral particle (be called producer cell with incasing cells this moment) that comprises the MPIF-1 gene.

To in producer's cell of transduction, adding fresh culture, subsequently by the dull and stereotyped culture medium of gathering in the crops of the 10cm of the producer's cell that converges. The culture medium that has consumed that will contain infectious viral particle to remove producer's cell of desorption, then infects fibroblast with this culture medium by the millopore membrane filtration. Converge flat board by fibroblastic Asia and remove culture medium, change to rapidly the culture medium from producer's cell. Remove this culture medium, change to fresh culture. If virus titer is high, will infects so in fact all fibroblasts, and need not to select. Very low if tire, must use the retroviral vector with selected marker (such as neo or his) so. In case effectively infect fibroblast, just be parsed into fibrocyte to determine whether to generate MPIF-1 albumen.

Then, improved fibroblast is transplanted among the host separately, or is grown at the cytodex3 microcarrier bead and to transplant after converging. Embodiment 26: the gene therapy of using endogenous MPIF-1 gene

Comprise by homologous recombination according to the another kind of method of gene therapy of the present invention endogenous MPIF-1 sequence and promoter can be operated combination, as for example described in the following document: U.S. Patent number 5,641,670 is disclosed on June 24th, 1997; International publication number WO 96/29411 is disclosed on September 26th, 1996; International publication number WO 94/12650 is disclosed on August 4th, 1994; The people such as Koller, Proc.Natl.Acad.Sci.USA, 86:8932-8935,1989; With the people such as Zijlstra, Nature, 342:435-438,1989. This method comprises activating and is present in the target cell but the gene of usually not expressing or expressing to be lower than aspiration level in cell.

Generation comprises the polynucleotides construction of promoter and target sequence, and this target sequence is positioned at the promoter flank, with 5 ' the non-coding sequence homology of endogenous MPIF-1. The target sequence will be neighbouring fully complementary with the 5 ' end of MPIF-1, thereby promoter will be operatively connected endogenous sequence after homologous recombination. Useful PCR increase promoter and target sequence. Preferably, the promoter of amplification comprises different restriction enzyme sites 5 ' with 3 ' end. Preferably, 3 ' end of first target sequence comprises promoter 5 ' the terminal identical restriction enzyme sites with amplification, and 5 ' end of second target sequence comprises the restriction enzyme sites identical with the promoter 3 ' end of amplification.

With the promoter of suitable restriction enzyme digest amplification and the target sequence of amplification, process with the calf intestinal phosphatase enzyme subsequently. In the situation that has the T4 dna ligase, that postdigestive promoter and postdigestive target sequence is added together. Keep the mixture that obtains being suitable for connecting under the condition of two fragments. Construction is carried out size separation at Ago-Gel, then take out with the ethanol precipitation by phenol and carry out purifying.

In this embodiment, use the polynucleotides construction by electroporation as exposed polynucleotides. Yet, also can use the polynucleotides construction with transfection promoter, such as liposome, virus sequence, virion, precipitating reagent, etc. These delivering methods are known in this area.

In case cell is transfected, homologous recombination will occur, cause promoter to be operatively connected endogenous MPIF-1 sequence. This will cause at cells MPIF-1. Can detect expression by any other method that immunology dyeing or this area are known.

Obtain fibroblast by Skin biopsy by the experimenter. The tissue that obtains is placed the DMEM+10% hyclone. With the fibroblast of Trypsin Induced exponential phase of growth or stationary phase morning, and washed next by the frosting upper punch with nutrient medium. Get a part of cell suspending liquid and be used for counting, carry out remaining cell centrifugal. Suck supernatant, and precipitation is resuspended in 5ml electroporation buffer solution (20mM HEPES pH7.3,137mM NaCl, 5mM KCl, 0.7mM Na₂HPO4,6mM dextrose). Cell is again centrifugal, suck supernatant, and cell is resuspended in the electroporation buffer solution that contains 1mg/ml acetylation bovine serum albumin. Final cell suspending liquid comprises about 3 * 10⁶Individual cell/ml. Should after resuspended, carry out immediately electroporation.

The secundum legem technology prepares DNA. For example, in order to make up the plasmid of target MPIF-1 locus, with HindIII digested plasmid pUC18 (MBI Fermentas, Amherst, NY). By pcr amplification CMV promoter, it has the XbaI site at 5 ' end, at 3 ' end the BamHI site is arranged. By two kinds of MPIF-1 non-coding sequences of pcr amplification: a kind of MPIF-1 non-coding sequence (MPIF-1 fragment 1) that increases, wherein 5 ' end contains the HindIII site, and 3 ' end contains the XbaI site; The another kind of MPIF-1 non-coding sequence (MPIF-1 fragment 2) that increases, wherein 5 ' end contains the BamHI site, and 3 ' end contains the HindIII site. With suitable enzymic digestion CMV promoter and MPIF-1 fragment (1 and 2) (CMV promoter-XbaI and BamHI; MPIF-1 fragment 1-XbaI; MPIF-1 fragment 2-BamHI) and connect together. Digest the connection product that obtains with HindIII, and connect the postdigestive pUC18 plasmid with HindIII.

DNA is added in the aseptic electric shock cup (BioRad) of electrode spacing 0.4cm. Final DNA concentration is at least 120 μ g/ml normally. Then add the 0.5ml cell suspending liquid in the electric shock cup and (comprise about 1.5 * 10⁶Individual cell), and with the gentle mixing of cell suspending liquid and dna solution. (BioRad) carries out electroporation with GenePulser equipment. Electric capacity and voltage are arranged to respectively 960 μ F and 250-300V. Along with voltage raises, cell survival reduces, but mixes genomic survivaling cell percentage and significantly increase the DNA that imports is stable. Given these parameters should obtain burst length of about 14-20 millisecond.

Cell behind the electroporation was kept about 5 minutes in room temperature, then with the gentle electric shock cup inclusion of drawing of aseptic pipettor. Cell directly is added in the 10ml preheating nutrient medium (DMEM that contains 15% calf serum) in the 10cm culture dish, and in 37 ℃ of insulations. Suck culture medium next day, changes the 10ml fresh culture, and continue to cultivate 16-24 hour.

Then improved fibroblast is expelled in the host separately, or the cytodex3 microcarrier bead grow to converge after again the injection. Fibroblast can generate described protein now. Then can as mentioned above fibroblast be imported the patient. Embodiment 27: the methods for the treatment of of using the vivo gene therapy

Another aspect of the present invention is to treat disorder, disease and situation with the vivo gene therapy. Gene therapy relates in exposed nucleic acid (DNA, RNA and antisense DNA or RNA) the MPIF-1 sequence importing animal body, with the expression of rising or reduction MPIF-1 polypeptide. The MPIF-1 polynucleotides can be operatively connected promoter or target tissue is expressed necessary any other genetic elements of MPIF-1 polypeptide. These gene therapies and delivery technology and method are known in this area, consult for example WO 90/11092, WO 98/11779; U.S. Patent number 5,693,622,5,705,151,5,580,859; The people such as H.Tabata, Cardiovasc.Res., 35 (3): 470-479,1997; The people such as J.Chao, Pharmacol.Res., 35 (6): 517-522,1997; J.A.Wolff, Neuromuscul.Disord, 7 (5): 314-318,1997; The people such as B.Schwartz, Gene Ther., 3 (5): 405-411,1996; The people such as Y.Tsurumi, Circulaion, 94 (12): 3281-3290,1996 (being collected herein by reference).

Can deliver MPIF-1 polynucleotides construction by any method that injectable materials is delivered to zooblast, such as being expelled in tissue (heart, muscle, skin, lung, liver, the intestines etc.) gap. Can in the acceptable liquid of pharmaceutics or aqueous carrier, deliver MPIF-1 polynucleotides construction.

Term " exposed " polynucleotides (DNA or RNA) refer to not contain the sequence of any delivery carrier, the effect that wherein said delivery carrier plays is auxiliary, promote or be convenient to enter cell comprises virus sequence, virion, Liposomal formulation, lipofectin reagent or precipitating reagent etc. Yet, also can be at Liposomal formulation (such as people such as P.L.Felgner, Ann.NY Acad.Sci., 772:126-139,1995 and the people such as B.Abdallah, Biol.Cell, 85 (1): 1-7, those Liposomal formulations of teaching in 1995) deliver the MPIF-1 polynucleotides in, described Liposomal formulation can prepare by the well-known method of those skilled in the art.

The MPIF-1 polynucleotide carrier construction that uses in gene therapy preferably neither is incorporated in the host genome, also do not comprise the construction that allows the sequence that copies. Any strong promoter that those skilled in the art will know that all can be used for driving the expression of DNA. Different from other gene therapy technology, the major advantage that exposed nucleotide sequence is imported target cell is the synthetic instantaneous person's character of polynucleotides in the cell. Studies show that can be with non-replicating dna sequence dna transfered cell, so that the generation of the expectation polypeptide that reaches 6 months to be provided.

MPIF-1 polynucleotides construction can be delivered to the tissue space of animal, comprise muscle, skin, brain, lung, liver, spleen, marrow, thymus gland, heart, lymph, blood, bone, cartilage, pancreas, kidney, gall-bladder, stomach, intestines, testis, ovary, uterus, rectum, nervous system, eye, body of gland and connective tissue. Tissue space comprises the mucopolysaccharide matrix that flows of the iuntercellular between the collagenous fibres of reticular fibre, vascular wall or room wall elastomer at organ-tissue, fibr tissue, perhaps wraps up in the connective tissue of muscle cell or the same matrix in the lacuna osseous. Similar, also can be blood plasma and the occupied gap of vasculolymphatic lymph liquid of circulation. Because following reason preferably is delivered to the musculature gap. Can deliver easily them by being injected to the tissue that comprises these cells. Preferably they are delivered to noble cells lasting, non-division and in wherein expression, also can in the cell that breaks up or not exclusively break up, realize delivery and expression, such as blood stem cell or SF. The ability of muscle cell picked-up and expression polynucleotides is strong especially in the body.

For the injection of exposed MPIF-1 polynucleotides, the effective dose of DNA or RNA will be about 0.05g/kg body weight-about 50mg/kg body weight. Preferred dose is the about 20mg/kg of about 0.005mg/kg-, more preferably about about 5mg/kg of 0.05mg/kg-. Certainly, the technical staff of ordinary skill will understand, and this dosage will change according to the tissue site of injection. Those of ordinary skills are easy to measure the suitable and effective dose of nucleotide sequence, and this dosage depends on situation to be treated and the path of using. Preferably using the path is the stomach and intestine outer pathway that is injected to tissue space. But also can use other stomach and intestine outer pathway, such as the suction of aerosol, especially for the delivery to lung or bronchial tissue, throat or schneiderian membrane. In addition, exposed MPIF-1 polynucleotides construction can be in the angioplasty process by flow process in used conduit be delivered to artery.

The following interior dose response effect of body that is determined at injection MPIF-1 polynucleotides in the muscle. The secundum legem recombinant DNA method prepares suitable MPIF-1 template DNA, is used for generating the mRNA of coding MPIF-1 polypeptide. Template DNA can be ring-type or linear, use as naked DNA, or with the liposome compound use. Then the template DNA with the difference amount is expelled in the mouse musculus quadriceps.

By intraperitoneal injection 0.3ml 2.5% avertin anesthesia the 5-6 female and male Balb/C mouse in age in week. Do the 1.5cm otch at front thigh, directly expose musculus quadriceps. In 1 minute, by No. 27 syringe needles the MPIF-1 template DNA in the 0.1ml carrier is expelled to knee from the about 0.5cm of distance muscle distally insertion point with the 1cc syringe, dark approximately 0.2cm. Suture placed on the injection site be used for later location, and with stainless steel clamp skin.

Suitably after the temperature retention time (such as 7 days), prepare the muscle extract by downcutting whole musculus quadriceps. 1 histochemical stain of carrying out the MPIF-1 protein expression is got in quricipital per 5 the 15 μ m section of sub-thread. Can carry out in a similar manner the time course research of MPIF-1 protein expression, but gather musculus quadriceps at different time by different mouse. Can after preparing total cell dna and HIRT supernatant by injection and control mice, measure rear the keep situation of MPIF-1 DNA in muscle of injection by the Southern engram analysis. The result of above-mentioned experiment can be used for extrapolating with the MPIF-1 naked DNA for the suitable dosage of people and other animal and other treatment parameter in the mouse. Embodiment 28:MPIF-1 transgenic animals

Also can in transgenic animals, express the MPIF-1 polypeptide. The animal of any species all can be used for generating transgenic animals, includes but not limited to mouse, rat, rabbit, hamster, cavy, pig, miniature pig, goat, sheep, ox and non-human primates, such as baboon, monkey and chimpanzee. In specific embodiment, in human body, express polypeptide of the present invention as the part of gene therapy scheme with other technology that technology described herein or this area are known.

Any technology that this area is known all can be used for transgenosis (being polynucleotides of the present invention) is imported animal to generate creator's strain of transgenic animals. These technology include but not limited to pronuclear microinjection (people such as Paterson, Appl.Microbiol.Biotechnol., 40:691-698,1994; The people such as Carver, Biotechnology (NY), 11:1263-1270,1993; The people such as Wright, Biotechnology (NY), 9:830-834,1991; With the people such as Hoppe, U.S. Patent number 4,873,191,1989); Entered system genitale (people such as Van der Putten, Proc.Natl.Acad.Sci.USA, 82:6148-6152,1985), blastocyst or embryo's transfer by the gene of retrovirus-mediated method; Gene targeting in the embryonic stem cell (people such as Thompson, Cell, 56:313-321,1989); Cell or embryo's electroporation (Lo, Mol.Cell.Biol., 3:1803-1814,1983); Use particle gun to import polynucleotides of the present invention (consulting such as people such as Ulmer Science, 259:1745,1993); Feed back in the blastocyst with nucleic acid construct thing importing embryonic pleuripotent stem cell and with stem cell; With by transgenosis (people such as Lavitrano, Cell, 57:717-723,1989) of Sperm-mediated etc. The summary of these technology is consulted Gordon, " transgenic animals ", Intl.Rev. Cytol., 115:171-229,1989 (complete being collected herein by reference).

Any technology that this area is known all can be used for generating the transgene clone that comprises polynucleotides of the present invention, for example, in the future the self-induction consideration convey that is in cultivation embryo, fetus or the mature cell of resting stage moves on in the enucleation oocyte (the people such as Campell, Nature, 380:810-813,1997).

The invention provides all cells and all carry genetically modified transgenic animals, and some but not all cells carries genetically modified animal, i.e. chimaeric animals. Transgenosis can single transgenosis or is integrated with the form of multicopy (such as concatermer, such as head to head series connection or head to the tail series connection). Method that also can be by teaching such as people such as Lasko people such as (, Proc.Natl.Acad. Sci.USA, 89:6232-3236,1992) Lasko selectively imports particular cell types with transgenosis and activates therein. This cell type specificity activates needed regulating and controlling sequence will depend on concrete purpose cell type, and will be apparent to those skilled in the art. When wishing that the polynucleotides transgenosis is incorporated into the chromosomal foci of endogenous gene, preferably carry out gene targeting.

In brief, when utilizing this technology, design comprises the carrier with some nucleotide sequences of endogenous gene homology, and purpose is to be incorporated in the nucleotide sequence of endogenous gene and to destroy its function by the homologous recombination with chromosome sequence. Method that also can be by teaching such as people such as Gu people such as (, Science, 265:103-106,1994) Gu selectively imports particular cell types with transgenosis, thus deactivation endogenous gene in the sort of cell type only. The needed regulating and controlling sequence of this cell type specificity deactivation will depend on concrete purpose cell type, and will be apparent to those skilled in the art. The content of every piece of document quoting in this paragraph is collected herein by reference.

Similar, can use will the encode DNA of total length MPIF-1 albumen of following primer to insert the carrier that is used for tissue specific expression.

Except express polypeptide of the present invention in transgenic animals with general or tissue-specific mode, the construction that generates the regulation and control expression of polypeptides by multiple other means (for example grow or chemical regulation is expressed) also will be conventional to those skilled in the art.

In case generated transgenic animals, then the expression of usable criterion technical Analysis recombination. Thereby can organize to confirm to have occured genetically modified integration by Southern engram analysis or round pcr analyzing animal and realize Preliminary screening. Can use multiple technologies assessment transgenic animals to organize the expression of transfer gene mRNA, include but not limited to Northern engram analysis, situ Analysis and reverse transcriptase-PCR (rt-PCR) by the tissue sample of animal acquisition. Also can use the transgene product specific antibody that the transgene expression tissue sample is carried out immunocytochemistry or immunohistochemical evaluation.

In case generated the creator animal, then can be with their breedings, inbreeding, outbreeding or hybridization, to generate the colony of particular animal. The example of these Reproductive Strategies includes but not limited to: will have the creator animal outbreeding of an above integration site, to set up independent strain; With independent strain inbreeding, to generate because of the compound transgenic strain of each genetically modified addition expression effect with the higher level express transgenic; With the transgenic animals hybridization of heterozygosis, the animal of isozygotying to generate the appointment integration site, thus not only increase expression but also eliminate the needs that screen animal by DNA analysis; With the incross that independently isozygotys, to generate compound heterozygosis strain or the strain of isozygotying; And breeding, render transgenic is in the unique background that is suitable for the purpose experimental model.

Transgenic animals of the present invention serve many purposes, and the biological function, the research that include but not limited to can be used for setting forth the MPIF-1 polypeptide are expressed the animal model that relevant situation and/or disorder and screening are effectively improved the compound of these situations and/or disorder with unusual MPIF-1. Embodiment 29:MPIF-1 knock-out animal

Can use the target practice homologous recombination (to consult such as people such as Smithies Nature, 317:230-234,1985 by the expression that deactivation or " knocking out " MPIF-1 gene and/or its promoter reduce endogenous MPIF-1 gene; Thomas and Capecchi, Cell 51:503-512,1987; The people such as Thompson, Cell, 5:313-321,1989; Complete being collected herein by reference). For example, can use flank is the cell of transfection expression polypeptide of the present invention in saltant nonfunctional polynucleotides of the present invention (or fully irrelevant dna sequence dna) (can have or not have selected marker and/or negative selectable marker) body with the DNA of endogenous polynucleotide sequence (code area of this gene or control region) homology. In another embodiment, use technology that this area knows to knock out containing but do not express to generate in the cell of genes of interest. Insert the deactivation that this DNA construction causes target gene by the target practice homologous recombination. These methods are particularly suitable for research and agriculture field, the modification of embryonic stem cell be can be used for generating the animal offspring (consult for example Tomas and Capecchi, 1987 and Thompson, 1989, see above) of target gene non-activity in these fields. Yet, if the suitable viral vectors that uses those skilled in the art to understand directly is applied to recombinant DNA constructs or the target body in the site that needs, so this method can routine be applicable to human body.

In other embodiments of the present invention, with the cell of genetically engineered rear expression polypeptide of the present invention or genetically engineered after do not express polypeptide of the present invention cell (for example knocking out) be applied in the patient body. These cells can derive from patient's (be animal, comprise the people) or the compatible donor of MHC, include but not limited to fibroblast, bone marrow cell, haemocyte (such as lymphocyte), adipocyte, muscle cell, endothelial cell etc. Usually use recombinant DNA technology to transform these cells at vitro genetic engineering, with the coded sequence transfered cell with polypeptide of the present invention, perhaps destroy polypeptid coding sequence of the present invention and/or the endogenous regulatory sequence relevant with it, for example (use viral vectors by transduction, preferably transgenosis is incorporated into the carrier in the cellular genome) or the transfection flow process, include but not limited to use plasmid, clay, YAC, naked DNA, electroporation, liposome etc. The coded sequence of polypeptide of the present invention can be placed under the control of strong composing type or inducible promoter or promoter/enhancer, to realize the expression of MPIF-1 polypeptide, preferred secretion. Can for example import in the circulation expressing and preferably secrete the engineered cells general importing patient of polypeptide of the present invention, perhaps in peritonaeum, import.

Perhaps, can and implant the cell doped matrix, a part that for example can be used as skin graft is implanted the fibroblast after genetically engineered; The endothelial cell that a part that can be used as lymph or blood vessel graft is implanted after genetically engineered (is consulted such as people such as Anderson U.S. Patent number 5,399,349; Mulligan and Wilson, U.S. Patent number 5,460,959, complete being collected herein by reference).

When cell right and wrong to be administered during from the compatible cell of body or non-MHC, can the host from forming for the immunoreactive technology of institute's transfered cell comes dosed cells with preventing of knowing of this area. For example, can packing form transfered cell, allow composition with near extracellular environment exchange in, the cell that does not allow host immune system identification import.

Knock-out animal of the present invention serves many purposes, and the biological function, the research that include but not limited to can be used for setting forth the MPIF-1 polypeptide are expressed the animal model system that relevant situation and/or disorder and screening are effectively improved the compound of these situations and/or disorder with unusual MPIF-1. Embodiment 30: the generation hybridoma technology of antibody

Can prepare antibody of the present invention (consulting " Current Protocols " (general scheme), the 2nd chapter) by several different methods. As the example of these methods, can use the cell of expressing polypeptide of the present invention to animal, to induce the generation of the serum that contains polyclonal antibody. In a preferred method, the preparation of preparation and purifying polypeptide of the present invention makes it to be substantially free of natural pollutant. Then this preparation is imported animal, to generate the more polyclonal antiserum of high specific acitivity of tool.

Use hybridoma technology to prepare monoclonal antibody specific (people such as K hler, Nature, 256:495,1975 of polypeptide of the present invention; The people such as K hler, Eur.J.Immunol., 6:511,1976; The people such as K hler, Eur.J.Immunol., 6:292,1976; The people such as Hammerling, in " Monoclonal Antibodies and T-Cell Hybridomas " (monoclonal antibody and T quadroma) book, Elsevier, New York, 563-681 page or leaf, 1981). Generally speaking, use polypeptide of the present invention, more preferably use the cellular immunity animal (preferred mouse) of expression-secretion type polypeptide. In any suitable culture medium for tissue culture, cultivate the cell that these express polypeptide, preferred add 10% hyclone (in about 56 ℃ of deactivations) and add about 10g/L nonessential amino acid, about 1, the EarleShi of 000U/ml penicillin and about 100 μ g/ml streptomysins revises the EagleShi culture medium.

Extract the splenocyte of these mouse and merge suitable myeloma cell line. Can adopt any suitable myeloma cell line according to the present invention; Yet preferably adopt parent myeloma cell lines (SP20) (can be obtained by ATCC). After the fusion, in the HAT culture medium, selectively keep the hybridoma of generation, then such as people such as Wands, Gastroenterology, 80:225-232,1981 describedly clone by limiting dilution. Then the hybridoma that this selection of upchecking obtains can be in conjunction with the clone of the antibody of polypeptide of the present invention to identify secretion.

Perhaps, can use anti-idiotype to generate in two step flow processs can be in conjunction with other antibody of polypeptide of the present invention. This method is utilized antibody self also to be antigen thereby might to obtain the fact in conjunction with the antibody of the second antibody. According to this method, protein-specific antibody is used for immune animal, preferred mouse. Then the splenocyte of this animal is used for generating hybridoma, and the screening hybridoma clone that can be blocked by polypeptide of the present invention with the ability of identifying the antibody binding proteins matter specific antibody that generated. These antibody comprise the anti-idiotype for described protein-specific antibody, and can be used for immune animal to induce the formation of other oroteins specific antibody.

For purposes in the body of antibody in the people, can antagonist " humanization ". Can generate these antibody with the genetic constructs of being derived by the hybridoma that generates monoclonal antibody mentioned above. Be used for generating chimeric and method humanized antibody is known in this area, and carried out in this article discussing that (summary is consulted Morrison, science, 229:1202,1985; The people such as Oi, BioTechniques, 4:214,1986; The people such as Cabilly, U.S. Patent number 4,816,567; The people such as Tanniguchi, EP 171,496; The people such as Morrison, EP 173,494; The people such as Neuberger, WO 86/01533; The people such as Robinson, WO 87/02671; The people such as Boulianne, Nature, 312:643,1984; The people such as Neuberger, Nature, 314:268,1985). By the antibody fragment of scFv library Separated pin to polypeptide

Will be by the gene constructed one-tenth antibody fragment of the natural generation V library of people PBL separation, its reactivity of comprising the polypeptide of the present invention of may or fail to expose for donor of described antibody fragment (is consulted for example U.S. Patent number 5,885,793, complete being collected herein by reference).

The redemption in library. Described in PCT publication number WO 92/01047, by the RNA structure scFv library of people PBL. In order to save the bacteriophage of showing antibody fragment, use about 10⁹The individual Escherichia coli inoculation 50ml that comprises phasmid contains the 2xYT (2xYT-AMP-GLU) of 1% glucose and 100 μ g/ml ampicillins and shakes and is cultured to O.D. and reaches 0.8. With this culture inoculation of 5ml 50ml 2xYT-AMP-GLU, add 2 * 10⁸The helper phage of TU missing gene 3 (M13 Δ gene III consults PCT publication number WO 92/01047), and with culture in 37 ℃ do not shake the insulation 45 minutes, then in 37 ℃ shake the insulation 45 minutes. With culture with 4,000rpm centrifugal 10 minutes, and precipitation is resuspended in 2 liters of 2xYT that contain 100 μ g/ml ampicillins and 50 μ g/ml kanamycins, overnight incubation. Described in PCT publication number WO 92/01047, prepare bacteriophage.

The elutriation in library. In PBS, spend the night with 4ml 100 μ g/ml or the coated immunity pipe of 10 μ g/ml polypeptide of the present invention (Nunc). Effective 2% Marvel-PBS in 37 ℃ of sealings 2 hours, is then cleaned 3 times with PBS. With about 10¹³The TU bacteriophage adds in the pipe, and in room temperature insulation 30 minutes, over and under rotating disk, then uprightly placed 1.5 hours. Effective PBS/0.1% polysorbas20 is cleaned 10 times, clean 10 times with PBS again. Add 1ml 100mM triethylamine, rotation is 15 minutes under rotating disk, then immediately with 0.5ml 1.0M Tris-HCl pH7.4 neutralization, thus wash-out bacteriophage. Then arise from 37 ℃ of insulations 30 minutes by bacteriophage and bacterium one with wash-out, and bacteriophage is used for infecting 10ml mid-log phase Escherichia coli TGl. Then Escherichia coli are coated on the TYE flat board that contains 1% glucose and 100 μ g/ml ampicillins. Then save the bacterium library that produces with Δ gene 3 helper phages as mentioned above, with for the preparation of the bacteriophage of screening wheel subsequently. Then this process is repeated altogether 4 to take turns affinity purification, take turns the 3rd and 4 and use the wash number of PBS, 0.1 % polysorbas20 to be increased to 20 times, use the wash number of PBS to be increased to 20 times.

The sign of bond. To take turns the bacteriophages of selecting wash-out by the 3rd and 4 and be used for ehec infection HB 2151, and generate soluble scFv people such as (, 1991) Marks by single bacterium colony and be used for measuring. Carry out ELISA with microtiter plate, described plate is coated with the 10pg/ml polypeptide of the present invention that is dissolved in 50mM bicarbonate pH9.6. Then check order by PCR fingerprint technique (consulting WO 92/01047) and further to characterize the clone of the ELISA positive. Can further characterize by the technology that this area is known these ELISA positive colonies, such as epitope mapping, binding affinity, receptor signal transduction, blocking-up or competitive ability and competitive activator or the antagonist activities that suppresses the antibody/antigen combination. Embodiment 31: the lethal exposure model

Figure 41 has shown the schematic diagram of the experimental program of embodiment 18. As shown in figure 42, MPIF-1 (Δ 23) strengthens the survival of lethal exposure mouse. The ability that MPIF strengthens survival changes with experiment condition.

The activity of research test MPIF-1 (Δ 23) shown in this embodiment. Yet those skilled in the art can be easy to revise illustrative research with test total length MPIF-1 or its fragment, and the activity of the polynucleotides of MPIF-1 (as passing through gene therapy), activator and/or antagonist. Embodiment 32: for marrow protection in the body of irradiation

Show that MPIF-1 increases the experiment (embodiment 18) of mouse survival behind the lethal exposure, illustrate that this chemotactic factor (CF) can take on radioprotector. In order further to study this activity, in the mouse of sublethal exposure, checked the variation of myeloid progenitor level, whether improve the recovery of rear these CFU-GMs of irradiation to determine MPIF-1.

Figure 43 has shown the schematic diagram of experimental program. This is the modification of the scheme of embodiment 17. Used MPIF-1 is MPIF-1 (Δ 23) batch HG00304-E11. Negative control group (IRR) is by only accepting carrier (HBSS+0.1% normal mouse serum i.p.) in the-3 to+3 days, and just mouse is before irradiation and had a rest afterwards 24 hours. In order to compare in the marrow analysis, control group (normally) mouse is not accepted irradiation.

The analysis of bone marrow cell. Put to death every group mouse at the 4th, 14,21 and 38 day, and gather marrow (BM) sample. Form the number that determination method is measured the myeloid progenitor (colony forming unit (CFU)-c and CFU-granulocyte, class red blood cell, megacaryocyte, macrophage (CFU-GEMM)) from marrow with soft agarose. Consult such as people such as Grzegorzewski Blood, 183:377,1994; And Metcalf, " The Hematopoietic Colony Stimulating Factors " (hematopoiesis colony stimulating factor), Amsterdam, Holland, Elsevier, 1984, the 27 pages. Use based on the mensuration system of individual layer agar and recombinant human erythropoietin (rhEpo) (8U/ml) and (100U/ml) cultured cell of recombined small-mouse interleukin-3 (rmIL-3). The multipotency colony that will comprise granulocyte, class red blood cell, megacaryocyte and macrophage pedigree is assessed as CFU-GEMM, and (the single pedigree colony that CFU-E and outburst form unit (BFU)-E) or granulocyte/monocyte (CFU-GM) precursory cell will be called CFU-c and will comprise monocyte (CFU-M), granulocyte (CFU-G), red blood cell. The CFU-GEMM determination method provides the information of recovering about multipotency CFU-GM in the marrow, and the CFU-c determination method provides the information about the CFU-GM recovery of more finalizing the design of every kind of marrow pedigree. Shown in Figure 44-45, MPIF-1 uses and causes maturation and immature myeloid level of hematopoietic progenitor cells significantly to rise.

The activity of research test MPIF-1 (Δ 23) described in this embodiment. Yet those skilled in the art can be easy to revise illustrative research with test total length MPIF-1 or its fragment, and the activity of the polynucleotides of MPIF-1 (as passing through gene therapy), activator and/or antagonist. The physics of embodiment 33:MPIF, chemistry and pharmaceutics characteristic

Dosage form, pharmacology type and use. Myeloid progenitor inhibitory factor is brachymemma (8.85kDa) recombinant human protein matter beta-chemokine. At expression in escherichia coli MPIF and be purified to homogeneous.

Provide MPIF with form aseptic, colourless solution, be intended to for injection. MPIF is mixed with the MPIF that comprises variable concentrations (2-8mg/ml), is buffered to the sterile solution of pH6.0 ± 0.2 with sodium acetate and sodium chloride. Solution is installed to 1 type once to be used in the vial. Proved 2 ℃ of-8 ℃ of preservations retains biological activity still after at least 1 year. MPIF uses intravenous.

MPIF albumen forms molecular weight 8.85kDa by 77 amino acid (Δ 23 adds N terminal M et residue).

Make the general description of specification. At expression in escherichia coli MPIF, and purifying thus. MPIF albumen is present in the protein structure that usually is called inclusion body. These inclusion bodys natural insoluble so that can before solubilising protein, remove the purge process of contaminative Escherichia coli composition.

Separate the albumen with purifying MPIF with filter method with chromatography. Following monitoring separation and purification step :-by 280nm UV absorption continuous monitoring by the resolution curve of the intermediate product of chromatographic column wash-out-measure by analytical RP-HPLC MPIF purity of protein and the content of different purification phase

Use analytical method to measure concentration, purity and the homogeneity of MPIF, and detect potential impurity, such as DNA, endotoxin and microbiology biological load.

Quality control. The MPIF drug products is clarification, colourless solution. All be viewed as single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reduction and non-reduced condition and the Coomassie blue stain subsequently. Size exclusion high performance liquid chroma-tography (SE-HPLC) has single eluting peak. Observe a main peak (〉=90%) and two secondary peaks by RPHPLC (RP-HPLC). Two secondary peaks all have the molecular weight identical with the main peak of MPIF and identical N end sequence (Edman degraded). Each batch MPIF as one man shows the peptide of similar number by the peptide mapping of Trypsin Induced, and they are just further characterized. MPIF molecular weight by mass spectroscopy is 8.85kDa.

Measure the activity of MPIF with two kinds of bioassary methods. What the calcium migration assay of THP-1 people's marrow tumour cell was measured is that MPIF is in conjunction with the direct result of its unique known receptor CCR1. The chemoproection determination method provides a kind of means for the ability that assessing compound protection mouse bone marrow cells CFU-GM avoids the cytotoxic effect of 5-FU.

What research was tested shown in this embodiment is the activity of MPIF-1 (Δ 23). Yet those skilled in the art can be easy to revise illustrative research with test total length MPIF-1 or its fragment, and the activity of the polynucleotides of MPIF-1 (as passing through gene therapy), activator and/or antagonist. Embodiment 34: non-clinical study

Foreword and general introduction. The marrow containment is the toxicity of a kind of modal dose limitation in the cytotoxicity chemotherapy. This containment is that the loss by myeloid progenitor in the marrow causes, when serious, can increase the risk of hemorrhagic and infection complication. Myeloid progenitor inhibitory factor (MPIF) is being developed to chemical protective agent. MPIF is characterized as being people's beta-chemokine. Chemotactic factor (CF) (chemoattracting cytoking) is to be replied damage, infected or immune system activation and the soluble protein secreted by the various kinds of cell type. These protein are structurally relevant because of sequence homology, and induce the ability of chemotactic response because of them in the leucocyte group and be correlated with in function.

MPIF makes it can make a distinction with all known chemotactic factor (CF)s and cell factor (B.A.Premack and U.Schall, Nature Med., 2 (11): 1174-1178,1996 to the biological action of myeloid progenitor; B.J.Rollins, Blood, 90 (3): 909-928,1997). Non-clinical study shows that this Novel Chemokine can limit propagation and/or the differentiation of CFU-GM, protects thus these cells to avoid cytotoxic effect people such as (, J.Exp. Med., 185:1163,1997) Patel of chemotherapeutics. MPIF-1 shows the effective external containment of low multiplication potentiality colony forming cell (LPP-CFC) from marrow. LPP-CFC is the dual intensity HPC that produces granulocyte and monocyte pedigree. The MPIF-1 also reversible inhibition granulocyte of being derived by murine stem cell and the colony of monocyte colony forming cell forms. External chemoproection experiment shows that MPIF Δ 23 can protect these HPCs to avoid the cytotoxic effect of antimetabolite (5-FU, Ara-C), antitumor antibiotics, topoisomerase I inhibitor and taxane (taxane).

Before each chemotherapy circulation, use immediately MPIF, can protect in vivo myeloid progenitor. Compare with the contrast of body chemotherapy model, MPIF processes and also causes myeloid progenitor and neutrophil cell and blood platelet peripheral cell group's faster quick-recovery. Unite the result from vitro study, these results show that MPIF might use clinically as protective agent, avoid the marrow containment of being induced by chemotherapy with the protection HPC.

(this moment predecessor group maximum) beginning MPIF therapy when chemotherapy begins can cause that progenitor cell obtains preserving in the chemotherapy circulation afterwards. In case in clinical, obtain confirming, this result has showed namely that with respect to more superior effects of growth factor such as granulocyte colony stimulating factor (G-CSF) or granulocyte and monocyte colony stimulating factor (GM-CSF) latter can not prevent the carrying out property loss in CFU-GM storehouse behind the chemotherapy treatment that repeats. In addition, MPIF after a plurality of circulation chemotherapy intervenes and will can be used for helping reducing disease and thrombopenia is recovered by neutrophil cell, or even having the CFU-GM storehouse, to reduce the patient of sign also be like this.

I phase placebo, dosage expansion research in the healthy volunteer who accepts repeated doses (n=6) (n=25), have been carried out. Take medicine by beginning according to the level of calculating than low 2 logarithms of mouse best protection dosage, and be extended to the dosage of 100 μ g/kg, this is lower 1 more than the logarithm than do not produce the maximum dose that can detect detrimental effect in clinical front toxicologic study. Process the healthy volunteer with 0.1,1,10,30 and 100 μ g/kg/ days and reach 6 days. Each dosage level has one group of 6 patient to participate in, and wherein accepts MPIF for 5, accepts placebo for 1. Do not observe and think and use relevant serious adverse events with MPIF. Generally speaking, the adverse events of report is slight and temporary transient, and mainly is slight headache, myalgia and fatigue. Only described an example serious adverse events (may be TIA) of observing after 52 hours of infusion the last time in 71 years old women, but this relation with research reagent it be unclear that. Patient's symptom has just solved after statement soon. Do not observe immune response, such as the formation of antibody. The analysis of convection type cell instrument data is presented at has CD14T monocyte instantaneous, dependent dose to reduce in the date of using MPIF. Yet, do not observe the minimizing of monocyte or neutrophil cell absolute counting. In the normal healthy volunteer of these hematopoiesis, do not observe the evidence of large variation or the inhibition hematopoiesis of blood count. It is relevant with dosage with the area (AUC) under the Cot curve that pharmacokinetic discloses Cmax (Cmax), processes the evidence (embodiment 34) that does not have drug accumulation after 6 days.

Pharmaceutics type and basic principle. MPIF, a kind of recombinant human protein's matter is that people's beta-chemokine is the clipped form of myeloid progenitor inhibitory factor-1 (MPIF-1). The a kind of of MPIF expects that indication is can protect myeloid progenitor in the patient who accepts marrow containment chemotherapy.

The systematic treatment that uses cytotoxic drug is the basis that the great majority of diffusive cancer are effectively treated. In addition, usually after by operation or radiotherapy in the treatment local disease, use NACT.

Because the marrow containment is a kind of modal, serious cytotoxicity relevant with chemotherapy, the patient who therefore is starved of clinically experience marrow containment therapy carries out further Supportive Care. Non-clinical study shows that MPIF has the protectant remarkable potentiality that avoid the cytotoxic effect of chemotherapeutics as protection hematopoiesis precursory cell.

Candidate stem cell and multipotency CFU-GM are responsible for the recovery of all hematopoietic lineages. In normal individual, the division of these cells is more not frequent, thereby the chemotherapeutics of the single course for the treatment of is had relative resistance. Yet after the chemotherapy of a plurality of circulations, myeloid progenitor replys to breed increase, and become to from the toxic action sensitivity of subsequently administration of chemotherapeutics and the course for the treatment of many. The cytotoxicity chemotherapy of a plurality of courses for the treatment of causes the carrying out property loss of myeloid progenitor, and it is poorer then to cause recovering delay and neutrophil cell and hematoblastic lowest count.

This protectant clinical value is to reduce the blood cell of being induced by chemotherapy to reduce the incidence of disease of disease and/or duration, reduce thus high dose or the continuous potentiality of chemotherapy circulation postoperative infection and hemorrhage possibility. In addition, MPIF will be dose intensity, the providing support property measure of in good time using of strong chemotherapy.

Present available therapy. The chemotherapy that colony stimulating factor (CSF) has been used for support standard and reinforcement dosage reaches almost 10 years. Can obtain non-clinical evidence explanation expansion chemotherapy dosage can significantly increase the destruction of tumour cell and the survival of the selected malignant tumor of improvement, but lacks the clinical affirmation of this viewpoint. Using after chemotherapy to stimulate granulocyte, macrophage and thrombopoietic CSF to reduce the duration of disease or thrombopenia to shorten neutrophil cell. On the contrary, MPFI by before being exposed to chemotherapy and among the propagation of temporary restriction myeloid progenitor protect them. This different binding mode has prevented the carrying out property loss in the CFU-GM storehouse that occurs after the chemotherapy of repeated doses, even also like this in the situation that associating CSF uses. The use of MPIF can increase the chemotherapy dose intensity thus.

Non-clinical study. MPIF can bring into play the function of the chemical protective agent of typing myeloid progenitor. Compare with other chemical protective agent that shows effect in external and/or animal model, MPIF suppresses CD34 based on it⁺The ability that the propagation of CFU-GM and colony forming unit-granulocyte and monocyte (CFU-GM) and colony forming unit-mixing (CFU-mix) cell colony form and show unique biological characteristics. In people's CFU-GM, MPIF forms with the CFU-GEMM colony CFU-GM and shows that similar inhibition is active. Thereby MPIF can suppress CD34⁺Multipotency CFU-GM, dual intensity CFU-GM and typing CFU-GM.

MPIF also can be used as effective, the reversible inhibitor performance function of the low multiplication potentiality colony forming cell (LPP-CFC) of mouse, and LPP-CFC comprises final generation on every side monocyte and granulocytic many typing myeloid progenitors. Find that also MPIF can suppress more original high proliferation potentiality colony forming cell (HPP-CFC), HPP-CFC shows the numerous characteristics of pluripotential hemopoietic stem cell.

The tissue expression of MPIF. Be the message that detects among HL-60 and the THP-1 according to original monocyte and myelomonocyte after lung, liver, marrow, activation, the mrna expression pattern of MPIF is complicated. Recently, in pancreas, heart, skeletal muscle and more among a small circle marrow, identified different transcripts.

Non-clinical pharmaceutics. Point out that from the result who surpasses 270 experiment in vitro MPIF does not have detectable effect to tumor cell line and 52 kinds of other tumor cell lines that tumour group of national cancer association (National Cancer Institute) is used for the screening antitumor and anticancer agent. Also have, set up based on the determination method that surpasses 75 kinds of cells and studied the MPIF activity. In the normal cell type, the BA of MPIF is confined to the specific cells (table 7) in the on every side immune system and HPC compartment. Particularly, find that MPIF has following effect :-in dormancy T cell and monocyte, induce in the chemotactic response-at monocyte, by the dendritic cells of monocyte derived and eosinophil and induce Ca²⁺Suppress people CD34 in the concentration range of migration-form at the colony that suppresses mouse multipotency and typing CFU-GM in the concentration range of 0.1-100ng/ml-at 0.1-100ng/ml⁺The in-vitro screening analysis of the propagation table 7:MPIF of cell

The cell tumour liver cell vascular endothelial cell neuronal cell propagation pulmonary epithelial cells osteoclast that the cell that the cell of being derived by entoderm based on the mensuration system of 75-80 kind cell is derived by mesoderm is derived by ectoderm/Gegenbaur's cell astrocyte apoptosis enterocyte cartilage cell cortical neuron differentiation

The migration of skin (fibroblast) GABAergic neuron

Smooth muscle (sustainer) Schwann cell

Myocyte's epidermal cell NCI antitumor screening

Immune system cell * * finds to have BA

The chemotactic activity of candidate stem cell * MPIF

MPIF is the lymphocytic effective chemotactic factor (CF) of dormancy T, is recorded to maximum at 10ng/ml and replys. MPIF does not stimulate chemotaxis in that broad scope (0.1-100ng/ml) is inner in the T cell that anti-CD3 activates. The monocyte of fresh separated is showed has chemotactic response to MPIF. In that quite to reply the high 10 times concentration of desired concn be that 100ng/ml observes maximum migration than causing in dormancy T cell. Weak chemotactic response occurs in neutrophil cell. MPIF does not induce chemotaxis in bone-marrow-derived lymphocyte, eosinophil, basophilic granulocyte, NK cell or blood platelet.

MPIF is to human blood cell's effect. MPIF has following interaction in vitro to the human blood cell.

Monocyte and by the macrophage of monocyte derived. In the monocyte of fresh separated, measured the impact that MPIF discharges lysosomal enzyme. In the scope of 0.5-500ng/ml, observe the release of the N-acetyl-β of low and variable level-D-Glucose glycosides enzyme. Although can detect, emission levels significantly is lower than the emission levels of being induced by MIP-1 β, MIP-1 α, RANTES or MCP-1. In other research, MPIF (1-1000ng/ml) is on the not impact of release of lysosomal enzyme elastoser, glucuronidase or myeloperoxidase. (0.1-100ng/ml) does not induce monocyte secretion IL-1 β, tumor necrosis factor-alpha (TNF-α), IL-10 or IL-2 to MPIF. In addition, do not detect the oxidative burst of activated macrophage or the impact of cellular cytoxicity activity.

Basophilic granulocyte. To be incubated 10 minutes with MPIF (1-1000ng/ml) by the basophilic granulocyte of peripheral blood purifying, and the supernatant that obtains will be measured histamine release. MPIF does not induce histamine release.

The NK cell. The PBMC of purifying is used for measuring MPIF to the impact on the cell killing of K562 cell by the NK mediation as the source. MPIF (1-100ng/ml) does not affect the K562 cell killing that is stimulated by IL-2, mediated by NK.

Blood platelet. Concentration is that the MPIF of 0.1-100ng/ml does not induce or regulates and control hematoblastic activation or gathering. MPIF suppresses people CD34⁺The propagation of CFU-GM

In order to assess MPIF to the impact of artificial blood progenitor cell proliferation, by Cord blood (cord blood) separation of C D34⁺Cell is with 5 * 10⁴Individual cell/ml is resuspended, and cultivates 4 days in the situation that has IL-3 and SCF. Then clean the myeloid progenitor group who obtains, and under four kinds of conditions, cultivate: only use culture medium; Culture medium adds MPIF; Culture medium adds the cytokine mixture of IL-3, GM-CSF and hematopoietin (EPO); Culture medium adds cell factor mixture and MPIF. Continue to cultivate after 6 days, measure the viable cell number in every kind of culture. As shown in figure 46, only adding when cultivating among the MPIF with culture medium or culture medium, myeloid progenitor can not be survived. On the contrary, cytokine mixture significantly improves the CFU-GM survival. Adding MPIF (1-1000ng/ml) causes remarkable (20-40%) of on cell proliferation to suppress. (these results have represented independent experiment 3 times. Numerical value is stated the average light absorption value ± SD of 3 parallel holes as. ) (Figure 46). MPIF suppresses people CFU-GM and CFU-Mix

For the inhibitory action of the determining MPIF specific CFU-GM of target whether, supporting that cultivation is by CD34 in the culture medium (Methocult TM semisolid culturemedium contains IL-3, GM-CSF, SCF, Epo and Tpo) that BFU-E, CFU-G, CFU-M, CFU-GM, CFU-Meg and CFU-Mix colony are grown⁺The precursory cell of deriving. Cultivate after 14 days, the colony that the colony that will generate under having the situation of MPIF generates with only with culture medium or control cells factor M IP-1 α or monocyte chemotactic protein-4 (MCP-4) time compares number and phenotype.

The result of twice representative experiment points out that MPIF suppresses the formation of (50-64%) CFU-GM and CFU-Mix. MIP-1 α and MCP-4 form not impact to the colony of any progenitor cell. These results verifications MPIF inhibitory action that the marrow precursory cell is grown, and MPIF is defined as the inhibitor (table 8) of human granular leukocyte and monocyte precursory cell. The impact that table 8:MPIF forms people CD34+ CFU-GM

Colony frequency (colony number of per 1000 cells)
Colony frequency (colony number of per 1000 cells)							Condition of culture *	BFU-E	CFU-G	CFU-M	CFU-GM	CFU-Meg	CFU-Mix
Experiment numbers							Condition of culture *	BFU-E	CFU-G	CFU-M	CFU-GM	CFU-Meg	CFU-Mix
Experiment numbers							1
Culture medium	13±2	15±2	14±3	16±3	11±3	11±2	1
Culture medium	13±2	15±2	14±3	16±3	11±3	11±2	MPIF-1	19±2	18±5	12±2	8±2	12±2	5±1
MPIF-1α	17±3	19±5	14±2	14±4	12±3	12±2	MPIF-1	19±2	18±5	12±2	8±2	12±2	5±1
MPIF-1α	17±3	19±5	14±2	14±4	12±3	12±2	Experiment numbers 2
Culture medium	14±3	13±3	13±1	12±3	13±1	11±2	Experiment numbers 2
Culture medium	14±3	13±3	13±1	12±3	13±1	11±2	MPIF-1	14±2	11±1	12±2	5±1	12±2	4±1
MPIF-1α	12±2	12±3	13±2	13±2	14±2	12±1	MPIF-1	14±2	11±1	12±2	5±1	12±2	4±1
MPIF-1α	12±2	12±3	13±2	13±2	14±2	12±1	MCP-4	12±1	14±2	12±1	12±2	11±1	12±2

External chemoproection

In order further to assess the protection potentiality of MPIF; separate pedigree loss cell (Lin-cell) by mouse bone marrow cells, and have the standard cell lines factor mixture that is formed by IL-3 (5ng/ml), SCF (50ng/ml), M-CSF (5ng/ml) and IL-1 α (10ng/ml) and be incubated under the situation that comprises or do not contain MPIF. After 60-70 hour, process culture with chemotherapeutics, and will be incubated continuation 3-24 hour, clone number that former determination method measure survival LPP-CFC by standard this moment. As shown in Figure 47, MPIF significantly reduces the cytotoxic effect of cell cycle specific reagent 5-FU, Ara-C, taxol and daunorubicin. On the contrary, MPIF can not protect CFU-GM to avoid the infringement of alkylating agent melphalan and thio-tepa. These results have supported relevant MPIF can bring into play the viewpoint of the effect of multipotency myeloid progenitor antiblastic, and help to understand effectively chemotherapeutics spectrum of this chemotactic factor (CF).

In another experiment, use two batches MPIF being shown as lot number 11 and lot number 9, use 0.1-100ng/ml the MPIF measurement of concetration MPIF for the Cytotoxic protective effect of being induced by 5-FU. Data have been indicated about using the dose response curve of MPIF and colony protection percentage. The data indication, 0.1ng/ml MPIF gives about 30% chemotactic protection, and 1000ng/ml MPIF gives the chemotactic protection (data do not show) more than 80%. The general introduction of research in the body

As shown in Figure 48, mainly in mouse, carry out the body inner analysis of MPIF, in rabbit and rat, carried out the supportive research about the compound security. Result's indication from these researchs :-MPIF is effective marrow protectant of HPC. The result of this protection is compared with the control myeloid progenitor and the faster quick-recovery of peripheral cell group after the treatment.-MPIF can use (intravenous) and not have remarkable toxicity in 14 days the time of growing up every day.-MPIF is on not impact of cardiovascular system.-MPIF does not have pyrogen.-MPIF is removed fast by circulation. The endogenous protective of myeloid progenitor

Whether test to measure MPIF protects myeloid progenitor to avoid the effect of chemotherapeutics in vivo. Mouse is injected MPIF (1.0mg/kg i.p.) every day, and interval 24 hours reached 3 days, then accepted the single injection (150mg/kg i.p.) of 5-FU at the 3rd day. Each time after 5-FU processes is put to death mouse, and in standard HPP-CFC or LPP-CFC determination method the marrow colony number is marked. Control group comprises the mouse of only accepting salt solution and the mouse of only accepting 5-FU. As shown in Figure 49 A, the marrow colony number that detects in MPIF processing mouse is used in 7 days at 5-FU and is returned to normal level. The mouse of only processing with 5-FU on the contrary, does not show that the marrow colony is formed with any recovery this moment. These results show, can be so that the faster quick-recoveries of colony forming cell with the MPIF preliminary treatment before the chemotherapy, and this might be that ability by MPIF protection myeloid progenitor realizes.

Proved the effect by the progenitor cell preservation of MPIF mediation in peripheral blood, wherein observing total white blood cell (WBC) counting in marrow namely increases after colony recovers soon. Data shown in Figure 49 B have been summarized 8 independently experiments. Shown in numerical value state the standard error of average WBC counting ± mean value as. Be lower than respectively 0.001 and 0.0001 the 6th with the relevant P value of the difference of observing in 8 days. MPIF is to the chemoproection effect of many circulations therapy

Although the presentation of results MPIF that shows so far can bring into play the protectant effect of marrow precursory cell, the protection in many circulations therapy is the clinical associated uses of MPIF. Figure 50 has showed the experimental result of measuring the chemoproection effect of MPIF in the treatment of 3 circulations. Use derives from normal mouse, only processes the mouse of (100mg/kg i.p.) with 5-FU or with the marrow of the mouse of 5-FU and MPIF (1.0mg/kg i.p.) processing; formation is analyzed to the marrow colony; the result shows that MPIF all protects the CFU-GM (people such as K.J.Grzegorzewski in the 5-FU of all 3 circulations processes; Blood; summary: supplementary issue, accept). MPIF dosage range kimonos regimen

It is broad that the non-clinical dosage of MPIF is replied, and scope is 0.01-10mg/kg. This broad replying is the basic principle of selecting for 3 log10 dose scopes of clinical testing.

Figure 51 has showed the different non-clinical scheme of taking medicine of test. Selection the-2 ,-1 is based on and observes this therapeutic scheme the most consistent and reproducible protective effect is provided in mouse in many testing schemes with the take medicine basic principle of scheme of 0 day people with respect to what chemotherapy was used. In addition, suppose in the body of MPIF that the serum half life is relatively short, the cell cycle time of stem cell CFU-GM long (people such as McNiece, Int.J.Cell Cloning, 8:146-160,1990; The people such as Bertoncello, Exp.Hematol., 19:174-178,1991), the MPIF that gives so patient's multi-agent is useful in theory, thereby makes CFU-GM expose maximization, thereby makes the possibility maximization of protection.

Integrate, show that with in vitro results the biological characteristics of MPIF is unique in the body. This protein bring into play effective marrow protectant function ability explanation it can use as chemical protective agent, and will protect early stage myeloid progenitor to avoid the effect of Common Chemotherapy agent. Reduce the blood cell of being induced by chemotherapy and reduce the incidence of disease of disease and the order of severity, reduce and infect and the potentiality of hemorrhage possibility thus because it has, the clinical value of this reagent is obvious. Non-clinical toxicology

Put into practice at excellent laboratories and to have carried out 3 times non-clinical toxicological studies under the policy (Good Laboratory Practice guidelines): dosage range research in 7 days, inferior chronic research in 14 days, and inferior chronic research in 25 days. Dosage up to 20mg/kg does not cause remarkable toxicity. The General Introduction of multiple dose research (Figure 52) and the general introduction of non-clinical study (Figure 53) clinical observation result have been showed among the figure. In addition, do not observing autonomous or cardiovascular impact up to the dosage of 10mg/kg. Non-clinical absorption, distribution, metabolism and drainage

In giving single dose intravenous or carried out the pharmacokinetic of MPIF in the BALB/c female mice of subcutaneous bolus MPIF (dosage is 20mg/kg). Put to death 3 mouse of each processed group at each time point and also take a blood sample, measure the concentration of MPIF by enzyme-linked immunosorbent assay. As shown in Figure 54, intravenous is used MPIF and is caused quick removing, but still keeps low but detectable level in rear 24 hours in injection. Subcutaneous administration produces similar curves, and Main Differences is that the appearance of serum peak level has postponed 30 minutes. After this, the observed result that removing situation and intravenous are processed in the mouse does not have difference. The removing of MPIF is consistent with the expection of small-sized protein.

Carried out the pharmacokinetic second time of MPIF in the mouse of bolus in giving single dose intravenous (20mg/kg). MPIF is removed fast by serum. Use and can detect MPIF in rear 8 hours, its level is 0.1% of application dosage.

The MPIF-1 relevant with the use in the chemotherapy (Δ 23) activity has been tested in the research of describing among this embodiment. Many researchs that are applied to MPIF-1 purposes in the radiotherapy that are equal in these schemes. In addition, those skilled in the art can be easy to revise illustrative research with test total length MPIF-1 or its fragment, and the activity of the polynucleotides of MPIF-1 (as passing through gene therapy), activator and/or antagonist. Embodiment 35: test with the previous people of clinical research before clinical

Clinical research 00304-CRX-HV-0l. Having carried out the I phase in the healthy volunteer studies with evaluate safety, pharmacokinetics (PK) and pharmacokinetics effect (people such as Louie, Blood, 90:1569A, summary; Supplementary issue, 1997). Enlarge (0.1,1,10,30,100 μ g/kg) at the successive doses that placebo is being arranged) in the blind test, 30 experimenter (14 male sex, 16 women, intermediate ages 46 years old, scope 21-73 year) accepts at random 6 doses of MPIF or placebo, in about 1 minute, used in continuous 6 days. Each group of taking medicine is comprised of 6 experimenters, and randomization ground was with active medicine-placebo ratio administration in 5: 1. In 4 time-of-weeks, estimate adverse events (AE). After 2 weeks, check preliminary data of safety, and make the decision of next group of beginning. Gather blood sample and be used for hematology, chemistry, PK, antigenicity and flow cytometry art to estimate the PBC group.

This is the I phase safety research of carrying out in the healthy volunteer, thereby main purpose is not to determine effect. Yet, estimated the differentiation of following laboratory parameters :-white blood cell (WBC) counting-absolute neutrophil cell counting (ANC)-red blood cell (RBC) counting-platelet count-measure PBC by the flow cytometry art to form

Do not observe between active medicine and the placebo treatment group and aspect the absolute neutrophil cell counting of absolute counting or percentage measure of the change, there are differences. Preclinical study result in these results and the intact animal is suitable. Same, do not observe white blood cell, red blood cell or blood platelet with respect to the marked change of baseline.

Obtain continuous flow type cell count observed result, further to describe MPIF to the impact of periphery white blood cell subgroup. Figure 55 illustrates the percentage of cell (granulocyte, monocyte, T and bone-marrow-derived lymphocyte) of peripheral blood mononuclear as the function of dosage and Study dates.

Any time in research process is not observed granulocyte, T lymphocyte or bone-marrow-derived lymphocyte with respect to the marked change of baseline. In the healthy volunteer granulocyte count not being had impact is to be consistent with the result who does not observe impact in the healthy animal of preclinical study.

In accepting tested group of 10-100 μ g/kg/ days, using the instantaneous minimizing of observing the peripheral mononuclear cells ratio Existence dependency dosage that carries CD14 in 6 days of MPIF.

Carried out analysis with respect to baseline with evaluation process during the conspicuousness that suppresses of monocyte CD14. Calculated gate CD14⁺Event is with respect to the variation percentage of baseline. Baseline value is considered every patient's contrast. Quite, minimum paired relatively p value is 0.18 (comparison between processing and the placebo) to baseline values between processed group.

The multianalysis of variate model all is significant at all time points. For

higher dosage group

10,30 and 100 μ g/kg, be different from 0 (p=0.0001) with respect to being changed significantly of baseline. Between placebo and this 3 processed group, also there is significant difference. Have significant difference between 100 μ g/kg group and 30 μ g/kg group and 10 μ g/kg group, indication has dose response.

For the impact that further Inspection Research medicine is showed monocyte, calculated absolute monocyte count (AMC). This is to carry out in the mode identical with calculating absolute neutrophil cell counting, uses following formula: AMC=WBC counting * % monocyte (from the differential counting) * 1000

Figure 56 has showed the result. The expection of not observing AMC in the MPIF processing procedure reduces, and by the morphology means monocyte is quantized simultaneously. In fact, during processing, see slight increase, betide MPIF dosage 1 μ g/kg and more than, and take medicine the last time and return to baseline counts in rear 2-3 days.

The simplicity of explanation of monocyte data is exactly that MPIF can cause that the monocyte chemotaxis of dependent dose changes the monocytic inflow that causes not carrying CD14. This soluble absolute monocyte count appropriateness of observing by the flow cytometry art increases and carries the result of the monocyte minimizing of CD14. All can obtain the clear evidence of relevant instantaneous and reversible BA by these two kinds of methods.

Security. A kind of predetermined indication of MPIF is protection marrow precursory cell in the patient who accepts marrow containment property chemotherapy. In the clinical testing of the dosage range of this reagent, placebo, estimate that the AE of frequent report is the marrow containment and uses relevant infectivity and bleeding complications with the chemotherapy of following. Before clinical and in the clinical research, MPIF can effect be instantaneous, reversible and be limited to the cell of marrow pedigree. Reduce frequency, the order of severity and the duration of the cytopenia of being induced by chemotherapy although estimate MPIF, the accident of effect prolongs can potentially make cytopenia worsen.

Study among the 00304-CRX-HV-01 I phase healthy volunteer, 18 experimenters (accept active medicine for 14, accept placebo for 4) have experienced and have added up to 32 adverse events (25 activity of medicine, 7 of placebos).

The researcher is divided into adverse events " using relevant with drugs ", " using irrelevant with drugs " or " correlation of using with drugs is unknown ". In order to press inducement with the adverse events reporting standards, the promoter thinks that those weave into the possible inducement of adverse events tool of " correlation of using with drugs is unknown " by the researcher, thereby it is lower to be reported in " relevant AE " group.

According to these standards, there are 11 to experience at least one and think and use relevant adverse events with MPIF among the experimenters that 25 are processed with MPIF. There are 3 to experience at least one and think the adverse events relevant with medicament administration among the experimenter that 5 are accepted placebo. Suitable with the adverse events incidence among the experimenter of active medicine or placebo treatment (being respectively 44% and 60%).

There are 29 to have

NCI CTC

1 or 2 grades of seriousness in 32 adverse events of report. 2 seriousness the unknowns. The adverse events of frequent report is headache (5 experimenters, 4 active medicine/1 placebos), drowsiness (3 experimenters, 2 active medicine/1 placebos) and leukopenia (3 experimenters, 2 active medicine/1 placebos). Table 9 showed the I phase study in the general introduction of all AE of report.

Adverse events is reported as serious and violent. It is dizzy that 72 years old white man women has experienced, and shows as dizziness, feels sick and vomiting, and use the last time MPIF (30 μ g/kg) instability of gait after about 56 hours. The experimenter is admitted to hospital spends the night, carry out O﹠A. Laboratory, Cranial Computed Tomography and the inspection of carotid ultrasound ripple fail to disclose reason. The experimenter long ago once had similar outbreak. Symptom disappears fast, and next day, the morning, the experimenter also left hospital, and does not have the sign of sequelae, and finishes research. The researcher reports into " the unknown " with this part adverse events with the correlation of drugs. Table 9: the general introduction of airframe systems COSTART adverse events and processing-processing intention

Airframe systems (COSTART)	Placebo		Process
	Placebo		Process														0.1μg/kg		1.0μg/kg		10μg/kg		30μg/kg		100μg/kg		All activity
	N	％	N	％	N	％	N	％	N	％	N	％	N	％			0.1μg/kg		1.0μg/kg		10μg/kg		30μg/kg		100μg/kg		All activity
	N	％	N	％	N	％	N	％	N	％	N	％	N	％	The weak back pain headache of whole health abdominal pain injection site edema injection site pain	2 1 0 0 1 0 0	40 20 0 0 20 0 0	0 0 0 0 0 0 0	0 0 0 0 0 0 0	4 0 1 1 4 0 0	80 0 20 20 80 0 0	0 0 0 0 0 0 0	0 0 0 0 0 0 0	0 0 0 0 0 0 0	0 0 0 0 0 0 0	1 0 0 0 0 1 1	20 0 0 0 0 20 20	5 0 1 1 4 1 1	20 0 4 4 16 4 4
Digestive system diarrhoea stomatitis	1 0 1	20 0 20	0 0 0	0 0 0	1 1 0	20 20 0	0 0 0	0 0 0	0 0 0	0 0 0	0 0 0	0 0 0	1 1 0	4 4 0		2 1 0 0 1 0 0	40 20 0 0 20 0 0	0 0 0 0 0 0 0	0 0 0 0 0 0 0	4 0 1 1 4 0 0	80 0 20 20 80 0 0	0 0 0 0 0 0 0	0 0 0 0 0 0 0	0 0 0 0 0 0 0	0 0 0 0 0 0 0	1 0 0 0 0 1 1	20 0 0 0 0 20 20	5 0 1 1 4 1 1	20 0 4 4 16 4 4
Digestive system diarrhoea stomatitis	1 0 1	20 0 20	0 0 0	0 0 0	1 1 0	20 20 0	0 0 0	0 0 0	0 0 0	0 0 0	0 0 0	0 0 0	1 1 0	4 4 0	Blood/lymphatic system anaemia extravasated blood leukocytosis leukopenia	1 0 0 0 1	20 0 0 0 20	2 0 1 0 1	40 0 20 0 20	1 1 0 0 1	20 20 0 0 20	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0	1 0 0 1 0	20 0 0 20 0	4 1 1 1 2	16 4 4 4 8
The musculoskeletal system myalgia	0 0	0 0	0 0	0 0	1 1	20 20	0 0	0 0	0 0	0 0	0 0	0 0	1 1	4 4		1 0 0 0 1	20 0 0 0 20	2 0 1 0 1	40 0 20 0 20	1 1 0 0 1	20 20 0 0 20	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0	1 0 0 1 0	20 0 0 20 0	4 1 1 1 2	16 4 4 4 8
The musculoskeletal system myalgia	0 0	0 0	0 0	0 0	1 1	20 20	0 0	0 0	0 0	0 0	0 0	0 0	1 1	4 4	The drowsiness blood vessel dilatation of the dizzy facial paralysis of nervous system is dizzy	1 1 0 1 1 0	20 20 0 20 20 0	0 0 0 0 0 0	0 0 0 0 0 0	2 0 0 2 1 0	40 0 0 40 20 0	0 0 0 0 0 0	0 0 0 0 0 0	2 1 0 0 0 1	40 20 0 0 0 20	1 0 1 0 0 0	20 0 20 0 0 0	5 1 1 2 1 1	20 4 4 8 4 4
The respiratory system pharyngitis	0 0	0 0	0 0	0 0	0 0	0 0	0 0	0 0	1 1	20 20	0 0	0 0	1 1	4 4		1 1 0 1 1 0	20 20 0 20 20 0	0 0 0 0 0 0	0 0 0 0 0 0	2 0 0 2 1 0	40 0 0 40 20 0	0 0 0 0 0 0	0 0 0 0 0 0	2 1 0 0 0 1	40 20 0 0 0 20	1 0 1 0 0 0	20 0 20 0 0 0	5 1 1 2 1 1	20 4 4 8 4 4
The respiratory system pharyngitis	0 0	0 0	0 0	0 0	0 0	0 0	0 0	0 0	1 1	20 20	0 0	0 0	1 1	4 4	Pruitus	0 0	0 0	1 1	20 20	0 0	0 0	0 0	0 0	0 0	0 0	0 0	0 0	1 1	4 4
Genitourinary metrorrhagia vaginitis	0 0 0	0 0 0	0 0 0	0 0 0	0 0 0	0 0 0	1 1 0	20 20 0	0 0 0	0 0 0	1 0 1	20 0 20	2 1 1	8 4 4	Pruitus	0 0	0 0	1 1	20 20	0 0	0 0	0 0	0 0	0 0	0 0	0 0	0 0	1 1	4 4

Pharmacokinetic parameter. In research 00304-CRX-HV-01, estimated pharmacokinetic parameter. Gathered plasma sample at baseline and after beginning to use in 5,10,15 and 30 minutes, 1,2,4 and 24 hour. In addition, use the last time before and afterwards collected specimens to estimate drug accumulation.

Figure 57 has summarized the data that obtain. In 0.1 and 1.0 μ g/kg group, be thirty minutes long respectively and be easy to detect the circulating level in 1 hour. More but high dose causes reaching 24 hours behind the infusion and has detection level. AUC and C_MAXProportional with application dosage. In the experimenter who accepts maximum dose level level (30 and 100 μ g/kg), T_1/2It is about 0.8 hour. Last study drug-administration blood plasma level indication before in the 6th day does not have the sign of drug accumulation.

These research indications MPIF does not cause the side effect relevant with G-CSF really, reduces heat pyrexia, catarrh, heating, fatigue, apocleisis etc. such as ostalgia, alopecia, diarrhoea, neutrophil.

What research was tested shown in this embodiment is the activity of MPIF-1 (Δ 23). Yet those skilled in the art can be easy to revise illustrative research with test total length MPIF-1 or its fragment, and the activity of the polynucleotides of MPIF-1 (as passing through gene therapy), activator and/or antagonist. Embodiment 36: the general introduction of data and to researcher's guidance

The general introduction of preclinical data. Studies show that in the body that MPIF is effective protective agent of HPC. Observe the inhibition of mouse multipotency with typing CFU-GM colony in the concentration range of 0.1-100ng/ml. Experiment in vitro shows that MPIF can provide the cytoprotection of the effect that avoids antimetabolite, antitumor antibiotics, topoisomerase enzyme inhibitor and taxane. In the research, the result of this protection is the faster quick-recovery of myeloid progenitor and peripheral cells group after the cytotoxicity chemotherapy in vivo.

MPIF can use (intravenous) and not have remarkable toxicity reaching in time of 14 days every day. In animal, be equal to dosage 1.67mg/kg up to the people and do not observe adverse effect. Dosage up to 20mg/kg in the animal of 28 days repeated doses toxicological studies is not observed adverse effect.

In surpassing 270 experiment in vitro, in tumor cell line, do not detect the effect that MPIF promotes growth.

The general introduction of clinical data. But safely use MPIF, and I phase placebo, dosage enlarge research (dosage range: accepting to tolerate among the healthy volunteer (n=25) of repeated doses fine 0.1-100 μ g/kg) :-in the healthy volunteer, do not observe and think and use relevant serious adverse events with MPIF.-the reduction of instantaneous, dependent dose of observing in PMBC that CD14 expresses by the flow cytometry art. Uninfluenced by absolute monocyte and neutrophil counting that the differential counting is measured.-until process and still can detect MPIF in rear 24 hours.-when being applied to the healthy volunteer, MPIF does not have immunogenicity.

Possible risk and side effect. Before clinical and in the clinical research, the effect of MPIF is instantaneous, reversible and is limited to the cell of marrow pedigree. Although estimate frequency, the order of severity and duration that MPIF can reduce the cytopenia of being induced by chemotherapy, the accident of effect prolongs can potentially make cytopenia worsen.

In the healthy volunteer's who accepts repeatedly intravenous dosages (up to 100 μ g/kg MPIF) initial clinical research, observe the suitable instantaneous adverse events of frequency and the order of severity and placebo. Do not observe and think and use relevant acute reaction with MPIF. MPIF does not have immunogenicity in the healthy volunteer.

Although it is relevant that adverse events seldom and MPIF use, the importing of exogenous proteins can potentially cause part or the general reaction of immunology mediation. The experimenter may experience acute allergy (such as heating, nettle rash, hypertension or low blood pressure and bronchial spasm) or other reaction (such as vasculitis or serum sickness). Although small, can not get rid of renal dysfunction, hepatosis, immune containment, coagulopathy and neuropathic possibility.

Should be during using and the sign of any acute adverse effect of close supervision experimenter afterwards. When acute reaction occured, the emergent supply (comprising that adrenaline, corticosteroid, hypertensor and the heart are except dirty defibrillation device) should be easy to use.

Taboo. Prove in clinical testing that not yet this product is used for the security of gestation or ursing mother. Prevention

Give patient's information. The same with all research products, should be only the clinical testing doctor or be familiar with using MPIF under researcher's the guidance of this product.

Prove in clinical testing that not yet this product is used for the security of gestation or ursing mother. Not yet assess the Animal Experimental Study about the security of growing before and after embryo or development of fetus, During Pregnancy and the childbirth. Any women who misses a menstrual period should suppose pregnancy, until verified really not so. Therefore, only when possible benefit surpasses the risk that mother and fetus may suffer, just can promptly study in the gestational period.

Still do not know whether MPIF is distributed in the milk. If find essential study drug-administration, should interrupt lactation so.

Laboratory tests. Should close supervision count with the patient's of MPIF processing peripheral blood. Should carry out laboratory tests according to appended clinical protocol.

Product interacts. Estimate that MPIF does not produce any serious interaction with other medicines.

Carcinogenesis, mutagenesis, fertility are impaired. The zooscopy of the carcinogenic of MPIF and the mutagenesis potentiality of not yet testing. In vitro study indication MPIF does not have detectable impact to tumor cell line.

Product abuse and dependence. Not yet product abuse and the dependence potential of check MPIF in non-clinical or clinical research, but estimate it is insignificant.

Excessive. There is not the excessive clinical experience of MPIF. In continuous 14 days give up to the animal of 20mg/kg dosage, do not observe toxicity. This is equivalent to the dosage of 1.67mg/kg among the people.

Dosage and using. Not yet determine the optimal dose kimonos regimen of MPIF. Primary Study has been estimated and has been taken (intravenous is used) 0.1-100 μ g/kg MPIF every day and reach 6 days security. The 2A phase that is used for mensuration BA and primary efficacy is studied the dosage range that will estimate 1-100 μ g/kg. To be determined by the result of these early stage efficacy studies follow-up dosage and dosage regimen.

Supply and storage. Form supply MPIF with sterile liquid formulations. This product must be stored in 2 ℃-8 ℃, only is authorized the come-at-able place of personnel. Show, be used for dilution medicine that the patient uses and place in room temperature and reach 12 hours and be still stable do not have the product degraded.

What research was tested shown in this embodiment is the activity of MPIF-1 (Δ 23). Yet those skilled in the art can be easy to revise illustrative research with test total length MPIF-1 or its fragment, and the activity of the polynucleotides of MPIF-1 (as passing through gene therapy), activator and/or antagonist. The solution structure of embodiment 37:MPIF-1 and dynamics general introduction

Myeloid progenitor inhibitory factor-1 (MPIF-1) is inhibitor and the monocytic activator of CFU-GM. By nuclear magnetic resonance (NMR) spectroscopy determining the solution structure of MPIF-1. Different from many CC chemotactic factor (CF)s, the structure of MPIF-1 holds itself out to be monomer. Except terminal residue, well determined its structure, it has the characteristic chemotactic factor (CF), i.e. 3 beta chains and top alpha-helix thereof. Except 4 cysteines that characterize most of chemotactic factor (CF)s, MPIF-1 also has 2 extra cysteines, forms 1 disulfide bond. The indication of main chain dynamics is compared the important dynamics of demonstration at residue and near the residue the disulfide bond of N terminal residue important on the function, N end ring with the core of protein. MPIF-1 is processed at N end by 99 amino acid whose front albumen, and the latter also has function, although activity is lower and be monomer under multiple solution condition. Therefore, MPIF-1 as all in other relevant chemotactic factor (CF) long proteinogen be unique. These researchs are consistent with the idea that monomer is enough to 7-TM acceptor on the activated leukocyte. Foreword

The biological process that chemotactic factor (CF) (chemoattracting cytoking) mediation is different, comprise that leucocyte transportation, hematopoiesis and blood vessel occur, and support the host and to bring into play basic role (people such as M.Baggiolini, Annu.Rev.Immunol. in the anti-infectious defence, 15:675-705,1997; B.J.Rollins, Blood, 90:909-928,1997; A.D.Luster, N.Engl. J.Med., 338:436-445,1998). So far about 40 kinds of chemotactic factor (CF)s have been identified; Length all is 70-100 amino acid, and is characterized as and has 4 conservative cysteines. Adjacent (CC) or (CXC) that separated by amino acid according to the first two cysteine, with chemotactic factor (CF) rough be divided into CC and CXC family. Gro-beta-T can further be divided into two subgroups, " ELR " and " non-ELR ". All ELR Gro-beta-Ts activate neutrophil cell, but not the ELR Gro-beta-T activates the lymphocyte of different subgroups. CC chemotactic factor (CF) activated mononuclear cell, macrophage, eosinophil, basophilic granulocyte, T cell, but do not activate neutrophil cell. In addition, a member (it only comprises 2 cysteines) and the CX of C family have also been identified₃A member of C family.

Myeloid progenitor inhibitory factor-1 (MPIF-1) (being also referred to as CK β 8), the member of CC family, certified in large scale sequencing work at first, and in liver, lung, pancreas and marrow the constructive expression (people such as V.P.Patel, J.Exp.Med., 185:1163-1172,1997). Except the colony of the bone marrow cell that can suppress to produce granulocyte and monocyte pedigree formed, it also had chemotaxis (people such as V.P.Patel, J.Exp. Med., 185:1163-1172,1997 to monocyte and eosinophil; The people such as B.-S.Youn, Blood, 91:3118-3126,1998). Alternative splicing produces the protein of two kinds of forms, is called CK β 8 and CK β 8-1, and length is respectively 99 and 116 amino acid people such as (, Blood, 91:3118-3126,1998) B.-S.Youn. What is interesting is that the clipped form (Δ 24-99) (after this being called MPIF-1) of observing 99 amino acid whose protein significantly more has activity (people such as B.Nardelli, J.Immunol., 162:435-444,1999; The people such as T.A.Berkhout, Biochem. Pharmacol., 59:591-596,2000). The intersection desensitization experiment indication MPIF-1 of monocyte and eosinophil is mainly in conjunction with the CCR1 acceptor, but in two kinds of situations all not exclusively desensitization also explanation may relate to the extra acceptor (people such as B.Nardelli, J.Immunol., 162:435-444,1999). MPIF-1 can induce by monocyte fast and the release of dependent dose [³H]-arachidonic acid, this relies on the outer calcium of born of the same parents and by phospholipase A₂(PLA ₂) the inhibitor blocking-up. In addition, PLA₂Activate and show it is that filamentous actin forms necessary in the monocyte.

Structure (people such as G.M.Clore, Biochemistry, 29:1689-1696,1990 of several CXC and CC chemotactic factor (CF) have been resolved by NMR spectroscopy and X-ray crystallography; The people such as W.J.Fairbrother, J.Mol.Biol., 242:252-270,1994; The people such as K.S.Kim, J.Biol.chem., 269:32909-32915,1994; The people such as M.G.Malkowski, J.Biol.Chem., 270:7077,1995; The people such as X.Zhang, Biochemistry, 33:8361-8366,1994; The people such as P.J.Lodi, Science, 263:1762-1767,1994; The people such as N.J.Skelton, Biochemistry, 34:5329-5342,1995; T.M.Handel and P.J.Domaille, Biochemistry, 35:6569-6584,1996; The people such as K.S.Kim, FEBS Lett., 395:277-282,1996; The people such as M.P.Crump, J.Biol.Chem., 273:22471-22479,1998; The people such as H.Sticht, Biochemistry, 38:5995-6002,1999). The chemotactic factor (CF) of most of initial characterization is dimers, and further observes CXC and use different protein zone dimerizations with the CC chemotactic factor (CF). In Gro-beta-T, the 1st β chain consists of dimer interface; And in the CC chemotactic factor (CF), the N terminal residue consists of dimer interface. These observed results and Gro-beta-T only activate neutrophil cell and the CC chemotactic factor (CF) activates other leukocytic observed result and causes it is believed that, it is the necessary view of leucocyte 7-TM receptors bind that dimer forms.

Follow-up discovery and the sign of chemotactic factor (CF)s such as SDF-1 (Gro-beta-T), MCP-3, eotaxin, HCC-2 and I-309 (CC chemotactic factor (CF)) have been eliminated these difference, because these chemotactic factor (CF)s mainly are the monomer (people such as K.S.Kim, FEBS Lett., 395:277-282,1996; The people such as M.P.Crump, J.Biol.Chem., 273:22471-22479,1998; The people such as H.St icht, Biochemistry, 38:5995-6002,1999; The people such as M.P.Crump, EMBO J., 16:6996-7007,1997). Solution research shows that also combination is responsive (K.H.Mayo and M.-J.Chen, BIochemistry, 28:9469,9478,1989 to ionic strength, buffer condition and pH; The people such as Y.Yang, J.Biol.Chem., 269:20110-20118,1994; The people such as H.B.Lowman, Protein Sci., 6:598-608,1997). Mutation research (people such as L.G.Czaplewski, J.Biol.Chem., 274:16077-16084,1999; The people such as J.S.Laurence, Biochemistry, 39:3401-3409,2000; The people such as C.D.Paavola, J.Biol.Chem., 273:33157-33165,1998) and the chemotactic factor (CF) of trapping free state (people such as K.Rajarathnam, Science, 264:90-92,1994; The people such as K.Rajarathnam, J.Biol.Chem., 272:1725-1729,1997) displaying monomer be enough in conjunction with and activated leukocyte on the 7-TM acceptor. Nearest research explanation, monomer form may be in conjunction with proteoglycans with set up in the concentration gradient (directed leucocyte transports necessary process) and play a role (people such as A.J.Hoogewerf, Biochemistry, 36:13570-13578,1997; W.Koopmann and M.S.Krangel, J.Biol.Chem., 272:10103-10109,1997).

In this research, by the NMR Spectroscopic Characterization solution structure and the main chain dynamics of MPIF-1. In addition, by sedimentation ultracentrifugation mensuration under multiple solution condition, studied albumen before MPIF-1 and the total length MPIF-1 in conjunction with tendency. In function aspects, structure and dynamic (dynamical) implication and binding characteristic have been discussed. The data of this research have consisted of the architecture basics of the structure auxiliary therapeutical agent of mutation research and immune correlated disease. Experiment flow

Protein expression and purifying. The codon of use for optimizing at expression in escherichia coli, chemical synthesis MPIF-1 gene order. Then gene is subcloned among the expression vector pHE4, this carrier comprises strong synthetic promoter (including two lac operators), effective ribosome bind site and is positioned at the synthetic transcription terminator that inserts the gene downstream. Expression plasmid is transformed among the bacterial strain SG13009 that is derived by e. coli k12. After inducing with IPTG, generated MPIF-1 with the form of insoluble protein, and in containing the 1.75mM guanidine hydrochloride of 5mM cysteine, extracted and again folding. The protein of expressing has extra Met (initiation codon) at the N end, in order to simplify, it is used as first residue of MPIF-1. In succession by strong cation (poros HS-50), anion (poros HQ-50) and cation (poros CM-20) exchange column, at last by size exclusion (Sephacryl S-100) post, with protein purification to homogeneous. As outline clone, expression and 99 amino acid whose ripe MPIF-1 of purifying total length about MPIF-1 albumen.

Sedimentation equilibrium. On Bechman XL-A type ultracentrifuge in 20 ℃ with rotating speed 23,000,28,000 and 40,000rpm carry out analytical ultracentrifugation experiments. With two kinds of different initial concentrations, in different buffer solutions and ionic strength, test, to study them to Dimerized impact (table 10). Measure the light absorption value of 280nm, and collect the data of 5 continuous radius scanning (step-length 0.003cm) mean values. With the equation below the data fitting:

C_{r} = C_{0} \exp (MHδ) + C_{0}^{2} K_{a} \exp (2 MHδ) + E

Wherein δ is (r²-r ₀ ²), H is (1-ν ρ) (ω²/2RT)，C _rAnd C₀Respectively to be positioned at radius r and r₀The concentration at place, M is the molecular weight of monomer, and ν is local specific volume, and ρ is solvent density, and ω is rotary head angular speed, and Ka is the binding constant of monomer-dimer balance, and E is needle position misalignment. Local specific volume is that the weighted average by each amino acid whose local specific volume calculates. Use Microcal Origin 4.1 softwares that Beckman provides as XL-A by nonlinear least squares method with this equation of data fitting. Pass through χ², residue quadratic sum and residue system deviation check to characterize the quality of match. With data fitting single kind or monomer-dimer model. The theoretical molecular of MPIF-1 and MPIF-1 (1-99) is respectively 8854.6 and 11367.6, and the data of two kinds of protein under all solution conditions all can fit to monomer (table 10). The sedimentation equilibrium ultracentrifugation research of table 10:MPIF-1 and total length MPIF-1

Protein	Buffer solution	pH	Temperature (℃)	Calculate molecular weight¹	State
Protein	Buffer solution	pH	Temperature (℃)	Calculate molecular weight¹	State	MPIF-1	20mM NaPi	5.0	23	8.8±0.6	Monomer
	20mM NaPi	7.0	23	9.0±0.8	Monomer	MPIF-1	20mM NaPi	5.0	23	8.8±0.6	Monomer
	20mM NaPi	7.0	23	9.0±0.8	Monomer		20mM NaPi、100mM NaCl	7.0	23	7.9±1.2	Monomer
	20mM NaPi、100mM NaCl	7.0	23	9.2±0.5	Monomer		20mM NaPi、100mM NaCl	7.0	23	7.9±1.2	Monomer
	20mM NaPi、100mM NaCl	7.0	23	9.2±0.5	Monomer	MPIF-1 (total length)	20mM 0ac、100mM NaCl	5.0	23	9.8±0.6	Monomer
	20mM NaPi、100mM NaCl	5.0	23	11.4±0.7	Monomer	MPIF-1 (total length)	20mM 0ac、100mM NaCl	5.0	23	9.8±0.6	Monomer
	20mM NaPi、100mM NaCl	5.0	23	11.4±0.7	Monomer		20mM NaPi	7.0	23	11.2±0.6	Monomer
	20mM NaPi、100mM NaCl	7.0	23	10.0±1.0	Monomer		20mM NaPi	7.0	23	11.2±0.6	Monomer

1 molecular weight is that (23,000,28,000 and 40, data 000rpm) are calculated by 3 kinds of rotating speeds of match.

NMR spectroscopy. All spectrum all are to collect at the Varian Unity Plus 600 that has been equipped with scanning field gradient annex or INOVA 500-MHz spectrometer in 35 ℃. Protein concentration is 2mM, is dissolved in the 20mM sodium acetate, 1mM sodium azide pH5.2 (is dissolved in 90%H₂O/10％ ²H ₂O (v/v) or 99.99%²H ₂O). Use the people's such as Wishart method people such as (, J.Biomol.NMR, 6:135-140,1995) D.S.Wishart, with chemical shift with reference to DSS. Test main chain NH, N, C according to HNCACB and CBCA (CO) NH_α, and C_βResonance assignment (D.R.Muhandiram and J.E.Kay, J.Magn.Reson., 103:203-216,1994). By¹⁵The total correlation spectroscopy (TOCSY) that N edits (people such as O.Zhang, J.Biomol. NMR, 4:845-858,1994) and the HCCH-TOCSY (people such as L.E.Kay, J.Magn.Reson. B, 101:333,1993) the chemical shift assignment of experiment offside chain atom. Use high-res two dimension¹H- ¹H nuclear Overhauser enhanced spectrum is learned (NOESY), TOCSY and DQF-COSY and is tested to the aromatic protons assignment. Between proton the distance from¹⁵The NOESY that N edits (incorporation time 50ms and 150ms) and¹⁵N/ ¹³The NOESY that C edits (incorporation time 75ms) experiment people such as (, J.Magn.Reson.B, 103:197-201,1994) S.M.Pascal. NOE intersection peak intensity is divided into strong, medium, faint or very weak, corresponds respectively to apart from the upper limit 2.8,3.5,4.0 and 5.0 dusts. On average roughly proofread and correct at the center that limits the use of on the methyl of non-stereospecificity assignment and the methene proton. In addition, add 0.5 dust relates to the distance of methyl proton with correction higher-strength to the upper bound. φ restriction derives from HNHA experiment people such as (, Nat.Struct.Biol., 2:768,1995) H.Kuboniwa, and the stereospecificity assignment of β proton gets that free HACAHB experiment derives³In J coupling constant (people such as S.Grzesiek, J.Am.Chem.Soc., 117:5312-5315,1995) and the NOESY spectrum by NH and C_αH to C_βThe relative intensity of the NOE of H proton. The stereospecificity assignment of Leu25 and 66 methyl protons is according to determining C behind 1 jiao of the χ_αH to CH₃The relative NOE intensity of proton draws.

The hydrogen bond restriction. At first according to protein is dissolved in²H ₂A series of 2D of record in 24 hours of O¹H- ¹⁵Slowly the potential candidate of hydrogen bond is identified in the observation that exchanges amide proton of N HSQC spectrum. For each hydrogen bond, use two kinds of distance limit (r_NH-OThe 1.8-2.3 dust, r_N-OThe 2.4-3.3 dust). Only after calculating one group of initial structure, use the hydrogen bond restriction. In Structure Calculation, only use hydrogen bond receptor to satisfy the amide proton of distance and angle limits.

Data processing and Structure Calculation. Use the NMRPipe program groups to process all NMR spectrum people such as (, J.Biomol.NMR, 6:277-293,1995) F.Delaglio. Service routine XPLOR is by heterozygosis distance geometry-dynamics simulation annealing method computation structure (A.T.Br ü nger, 3.1 editions handbooks of XPLOR, Yale University, New Haven, CT, 1993). Altogether 713 irredundant NOE distance limit (320 intra-residue, 178 continuous, 84 middle distances and 132 remote NOE) have been used. In addition, 82 dihedral angles (53 φ and 29 χ in last Structure Calculation, have been used₁) and 36 hydrogen bond restrictions (from 18 hydrogen bonds). Only generate preliminary structure with the NOE restriction, in Structure Calculation subsequently, comprised the restriction of dihedral angle and hydrogen bond. Using standard force field parameter setting among the XPLOR 3.1 editions and topological file to carry out simulated annealing calculates. Altogether generate 50 kinds of structures, better selected 30 kinds of best structures according to minimum energy and spatial chemistry quality.

Dynamics. In 35 ℃ at the homogeneous mark¹⁵Use the pulse train record of gradient type on the N MPIF-1¹⁵N-T ₁、T ₂, and [¹H]- ¹⁵N NOE experiment people such as (, Biochemistry, 33:5984-6003,1994) N.A.Farrow. With 544 (t₂) and 128 (t₁) real point obtained all spectrum, T₂Experiment uses recirculation to postpone 3 seconds, T₁Experiment uses recirculation to postpone 1.2 seconds. By record have or do not have the saturated HSQC spectral measurement of proton [¹H]- ¹⁵N NOE. Postponing 5 seconds records does not have the spectrum of NOE, postpones 2 seconds and the saturated spectrum that recorded NOE in 3 seconds of proton, with the same delay between the generation transition 5 seconds. Use NMRPipe processing spectrum, first two dimension of service routine PIPP artificial assignment¹⁵N-T ₁And T₂Spectrum. Service routine CAPP selects the subsequent optical spectrum automatically. The exponential damping of two parameters of intersection peak nonlinear least square match is obtained T₁And T₂Value. With T₁And T₂The uncertainty of value is as the standard deviation of match. By having or do not have proton saturated and the strong ratio in peak record obtains the NOE value. Estimated the uncertainty of NOE value by the baseline of the spectrum of the people such as Farrow people such as (, Biochemistry, 33:5984-6003,1994) N.A.Farrow definition. The result

Sedimentation equilibrium. The binding ability of chemotactic factor (CF) relies on solution condition, such as pH and ionic strength. Use supercentrifugation to study the binding characteristic of MPIF-1 and total length MPIF-1 in different pH and ionic strength. Table 10 has shown result's general introduction. Two kinds of protein of data indication all are monomer under experiment condition, and do not show in conjunction with tendency. The free state of MPIF-1 and NMR structure and¹⁵Dynamic (dynamical) calculating correlation time of N is consistent.

The structures statistics of MPIF-l. Table 11 has shown the statistics of 30 kinds of final simulation (SA) structures, and Figure 58 A has shown superimposed on average structure of various structures. Except terminal residue 1-10 position and 67-77 position, well determined the structure of this protein. Service routine PROCHECK (people such as R.A.Laskowski, J.Appl.Crystallogr., 26:283-291,1993) and VADAR (the VADAR structural analysis of protein, can byhttp：//www.pence/ualberta.caThe quality of the structure that acquisition) test generates according to multiple standards is such as the zone that occupies in spatial chemistry, hydrogen bond, the Ramachandran collection of illustrative plates, Van der Waals contact, the charged residue of burying, residue number and the packing defect of burying. All 30 kinds of structures have all reached the desired above-mentioned standard of high-res structure.

Table 11:30 kind is calculated structures statistics and the atom r.m.s. difference of MPIF-1 structure
		Energy (kcalmol^-1)
NOE ^a	3.21±0.48	Energy (kcalmol^-1)
NOE ^a	3.21±0.48	Dihedral angle a	0.01±0.01
Key	4.31±0.09	Dihedral angle a	0.01±0.01
Key	4.31±0.09	Van der Waals	1.54±0.30
With respect to the geometric deviation of ideal^b		Van der Waals	1.54±0.30
With respect to the geometric deviation of ideal^b		Key ()	0.0019±0.0001
The angle (°)	0.536±0.001	Key ()	0.0019±0.0001
The angle (°)	0.536±0.001	Improper (improper) (°)	0.305±0.001
Atom r.m.s. difference ()^c		Improper (improper) (°)	0.305±0.001
Atom r.m.s. difference ()^c		Backbone atoms (11-66 position)	0.57±0.08
Heavy atom (11-66 position)	1.09±0.08	Backbone atoms (11-66 position)	0.57±0.08

The numerical value of a NOE and torsion angle is respectively to be 50kcalmol by force constant^-1· ²And 200kcalmol^-1·rad ^-2Square potential well (square well potential) calculate. B key, angle and inappropriate numerical value show with respect to the deviation based on the stereochemical ideal value of perfection. The r.m.s. difference of 30 kinds of final structures of c is superimposed on the average structure.

All structures and energy minimization average structure are showed preferably covalency geometry (table 11) and minimum NMR Constraint Violation. The NOE of 30 kinds of SA structures is no more than 0.2 dust in violation of rules and regulations, and dihedral angle is no more than 2 degree in violation of rules and regulations. 11-66 position residue r.m.s. about all 30 kinds of structures and average structure distributes, and backbone atoms is 0.57 dust, and heavy atom is 1.09 dusts (Figure 59 A and 59B). Assessed the precision (people such as S.G.Hyberts, Protein Sci., 1:736-751,1992) of torsion angle according to order parameter S. The order parameter of φ and φ torsion angle is greater than 0.95 in the 11-66 position residue that makes up, and the main chain of indicating structure has obtained well determining (Figure 59 C and 59D). Figure 59 E has shown χ₁Order parameter. Figure 60 F has shown the solvent accessible area, and the buried and large-scale solvent of low numerical value indication side chain can not approach. These residues tend to have hydrophobicity, and usually by high order parameter χ₁Show it is highly organized (Figure 59 E) greater than 0.9. An exception that shows the large-scale hydrophobe of low S is Ile13. It is exposed to solvent, and guards in several CXC and CC chemotactic factor (CF), and structure-function studies show that this residue brings into play key effect in receptors bind. All torsion angles of observing all 30 kinds of structures all fall into the preference district of Ramachandran collection of illustrative plates. 72% residue falls into core (having a preference for most) district, and 23 % fall into and allow the district, and 1% falls into tolerant district.

The liquid structure that falls of MPIF-l. The structure of MPIF-1 takes typical chemotactic factor (CF) folding, stretches ring and 3 β chains subsequently and C end α spiral by the N end and consists of (Figure 58 B). Front 10 residues before the CC motif do not show NOE or only show N continuous OE that φ and ψ have low order parameter, therefore lack the structure of determining. Be the N end ring that comprises a series of corners (13-20 position residue) subsequently, lead to 3₁₀Spiral (21-24 position residue). Article one, β chain (27-31 position residue) connects second β chain (39-44 position residue) by III type corner, and the latter connects the 3rd β chain (48-52 position residue) by I type corner again. Article three, chain warp III type corner leads to spiral (56-66 position residue). 67-77 position residue is loose to a great extent, and this is consistent with long hanging down apart from NOE shortage, middle distance NOE deficiency and order parameter. Lay respectively at the Thr31 of article one chain and second chain end and the hydroxyl proton of Thr44 and form hydrogen bond with the main chain carbonyl of crossing over chain, therefore bring into play structure function. In 6 cysteines, 4 cysteines are that the feature of all CC chemotactic factor (CF)s: Cys11 and Cys35 form disulfide bond, and this is a part that connects the corner of article one and second β chain; Cys51 in Cys12 and the 3rd the β chain forms disulfide bond. MPIF-1 has two extra cysteines, i.e. Cys22 in 310 spirals and the Cys62 in the α spiral. Produce the data basis of cysteine chemical shift, and observed C_βDisplacement is unique different (result who does not deliver) free closing in the form with disulfide bond. They relate to disulfide bond formation the displacement indication of Cys22 and Cys62. This structure has disclosed cysteine near the formation disulfide bond, and NMR characteristic such as coupling constant is little, the exchange amide proton is slow and also shown this point as the NOE pattern of the Cys62 of spiral residue feature. The Cys12-Cys51 disulfide bond is taked the left hand distortion in most of structures, and other two disulfide bond are loose. Well determined structural core by the α spiral with a large amount of long hydrophobic contacts of distance (Ile20, Leu25, Tyr28, Phe29, Val40, Phe42, Phe50, Ala52, Val59, Met63 and Leu66) between β chain residue. Except disulfide bond, the length between Thr31, Gly39, Tyr15, Cys11, Cys12 and the Cys51 makes N end ring and N terminal residue towards core texture apart from NOE, and this function for them may be important.

Dynamics. Can obtain in 73 expection resonance 60¹⁵NT ₁、T ₂, and NOE relaxation data. Since chemical shift overlapping or for the reliable quantized of intensity signal too a little less than, thereby can not obtain the data of 4 proline, and can not obtain to remain the data of residue Met1, Asp2, His5, Ala6, Ser8, Ile13, Ser19, Ser33, Glu34, Ser36, Lys46, Leu66 and Lys67. Use Model Independent system (G.Lipari and A.Szabo, J.Am.Chem.Soc., 104:4546-4559, the 1982a of Lipari-Szabo; G.Lipari and A.Szabo, J.Am.Chem.Soc., 104:4559-4570,1982b) analyze¹⁵N T1, T2 and NOE relaxation data are to describe the Internal dynamics of MPIF-1. The model that the relaxation data match of each residue is different comprises S²-τ _c(model 1), S²-τ _c-τ _e(model 2), S²-τ _c-R _ex(model 3), S²-τ _c-τ _e-R _ex(model 4) and allow between two different time yardsticks, to occur the dual-time Scale Model (model 5) people such as (, J.Am.Chem.Soc., 112:4989-4991,1990) G.Clore of internal motion. Select suitable model by estimating the match quality for each residue. The T that will test and calculate by use isotropism spectral concentration function at first₁、T ₂, and the NOE value minimizes and calculate best τ on each residue basis_c Be slower than 100ps and T because of the mobility internal motion₂Under the condition that significantly shortens, think T₁/T ₂Ratio (Figure 60 D) does not rely on order parameter or internal motion, and integral body τ correlation time can be provided_cEstimated value. Such residue is accredited as¹⁵N[ ¹H] the NOE value is less than 0.65 and T₁/T ₂Those residues of ratio 1SD beyond mean value are such. On this basis, get rid of 26 residues, and calculated τ according to remaining 34 residues_cBe 4.6 ± 0.2ns.

Comprehensive order parameter (S²), Chemical Exchange (Rex) and local correlation time (τ_c) as the function of amino acid sequence, be shown in Figure 60 E-60G. Comprehensive order parameter provides the measurement of the amplitude of internal motion, wherein S²=1 means that specifying N-H key carrier is rigidity, and S²=0 indication motion is unrestricted. 1-10 position residue before the terminal 67-77 position residue of C and terminal first cysteine of N shows low order parameter (S²＜0.7). Two ends fail all in the NMR structure to determine well that dynamics data has confirmed that these residues are variable in essence, and the shortage of structure is not owing to lack the experiment restriction. End is foreclosed the evaluation S of 11-66 position residue²0.84 ± 0.06. Other residue of showing low order parameter is Arg18 and the Ile20 (0.7) in the N end ring. The relaxation data of residue Tyr15, Cys22, Thr44, Cys51, Ala52, Asn77 need to exchange the time limit (model 3 and 4) (Figure 60 G), and C terminal residue 69-73 position needs the dual-time Scale Model. Residue A rg3, Phe4 and Thr74 can not any models of match. But all other residue match naive models. Discuss

MPIF-1 is monomer in the liquid that falls. The NMR structure determination and from¹⁵The calculating of the dynamic (dynamical) spin correlation time of N indication MPIF-1 is that the form with monomer exists under experiment condition (50mM acetate pH5.2). It is (table 10) that the form with monomer exists that MPIF-1 is also indicated in the research of ultracentrifugation under the multiple solution condition.

Structure and solution research provide some understanding about the binding characteristic of chemotactic factor (CF), but produce still unpredictable of interactional specific molecular characterization. The CC chemotactic factor (CF) shows maximum difference in their binding ability, and can both be existed by very high orderly polymer to monomer. RANTES (the people such as N.J.Skelton, Biochemistry, 34:5329-5342,1995), MIP-1 α (people such as I.G.Czaplewski, J.Biol.chem., 274:16077-16084,1999), (263:1762-1767 for the people such as P.J.Lodi, Science for MIP-1 β, 1994) the neutral pH height in conjunction with (＞100kDa), but reversibly be dissociated into dimer at low pH; And I-309, HCC-2 and MCP-3 are monomer (people such as J.F.Paolini, J.Immunol., 153:2704-2717,1994; The people such as H.Sticht, Biochemistry, 38:5995-6002,1999; The people such as K.S.Kim, FEBS Lett., 395:277-282,1996).

On the other hand, the bonding behavior of Gro-beta-T is restricted more, only forms dimer and the tetramer (people such as I.Clark-Lewis, J.Leukocyte Biol., 57:703-711,1995). Think that according to structure in fact Dimerized vital some residue is only had slight influence or not impact, on the contrary, the sudden change away from the residue of dimer interface but produces monomer (people such as L.G.Czaplewski, J.Biol.Chem., 274:16077-16084,1999; The people such as J.S.Laurence, Biochemistry, 39:3401-3409,2000; The people such as C.D.Paavola, J.Biol.Chem., 273:33157-33165,1998). Obviously, in primary sequence away from residue plurality of stable strength promote dimer and tripolymer in conjunction with in play a role.

Yet, exist compellent evidence show as the CXC of monomer and CC chemotactic factor (CF) all be enough in conjunction with and activated leukocyte on the 7-TM acceptor. Previous 3 kinds of neutrophil cells activation chemotactic factor (CF)s (IL-8, NAP-2 and MGSA) by the trapping free state make studies show that they can not be Dimerized, monomer has the activity (people such as K.Rajarathnam as native protein in external functional examination method, Science, 264:90-92,1994; The people such as K.Rajarathnam, J.Biol.Chem., 272,1725-1729,1997). Similar, the mutation research of RANTES, MCP-1, MIP-1 α and MIP-1 β also displaying monomer is receptors bind kind (people such as L.G.Czaplewski, J.Biol.Chem., 274:16077-16084,1999; The people such as J.S.Laurence, Biochemistry, 39:3401-3409,2000; The people such as C.D.Paavola, J.Biol.Chem., 273:33157-33165,1998).

That has just produced such problem, is what (if any) the definite effect that is dimer and the orderly oligomer of Geng Gao? nearest research illustrates that this effect may be in conjunction with proteoglycans and set up concentration gradient (transportation leucocyte necessary the process) (people such as A.J.Hoogewerf, Biochemistry, 36:13570-13578,1997; W.Koopmann and M.S.Krangel, J.Biol.Chem., 272:10103-10109,1997). For being alkaline residue in conjunction with the vital residue of proteoglycans, such as arginine, lysine and histidine, they tend to that cluster exists and away from in conjunction with the vital zone of 7-TM acceptor in CXC and CC chemotactic factor (CF). In this research, MPIF-1 is not presented at and forms dimeric any tendency in the solution. Whether studying when having the cell surface protein glycan MPIF-1 and other monomer chemotactic factor (CF) I-309 and HCC-2 Dimerized.

The attractive characteristic of chemotactic factor (CF) is the multiple kind that has N end or the processing of C end difference. Some chemotactic factor (CF) as MPIF-1, as large-scale precursor secretion, has the protein of function by the proteolysis processing generation of N end or C end. NAP-2, β-thromboglobulin (β TG) and connective tissue activating protein-III (CTAP-III) are the N end products of platelet basic protein matter (PBP), only having NAP-2 is effective activator of neutrophil cell, and thinks that other is the precursor of non-activity. All four kinds all form dimer and the tetramer (people such as Y.Yang, J.Biol. Chem., 269:20110-20118,1994); In addition, also separated at the NAP-2 of C end difference processing and CTAP-III people such as (, J. Immunol., 161:4975-4982,1998) J.E.Ehlert by cell culture.

About 10 Amino acid profiles of the N end regions of functional chemotactic factor (CF) before by first conservative cysteine, mutation research show these residues in conjunction with and activated receptor have substantial role. The CC chemotactic factor (CF) of brachymemma is showed difference in height and uncommon characteristic people such as (, J.Leukocyte Biol., 57:703-711,1995) I.Clark-Lewis. For example, in MCP-1, lack front 4 residues and cause loss of activity, cause active basic recovery and lack front 5 residues. Further disappearance causes afunction, but has the remaining ability (J.-H.Gong and I.Clark-Lewis, J.Exp. Med., 181:631-640,1995) of the function of bind receptor and performance antagonist. The N of IL-8 (a kind of Gro-beta-T) end consecutive miss causes active increase, loss of activity, then is transformed into antagonist people such as (, J.Biol.Chem., 268:7125-7128,1993) B.Moser. Also observe, the brachymemma of the front N end of first cysteine residue is conducive to form monomer in the CC chemotactic factor (CF), and length of this explanation N end residue plays a role in Dimerized.

The alternative splicing of MPIF-1 gene causes the protein of two kinds of forms, is respectively 99 and 116 amino acid (being called CK β 8 and CK β 8-1) (people such as B.-S.Youn, Blood, 91:3118-3126,1998). This observed result is exception in the CC chemotactic factor (CF), because this difference in length is N end regions (being respectively 32 and 49 amino acid before first cysteine). The protein of two kinds of forms be presented at myeloid progenitor suppress with monocyte chemotaxis in have similar activity. 99 expression of amino acid whose protein in baculoviral produce the protein of 3 kinds of forms, i.e. full-length and two kinds of truncated-types. Find that truncated-type suppresses all more effective in the determination method at monocyte chemotaxis and myeloid progenitor. It is monomers that MPIF-1 and the total length MPIF-1 data (table 10) under multiple solution condition are indicated them, and does not have related between N end residue length and the dimeric ability of formation. The effectiveness difference of MPIF-1 variant as other chemotactic factor (CF), plays a role in vivo, namely spatially and in time regulates and control leukocyticly to raise and activate. Thereby an aspect of leukocyte activation is release action regulates and control its peptase and protease active and function in chemotactic factor (CF).

The description of structure and with the comparison of other chemotactic factor (CF). MPIF-1 takes typical chemotactic factor (CF) folding, i.e. three β chains and a top α spiral thereof. Well determined the core of this structure, shown the minimum rmsd of main chain and heavy atom, it is those acid amides and those acid amides in the α spiral that form hydrogen bond at interchain that the high order parameter of φ, φ and χ 1, great majority exchange acid amides slowly. Illustrate several CC chemotactic factor (CF) structures, comprised MIP-1 β, RANTES, MCP-1, MCP-3, eotaxin and HCC-1 (people such as P.J.Lodi, Science, 263:1762-1767,1994; The people such as N.J.Skelton, Biochemistry, 34:5329-5342,1995; T.M.Handel and P.J.Domaille, Biochemistry, 35:6569-6584,1996; The people such as K.S.Kim, FEBS Lett., 395:277-282,1996; The people such as M.P.Crump, J.Biol.Chem., 273:22471-22479,1998; The people such as H.Sticht, Biochemistry, 38:5995-6002,1999). The secondary of MPIF-1 and tertiary structure element in other CC chemotactic factor (CF) (Figure 61), observe similar, although the sequence homogeneity between MPIF-1 and other CC chemotactic factor (CF) is approximately changing (Figure 62) between the 25%-60%. Be the 1.5-2.0 dust with the superimposed demonstration of the main chain rmsd of structured region among the MPIF-1 (11-66 position residue) and other CC chemotactic factor (CF). In structured region (chain and spiral), observe minimum rmsd, in N end residue, N end ring and 30s corner, observe higher rmsd. In these zones of protein, observe maximal sequence difference, and they also be relatively uncertain, variable and on function important zone.

Except 4 cysteines (Cys11, Cys12, Cys35, Cys51), residue Ile13, Tyr15, Ile20 (N end ring), Leu25, Tyr28, Phe29, Thr31 (article one β chain), Val40, Ile41, Phe42, Thr44 (second β chain), Phe50, Ala52 (the 3rd β chain), Val59, Met63 and Leu66 (α spiral) (according to the MPIF-1 sequence numbering) also are (Figure 62) that substitutes that guard or conservative. Great majority are the large-scale hydrophobes that are arranged in (β) chain or spiral, and take the same side chain conformation in different structures, consist of the support of structure. Thr31 and Thr44 are the polar residues of burying fully, and structure discloses its hydroxyl proton and forms hydrogen bond with the main chain carbonyl of crossing over chain, brings into play thus structure function.

Structure discloses the Cys12-Cys51 disulfide bond and mainly takes the left hand helix conformation.¹⁵The N dynamics data indicates near the residues some cysteine and the 3 pairs of disulfide bond to show mobilities, and these zones that show protein are variable and the experience mobility. The Cys11-Cys35 disulfide bond shows maximum segmentation movement, to cause function significantly loss and structure will not change (the people such as K.Rajarathnam such as faint the shaking that in IL-8, has shown disulfide bond, Biochemistry, 38:7653-7658,1999). The dynamics that someone proposes N end regions, disulfide bond and 30s corner is brought into play integration people such as (, J. Leukocyte Biol., 57:703-711,1995) I.Clark-Lewis at specific binding with in activating. MPIF-1 shows with HCC-2 the highest serial homology, and the latter also has 6 cysteines, can and be the inhibitor of stem cells hyperplasia in conjunction with CCR1. A difference between MPIF-1, HCC-2 and other CC chemotactic factor (CF) is the Trp residue that is arranged in the 58th of CC chemotactic factor (CF), is Gln in MPIF-1, is Gly in HCC-2. The space size that the structure of MPIF-1 and HCC-2 discloses the indoles side chain has hindered disulfide bond.

Except conservative hydrophobe, some charged residue is also guarded; Lys45 and the Arg48 of the Arg18 of N end ring and 40s corner are exposed to solvent, and relate in conjunction with electronegative proteoglycans (people such as L.Chakravarty, J.Biol.Chem., 273:29641-29647,1998; The people such as W.Koopmann, J.Immunol., 163:2120-2127,1999). Yet the relative importance of the location of proteoglycans binding structural domain and the charged residue that relates to combination changes along with the different of chemotactic factor (CF). IL-8 (the people such as G.S.V.Kuschert, Biochemistry, 37:11193,1998), the PF-4 (people such as K.H.Mayo, Biochem. J., 312:357-365,1995) and MCP-1 (people such as L.Chakravarty, J.Bol. Chem., 273:29641-29647,1998) charged residue in the C end spiral, the SDF-1 (people such as A.Amara, J.Biol.Chem., 274:239 16-23925,1999) in residue and MIP-1 α (W.Koopmann and the M.S.Kvangel of article one β chain, J.Biol. Chem., 272:10103-10109,1997) and MIP-1 β (people such as W.Koopmann, J.Immunol., 163:2120-2127,1999) the 40s corner in and the residue of N end ring relate to the combination with proteoglycans. MPIF-1 is highly alkaline protein, and has extra alkaline residue in 40s ring (Lys46) and α spiral (Lys57, Arg64 and Lys67), shows that it is more closely in conjunction with proteoglycans (Figure 63). Structure-function

Monocytic activation. MPIF-1 in conjunction with CCR1, and shows it is monocytic effective activator (people such as B.Nardelli, J.Immunol., 162:435-444,1999 with high-affinity; The people such as T.A.Berkhout, Biochem.Pharmacol., 59:591-596,2000). MCP-3, RNATES, MIP-1 α and HCC-2 also in conjunction with and activate CCR1. Mutagenesis research indication, in CXC and CC chemotactic factor (CF), the N end residue before first cysteine and N end ring (second cysteine and 3₁₀Between the spiral) residue in receptors bind, play a significant role (people such as I.Clark-Lewis, J.Leukocyte Biol., 57:703-711,1995; J.-H.Gong and I.Clark-Lewis, J.Exp.Med., 181:631-640,1995; The people such as B.Moser, J.Biol.Chem., 268:7125-7128,1993; The people such as D.R.Pakianathan, Biochemistry, 36:9642-9648,1997). Someone proposes, and consists of the initial N end residue of stopping the site in the N end ring residue of chemokine ligand and the acceptor and interacts; This interaction has and the interactional Optimal orientation of acceptor residue part N end residue, causes that conformation change and function reply (people such as M.P.Crump, EMBO J., 16:6996-7007,1997; The people such as I.Clark-Lewis, J.Leukocyte Biol., 57:703-711,1995).

In the structure of MPIF-1, N end ring residue has obtained fine definition (S of φ, ψ＞0.95), and takes unique conformation. On the other hand,¹⁵This zone of N dynamics data indicator protein matter is relatively variable, and shows the mobility of the fluctuation of subnanosecond and slow millisecond time scale. In eotaxin (people such as M.P.Crump, Protein Sci., 8:2041-2054,1999; The people such as J.Ye, J.Biomed.NMR, 15:115-124,1999), vMIPII (in conjunction with a kind of viral monomer CC chemotactic factor (CF) of multiple CxC and the CC-chemokine receptor) (people such as A.C.LiWang, Biochemistry, 38:442-453,1999) and fractalkine (a kind of monomer CX₃The C chemotactic factor (CF)) in (people such as L.S.Mizoue, Biochemistry, 38:1402-1414,1999) similar observed result is arranged. N end ring structure territory is near all three pairs of disulfide bond. The residue that sequence analysis also discloses corresponding to Ile13, Tyr15, Arg18 and Ile20 (according to the numbering of MPIF-1) is that guard or conservative substituting in other CC chemotactic factor (CF), and the mutagenesis of these residues causes the combination of acceptor corresponding with it to weaken. Ile13 (second cysteine after first residue) is exposed to solvent in all CXC and CC chemotactic factor (CF), be most likely at and bring into play direct effect in the activated receptor. In RANTES, the importance of observing N end ring residue has receptor-specific: Arg17 is necessary in conjunction with CCR1, Phe12 is necessary in conjunction with CCR3, Phe12 and Ile15 are in conjunction with the necessary (people such as D.R.Pakianathan of CCR5, Biochemistry, 36:9642-9648,1997). In most of structures, Tyr15 and Ile20 bury, and take similar conformation, and suffer the parcel of other hydrophobic residue, the indicating structure effect. These observed results show that in MPIF-1, Ile13 and Arg18 bring into play structure function, and are directly involved in receptors bind; Tyr15 and Ile20 are as the part performance function of structure stand.

The NMR structure of MPIF-1 and dynamics data indication, the N end residue before first cysteine is loose. Obtain similar observed result in all monomer NMR structures and dimer CXC structure, it is necessary that the mutability that shows these residues interacts for the best with acceptor. All Gro-beta-Ts that activate neutrophil cell have combination and activate necessary characteristic " ELR " sequence people such as (, J.Leukocyte Biol., 57:703-711,1995) I.Clark-Lewis before first cysteine. Do not have this characteristic sequence in the CC chemotactic factor (CF), and sequence analysis fails to provide any information about receptor-specific. In addition, the CC chemotactic factor (CF) shows complicated ligand-receptor characteristic. Most of CC chemotactic factor (CF)s of activated mononuclear cell, macrophage, eosinophil and T cell can be in conjunction with multiple acceptor, and most receptors can be in conjunction with multiple chemotactic factor (CF). On the other hand, a known part example (LARC and CCR6) in conjunction with a kind of acceptor. The N of MPIF-1 end residue shows with very little in conjunction with the similitude of other CC chemotactic factor (CF) (HCC-2, MCP-3, RANTES and MIP-1 α) of CCR1 or do not have. HCC-2 shows with MPIF-1 the highest whole sequence homology (about 60%), but the N end does not have homology. Recently, except CCR1 also the structure-function in conjunction with the RANTES of multiple acceptor studies show that have receptor-specific to reply to some N end residue that suddenlys change; Pro2, Asp6 and Thr7 are for being necessary in conjunction with CCR1; Pro2 and Tyr3 are for being necessary in conjunction with CCR3; Tyr3 and Asp6 are for being necessary in conjunction with CCR5. It is identical not having which N end residue between MPIF-1 and RANTES, and to only have a residue be conservative (Tyr3 among the RANTES and the Phe4 among the MPIF-1) that substitutes. Tyr3 is presented at RANTES in conjunction with playing a significant role among the CCR3, and that whether MPIF-1 can have in conjunction with CCR3 is to be tested. What is interesting is, show recently, is that the character of the length of N end residue but not side chain is vital people such as (, Biochemistry, 38:16167-16177,1999) Jarnagin for MCP-1 in conjunction with CCR2.

The containment of progenitor cell proliferation. The MPIF-1 of form of ownership shows the containment progenitor cell proliferation, and it is more effective than front albumen to observe aspect inhibition truncated-type. Strikingly, CC and Gro-beta-T, such as MIP-1 α, IL-8, GRO-β, PF-4, IP-10 and MCP-1, demonstration can be contained progenitor cell proliferation; And relevant chemotactic factor (CF) is not contained effect such as GRO-α, NAP-2, MIP-1 β and RNATES. The chemotactic factor (CF) characteristic shows that the ability of regulation and control progenitor cell proliferation and the activation of its homology chemokine receptors have nothing to do. In addition, the monomeric form of MIP-1 α is than the aggregated forms of native protein significantly more effective (people such as L.G.Czaplewski, J.Biol. Chem., 274:16077-16084,1999), and MPIF-1 can play a role with monomeric form. These molecular basis researchs of MPIF-1 function have clinical relevance with the patient (such as the patient of experience chemotherapy) of various diseases.

Obviously, can implement to put into practice the present invention by other method beyond above description and embodiment specifically describe.

According to teaching above, many modifications of the present invention and change are possible, therefore belong within the scope of claims.

All patents, patent application and the content of delivering thing are collected herein by reference. Complete being collected herein by reference of open book with the Application No. 08/941,020 submitted on September 30th, 1997.

Sequence table＜110〉Grzegorzewski, Krzysztof J.

Rosen，Craig A.

Patel, Vikram＜120〉use CKBeta8 (MPIF-1) to treat or prevent cell, tissue, with the method for organ damage＜130〉1488.033PC0P＜150〉US 60/159,362＜151〉1999-10-14＜150〉US 60/164,059＜151〉1999-11-08＜150〉US 60/172,063＜151〉1999-12-23＜150〉US 60/189,048＜151〉2000-03-14＜150〉US 60/199,142＜151〉2000-04-24＜150〉US 60/211,458＜151〉2000-06-13＜150〉US 60/212,658＜151〉2000-06-19＜160〉42＜170〉PatentIn 3.0 editions＜210〉1＜211〉363＜212〉DNA＜213〉human (Homo sapiens)＜220〉＜221〉CDS＜222〉(1) .. (360)＜400〉1 atg aag gtc tcc gtg gct gcc ctc tcc tgc ctc atg ctt gtt act gcc, 48 Met Lys Val Ser Val Ala Ala Leu Ser Cys Leu Met Leu Val Thr Ala, 15 10 15 ctt gga tcc cag gcc cgg gtc aca aaa gat gca gag aca gag ttc atg, 96 Leu Gly Ser Gln Ala Arg Val Thr Lys Asp Ala Glu Thr Glu Phe Met

20 25 30 atg tca aag ctt cca ttg gaa aat cca gta ctt ctg gac aga ttc cat 144 Met Ser Lys Leu Pro Leu Glu Asn Pro Val Leu Leu Asp Arg Phe His

35 40 45 gct act agt gct gac tgc tgc atc tcc tac acc cca cga agc atc ccg 192 Ala Thr Ser Ala Asp Cys Cys Ile Ser Tyr Thr Pro Arg Ser Ile Pro

50 55 60 ggt tca ctc ctg gag agt tac ttt gaa acg aac agc gag tgc tcc aag 240 Gly Ser Leu Leu Glu Ser Tyr Phe Glu Thr Asn Ser Glu Cys Ser Lys 65 70 75 80 ccg ggt gtc atc ttc ctc acc aag aag ggg cga cgt ttc tgt gcc aac 288 Pro Gly Val Ile Phe Leu Thr Lys Lys Gly Arg Arg Phe Cys Ala Asn

85 90 95 ccc agt gat aag caa gtt cag gtt tgc atg aga atg ctg aag ctg gac 336 Pro Ser Asp Lys Gln Val Gln Val Cys Met Arg Met Leu Lys Leu Asp

100 105 110 aca cgg atc aag acc agg aag aat tga 363 Thr Arg Ile Lys Thr Arg Lys Asn

115 120＜210〉2＜211〉120＜212〉PRT＜213〉mankind＜400〉2 Met Lys Val Ser Val Ala Ala Leu Ser Cys Leu Met Leu Val Thr Ala 15 10 15 Leu Gly Ser Gln Ala Arg Val Thr Lys Asp Ala Glu Thr Glu Phe Met

20 25 30 Met Ser Lys Leu Pro Leu Glu Asn Pro Val Leu Leu Asp Arg Phe His

35 40 45 Ala Thr Ser Ala Asp Cys Cys Ile Ser Tyr Thr Pro Arg Ser Ile Pro

50 55 60 Gly Ser Leu Leu Glu Ser Tyr Phe Glu Thr Asn Ser Glu Cys Ser Lys 65 70 75 80 Pro Gly Val Ile Phe Leu Thr Lys Lys Gly Arg Arg Phe Cys Ala Asn

85 90 95 Pro Ser Asp Lys Gln Val Gln Val Cys Met Arg Met Leu Lys Leu Asp

100 105 110 Thr Arg Ile Lys Thr Arg Lys Asn

115 120＜210〉3＜211〉100＜212〉PRT＜213〉mankind＜400〉3 Met Arg Val Thr Lys Asp Ala Glu Thr Glu Phe Met Met Ser Lys Leu 15 10 15 Pro Leu Glu Asn Pro Val Leu Leu Asp Arg Phe His Ala Thr Ser Ala

20 25 30 Asp Cys Cys Ile Ser Tyr Thr Pro Arg Ser Ile Pro Cys Ser Leu Leu

35 40 45 Glu Ser Tyr Phe Glu Thr Asn Ser Glu Cys Ser Lys Pro Gly Val Ile

50 55 60 Phe Leu Thr Lys Lys Gly Arg Arg Phe Cys Ala Asn Pro Ser Asp Lys 65 70 75 80 Gln Val Gln Val Cys Met Arg Met Leu Lys Leu Asp Thr Arg Ile Lys

85 90 95 Thr Arg Lys Asn

100＜210〉4＜211〉76＜212〉PRT＜213〉mankind＜400〉4 Met Arg Phe His Ala Thr Ser Ala Asp Cys Cys Ile Ser Tyr Thr Pro 15 10 15 Arg Ser Ile Pro Cys Ser Leu Leu Glu Ser Tyr Phe Glu Thr Asn Ser

20 25 30 Glu Cys Ser Lys Pro Gly Val Ile Phe Leu Thr Lys Lys Gly Arg Arg

35 40 45 Phe Cys Ala Asn Pro Ser Asp Lys Gln Val Gln Val Cys Met Arg Met

50 55 60 Leu Lys Leu Asp Thr Arg Ile Lys Thr Arg Lys Asn 65 70 75＜210〉5＜211〉78＜212〉PRT＜213〉mankind＜400〉5 His Ala Ala Gly Phe His Ala Thr Ser Ala Asp Cys Cys Ile Ser Tyr 15 10 15 Thr Pro Arg Ser Ile Pro Cys Ser Leu Leu Glu Ser Tyr Phe Glu Thr

20 25 30 Asn Ser Glu Cys Ser Lys Pro Gly Val Ile Phe Leu Thr Lys Lys Gly

35 40 45 Arg Arg Phe Cys Ala Asn Pro Ser Asp Lys Gln Val Gln Val Cys Met

50 55 60 Arg Met Leu Lys Leu Asp Thr Arg Ile Lys Thr Arg Lys Asn 65 70 75＜210〉6＜211〉599＜212〉DNA＜213〉human＜220〉＜221〉CDS＜222〉(35) .. (445)＜400〉6 gtcctccggc cagccctgcc tgcccaccag gagg atg aag gtc tcc gtg gct gcc 55

Met Lys Val Ser Val Ala Ala

1 5 ctc tcc tgc ctc atg ctt gtt act gcc ctt ggc tcc cag gcc cgg gtc 103 Leu Ser Cys Leu Met Leu Val Thr Ala Leu Gly Ser Gln Ala Arg Val

10 15 20 aca aaa gat gca gag aca gag ttg acg atg tca aag ctt cca ttg gaa 151 Thr Lys Asp Ala Glu Thr Glu Leu Thr Met Ser Lys Leu Pro Leu Glu

25 30 35 aat cca gta ctt ctg gac atg ctc tgg agg aga aag att ggt cct cag 199 Asn Pro Val Leu Leu Asp Met Leu Trp Arg Arg Lys Ile Gly Pro Gln 40 45 50 55 atg acc ctt tct cat gcc gca gga ttc cat gct act agt gct gac tgc 247 Met Thr Leu Ser His Ala Ala Gly Phe His Ala Thr Ser Ala Asp Cys

60 65 70 tgc atg tcc tac acc cca cga agc atc ccg tgt tca ctc ctg gag agt 295 Cys Met Ser Tyr Thr Pro Arg Ser Ile Pro Cys Ser Leu Leu Glu Ser

75 80 85 tac ttt gaa acg aac agc gag tgc tcc aag ccg ggt gtc atc ttc ctc 343 Tyr Phe Glu Thr Asn Ser Glu Cys Ser Lys Pro Gly Val Ile Phe Leu

90 95 100 acc aag aag ggg cga cgt ttc tgt gcc aac ccc agt gat aag caa gtt 391 Thr Lys Lys Gly Arg Arg Phe Cys Ala Asn Pro Ser Asp Lys Gln Val

105 110 115 cag gtt tgc atg aga atg ctg aag ctg gac aca cgg atc aag acc agg, 439 Gln Val Cys Met Arg Met Leu Lys Leu Asp Thr Arg Ile Lys Thr Arg, 120 125 130 135 aag aat tgaacttgtc aaggtgaagg ggacacaagt tgccagccac caactttctt, 495 Lys Asn gcctcaacta acttcctgaa ttcttttttt aagaagcatt tattcttgtg ttctggattt, 555 agagcaattc atcttttctc acctttaaaa aaaaaaaaaa aaaa 599＜210〉7＜211〉137＜212〉PRT＜213〉mankind＜400〉7 Met Lys Val Ser Val Ala Ala Leu Ser Cys Leu Met Leu Val Thr Ala 15 10 15 Leu Gly Ser Gln Ala Arg Val Thr Lys Asp Ala Glu Thr Glu Leu Thr

20 25 30 Met Ser Lys Leu Pro Leu Glu Asn Pro Val Leu Leu Asp Met Leu Trp

35 40 45 Arg Arg Lys Ile Gly Pro Gln Met Thr Leu Ser His Ala Ala Gly Phe

50 55 60 His Ala Thr Ser Ala Asp Cys Cys Met Ser Tyr Thr Pro Arg Ser Ile 65 70 75 80 Pro Cys Ser Leu Leu Glu Ser Tyr Phe Glu Thr Asn Ser Glu Cys Ser

85 90 95 Lys Pro Gly Val Ile Phe Leu Thr Lys Lys Gly Arg Arg Phe Cys Ala

100 105 110 Asn Pro Ser Asp Lys Gln Val Gln Val Cys Met Arg Met Leu Lys Leu

115 120 125 Asp Thr Arg Ile Lys Thr Arg Lys Asn

130 135＜210〉8＜211〉26＜212〉DNA＜213〉＜400〉8 tcaggatccg tcacaaaaga tgcaga 26＜210〉9＜211〉26＜212〉DNA＜213〉＜400〉9 cgctctagag taaaacgacg gccagt 26＜210〉10＜211〉27＜212〉DNA＜213〉＜400〉10 cccgcatgcg ggtcacaaaa gatgcag 27＜210〉11＜211〉27＜212〉DNA＜213〉＜400〉11 aaaggatcct caattcttcc tggtctt 27＜210〉12＜211〉48＜212〉DNA＜213〉＜400〉12 acatgcatgc guguuaccaa agacgcugaa accgaauuca ugaugucc 48＜210〉13＜211〉36＜212〉DNA＜213〉＜400〉13 gcccaagctt tcagttttta cgggttttga tacggg 36＜210〉14＜211〉88＜212〉DNA＜213〉＜400〉14 gcatgcgugu uaccaaagac gcugaaaccg aauucaugau guccaaacug ccgcuggaaa 60 acccgguucu gcuggaccgu uuccacgc 88＜210〉15＜211〉104＜212〉DNA＜213〉＜400〉15 gcuggaaucc uacuucgaaa ccaacuccga augcuccaaa ccggguguua ucuuccugac 60 caaaaaaggu cgucguuucu gcgcuaaccc guccgacaaa cagg 104＜210〉16＜211〉89＜212〉DNA＜213〉＜400〉16 aagctttcag tttttacggg tgggcagacg ggtgtccagt ttcagcatac gcatacaaac 60 ctgaacctgt ttgtcggacg gcttagcgc 89＜210〉17＜211〉94＜212〉DNA＜213〉＜400〉17 ggtttcgaag taggattcca gcagggagca cgggatggaa cgcggggtgt aggagatgca 60 gcagtcagcg gaggtagcgt ggaaacggtc cagc 94＜210〉18＜211〉32＜212〉DNA＜213〉＜400〉18 gcgcagccat ggaaaacccg gttctgctgg ac 32＜210〉19＜211〉83＜212〉PRT＜213〉＜400〉19 Met Glu Asn Pro Val Leu Leu Asp Arg Phe His Ala Thr Ser Ala Asp 1 5 10 15 Cys Cys Ile Ser Tyr Thr Pro Arg Ser Ile Pro Cys Ser Leu Leu Glu

20 25 30 Ser Tyr Phe Glu Thr Asn Ser Glu Cys Ser Lys Pro Gly Val Ile Phe

35 40 45 Leu Thr Lys Lys Gly Arg Arg Phe Cys Ala Asn Pro Ser Asp Lys Gln

50 55 60 Val Gln Val Cys Met Arg Met Leu Lys Leu Asp Thr Arg Ile Lys Thr, 65 70 75 80 Arg Lys Asn＜210〉20＜211〉35＜212〉DNA＜213〉primer＜400〉20 gccatggcat gctggaaaac ccggttctgc tggac 35＜210〉21＜211〉84＜212〉PRT＜213〉mankind＜400〉21 Met Leu Glu Asn Pro Val Leu Leu Asp Arg Phe His Ala Thr Ser Ala 15 10 15 Asp Cys Cys Ile Ser Tyr Thr Pro Arg Ser Ile Pro Cys Ser Leu Leu

20 25 30 Glu Ser Tyr Phe Glu Thr Asn Ser Glu Cys Ser Lys Pro Gly Val Ile

35 40 45 Phe Leu Thr Lys Lys Gly Arg Arg Phe Cys Ala Asn Pro Ser Asp Lys

50 55 60 Gln Val Gln Val Cys Met Arg Met Leu Lys Leu Asp Thr Arg Ile Lys, 65 70 75 80 Thr Arg Lys Asn＜210〉22＜211〉32＜212〉DNA＜213〉primer＜400〉22 gcgcagccat ggaccgtttc cacgctacct cc 32＜210〉23＜211〉77＜212〉PRT＜213〉mankind＜400〉23 Met Asp Arg Phe His Ala Thr Ser Ala Asp Cys Cys Ile Ser Tyr Thr 15 10 15 Pro Arg Ser Ile Pro Cys Ser Leu Leu Glu Ser Tyr Phe Glu Thr Asn

20 25 30 Ser Glu Cys Ser Lys Pro Gly Val Ile Phe Leu Thr Lys Lys Gly Arg

35 40 45 Arg Phe Cys Ala Asn Pro Ser Asp Lys Gln Val Gln Val Cys Met Arg

50 55 60 Met Leu Lys Leu Asp Thr Arg Ile Lys Thr Arg Lys Asn, 65 70 75＜210〉24＜211〉29＜212〉DNA＜213〉primer＜400〉24 gccatggcat gcgtttccac gctacctcc, 29＜210〉25＜211〉32＜212〉DNA＜213〉primer＜400〉25 gcgcagccat ggctacctcc gctgactgct gc 32＜210〉26＜211〉73＜212〉PRT＜213〉mankind＜400〉26 Met Ala Thr Ser Ala Asp Cys Cys Ile Ser Tyr Thr Pro Arg Ser Ile 15 10 15 Pro Cys Ser Leu Leu Glu Ser Tyr Phe Glu Thr Asn Ser Glu Cys Ser

20 25 30 Lys Pro Gly Val Ile Phe Leu Thr Lys Lys Gly Arg Arg Phe Cys Ala

35 40 45 Asn Pro Ser Asp Lys Gln Val Gln Val Cys Met Arg Met Leu Lys Leu

50 55 60 Asp Thr Arg Ile Lys Thr Arg Lys Asn, 65 70＜210〉27＜211〉21＜212〉DNA＜213〉primer＜400〉27 ttcgaagtag gcttccagca g, 21＜210〉28＜211〉21＜212〉DNA＜213〉primer＜400〉28 ctgctggaag cctacttcga a, 21＜210〉29＜211〉35＜212〉DNA＜213〉primer＜400〉29 gccatggcat gcgtgttacc aaagacgctg aaacc 35＜210〉30＜211〉100＜212〉PRT＜213〉mankind＜400〉30 Met Arg Val Thr Lys Asp Ala Glu Thr Glu Phe Met Met Ser Lys Leu 15 10 15 Pro Leu Glu Asn Pro Val Leu Leu Asp Arg Phe His Ala Thr Ser Ala

20 25 30 Asp Cys Cys Ile Ser Tyr Thr Pro Arg Ser Ile Pro Cys Ser Leu Leu

35 40 45 Glu Ala Tyr Phe Glu Thr Asn Ser Glu Cys Ser Lys Pro Gly Val Ile

85 90 95 Thr Arg Lys Asn

100＜210〉31＜211〉36＜212〉DNA＜213〉＜400〉31 gcccaagctt tcagttttta cgggttttga tacggg 36＜210〉32＜211〉27＜212〉DNA＜213〉＜400〉32 ggaaagctta tgaaggtctc cgtggct 27＜210〉33＜211〉59＜212〉DNA＜213〉＜400〉33 cgctctagat caagcgtagt ctgggacgtc gtatgggtaa ttcttcctgg tcttgatcc 59＜210〉34＜211〉33＜212〉DNA＜213〉＜400〉34 aaaggatccg ccaccatgaa ggtctccgtg gtc 33＜210〉35＜211〉27＜212〉DNA＜213〉＜400〉35 aaaggatcct caattcttcc aggtctt 27＜210〉36＜211〉92＜212〉PRT＜213〉＜400〉36 Met Gln Val Ser Thr Ala Ala Leu Ala Val Leu Leu Cys Thr Met Ala 1 5 10 15 Leu Cys Asn Gln Phe Ser Ala Ser Leu Ala Ala Asp Thr Pro Thr Ala

20 25 30 Cys Cys Phe Ser Tyr Thr Ser Arg Gln Ile Pro Gln Asn Phe Ile Ala

35 40 45 Asp Tyr Phe Glu Thr Ser Ser Gln Cys Ser Lys Pro Gly Val Ile Phe

50 55 60 Leu Thr Lys Arg Ser Arg Gln Val Cys Ala Asp Pro Ser Glu Glu Trp 65 70 75 80 Val Gln Lys Tyr Val Ser Asp Leu Glu Leu Ser Ala

85 90 <210>37 <21l>4208 <212>DNA <213> <400>37 aagcttaaaa aactgcaaaa aatagtttga cttgtgagcg gataacaatt aagatgtacc 60 caattgtgag cggataacaa tttcacacat taaagaggag aaattacata tggaccgttt 120 ccacgctacc tccgctgact gctgcatctc ctacaccccg cgttccatcc cgtgctcgct 180 gctggaatcc tacttcgaaa ccaactccga atgctccaaa ccgggtgtta tcttcctgac 240 caaaaaaggt cgtcgtttct gcgctaaccc gtccgacaaa caggttcagg tttgtatgcg 300 tatgctgaaa ctggacaccc gtatcaaaac ccgtaaaaac tgataaggta cctaagtgag 360 tagggcgtcc gatcgacgga cgcctttttt ttgaattcgt aatcatggtc atagctgttt 420 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag 480 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg 540 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg 600 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc 660 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc 720 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg 780 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat 840 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag 900 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga 960 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg 1020 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt 1080 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac 1140 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc 1200 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt 1260 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc 1320 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc 1380 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg 1440 aacgaaaact cacgttaagg gattttggtc atgagattat cgtcgacaat tcgcgcgcga 1500 aggcgaagcg gcatgcattt acgttgacac catcgaatgg tgcaaaacct ttcgcggtat 1560 ggcatgatag cgcccggaag agagtcaatt cagggtggtg aatgtgaaac cagtaacgtt 1620 atacgatgtc gcagagtatg ccggtgtctc ttatcagacc gtttcccgcg tggtgaacca 1680 ggccagccac gtttctgcga aaacgcggga aaaagtggaa gcggcgatgg cggagctgaa 1740 ttacattccc aaccgcgtgg cacaacaact ggcgggcaaa cagtcgttgc tgattggcgt 1800 tgccacctcc agtctggccc tgcacgcgcc gtcgcaaatt gtcgcggcga ttaaatctcg 1860 cgccgatcaa ctgggtgcca gcgtggtggt gtcgatggta gaacgaagcg gcgtcgaagc 1920 ctgtaaagcg gcggtgcaca atcttctcgc gcaacgcgtc agtgggctga tcattaacta 1980 tccgctggat gaccaggatg ccattgctgt ggaagctgcc tgcactaatg ttccggcgtt 2040 atttcttgat gtctctgacc agacacccat caacagtatt attttctccc atgaagacgg 2100 tacgcgactg ggcgtggagc atctggtcgc attgggtcac cagcaaatcg cgctgttagc 2160 gggcccatta agttctgtct cggcgcgtct gcgtctggct ggctggcata aatatctcac 2220 tcgcaatcaa attcagccga tagcggaacg ggaaggcgac tggagtgcca tgtccggttt 2280 tcaacaaacc atgcaaatgc tgaatgaggg catcgttccc actgcgatgc tggttgccaa 2340 cgatcagatg gcgctgggcg caatgcgcgc cattaccgag tccgggctgc gcgttggtgc 2400 ggatatctcg gtagtgggat acgacgatac cgaagacagc tcatgttata tcccgccgtt 2460 aaccaccatc aaacaggatt ttcgcctgct ggggcaaacc agcgtggacc gcttgctgca 2520 actctctcag ggccaggcgg tgaagggcaa tcagctgttg cccgtctcac tggtgaaaag 2580 aaaaaccacc ctggcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt 2640 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta 2700 atgtaagtta gcgcgaattg tcgaccaaag cggccatcgt gcctccccac tcctgcagtt 2760 cgggggcatg gatgcgcgga tagccgctgc tggtttcctg gatgccgacg gatttgcact 2820 gccggtagaa ctccgcgagg tcgtccagcc tcaggcagca gctgaaccaa ctcgcgaggg 2880 gatcgagccc ggggtgggcg aagaactcca gcatgagatc cccgcgctgg aggatcatcc 2940 agccggcgtc ccggaaaacg attccgaagc ccaacctttc atagaaggcg gcggtggaat 3000 cgaaatctcg tgatggcagg ttgggcgtcg cttggtcggt catttcgaac cccagagtcc 3060 cgctcagaag aactcgtcaa gaaggcgata gaaggcgatg cgctgcgaat cgggagcggc 3120 gataccgtaa agcacgagga agcggtcagc ccattcgccg ccaagctctt cagcaatatc 3180 acgggtagcc aacgctatgt cctgatagcg gtccgccaca cccagccggc cacagtcgat 3240 gaatccagaa aagcggccat tttccaccat gatattcggc aagcaggcat cgccatgggt 3300 cacgacgaga tcctcgccgt cgggcatgcg cgccttgagc ctggcgaaca gttcggctgg 3360 cgcgagcccc tgatgctctt cgtccagatc atcctgatcg acaagaccgg cttccatccg 3420 agtacgtgct cgctcgatgc gatgtttcgc ttggtggtcg aatgggcagg tagccggatc 3480 aagcgtatgc agccgccgca ttgcatcagc catgatggat actttctcgg caggagcaag 3540 gtgagatgac aggagatcct gccccggcac ttcgcccaat agcagccagt cccttcccgc 3600 ttcagtgaca acgtcgagca cagctgcgca aggaacgccc gtcgtggcca gccacgatag 3660 ccgcgctgcc tcgtcctgca gttcattcag ggcaccggac aggtcggtct tgacaaaaag 3720 aaccgggcgc ccctgcgctg acagccggaa cacggcggca tcagagcagc cgattgtctg 3780 ttgtgcccag tcatagccga atagcctctc cacccaagcg gccggagaac ctgcgtgcaa 3840 tccatcttgt tcaatcatgc gaaacgatcc tcatcctgtc tcttgatcag atcttgatcc 3900 cctgcgccat cagatccttg gcggcaagaa agccatccag tttactttgc agggcttccc 3960 aaccttacca gagggcgccc cagctggcaa ttccggttcg cttgctgtcc ataaaaccgc 4020 ccagtctagc tatcgccatg taagcccact gcaagctacc tgctttctct ttgcgcttgc 4080 gttttccctt gtccagatag cccagtagct gacattcatc cggggtcagc accgtttctg 4140 cggactggct ttctacgtgt tccgcttcct ttagcagccc ttgcgccctg agtgcttgcg 4200 gcagcgtg 4208 <210>38 <211>112 <212>DNA <213> <400>38 aagcttaaaa aactgcaaaa aatagtttga cttgtgagcg gataacaatt aagatgtacc 60 caattgtgag cggataacaa tttcacacat taaagaggag aaattacata tg 112 <210>39 <211>74 <212>PRT <213> <400>39 Met His Ala Thr Ser Ala Asp Cys Cys Ile Ser Tyr Thr Pro Arg Ser 1 5 10 15 Ile Pro Cys Ser Leu Leu Glu Ser Tyr Phe Glu Thr Asn Ser Glu Cys

20 25 30 Ser Lys Pro Gly Val Ile Phe Leu Thr Lys Lys Gly Arg Arg Phe Cys

35 40 45 Ala Asn Pro Ser Asp Lys Gln Val Gln Val Cys Met Arg Met Leu Lys

50 55 60 Leu Asp Thr Arg Ile Lys Thr Arg Lys Asn 65 70＜210〉40＜211〉79＜212〉PRT＜213〉mankind＜400〉40 Met His Ala Ala Gly Phe His Ala Thr Ser Ala Asp Cys Cys Met Ser 15 10 15 Tyr Thr Pro Arg Ser Ile Pro Cys Ser Leu Leu Glu Ser Tyr Phe Glu

20 25 30 Thr Asn Ser Glu Cys Ser Lys Pro Gly Val Ile Phe Leu Thr Lys Lys

35 40 45 Gly Arg Arg Phe Cys Ala Asn Pro Ser Asp Lys Gln Val Gln Val Cys

50 55 60 Met Arg Met Leu Lys Leu Asp Thr ArgIle Lys Thr Arg Lys Asn 65 70 75＜210〉41＜211〉85＜212〉PRT＜213〉mankind＜400〉41 Met Pro Gln Met Thr Leu Ser His Ala Ala Gly Phe His Ala Thr Ser l 5 10 15 Ala Asp Cys Cys Met Ser Tyr Thr Pro Arg SerIle Pro Cys Ser Leu

20 25 30 Leu Glu Ser Tyr Phe Glu Thr Asn Ser Glu Cys Ser Lys Pro Gly Val

35 40 45 Ile Phe Leu Thr Lys Lys Gly Arg Arg Phe Cys Ala Asn Pro Ser Asp

50 55 60 Lys Gln Val Gln Val Cys Met Arg Met Leu Lys Leu Asp Thr Arg Ile 65 70 75 80 Lys Thr Arg Lys Asn

85＜210〉42＜211〉733＜212〉DNA＜213〉＜400〉42 gggatccgga gcccaaatct tctgacaaaa ctcacacatg cccaccgtgc ccagcacctg 60 aattcgaggg tgcaccgtca gtcttcctct tccccccaaa acccaaggac accctcatga 120 tctcccggac tcctgaggtc acatgcgtgg tggtggacgt aagccacgaa gaccctgagg 180 tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa tgccaagaca aagccgcggg 240 aggagcagta caacagcacg taccgtgtgg tcagcgtcct caccgtcctg caccaggact 300 ggctgaatgg caaggagtac aagtgcaagg tctccaacaa agccctccca acccccatcg 360 agaaaaccat ctccaaagcc aaagggcagc cccgagaacc acaggtgtac accctgcccc 420 catcccggga tgagctgacc aagaaccagg tcagcctgac ctgcctggtc aaaggcttct 480 atccaagcga catcgccgtg gagtgggaga gcaatgggca gccggagaac aactacaaga 540 ccacgcctcc cgtgctggac tccgacggct ccttcttcct ctacagcaag ctcaccgtgg 600 acaagagcag gtggcagcag gggaacgtct tctcatgctc cgtgatgcat gaggctctgc 660 acaaccacta cacgcagaag agcctctccc tgtctccggg taaatgagtg cgacggccgc 720 gactctagag gat 733

Claims

1. the method for the cellular damage for the treatment of or preventing to be induced by cytotoxic agent, comprise polypeptide or its active variant or the fragment of individuality being used the SEQ ID NO:2 of effective dose, wherein said cell is selected from lower group: (a) cell of connective tissue, except stem cell, hematopoietic cell and multipotency CFU-GM; (b) epithelial cell; (c) myocyte; (d) neural cell.

2. the process of claim 1 wherein that the cell of described connective tissue is selected from lower group: the cell of skeletal system, Gegenbaur's cell, osteoclast, osteocyte, cartilage cell, adipocyte, periosteum cell, bone inner cell, odontoblast, haemocyte, red blood cell, leucocyte, eosinophil, basophilic granulocyte, neutrophil cell, lymphocyte, monocyte, blood platelet, tissue macrophages, organ specificity phagocyte, bone-marrow-derived lymphocyte, T lymphocyte, megaloblast, monoblast, myeloblast, lymphoblast, primitive erythroblast, megakaryoblast, premonocyte, promyelocyte, young lamphocyte, early stage metarubricyte, megacaryocyte, mid-term metarubricyte, juvenile cell and late period metarubricyte.

3. the process of claim 1 wherein that described individuality accepting to kill and wound the therapy of cell in the division.

4. the method for claim 3, wherein said therapy is selected from chemotherapy, radiotherapy or targeted radiotherapy.

5. the process of claim 1 wherein that described individuality is exposed to cytotoxic agent because of occupation or accident.

6. the method for claim 4 was wherein used described polypeptide before described therapy processes.

7. the method for claim 4 is wherein used described polypeptide in described therapy processes.

8. the method for claim 4 is wherein used described polypeptide after described therapy.

9. the method for claim 5 was wherein used described polypeptide before described exposure.

10. the method for claim 5 is wherein used described polypeptide in described process-exposed.

11. the method for claim 5 is wherein used described polypeptide after described exposure.

12. the method for claim 4, wherein said therapy is targeted radiotherapy.

13. the method for claim 12, wherein said targeted radiotherapy is radioimmunotherapy.

14. the process of claim 1 wherein that described individuality having a mind to be exposed to cytotoxic agent.

15. the method for claim 14, wherein said cytotoxic agent are radiation.

16. the method for claim 15 was wherein used described polypeptide before described exposure.

17. the method for claim 15 is wherein used described polypeptide in described process-exposed.

18. the method for claim 15 is wherein used described polypeptide after described exposure.

19. the method for claim 15, wherein said radiation is attributed to nuclear explosion.

20. the method for claim 19, wherein said individual need is for protection or the treatment of radiation sickness or radiation burn.

21. suppress the method for Leukemia Cell Proliferation, comprise polypeptide or its active variant or the fragment of the SEQ ID NO:2 that uses effective dose, wherein said polypeptide, variant or fragment have been puted together the therapeutic part.

22. the method for claim 21, wherein said therapeutic partly are radio isotopes.