CN1871255A - Novel secretory protein and production process and use thereof - Google Patents

Abstract

The objective is to provide a polypeptide having a novel structure and showing an activity of inhibiting angiogenesis or an activity of inhibiting osteoclastogenesis, and to provide a recombinant protein by constructing a method of purifying the above protein. To provide an ingredient useful in designing remedies for tendinitis, rheumatoid arthritis, arthritis deformans, malignant tumor, etc. A novel soluble polypeptide protein.

Description

Novel secretory protein and its production and use

Technical field

The present invention relates to have active solubility novel protein of inhibition of bone resorption and preparation method thereof, use this proteinic novel diagnosis medicine and curative because of the activity and/or the osteoclast formation inhibition activity that suppress the blood vessel generation.

Background technology

So-called " blood vessel generation " is meant in vivo and can forms blood vessel again.Exist promotion material and inhibitory substance for blood vessel, this balance adjustment blood vessel and is taken place.Adult blood guard system total length is 10km in general, and the surface-area of vascular endothelial cell is 7000m ², weight is 1kg, it is generally acknowledged that it is the organ that is distributed in the human body maximum of intravital all sites.In fetus period and developmental process, blood vessel takes place active, but for adult, thinks that only at ovulation and trauma care and so on blood vessel taking place just in particular cases takes place.Vascular system is indispensable for earning a bare living, and above-mentioned blood vessel is the reaction of normal organism.

On the other hand, the reason of various diseases takes place also to become in the unusual blood vessel outside the above-mentioned situation.The example of its representative has the cancer blood vessel to take place.The cancerous tissue medium vessels causes enlarging markedly of tumour, and transfer ability strengthens.Therefore, if suppress to supply with the new life of the blood vessel of nutritive ingredient, just can make cancer remain on dormant state to cancer cells.The people such as Folkman that propose the tumour dormancy identify the supressor of the blood vessel generation of cancer cells generation, called after angiostatin and endostatin (non-patent literature 1, non-patent literature 2).After having known that these blood vessel generation supressors almost completely make the cancer degeneration in mouse, Study of Angiogenesis is able to positive carrying out.Now, many pharmacy corporations are devoted to the exploitation of angiogenesis inhibitor, implement clinical trial with cancer as object, but do not realize capturing cancer, place hope on novel angiogenesis inhibitor.

Disease beyond the cancer, for example unusual blood vessel also takes place and takes place in diabetic retinopathy in the rheumatoid arthritis etc., has reported many data now, and these Notes of Key Datas take place and might can treat these diseases by suppressing these blood vessels.Therefore, think that angiogenesis inhibitor becomes at cancer and other treatment of diseases medicine of following blood vessel to take place.

Vascular system is distributed in intravital all sites, but also has the tissue that lacks plexus vasculosus.Tissue as lacking plexus vasculosus can have cartilage, tendon, ligament, eyeball etc. for example.In the mescenchymal tissue, bone and muscle etc. is rich in blood vessel, and therefore producing fracture and muscle injury also has the regenerated ability.

Relative with it, the cartilage of mescenchymal tissue, tendon, ligament are the tissues that lacks plexus vasculosus, when damage or fracture take place, regenerate, cure naturally very difficulty.Therefore on the other hand,, can cause disorganization, think to have endogenic blood vessel generation supressor in these tissues, suppress blood vessel from intrusion on every side in case blood vessel is invaded in these vesselless tissues.

Cartilage regulatory factor (Chondromodulin)-I (ChM-I) is as the blood vessel generation supressor that exists in the cartilage, be by ox fetus cartilage purifying, be the sugar-protein that size is about 25kDa, think that now it controls the intrusion (non-patent literature 3) of blood vessel to cartilage.

In addition, found to have the II type transmembrane protein ChM1L (non-patent literature 4, patent documentation 1) of homology with ChM-I.ChM1L is the gene of specifically expressing in tough conjunctive tissues such as tendon, ligament, thinks that now its control blood vessel is to these tissues intrusions (patent documentation 2, non-patent literature 5, non-patent literature 6).

As mentioned above, tendon and ligament also are all the tissue that lacks plexus vasculosus with cartilage.Tendon and ligament are to link bone and the important tissue of muscle, and their damage or fracture also can the constrained motion players and common people's body kinematics, therefore are considered to serious disease.This tendon and ligament tissue are important tissues, but compare with cartilage, and basis and clinical study up to now also do not have very big progress.Its reason can exemplify the security of materials such as cell, does not have the tagged molecule of tendon or ligament specifically expressing.Because this background, therefore the hope existence can be estimated the damage of tendon or ligament or the tagged molecule of the degree of reparation.

Owing to find ChM1L specifically expressing in tendon and ligament, therefore think and to utilize it as the damage of estimating these tissues or the tagged molecule of reparation.In addition, by the activity of control ChM1L, the possibility of treatment tendon or ligament injury is arranged also.

But, take place for protein such as ChM-I and secretary protein and ChM1L being used to suppress blood vessel, still open question is also a lot.

When recombinant protein is developed as pharmaceutical preparation, need a large amount of active proteins of preparation.In general, but in plant-scale protein production widespread use adopt the especially colibacillary expression system of microorganism.Favourable part at expression in escherichia coli is, but by using the carrier and the high-density culture of high level expression, can obtains high levels of recombinant proteins matter.

But, be that often intestinal bacteria form the inclusion body that contains recombinant protein easily as there being major issue in this situation.In fact, when expression in escherichia coli, also form inclusion body, make it reclaim difficulty (non-patent literature 2), therefore now also at research modification method (non-patent literature 3) owing to implement the endostatin of clinical trial as angiogenesis inhibitor.

People also study the ChM-I that the activity that suppresses the blood vessel generation because of it is developed to cancer therapy drug, but it is reported, form inclusion body when escherichia coli expression ChM-I, make it reclaim difficulty (non-patent literature 7).In addition, even adopt Chinese hamster ovary (CHO) cell as host cell, ChM-I forms to assemble also needs the process that reclaims, therefore obtains a large amount of active proteins have any problem (non-patent literature 8).

It is reported, the same with ChM-I for ChM1L, when expression in escherichia coli ChM1L, also form inclusion body, make and reclaim difficulty (non-patent literature 9).In addition,, can from nutrient solution, obtain active protein, but its expression amount is low, equally with ChM-I forms gathering, can't obtain a large amount of active protein (patent documentation 4) by in the nutrient solution of COS7 cell, expressing ChM1L.

In addition, thus the zone, extracellular of transmembrane protein is cut off and makes it be secreted into the extracellular.For example, it is synthetic that tumor necrosis factor-alpha (TNF-α) is used as II type transmembrane protein, play the function of transmembrane protein, but it cut off by proteolytic enzyme such as TNF-α saccharases as can be known now, also plays the function of secreted protein.For ChM-I, also have II type membrane protein structure, but processed by the site (RERR) of proteolytic enzyme such as furin identification as can be known, 120 amino acid of C-terminal can be secreted into extracellular (non-patent literature 3 and non-patent literature 8).

But, the secretor type ChM-I that above-mentioned 120 amino-acid residues constitute, even with its recombinant production do not demonstrate enough solubilities yet, do not solve foregoing problems.

In addition, for ChM1L, though have amino acid sequence homology with ChM-I, but there is not the so typical proteolytic enzyme recognition site of ChM-I, infer its function, do not have the report (patent documentation 4:WO00/12708, patent documentation 5:WO00/29579, patent documentation 6:WO01/23557, patent documentation 7:WO01/48203, patent documentation 8:WO01/53344, non-patent literature 4, non-patent literature 5, non-patent literature 6) of secretary protein as transmembrane protein.

Patent documentation 1:WO01/23557

Patent documentation 2:WO01/53344

Patent documentation 3: special table 2002-504494 communique

Patent documentation 4:WO00/12708

Patent documentation 5:WO00/29579

Patent documentation 6:WO01/23557

Patent documentation 7:WO01/48203

Patent documentation 8:WO01/53344

People Cell (USA) such as non-patent literature 1:O ' Reilly on October 21st, 1994 published the 79th volume the 2nd phase p315-328

People Cell (USA) such as non-patent literature 2:O ' Reilly on January 24th, 1997 published the 88th volume the 2nd phase p277-285

Non-patent literature 3: The Journal of Biological Chemistry (USA) such as open and on December 19th, 1997 published the 272nd volume the 51st phase p32419-32426

Non-patent literature 4: Biochemical and Biophysical ResearchCommunications (USA) such as mountain name publishes the 280th volume the 4th phase p1101-1106 on February calendar year 2001 2

Developmental dynamics:an officialpublication of the American association of anatomists (USA) such as non-patent literature 5:Brandau publishes the 221st volume the 1st phase p72-80 May calendar year 2001

Biochemical and biophysical researchcommunications (USA) such as non-patent literature 6: Su Nan publishes the 280th volume the 5th phase p1323-1327 on February calendar year 2001 2

Non-patent literature 7: Japanese molecular biosciences such as mountains and rivers association the 25th annual meeting lecture summary collects publishes 2P-0206 November calendar year 2001

The Journal of biological chemistry (USA) such as non-patent literature 8:Azizan publishes the 276th volume the 26th phase p32419-32426 on June calendar year 2001 29

Non-patent literature 9: Japanese molecular biosciences association such as Chang Gu river the 25th annual meeting lecture summary collects publishes 2P-0770 November calendar year 2001

Disclosure of an invention

The problem that invention will solve

Like this, because wild-type ChM-I and ChM1L are its a large amount of preparations aspect in the disadvantage of the utilization of aspects such as medicine, therefore need the different material of exploitation or need to develop the method for preparing wild-type ChM-I or ChM1L easily at least.

The method that is used to deal with problems

The inventor is to natural transmembrane ChM1L various researchs in addition, it found that a kind of new having suppresses the active soluble polypeptide (S-ChM1L) that blood vessel takes place, it should be called the ChM1L secretary protein of never reporting up to now, and then has identified S-ChM1L and its modification type polypeptide (MS-ChM1L) and have and suppress the activity that blood vessel takes place and suppress activity by the bone resorption due to the osteoclast.

In other words, the present invention relates to 1) comprise the aminoacid sequence shown in the sequence number 9 or have the aminoacid sequence of at least 70% homology with the aminoacid sequence shown in the sequence number 9, and have suppress that blood vessel takes place active and/or suppress the active polypeptide of bone resorption.In addition, also relate to 2) according to 1) polypeptide, its N-terminal and/or C-terminal also are added with amino-acid residue, 3) according to 2) polypeptide, the amino-acid residue that its N-terminal adds is for to be made of the initial aminoacid sequence of methionine(Met), 4) according to 2) or 3) shown in polypeptide, the amino-acid residue that its N-terminal and/or C-terminal add is that successive 6-8 histidine mark sequence and/or the aminoacid sequence that contains the FLAG flag sequence constitute, 5) according to 2) or 3) shown in polypeptide, the amino-acid residue that this N-terminal and/or C-terminal add is fluorescence protein or its analogue that jellyfish (Aequorea victoria) obtains, or the aminoacid sequence of secretor type alkaline phosphatase or its analogue constitutes.Also relate to 6) according to 1) polypeptide; the amino-acid residue that its N-terminal and/or C-terminal add contains modified amino-acid residue, 7) according to 6) polypeptide; the amino-acid residue that its N-terminal adds is glutamine or pyroglutamyl amine residue, 8) according to 6) polypeptide, it is characterized in that described modified amino-acid residue has to be selected from least a modification group in ethanoyl, formyl radical, biotinyl, Boc base or the Fmoc base.

In addition, the invention still further relates to 9) have a nucleic acid molecule of the nucleotide sequence of the aminoacid sequence shown in the encoding sequence numbers 9,10) have the nucleic acid molecule of nucleotide sequence of the 4-243 position of sequence number 3, perhaps under stringent condition with the complementary sequence hybridization of the nucleotide sequence of the 4-243 position of sequence number 3, and has the nucleic acid molecule that coding has the nucleotide sequence of the active polypeptide that suppresses the active of blood vessel generation and/or suppress bone resorption, 11) have coding 2)-6) nucleic acid molecule of the nucleotide sequence of the polypeptide shown in each, 12) contain 9)-11) carrier of the nucleic acid molecule shown in each, 13) by 12) the carrier transformed host cells.

And then, aforementioned polypeptides can adopt suitable recombinant host cell to be prepared into soluble polypeptide, also find by denaturing agent and the specific tensio-active agent of coexisting in the preparation, can with in the impurity such as intracellular toxin of this soluble protein from host cell easily purifying come out, thereby finish the present invention.

In other words, the invention still further relates to 14) according to aforementioned 1)-8) preparation method of the polypeptide shown in each, it is characterized in that it has cultivates aforementioned 13) transformed host cell step and reclaim the step of the polypeptide that generates, 15) according to 14) the preparation method of polypeptide, it is characterized in that in the presence of protein denaturant, from transformed host cell, reclaiming the extracting solution that contains polypeptide, 16) according to 14) or 15) preparation method of the peptide shown in each, it comprises the extracting solution that reclaims with the TritonX-114 processing from host cell after, carrying out centrifugation handles to remove the step of thermal source, 17) aforementioned 14)-16) preparation method of the polypeptide shown in each, it is characterized in that after reclaiming the extracting solution contain polypeptide from host cell institute in steps, the pH that will contain the solution of this polypeptide is adjusted in the scope of 8.0-8.5,18) prepare the method for recombinant human ChM-I or recombinant human ChM1L, its be adopt can expressing human ChM-I or the recombinant host cell of people ChM1L prepare the method for recombinant human ChM-I or recombinant human ChM1L, this method is included in protein denaturant and has the process that reclaims the extracting solution that contains recombinant human ChM-I or recombinant human ChM1L down from the host cell of reorganization, and carry out centrifugation to remove the step of thermal source after with TritonX-114 this extracting solution being handled, 19) according to aforementioned 18) the preparation method, it is characterized in that after reclaiming the step of extracting solution in steps in, the pH that will contain the solution of this polypeptide is adjusted in the scope of 8.0-8.5.

In addition, the invention provides and contain aforementioned 1) to 8) pharmaceutical composition of the polypeptide shown in each, especially 21) the pharmaceutical composition shown in the claim 20, it is angiogenesis inhibitor and/or osteoclast activation inhibitor, and is used to diagnose and takes place with tendonitis, rheumatoid arthritis, osteoarthritis, malignant tumour, diabetic retinopathy, glaucoma, psoriasis, keloid, arteriosclerosis etc. and blood vessel or the composition for diagnosis of bone resorption associated conditions.

And then, also provide and contained aforementioned 9)-11) transgenic nonhuman animal through genetic manipulation of the nucleic acid molecule shown in each.

The effect of invention

Soluble protein of the present invention is as mentioned below, have and suppress blood vessel activity that takes place and the activity that suppresses by the bone resorption that activation produced of osteoclast, be expected to the time as drug use, blood vessel takes place and the activation of osteoclast by suppressing, and acquisition is especially at the obvious treatment effect of rheumatoid arthritis and bone metastatic tumo(u)r.

And then, because protein so antigenicity that soluble protein of the present invention is the partial sequence by naturally occurring ChM1L to be constituted are low, in adopting reconstitution cell production, not needing folding again process, therefore can prepare in a large number.

And then, can remove the intracellular toxin that derives from host cell easily among the preparation method of soluble protein of the present invention, can make soluble proteins of the present invention directly be applied to the level of organism to reach, this is to use in the preparation of reconstitution cell required.This in addition method is also effective for the purifying of the wild-type ChM1L that is difficult to usefulness ordinary method recombinant production.

The preferred forms of invention

＜soluble polypeptide S-ChM1L, MS-ChM1L or their nucleic acid molecule of encoding 〉

The present invention relates to have the active soluble polypeptide that suppresses blood vessel activity that takes place and the bone resorption that suppresses to cause by osteoclast.

Known what is called forms the phenomenon experience following steps of capillary vessel " blood vessel take place (angiogenesis) " again: in (1) basement membrane of blood vessel and the extracellular matrix around it, the digestion that causes by matrix metalloproteinase (MMP) etc., the migration of (2) vascular endothelial cell, the propagation of (3) vascular endothelial cell, the stage that forms of (4) capillary vessel.Alleged blood vessel takes place to suppress to be meant relevant with at least one stage in the above-mentioned stage among the present invention, suppresses the activity of blood vessel generation basically.

Shown in the embodiment described as follows, the polypeptide with the aminoacid sequence shown in the sequence number 9 has the activity in all stages of suppressing above-mentioned (1)-(4).Therefore, think that polypeptide of the present invention can be used as the treatment of diseases drug use that cancer, rheumatoid arthritis, psoriasis, diabetic retinopathy etc. follow blood vessel to take place.

In addition, find that surprisingly soluble polypeptide of the present invention also has the function of the absorption of the bone that inhibition causes by osteoclast.Alleged bone resorption suppresses to be meant to the inhibition activity by the absorption of the bone that activation produced of osteoclast among the present invention.

Generally speaking, the balancing control bone metabolism by the bone resorption that is adjusted to bone forming that osteocyte produces and produces by osteoclast.As the disease that causes unusually by bone metabolism, known have osteoporosis, rheumatoid arthritis, bone Paget disease, hypercalcemia, the forfeiture of tooth rim bone, kidney osteotrophy abnormality disease (renal osteodystrophy), bone solvability tumour and a bone metastatic tumo(u)r etc.Therefore, soluble polypeptide of the present invention also can be used at the relevant above-mentioned treatment of diseases medicine of bone resorption that produces by osteoclast.Especially, because being problem, rheumatoid arthritis is to follow the synovial cell's that blood vessel takes place inflammation, propagation, and the disease of the problem of the bone/cartilage destruction that causes by osteoclast, and the bone metastatic tumo(u)r is a kind of like this disease, its medium vessels takes place with the increase of tumour and shifts closely related, osteoclast is closely related with the dissolving of the bone that is caused by bone resorption, therefore compares with existing medicament, and soluble polypeptide of the present invention has advantage.

Soluble polypeptide of the present invention, have the polypeptide (S-ChM1L) of the aminoacid sequence shown in the sequence number 9 typically, also can be that one or more than one amino-acid residue have further been added in its two ends, especially added the polypeptide (MS-ChM1L) that constitutes the amino-acid residue of other peptide except that ChM1L at described polypeptide two ends.For example, the polypeptide that has added glutamine or pyroglutamyl amine on the N-terminal of the aminoacid sequence shown in the sequence number 9 also has and suppresses activity that blood vessel takes place and the activity that suppresses bone resorption.In addition, even add suitable flag sequence on the N-terminal of the aminoacid sequence shown in the sequence number 9, being typically the polypeptide that adds histidine mark or FLAG mark still has activity that suppresses the blood vessel generation and the activity that suppresses bone resorption.And then even replace flag sequence, the polypeptide that adds from the fluorescence protein of jellyfish or the aminoacid sequence of secretor type alkaline phosphatase etc. still keeps desirable function.

Therefore, it should be understood that polypeptide (S-ChM1L), its one or both ends with the aminoacid sequence shown in the sequence number 9 have added other proteinic amino acid whose polypeptide (MS-ChM1L) and also belonged in the scope of the present invention.

In addition, a part of amino-acid residue in the aminoacid sequence shown in the sequence number 9 lacked or replace but still the active polypeptide that has suppress that blood vessel takes place active and/or suppress bone resorption within the scope of the invention, it also can be regarded as MS-ChM1L.

For example, the soluble polypeptide that also can obtain in S-ChM1L, to have and more than one amino-acid residue to replace with the amino-acid residue that has similar chemical property or structure with it.This amino acid whose replacement that is replaced into chemical property or structural similitude, the concrete form that is the replacement of high conservative property is that those skilled in the art are well-known, for example can exemplify glycine (Gly) and proline(Pro) (Pro), Gly and L-Ala (Ala) or Xie Ansuan (Val), leucine (Leu) and Isoleucine (Ile), L-glutamic acid (glu) and glutamine (Gln), aspartic acid (Asp) and l-asparagine (Asn), halfcystine (Cys) and Threonine (Thr), Thr and Serine (Ser) or ala, Methionin (Lys) and arginine (Arg) etc.

Therefore, even soluble polypeptide is to be made of the sequence different with the aminoacid sequence shown in the sequence number 9, as long as the replacement of its difference and high conservative property as indicated above quite and this polypeptide have the activity that suppresses the active and/or inhibition bone resorption that blood vessel takes place, then this soluble polypeptide also is a kind of of MS-ChM1L.

Aminoacid sequence shown in the aminoacid sequence of above-mentioned MS-ChM1L and the sequence number 9 have 70% or more than, preferred 80% or more than, or more preferably 90% or above homology.

The MS-ChM1L of the invention described above can adopt chemical synthesis or common genetic recombination techniques easily to be prepared.

As preparation by gene engineering method, can exemplify in appropriate host cell and to express the DNA that the base sequence by the aminoacid sequence of coding soluble polypeptide of the present invention constitutes, reclaim the method for this polypeptide.For example, typically can prepare the DNA of the aminoacid sequence N-terminal interpolation of coding shown in sequence number 9 from the base sequence formation of the aminoacid sequence of the initial aminoacid sequence of methionine(Met), it is reconstituted in transformed host cell on the suitable carriers, makes its expression again.

In addition, for efficient and the expression efficiency that improves stability, solubility, purifying, the molecule that detection is produced etc., can prepare and be coded in the DNA that has added the aminoacid sequence of any peptides formation to the aminoacid sequence shown in the sequence number 9, it is reconstituted in transformed host cell on the suitable carriers, makes its expression again.

As an example, soluble polypeptide of the present invention can be prepared as follows, and promptly comprises his flag sequence that contains the successive histidine residues that has more than 6 or 6 or FLAG flag sequence by the nucleotide sequence preparation of coded markings sequence.Soluble polypeptide with this structure has the advantage that is easy to by metallo-chelate carrier or antibody purification.

In addition as other example, also can be to have added the formal representation that constitutes from the so-called fused protein of the aminoacid sequence of the fluorescence protein of jellyfish or secretor type alkaline phosphatase.For with fused protein from the fluorescence protein of jellyfish, can measure its fluorescence intensity, for measuring the colour developing that produces by this enzyme and its substrate reactions, luminous or intensity of fluorescence, easily detect the existence of these fused proteins with the fused protein of secretor type alkaline phosphatase.

This situation, the fused protein of these soluble polypeptides of the present invention, gene order that typically can be by the polypeptide (S-ChM1L) that constitutes at the amino acid shown in the encoding sequence numbers 95 ' or 3 ' terminal in conjunction with coding from the fluorescence protein of jellyfish or the base sequence of secretor type alkaline phosphatase, be inserted into expression vector and in suitable host, express acquisition and translate into above-mentioned each proteinic aminoacid sequence.

In addition, the soluble protein of the present invention that the expression of arbitrary amino acid sequence form is arranged with fusion, can obtain promptly suitable polypeptide and the sequence bonded form that is added on this polypeptide, the form of perhaps removing the aminoacid sequence that it added as follows with S-ChM1L.An example as the method for the aminoacid sequence of removing interpolation, can be by in the sequence that contains soluble polypeptide and be added on and insert Methionin between the sequence on these sequences and carry out gene constructed, handle the protein of expressing acquisition with endopeptidase Lys-C (EC.3.4.21.50), partly carry out purifying by cutting off this lysine residue C-terminal, reclaim the polypeptide that contains soluble polypeptide sequence part.

The whole bag of tricks that above-mentioned operation can utilize those skilled in the art to use always carries out, in addition, also can utilize the known method such as method (people's such as hermanson biological combination technology (bioconjugate technique) (USA) published in 1996 Academic Press) of the synthetic polypeptide of enzyme or chemistry.

In addition, behind the soluble polypeptide that obtains to obtain or produce,, its structure can obtain soluble polypeptide of the present invention by being modified also by method of gene recombination by living tissue.For intravital protein of biology or peptide hormone etc., known its N-terminal takes to be converted to the form of Pyrrolidonecarboxylic acid or the form of modifying through ethanoyl or formyl radical etc., and the raising while activity of the stability of protein or peptide hormone is changed.Thus, can also adopt the N-terminal of posttranslational modification soluble polypeptide molecule of the present invention as required.An example as the method that obtains this molecule, expression contains the soluble polypeptide molecule and N-terminal is the sequence of glutamine residue, by under acidic conditionss such as 5-10% acetum, handling the gained polypeptide, can obtain the molecule that N-terminal is converted to pyroglutamyl amine (Proceedings of theNational Academy of Science of the United States of America (USA) such as park in March, 1991 publish p22046-2050).In addition as other example, to contain N-terminal be the soluble polypeptide polypeptide of sequence of the present invention of expression with arbitrary amino acid residue etc. of α amino by handling with sulfo--NHS-acetic acid or Glacial acetic acid, can obtain the peptide that N-terminal is acetylation.In addition, also can modify with compound treatment such as fluorescent substances and contain gained soluble polypeptide polypeptide of sequence.

The molecule of modifying ChM1L and having homology through aminoacid replacement for preparing that above method obtains by the method identical with ChM1L with the method that obtains following recombinant protein, by the method shown in the embodiment 5-13, can confirm to suppress blood vessel activity that takes place and the activity that suppresses bone resorption.

＜recombinant protein 〉

The expression of soluble polypeptide of the present invention and purifying typically can be undertaken by following manner, and preparation can be expressed the recombinant DNA of the gene of coded polypeptide in host cell, it are imported in the host cell transform, and cultivate transformant.As this host cell, can use any of eukaryotic host cell and prokaryotic host cell.

In the eukaryotic host cell, can contain vertebrates, yeast and insect cell etc.Chinese hamster ovary celI, 293T cell and COS7 cell etc. can have been exemplified as vertebrate cells.

As vertebrate expression vector, can use promotor usually with the upstream position that is positioned at gene to be expressed, treat the carrier of the polyadenylation position in expressing gene downstream and transcription termination sequence etc.As this expression vector, can exemplify for as have pSV2dhfr (Mol.Cell.boil., 854,1981), the pcDNA3.1 (+) (Invitrogen company) of the early promoter of SV40 and a pCAGGS (Gene, 108,193-200,1991) etc.

It is known that the method for expressing target protein matter in the eukaryotic cell has many systems in the art.For example, can there be the spy to open " protein expression in the yeast " shown in the clear 57-159489 communique for example as expression system in the yeast, can there be the spy to open " preparation method of recombination bacillary viral vector " shown in the clear 60-37988 communique for example as the expression system in the insect cell, can exemplify the spy as the expression system in the mammalian cell and open " improvement of eukaryotic expression " shown in the flat 2-171198 communique, in addition also exist a lot certainly.

For example also can express the gene that obtains coding soluble polypeptide of the present invention in the prokaryotic host cells such as intestinal bacteria, Bacillus subtilus and streptomycete.For example, can preferably use bacterial strains such as intestinal bacteria (Echerichia coli) K12 as above-mentioned host's intestinal bacteria, can use pBR322 and improved carrier thereof, but also can use known various bacterial strains and the carrier that is not limited to these carriers as carrier.As promotor, for example promotors such as intestinal bacteria lactose (lac), intestinal bacteria trp can be arranged for example, but be not limited to these promotors.In addition, above-mentioned promotor all is well known to those skilled in the art, can be that synthetic also can be by known plasmid construction.

Encode and to introduce many modifications or change in the nucleotide sequence of the gene of soluble polypeptide of the present invention, the recombinant plasmid that contains this gene or recombinant virus.For example, by the degeneracy of genetic code,, can Nucleotide be replaced not changing under its coded amino acid whose situation for whole cryptographic zone of polypeptide.Such sequence can be inferred according to proteinic aminoacid sequence, also can make up by the synthetic method of following routine.This synthetic method can be carried out according to people's such as people's such as Itakura method (people such as Itakura, Science198,1059,1977) and Crea method (people such as Crea, Proc.Natl.Acad.Sci.USA75,5765,1978) basically.Therefore, the present invention is not limited to base sequence, plasmid and the virus in the illustration especially.

Method and consequent method for transformation as the goal gene importing host cell that will so obtain can adopt conventional the whole bag of tricks.In addition, the transformant of acquisition can be cultivated according to ordinary method, can produce soluble polypeptide of the present invention.As employed substratum in cultivating, the various substratum commonly used that the host cell that can suitably select to be adopted needs are cultivated under the condition that is suitable for the host cell growth.

By foregoing, can be in the cell of transformant, produce this protein on extracellular or the cytolemma.Soluble polypeptide of the present invention, as required, (compile " biochemical data book II " the 1st edition the 1st printing, Tokyo KCC with reference to Japanese biological chemistry association published people Biochemistry (USA) such as p1175-1259 Arakawa and published the 25th on December 16th, 1986 and roll up the 25th phase p8274-8277 (1986) various lock out operation that can be by utilizing character such as its physics character, chemical property on June 23rd, 1980 with the people; People EuropianJouanal of Biochemistry (Germany) such as Langley on March 2nd, 1987 published the 163rd volume the 2nd phase p313-321 etc.) separate purifying.As this method, can exemplify for example common reconstruction process, handle (salting-out process), centrifugation, osmotic shock by protein precipitant, various liquid chromatographies such as ultrasonic disruption, ultrafiltration, gel-filtration, adsorption chromatography, ion exchange chromatography, affinity chromatography, high performance liquid chromatography (HPLC), dialysis method, their combination etc.In addition, merge the protein that affinity tag is arranged on this protein, just may utilize this mark to implement protein affinity purification if be expressed in.As this affinity tag, polyhistidine tag (Journal of Virology (USA) such as His mark, Sisk in February, 1994 publish the 68th volume the 2nd phase p766-775) and FLAG mark Biotechnology such as (published the 6th in 1988 roll up p1204-1210) Hopp for example can be arranged for example.Merge these affinity tags soluble polypeptide of the present invention (MS-ChM1L) expression and detect, can implement according to embodiment 1 described mode, use the purifying of the MS-ChM1L of this mark also to implement to obtain according to embodiment 3 described modes.The preparation method of soluble polypeptide of the present invention more specifically is specified in embodiment 3.

＜synthetic peptide 〉

As the method for the soluble polypeptide of the present invention that obtains, can exemplify the method for this polypeptide of chemosynthesis.

This situation can be utilized the general method of peptide synthesis that adopts such as solid-phase synthesis or liquid phase synthesizing method.During peptide is synthetic; can utilize known method (peptide synthetic basis such as spring paddy and the kind Co., Ltd. of experiment ball published in 1975, vow the biochemical Biochemistry Experiment lecture 1 proteinic chemical IV Tokyo chemistry of can compiling of island etc.-Japan published in 1977) for the disengaging of the protection of condensation method or amino-acid residue and synthetic back protecting group with people Co., Ltd..In addition, also can the whole peptide sequence of synthesizing soluble polypeptide protein, but also can adopt synthetic respectively this proteinic partial peptide, the method for this partial peptide of condensation (the Japan proteinic chemical IV of biochemical meeting volume Biochemistry Experiment lecture synthesizes and expresses the Tokyo chemistry and published in 1991 with people Co., Ltd.).The amino acid whose α amino that uses in peptide is synthetic is subjected to tBoc base or the protection of Fmoc base usually, for the peptide of final acquisition, can be to have the form of these protecting groups or the form of deprotection still.In addition as required, also can form the amino-terminal end of deprotection of this peptide through the form of chemically modifieds such as enzyme modification or Pyrrolidonecarboxylic acid or ethanoyl or formyl radical (people's such as Hermanson biological combination technology (USA) Academic Press1996 publishes).The peptide of sequence that can contain soluble polypeptide as concrete example is synthetic in the form that N-terminal has glutamine, becomes Pyrrolidonecarboxylic acid (Proceedings of the National Academy of Science of the United States ofAmerica (USA) such as Park in March, 1991 publish p22046-2050) by make its cyclisation with the processing of diluted acids such as 5-10% acetum.

＜antibody 〉

Can be used for the diagnosis/treatment of bone/joint disease at the antibody of soluble polypeptide of the present invention.For example in diagnosis, can utilize Western trace, immuno-precipitation, ELISA method of antibody etc.

The antibody of above-mentioned employing can use the method that well known to a person skilled in the art to obtain.The antibody that uses among the present invention can be polyclonal antibody or monoclonal antibody (Nature (England) such as Milstein on October nineteen eighty-three 6 published the 305th volume the 5934th phase p537-540).For example, at the polyclonal antibody of soluble polypeptide of the present invention can from the phase homopolypeptide as antigen, from reclaiming by the mammiferous serum of its sensitization etc.At the monoclonal antibody of described polypeptide, can from Mammals, take out immunocyte by described antigen sensibilization, wait to merge with the myeloma cell who clones to obtain hybridoma, from its culture, reclaim.

In the proteinic detection of soluble polypeptide of the present invention or ChM1L, can be to the suitable mark of these antibody.In addition, not these antibody of mark and mark and this antibodies specific bonded material, for example a-protein or protein G indirect detection go out described polypeptide or protein.

Can be by for example in expression vector, inserting gene or its part of encoded peptide, import appropriate host cell, make transformant, thereby cultivate this transformant express recombinant protein matter, from culture or culture supernatant, be purified into the recombinant protein of expression, obtain as antigenic soluble polypeptide of the present invention or its partial peptide.Perhaps, the oligopeptides that also can chemosynthesis constitutes by aminoacid sequence or its partial amino-acid series of described genes encoding, with it as immunogen.As by the animal of immunity, can use mouse, rat, rabbit, goat, horse, hamster etc.

＜diagnostic method 〉

For diagnostic method of the present invention, usually with the organism sample collected from the tester as sample.As biological sample, preferred blood sample.So-called blood sample, the blood plasma or the serum that can use whole blood or obtain by whole blood.In addition, as the biological material among the present invention, except blood, can also use synovia, joint cartilage sheet, synovial tissue, tendinous tissue, ligament tissue, muscle tissue, tear etc. by the examination of living tissue collection.The collection method of these biological samples is known.

If prepare lysate, can make the sample of the immunologic mensuration that is used for soluble polypeptide of the present invention or ChM1L by above-mentioned organism sample.The extracting of the lysate of organism sample can utilize commercially available test kit to carry out easily.If soluble polypeptide of the present invention in addition or ChM1L are secreted in the blood or in the synovia, just can determine soluble polypeptide of the present invention contained in the body fluid samples such as examinee's blood or serum or the amount of ChM1L.Can use as required said sample in the method for the present invention with dilutions such as damping fluids.

And then the invention provides and be used for taking place or the reagent of the diagnostic method of the disease that bone resorption is relevant with blood vessel.Promptly the present invention relates to a kind ofly comprise that identification contains the antibody of polypeptide of the aminoacid sequence of soluble polypeptide of the present invention, be used to diagnose and follow that disease that blood vessel takes place and ChM1L are active to be weakened or the enhanced disease reagent of rheumatoid arthritis and tendonitis for example.

The antibody that constitutes reagent of the present invention can be according to the form of test, in conjunction with suitable marker.The antibody that perhaps constitutes reagent of the present invention also can be according to the form predetermined fixed of testing on suitable upholder.Reagent of the present invention in addition, except aforementioned antibody, the diagnosis test kit of the composition array configuration that also can make and check or need add when preserving.As the composition of the interpolation that can constitute test kit, can be useful on damping fluid, positive control, the negative control that dilutes reagent or organism sample, the substrate that is used to measure marker for example.These compositions also can be pre-mixed as required.In addition, can in each composition, add preservatives or sanitas as required.Test kit can also contain the specification sheets of reaction vessel, record test procedure etc. in addition.

The diagnosis of so-called disease comprises for example following diagnosis among the present invention.For the disease of following blood vessel to take place with ChM1L is active weakens or the enhanced disease, for example cancer, rheumatoid arthritis and tendonitis etc., even, whether be the patient who suffers from these diseases if carry out just can easily judging according to inspection of the present invention by the patient that general inspection can't be diagnosed.More specifically be, in the patient who demonstrates doubtful tendonitis symptom, the raising of ChM1L protein expression or reduction, the reason that shows this symptom is the possibility height of tendonitis etc.

Perhaps, be used in and judge and to follow the disease that blood vessel takes place and ChM1L is active weakens or the enhanced disease, for example whether cancer, rheumatoid arthritis and tendonitis etc. have the inspection of the tendency of improvement to become possibility.That is to say, can be used for judging these treatment of diseases effects.More specifically be that for the patient who demonstrates doubtful tendonitis symptom, the raising of ChM1L protein expression or reduction show the possibility height that tendonitis is further carried out or improved.

And then, according to the difference of expression level, also can judge and follow the disease that blood vessel takes place and ChM1L is active weakens or the enhanced disease, for example severities such as cancer, rheumatoid arthritis and tendonitis.That is to say that ChM1L protein expression degree may be relevant with the light and heavy degree of these diseases.

＜medicine 〉

At the disease of following blood vessel to take place with ChM1L is active weakens or the enhanced disease, the medicine of cancer, rheumatoid arthritis and tendonitis (blood vessel generation when psoriasis, vascular tumor, diabetic retinopathy, corneal injury or corneal transplantation etc.) for example, contain soluble polypeptide of the present invention as effective constituent, it can be prepared by mixing with acceptable carrier, activator or thinner etc. on the physiology.Medicine of the present invention is a purpose to improve symptom, can oral or non-oral administration.

As oral preparation, can be selected from formulations such as granule, pulvis, pill, capsule, emulsion or suspensoid.Comprise subcutaneous injection agent, intramuscular dose, joint cavity injection agent or intraperitoneal injection agent etc. as injection.

In addition, can adopt eye drops, ointment, contain contact lens that composition is arranged etc. as the form of administration in the ophtalmic treatments.And then the internal blood vessel site can also be to contain or to be coated with the form use of support (stent) or vascular peg stay suppository.

In addition, the effective constituent that is fit to the medicine of administration is when being made of protein, imports this proteinic gene of coding in vivo by adopting gene therapy method, can reach result of treatment.Bring the proteinic gene of result of treatment by importing coding in vivo, express, the method for treatment disease is known (the Japanese pharmacology impurity of golden field work calendar year 2001 publish the 117th volume p299-306).

Dosage, have nothing in common with each other according to patient's age, sex, body weight and symptom, result of treatment, medication, treatment time or the kind etc. that contains the activeconstituents of this medical composition, a common average adult, everyone 0.1mg to 500mg scope at every turn is preferably with the administration of 0.5mg to 20mg scope.But, because dosage changes according to the difference of various conditions, also exist than above-mentioned dosage amount still less with regard to enough situations, perhaps also there is the situation of the dosage that needs to surpass above-mentioned scope.

Below, with embodiment the present invention is described in more detail, but these embodiment do not limit the scope of the invention.

And, in the following embodiments, to various operations unless otherwise specified all according to Sambrook J, Fritsch EF, Maniatis T. shows " molecular cloning: laboratory manual the 2nd edition " (Molecula Cloning:a Laboratory Manual, 2nd edn. (USA) Cold SpringHarbor Laboratory Press 1989) shown in method carry out, perhaps the specification sheets according to the commercial goods uses when reagent that uses or test kit.

(embodiment)

Among the following embodiment, so-called COS7 cell is the cell that is obtained by the cercopithecus aethiops kidney, it is can be by the cell (No.CRL-1651) of ATCC (American type culture collection) acquisition, so-called 293T cell is the cell that is obtained by fetal kidney, it can obtain (Cat.No.Q401) by Gene Hunter company, so-called MRC-5 cell is the cell that is obtained by the fetus normal fibroblast, it can grind the Biological resources center by reason and obtain (No.RCD0211), the B16F10 cell is to obtain cell by mouse black cell knurl, it is can be by the cell (No.CRL-6475) of ATCC acquisition, so-called Lewis lung cancer (LLC) cell is the cell that is obtained by mice lung cancer, and it can obtain (No.CRL-1642) by ATCC.In addition, among the embodiment, HUVEC (human umbilical vein's endotheliocyte) cell and HMVEC (human skin venule endotheliocyte) can obtain from Clontics company, and perhaps the NHDF cell can obtain by the pure medicine of three light.

Embodiment 1

The proteinic detection of soluble polypeptide

＜method〉C end by PCR method amplification coding people ChM1L (amino acid 1-317 position) merges the proteinic cDNA (sequence number 1) that the FLAG mark is arranged, is cloned in the pCAGGS carrier (Gene (Holland) such as Miwa on December 15th, 1991 published the 108th roll up the 2nd phase p193-200) (pCAGGS-hChM1L-FLAG).However, as the FLAG mark described in the present embodiment (sigma company), be the hydrophilic mark peptide (AspTyr Lys Asp Asp Asp Asp Lys) that constitutes by 8 amino acid.Adopt Lipofectamine plus reagent (Lifetechnologies company), according to product description, with pCAGGS and pCAGGS-hChM1L-FLAG rotaring redyeing COS 7 cell and 293T cell.Reclaim culture supernatant from transfection after about 48 hours, carry out immunoprecipitation with the M2 agarose (Sigma company) of anti-FLAG.The sample of gel with 15% after to immunoprecipitation carries out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), transfers on the nitrocellulose membrane.One-level antibody adopts the polyclonal antibody (non-patent literature 5) of anti-ChM1L, secondary antibody adopts the anti-tame rabbit igg antibody (Dako company) with horseradish peroxidase-labeled, and (amersham pharmacia biotech company) carries out color reaction according to product description by ECLplus reagent.

＜result〉Fig. 1 represents by the Western blot method detected result (swimming lane 1:pCAGGS (COS7 cell), swimming lane 2:pCAGGS-ChM1L-FLAG (COS7 cell), swimming lane 3:pCAGGS (293T cell), swimming lane 4:pCAGGS-ChM1L-FLAG (293T cell)) to soluble polypeptide.In pCAGGS-hChM1L-FLAG institute cells transfected, be clear that the soluble polypeptide that has about 15kDa in the nutrient solution.

Embodiment 2

The analysis of the purifying of soluble polypeptide and N-terminal aminoacid sequence

＜method〉adopt lipofectamineplus reagent (Life technologies company), according to product description,, reclaim culture supernatant after about 48 hours with pCAGGS-hChM1L-FLAG transfection 293T cell.With the M2 agarose (Sigma company) of anti-FLAG, the preparation affinity chromatographic column is with the culture supernatant upper prop.With 25mM Tris hydrochloride buffer, after 150mM NaCl (pH7.4) washes post,, be among 1/20 the 1MTris-HCl (pH9.5) with volume and elutant with 0.1M glycine-HCl (pH3.5) wash-out.Carry out SDS-PAGE with elutant with 15% gel, transferring to Sequi-blot ^TMAfter pvdf membrane (bio-rad company) is gone up, with GelCode Bluestain reagent (Pierce company) dyeing.Cut out the band of about 15kDa, after handling with pyroglutamate aminopeptidase (Takara company), analyze the N-terminal aminoacid sequence by the terminal edman degradation Edman of Edman.

＜result〉(A) in expression be that each fraction of the purge process of soluble polypeptide is implemented SDS-PAGE, by the painted result of GelCode Bluestain reagent (Pierce company) (swimming lane 1: culture supernatant, swimming lane 2: be not adsorbed onto fraction, swimming lane 3 on the post: clean fraction, swimming lane 4-11: elutriated fraction).The fraction of wash-out from post, clearly there is the soluble polypeptide of about 15kDa in proof.Cut out the band of about 15kDa, analyze, can not read aminoacid sequence, think that N-terminal is closed by the Edman edman degradation Edman.After the pyroglutamate aminopeptidase processing, analyze the N-terminal aminoacid sequence, find out that sequence is ASEEELP.Therefore, find out that soluble polypeptide is to be made of 81 amino acid (sequence number 4) of the 237th to the 317th of film mating type ChM1L (317 amino acid), be clear that amino acid whose the 237th glutamine of N-terminal is converted into Pyrrolidonecarboxylic acid (B: with reference to the point of contact of ChM1L and ChM-I relatively).

Embodiment 3

Expression and the purifying of MS-ChM1L in intestinal bacteria

＜method〉PCR method amplification coding has Met and comprises the His mark and proteic cDNA (sequence number 5) that the N-terminal of the FLAG mark and 237-317 bit position aminoacid sequence people ChM1L merges, be cloned in pET (Novagen company) carrier (pET-shChM1L).With pET-shChM1L transformed into escherichia coli Origami B (DE3) pLyS (Novagen company).Cultivating intestinal bacteria in the LB substratum spends the night, after its part cultivated 3 hours again, add the expression that isopropyl-(isopropy-1-thio-β-D-thiogalactoside, IPTG) is induced recombinant protein by ultimate density, continue to cultivate 4 hours with 1mM.Make the intestinal bacteria granulating with 5000 * g centrifugation medium, with 6M guanidine, 0.1MNaH ₂PO ₄, behind the 0.01M Tris hydrochloride buffer wash-out of pH8.0, remove insoluble fraction, last nickel-nitrilotriacetic acid(NTA) agarose (Qiagen) post by centrifugation.With containing 8M urea, 0.1MNaH ₂PO ₄, 0.01M Tris hydrochloride buffer pH8.0 clean post, after further cleaning, with the buffer solution elution recombinant protein that adds the 200mM imidazoles with the imidazole concentration that slowly increases.With PD-10 post on the elutriated fraction (Amersham Pharmacia biotech company), use 25mM HEPES, 0.15M NaCl, the buffer-exchanged of pH8.3.Remove intracellular toxin in the recombinant protein solution by the method shown below of the people's such as Aida that adopt TritonX-114 method (people Journal of ImmunologicalMethods (Holland) such as Aida on September nineteen ninety 14 published the 132nd volume the 2nd phase p191-195) being carried out the part change.In recombinant protein solution, add Triton X-114, at 30 minutes, 37 ℃ following incubations of incubation on ice after 10 minutes, under 25 ℃ centrifugal 10 minutes, reclaim supernatant with 2000 * g with 1% ultimate density.Ultimate density with 1% in the described supernatant adds Triton X-114, repeats aforesaid operations once more.Clean the PD-10 post with after removing the intracellular toxin in the post with 1% Sodium desoxycholate, replace with through Posidain Filter (Pole company) and remove endotoxic 25mM HEPES, 0.15M NaCl is behind the pH8.3, with recombinant polypeptide solution upper prop, remove residual Triton X-114.(Limulus amebocyte lysate assay) (Biowhittacker company) measures endotoxic concentration with the tachypleus amebocyte lysate assay method.Use bovine serum albumin (BSA) as standard protein, measure protein concn with BCA protein test agent (Pierce company).With 15% gel the recombinant polypeptide of purifying is carried out SDS-PAGE, with GelCode Bluestain reagent (Pierce company) dyeing.

＜result〉recombinant polypeptide of purifying implemented SDS-PAGE after, be shown in (swimming lane 1: non-reducedization (2 mercaptoethanol), swimming lane 2: reducing (+2 mercaptoethanol)) among Fig. 3 with the painted result of GelCodeBluestain reagent (Pierce company).The endotoxic concentration of the recombinant polypeptide of purifying is less than 5EU/mL/mg protein, and productivity is the 15-30mg/L culture.By aforesaid method, can obtain the recombinant polypeptide that to use for cell and organism in a large number.

Embodiment 4

The analysis of the inhibited proliferation of vascular endothelial cell

＜method〉analysis of cell proliferation is that synthetic (cell is to the absorption of BrdU) with DNA is that index is carried out.On 96 orifice plates with the concentration culturing cell in 3000/hole, after this, under the condition that serum-free exists, cultivate 24 hours (37 ℃, CO ₂Exist down).After each hole cleaning, the MS-ChM1L of various concentration exists down, with 10ng/ml FGF-2 (fibroblast growth factor 2), 10ng/ml VEGF (blood vessel endothelial factor), 10ng/ml HGF (stem cell proliferation factor), 10%FBS (fetal bovine serum) irritation cell 24 hours (Fig. 4 B-4E) or 48 hours (Fig. 4 A).Be absorbed in the cell at 3 hours last BrdU.

＜result〉being shown among Fig. 4 of MS-ChM1L for the synthetic activity that suppresses of the DNA of various cells.A among Fig. 4 represents that the DNA that is caused by various stimulations of MS-ChM1L inhibition HUVEC synthesizes, B represents that MS-ChM1L concentration dependent ground inhibition HUVEC's stimulates the DNA that causes synthetic by FGF-2, C represents that the DNA that MS-ChM1L suppresses among the HMVEC synthesizes, D represents that the DNA that MS-ChM1L does not suppress among the NHDF synthesizes, and E represents that the DNA that MS-ChM1L does not suppress among the MRC-5 synthesizes.Each value representation mean value ± standard deviation, " * * " and " * * * " represents with respect to control value (vehicle) significant difference (* * is that P＜0.01, * * * are P＜0.001).

The MS-ChM1L of 100 μ g/ml almost completely suppresses FGF-2, VEGF in human umbilical vein's endotheliocyte (HUVECs can available from Clonetics), the dependent DNA of HGF, FBS synthetic (Fig. 4 A).In addition, dependent DNA synthetic inhibition is (Fig. 4 B) of concentration dependent to FGF-2 to have observed MS-ChM1L.(human skin capillary endothelium: HMVEC, clonetics company) found that also the DNA synthetic that is caused by MS-ChM1L suppresses (Fig. 4 C) in other vascular endothelial cell.But in human fibroblasts's normal human skin inoblast (NHDF, three pure pharmacy), MRC-5 cell (normal lung inoblast), the MS-ChM1L that observes 100 μ g/ml does not cause synthetic be suppressed (Fig. 4 D and the 4E) of DNA yet.Therefore, it is synthetic to think that the MS-ChM1L vascular endothelial cell suppresses DNA specifically.

Embodiment 5

The inhibiting analysis that the vascular endothelial cell capillary vessel forms

＜method〉in 24 orifice plates, add Growth-factor-reducedMatrigel (Becton Dickinson company) with 320 μ l/ holes, cultivated 30 minutes down for 37 ℃.Use and pass through EBM-2 substratum (Clonetics company) with the substratum of EGM-2 substratum (clonetics company) with 1/8 dilution, preparation contains the cell suspending liquid of 50000 HUVEC/mL.In the cell suspending liquid of 1/mL, add 100 in (50000 cells), 25,12.5 the reorganization MS-ChM1L of μ g/mL or damping fluid (25mM HEPES, 0.15M NaCl, pH8.3), be seeded on 24 orifice plates of Growth-factor-reduced Matrigel (Becton Dickinson company) bag quilt, observe capillary vessel formation after 6 hours and take pictures.

＜result〉MS-ChM1L is shown among Fig. 5 the inhibition that the capillary vessel of HUVEC forms.A represents 25mM HEPES, 0.15M NaCl, and capillary vessel forms under the pH8.3 condition, and B represents 100 μ g/mL MS-ChM1L, and C represents 25 μ g/mL MS-ChM1L, and D represents the situation of 12.5 μ g/mL MS-ChM1L, the calibration lines are represented 100 μ m.Show that MS-ChM1L forms with the capillary vessel that concentration dependent suppresses HUVEC.

Embodiment 6

The inhibiting analysis of the migration of vascular endothelial cell

＜method〉vitronectin (Sigma company) bag by using 1 μ g/mL carries out the migration experiment of vascular endothelial cell as the Transwell (coster company) of 8mM by the aperture of the upper and lower of filter membrane.That is to say, add the substratum that contains 0.1% serum of 600 μ l altogether, the substratum that contains the substratum of 10ng/mlVEGF or contain 10ng/ml FGF-2 at each Kong Zhongjun of 24 orifice plates.After on each hole of plate filter membrane being installed, with human umbilical vein's endotheliocyte (the human umbilical veinendothelial cells:HUVEC that cultivates 24 hours under the serum-free existence condition, clonetics company) is suspended in the nutrient solution of the MS-ChM1L that has various concentration, adds the upper strata of filter membrane with the density in 50000/hole to.37 ℃, CO ₂Culturing cell took off filter membrane after 4 hours under existing, and used the methyl alcohol fixed cell.To after the filter membrane dyeing, remove the cell on filter membrane upper strata with Diff-Quick staining fluid (Dade Behring company) with cotton rod.Cut filter membrane with blade, enclose slide glass, microscopically is to the migrating cell counting number.Obtain the cell count of migrating cell number as an average visual field.

＜result〉MS-ChM1L is shown among Fig. 6 the inhibition of the migration of HUVEC.A represents that MS-ChM1L suppresses the migration for VEFG of HUVEC, and B represents the migration for FGF-2 of the inhibition HUVEC of MS-ChM1L concentration dependent.Each value representation mean value ± standard deviation shows with respect to control value (vehicle) significant difference (* is that P＜0.05, * * are P＜0.01).The migration result of experiment is observed MS-ChM1L and has been produced inhibition (Fig. 6 A and 6B) for the migration of VEFG, FGF-2.The strongest when this migration is suppressed at MS-ChM1L and is 100 μ g/mL, found concentration dependent.That is to say, show that MS-ChM1L suppresses the migration of vascular endothelial cell.

Embodiment 7

The inhibiting analysis of the cell adhesion of vascular endothelial cell

＜method〉with 1 μ g/mL fibronectin (Sigma company), 1 μ g/mL vitronectin (Sigma company), 1 μ g/mL type i collagen bag by 96 orifice plates.After cleaning each hole, block with the PBS that contains 1%BSA.HUVEC is suspended in the substratum that does not contain serum with 1000000/mL concentration, with fluorexon AM (Molecular probes company) fluorescent mark.After cleaning cell, in each hole with 100000/hole add MS-ChM1L to its concentration be 100 μ g/mL.37 ℃, CO ₂Culturing cell was measured fluorescence intensity with the fluorescence plate reader after 1 hour under existing.

＜result〉show that MS-ChM1L suppresses the adhesion of HUVEC for vitronectin among Fig. 7.Each value representation mean value ± standard deviation, * * are represented with respect to control value (vechile) significant difference (* * is P＜0.01).Observing MS-ChM1L inhibition HUVEC adheres for vitronectin.On the other hand, there be not the influence of discovery for the adhesion of fibronectin, type i collagen.That is to say, understood fully that MS-ChM1L suppresses the cell adhesion for vitronectin.In addition, known this experiment be can be easy the method for investigation reorganization MS-ChM1L protein active.And then, because the known glass Fibronectin combines with beta 2 integrin alpha v β m, known that therefore MS-ChM1L suppresses the interaction of vitronectin and beta 2 integrin alpha v β m.In addition, infer its mechanism be MS-ChM1L by combining with beta 2 integrin alpha v β m, suppress its activity expression blood vessel generation restraining effect.

Embodiment 8

Analysis about cell cycle of vascular endothelial cell

＜method〉by carrying out the analysis of cell cycle by the DNA amount of flow cytometer (flow cytometer) monitoring cell.Because cell migration regularly as M phase → G1 phase → S phase → G2 phase → (the M phase), therefore the DNA amount can identify the cell cycle and is in which stage by inquiry.In the 10cm ware, cultivate HUVEC with the concentration in 1000000/hole, and then cultivation 24 hours in the presence of serum-free (37 ℃, CO ₂Exist down).After the cleaning, 100 μ g/mL MS-ChM1L existed down, with EGM-2 substratum irritation cell 24 hours.Behind Triton-X100, Rnase processing cell, dye with PI (propidium iodide), analyze by flow cytometer.

＜result〉to compare with the situation (Fig. 8 A) that only stimulates with the EGM-2 substratum, in the condition with EGM-2 substratum and MS-ChM1L stimulation, the cell count of G1 phase increases, and the cell count of S phase and G2/M phase reduces (Fig. 8 B).This result shows that the cell of G1 phase do not move to the S phase.Therefore, manifest HUVEC stops at the cell cycle by the effect of MS-ChM1L the G1 phase (G1 stops).

Embodiment 9

Analysis about the generation of the matrix metallo-proteinase in the vascular endothelial cell (MMPs)

＜method〉in 24 orifice plates, cultivate HUVEC, then cultivation 24 hours in the presence of serum-free (37 ℃, CO ₂Exist down).After the cleaning, 100 μ g/mL MS-ChM1L exist down, do not stimulate or with 10ng/ml tnf-α irritation cell 24 hours.Extract total RNA with Rneasy Mini test kit (Qiagen company) and Dnase, synthesize cDNA by reverse transcription reaction with Omniscript RT test kit (Qiagen company).Sense primer by containing each 0.1 μ M and antisense primer, 5 μ l, 2 * SYBR Green PCR Master Mix (Applied Biosystems company), 2 μ lcDNA, total amount is that the reaction solution of 10 μ l carries out PCR in real time.As reaction conditions, carry out 1) sex change (95 ℃ are following 15 seconds), 2) annealing and the circulation of extension (60 ℃ are following 1 minute).Adopt GeneAmp 5700 sequence detection systems (Applied Biosystems company) that the expression amount of each goal gene is carried out quantitatively.

By measure the fluorescence signal intensity with amplification PCR products (double-stranded DNA) bonded SYBR Green at each circulation time of PCR on time, be made into the amplification curve of PCR product with respect to cycle number, it is (common with any thresholding to calculate this amplification curve then, select near the mid point of index amplification region of amplification curve) circulation thresholding (the threshold cycle that reports to the leadship after accomplishing a task, ct) value is carried out the quantitative of amplification PCR products amount.By calculating formula 2- ^{(ct of the ct-18s of purpose)}Calculate purpose mRNA with respect to relative expression quantity as internal standard 18S.

＜result〉analyze with regard to the expression of the mRNA of MMP-2, MT1-MMP.Fig. 9 has shown the influence of MS-ChM1L for the expression of the mRNA of the matrix metallo-proteinase among the HUVEC.A represents that MS-ChM1L suppresses the expression of MMP-2mRNA, and B represents that MS-ChM1L suppresses the expression of MT1-MMP mRNA.Each value is still represented mean value ± standard deviation, and * and * * represent with respect to control value (vechlie) significant difference (* is that P＜0.05, * * are P＜0.01).

Known, MMP-2, MT1-MMP express in the situation that stimulates vascular endothelial cell with TNF-α increases (J.Cell Sci., January calendar year 2001; 114 (pt 1): 131-139, blood., on March 1st, 2003; 101 (5): 1810-7, Biochem.J., on December 15th, 1993; 296 (pt 3): 803-9).Under this experiment condition, do not identify the increase of the expression that causes by TNF-α, no matter whether there is TNF-α, MS-ChM1L suppresses the expression of the mRNA of MMP-2 and MT1-MMP.

(Fig. 9 A and B).MMP-2 is to destroy very relevant MMP with basilar membrane with MMP-9, and they decompose the main component IV Collagen Type VI of basilar membrane forcefully.In addition, MT1-MMP not only is converted into precursor MMP-2 the MMP of activated form on cytolemma, but also decomposes own extracellular matrix.Therefore, the results suggest MS-ChM1L of expression that suppresses the mRNA of MMP-2, MT1-MMP by the effect of current MS-ChM1L has the basilar membrane destructive possibility when suppressing blood vessel and taking place.

Embodiment 10

Inhibiting analysis takes place in blood vessel in the body in the FGF-2 inductive sponge blood vessel generation model

＜method〉usefulness FGF-2 inductive sponge blood vessel generation model (Br JPharmacol.2000,399,2-3,233-237) the intravital angiogenesis restraining effect of research MS-ChM1L.In male SD rabbit (seven week big) at circular sponge disc (the circular sponge disk of back side subcutaneous transplantation, thick 5mm * diameter 15mm), rise next day once a day in sponge with the people FGF-2 (500ng/50 μ l/ site) that recombinates and use and took place with induction of vascular in 3 days.In sponge, used 3 days once a day with MS-ChM1L (5ng/50 μ l/ site).Take out sponge and surrounding tissue in the time of the 4th day, implement visual inspection and Histological research.

＜result〉observe by using formation and the generation of blood vessel in granulation tissue that FGF-2 has produced granulation tissue on every side, (Figure 10) takes place in formation and blood vessel that MS-ChM1L has suppressed granulation tissue in this model significantly.Observe active inflammation at transplant early in this model, propagation and the blood vessel of observing sponge granulation tissue on every side take place.Therefore, prompting MS-ChM1L suppresses propagation, the blood vessel generation of proliferative inflammation and granulation tissue.

Embodiment 11

Osteoclast forms inhibiting analysis (with the dependent scavenger cell research of M-CSF)

＜method〉adopt the dependent scavenger cell of M-CSF (scavenger cell clone stimulating factor), to people's such as east method (The Journal of Biological Chemistry (USA) such as east on February 18th, 2000 disclose the 275th volume the 7th phase p4858-4864) thus carry out the formation that part changes the enforcement osteoclast.From the Thigh bone of big male ddY mouse of 7-8 week (Japanese SLC one company) and shin bone, take out medullary cell, after destroying red blood corpuscle, containing α-MEM, 10%FBS, cultivate in the substratum of 100ng/ml human M-CSF (Pepro-Tech Ec company limited), carry out the cultivation of going down to posterity for 2 times, obtain the scavenger cell that derives from medullary cell of M-CSF dependency propagation.This cell is interspersed among on 48 orifice plates with 10000/hole, (confirm to take place to adhere to the back) after about 6 hours, add 100ng/ml human M-CSF, 50ng/ml people sRANKL (solubility RANK part, Pepro-Tech Ec company limited) and MS-ChM1L or ChM--I.Laggard stone acid resistance Phosphoric acid esterase (TRAP) dyeing of serving a round of liquor to the guests in 5 days.

＜result〉MS-ChM1L and ChM--I suppress to express TRAP (Nature, on May 15th, 2003 of known mark as mature osteoclast significantly; 423 (6937): 337-42, the formation of osteoclast Review) (Figure 11).

Embodiment 12

Osteoclast forms inhibiting analysis (studying with the medullary cell culture systems)

＜method〉from the Thigh bone of big male ddY mouse of 9-10 week (Japanese SLC one company) and shin bone, take out medullary cell, after destroying red blood corpuscle, be suspended in and contain α-MEM, 10%FBS, nutrient solution in, intersperse among on 48 orifice plates with 500000/hole, after 24 hours, add known 1,25 (OH) 2D3 (Nature, on May 15th, 2003 of inducing osteoclast to form; 423 (6937): 337-42, Review) with MS-ChM1L or ChM--I.Carry out TRAP dyeing after 8 days.

＜result〉show that MS-ChM1L and ChM--I suppress medullary cell and form osteoclast among Figure 12.A represents 1,25 (OH) ₂D ₃(10 ^-8M)+Vehicle, B represents 1,25 (OH) 2D3 (10-8M)+10 μ g/mL MS-ChM1L, C represents 1,25 (OH) 2D3 (10-8M)+100 μ g/mL MS-ChM1L, D represent 1,25 (OH) 2D3 (10-8M)+10 μ g/mL ChM--I, E represents 1,25 (OH) 2D3 (10-8M)+25 μ g/mL ChM--I.The result shows that MS-ChM1L and ChM--I significantly suppress the formation (Figure 12) of the positive osteoclast of TRAP.

Embodiment 13

Osteoclast forms inhibiting Analysis on Mechanism

＜method〉on 96 orifice plates, scatter the dependent bone marrow macrophage of M-CSF by the method preparation identical with embodiment 11 with 10000/hole, (confirm that the back takes place to adhere to cell) after about 6 hours, add 100ng/ml human M-CSF, 50ng/ml people sRANKL (solubility RANK part, Pepro-Tech Ec company limited) and MS-ChM1L.0-5 days, 0-3 days, 3 time periods of 3-5 days handle with MS-ChM1L.Identify osteoclast by tartrate resistance Phosphoric acid esterase (TRAP) dyeing back after 5 days, research forms number.Before MS-ChM1L handles, handle the back and removed nutrient solution on the 1st, 3,5 day, extract total RNA with Rneasy Mini test kit (Qiagen company) and Rnase-Free Dnase set (Qiagen company).Then, (Qiagen company) carries out reverse transcription reaction with the OmniscriptRT test kit, synthetic cDNA.With SYBR GreenPCR Master Mix (Applied Biosystems company) and following shown in primer implement PCR in real time by ABI PRISM-TM7000 (Applied Biosystems company).Primerrodent GAPDH primer is bought from Applied Biosystems company.

And shown in hereinafter, differentiation, the maturation of each gene of Ce Dinging and osteoclast have confidential relation as can be known.

Present known Calcitonin Receptor (CTR) is mark (Nature, on May 15th, 2003 of multinuclear mature osteoclast; 423 (6937): 337-42, Review).M-CSF is the necessary cytokine of the existence of osteoclast precursor cell, and its acceptor is c-fms (Nature, on May 15th, 2003; 423 (6937): 337-42, Review).RANKL is osteoclast formation and activates necessary cytokine that its acceptor is RANK (Nature, on May 15th, 2003; 423 (6937): 337-42, Review).NFATc1 is that osteoclast forms necessary transcription factor, does not just form osteoclast (Nature, on May 15th, 2003 if suppress now the function of NFATc1 as can be known; 423 (6937): 337-42, Review.dev.cell, in December, 2002,3 (6): 889-901).

CTR

F5’-GTGCTCCTCGGGCTGTAGC

R5’-GAGGATTCCGTGGTTCCTGAT

TRAP

F5’-GATCCCTCTGTGCGACATCA

R5’-CCAGGGAGTCCTCAGATCCA

c-fms

F5’-TGGCATCTGGCTTAAGGTGAA

R5’-GAATCCGCACCAGCTTGCTA

RANK

F5’-ATGAGTACACGGACCGGCC

R5’-GCTGGATTAGGAGCAGTGAACC

NFATc1

F5’-AGGCTGGTCTTCCGAGTTCA

R5’-ACCGCTGGGAACACTCGAT

＜result〉using the dependent medullary cell of M-CSF in the osteoclast formation system of M-CSF, RANKL effect, add 10 μ g/mL, 25 μ g/mL, 50 μ g/mL MS-ChM1L cultivation 5 days, the consumption of 25 μ g/mL, 50 μ g/mL has suppressed osteoclast formation (Figure 13) significantly.In addition, if the initial stage (0-3 days) of cultivating with MS-ChM1L effect, cultivate and also can not form osteoclast even remove MS-ChM1L after this, the situation at late stage of culture (3-5 days) interpolation MS-ChM1L has just formed osteoclast in contrast.According to these results, known initial stage effect and inhibition thereafter the differentiation of MS-ChM1L in the osteoclast differentiation, do not suppress the formation of osteoclast for the cell that evolves to differentiation degree.In the situation of handling MS-ChM1L, known Calcitonin Receptor as the osteoclast mark (CTR) has suppressed the expression of tartrate resistance Phosphoric acid esterase (TRAP).In addition, the expression of necessary factor NFATc1 of the formation of osteoclast and RANK is also suppressed significantly.On the other hand, in the expression of the acceptor c-fms of the necessary factor M-CSF of existence of the aforementioned cell of osteoclast, do not find to have significant difference (Figure 14).

According to these results, known that MS-ChM1L does not suppress the existence signal of osteoclast, and suppressed the formation of osteoclast.

Embodiment 14

By the analysis of using the melanomatous inhibited proliferation of B16F10 that MS-ChM1L causes

＜method〉at the back of C57BL6/J mouse (male, 6 week big) subcutaneous with 500000/only transplant the B16F10 cell, measure the tumour size after 3 days and divide into groups.After dividing into groups, 1 day 1 time around the cancer cells subcutaneous with 50 μ l/ sites use MS-ChM1L (3mg/mL) or vehicle (25mM HEPES, 0.15M NaCl, pH8.3).Measure the size (length x width of cancer cells every day with calipers (caliper) ²* 0.52).With DNA synthetic (cell is to the absorption of BrdU) is the proliferation assay of the B16F10 cell that index is carried out external (in vitro).With concentration culturing cell on 96 orifice plates in 3000/hole, and then cultivation 24 hours in the presence of serum-free (37 ℃, CO ₂Exist down).After each hole cleaning, the MS-ChM1L of various concentration exists down, uses 10%FBS irritation cell 24 hours.Be absorbed in the cell at 3 hours last BrdU.

＜result〉MS-ChM1L suppress in vivo the B16F10 cell propagation (Figure 15 A, C).On the other hand, in the external propagation (Figure 15 B) that does not suppress the B16F10 cell.Therefore, think that MS-ChM1L suppresses the propagation of intravital B16F10 cell by blood vessel generation restraining effect.

Embodiment 15

By the analysis of using the melanomatous lung metastasis inhibition of the B16F10 effect that MS-ChM1L causes

＜method〉(male the C57BL6/J mouse, 6 weeks are big) intravenously with 50000/only transplant the B16F10 cell, 1 day 1 time at the back subcutaneous with 50 μ l/ sites (150 μ g/ site) use MS-ChM1L (3mg/mL) or vehicle (25mM HEPES, 0.15M NaCl, pH8.3).After the transplanting, took out lung on the 21st day, at clone's counting number of microscopically to the cell of transfer.

＜result〉the MS-ChM1L lung that suppressed the B16F10 cell shifts (Figure 16).Each value is still represented mean value ± standard deviation, and * * * represents with respect to control value (vehicle) significant difference (P＜0.001).

Embodiment 16

The analysis of the apoptosis-inducing effect of vascular endothelial cell

＜method〉knownly change in apoptosis incipient cell membrane structure, the phosphatidylserine that is present in the inboard of bilayer lipid membrane in the normal cell is exposed at the outside of cytolemma.Therefore, adopt the analysis that utilizes annexin V (Annexin V) and phosphatidylserine bonded method to carry out the apoptosis-inducing effect.Concrete is, on 6 orifice plates with the concentration cultivator dermal microvascular endothelial cell (HMVECs) in 100000/hole, then cultivation 24 hours in the presence of serum-free (37 ℃, CO ₂Exist down).After the cleaning, the MS-ChM1L of various concentration exists down, with 10ng/ml FGF-2 irritation cell.After 48 hours, peel off cell, after annexin V-FITC apoptosis test regent box (Biovision company) dyeing, analyze by flow cytometer with trypsinase-EDTA.

＜result〉MS-ChM1L induces the apoptosis of HMVECs in the concentration dependent mode.Therefore, think that MS-ChM1L has the effect of cell death inducing (Figure 17) in vascular endothelial cell.Each value is still represented mean value ± standard deviation, and * * * represents with respect to control value (vehicle) significant difference (P＜0.001).

Embodiment 17

The proteinic detection of living tissue soluble M S-ChM1L

＜method〉from DBA1/J mouse (male, 13 weeks are big), take out heel string, eyeball, kidney, liver, behind liquid nitrogen freezing, pulverize with pestle and mortar.With the histolysis of 4M guanidine, 50mM sodium-acetate pH5.8 and homogenizer, after ultrasonication, with 15000rpm centrifugation 10 minutes with pulverizing.100% ethanol that adds 9 times of amounts in the supernatant after centrifugal is placed after 5 minutes, with 15000rpm centrifugation 10 minutes down for-80 ℃.After removing supernatant,, add 100% ethanol of 9 times of amounts again, with 15000rpm centrifugation 10 minutes with 50mM sodium-acetate pH5.8 suspension post precipitation.With 8M urea, 20Mm Tris pH8.0 dissolution precipitation.Study plot uses bovine serum albumin to measure protein concn by BCA protein detection reagents (Pierce company).With the 10-20% gel sample is carried out SDS-PAGE with 35 μ g/ swimming lanes, transfer on the nitrocellulose filter.One-level antibody is anti-ChM1L polyclonal antibody (non-patent literature 6), secondary antibody adopts the anti-tame rabbit igg antibody (Dako company) with horseradish peroxidase-labeled, and (Amersham Pharmacia biotech company) carries out color reaction according to product description by ECLplus reagent.

＜result〉known the film mating type ChM1L protein that has about 40kDa in tendon and the eyeball, and also have the solubility ChM1L protein (Figure 18) of about 14kDa in the tendon.According to above result, known that solubility ChM1L protein exists for the first time on physiology.

Embodiment 18

The tissue specificity analysis that ChM1L mRNA expresses

＜method〉with regard to the tissue specificity that ChM1L mRNA expresses, from each tissue of mouse, extract total RNA after, by carrying out the PCR in real time analysis.That is to say, implement by operation shown below.Behind the total RNA of extraction, purify from each tissue of C57BL6/J mouse with ISOGEN (Japanese genome company system) with Rneasy Mini test kit (Qiagen company) and Rnase-Free Dnase set (Qiagen company).By using the reverse transcription reaction of Omniscript RT test kit (Qiagen company), RnaseOUT reorganization ribonuclease inhibitor (invitro system) and random primer (Takrabio system), carry out the synthetic of cDNA.Carry out PCR in real time with the reaction solution that contains at the sense primer of mouse ChM1L gene and antisense primer, SYBR Green PCR MasterMix, cDNA.Primer uses (sense primer) 5 '-AAACACTTCTGGCCCGAGGTAT-3 ', (instead with primer) 5 '-AGTGTGCTCCATGTCATAGGTTTTC-3 ' to be used for the mensuration of mouse ChM1LmRNA.In addition, mouse GAPDH mRNA measures the sense primer of usefulness, the anti-primer of buying from Appliedbiosystems company limited that uses with primer.PCR is reflected at 1) sex change (95 ℃ are following 15 seconds), 2) carry out 40 circulations under annealing and the condition of extension (60 ℃ are following 1 minute).Adopt GeneAmp 5700 sequence detection system softwares (Applied Biosystems company) that the expression amount of each goal gene is carried out quantitatively.That is to say, by measure the fluorescence signal intensity with amplification PCR products bonded SYBR Green at each circulation time of PCR on time, be made into the amplification curve of PCR product with respect to cycle number, it is (common with any thresholding to calculate this amplification curve then, select near the mid point of index amplification region of amplification curve) circulation thresholding (ct) value of reporting to the leadship after accomplishing a task, carry out quantitative.Calculate ChM1L mRNA respectively with respect to relative expression quantity by calculating formula shown below as internal standard GAPDH.

Relative expression quantity=2-of ChM1L mRNA ^{(the ct of GAPDH? the ct of ChM1L)}

＜result〉being expressed in of ChM1L mRNA the highest in the heel string (Figure 19).In eye, brain, lung, thymus gland, diaphragm, stomach, pancreas, muscle, skin, rib, also found the expression of ChM1LmRNA, but to compare expression amount low with heel string.In addition, in spleen, heart, liver, kidney, small intestine, fatty tissue, do not detect the expression of ChM1L mRNA.Therefore, pointed out ChM1L specific expressed with tendon.

Embodiment 19

The cell-specific analysis that the separation of mouse tendon cell and ChM1LmRNA express

＜method〉separate the tendon cell of mouse by operation shown below.Just, do not sneak into skin, muscle, fatty the tendon that extracts mouse, and digestion 3 hours in the DMEM/10%FBS of the collagenase that contains 2mg/mL (37 ℃, 5%CO ₂).After centrifugal, will obtain cell mass and be suspended among the DMEM/10%FBS, in the 10cm ware, cultivate (37 ℃, 5%CO ₂).After 7 days, breed fully owing to observed culturing cell, the cultivation of going down to posterity obtains the mouse tendon cell.

For the cell-specific that ChM1LmRNA expresses, be to analyze by carrying out PCR in real time after extracting total RNA with the cell strain that derives from mouse from the mouse tendon cell.Just, implement by operation shown below.Extract total RNA by Rneasy Mini test kit (Qiagen company) and Rnase-Free Dnase set (Qiagen company), by using the reverse transcription reaction of OmniscriptRT test kit (Qiagen company), RnaseOUT reorganization ribonuclease inhibitor (invitro system) and random primer (Takrabio system), carry out the synthetic of cDNA.Carry out PCR in real time with the reaction solution that contains at the sense primer of mouse ChM1L gene and antisense primer, SYBR Green PCRMaster Mix, cDNA.Primer uses (sense primer) 5 '-AAACACTTCTGGCCCGAGGTAT-3 ', (instead with primer) 5 '-AGTGTGCTCCATGTCATAGGTTTTC-3 ' to be used for the mensuration of mouse ChM1LmRNA.In addition, mouse GAPDH mRNA measures the sense primer of usefulness, the anti-primer of buying from Appliedbiosystems company limited that uses with primer.PCR is reflected at 1) sex change (95 ℃ are following 15 seconds), 2) carry out 40 circulations under annealing and the condition of extension (60 ℃ are following 1 minute).Adopt GeneAmp 5700 sequence detection system softwares (Applied Biosystems company) that the expression amount of each goal gene is carried out quantitatively.That is to say, by measure the fluorescence signal intensity with amplification PCR products bonded SYBR Green at each circulation time of PCR on time, be made into the amplification curve of PCR product with respect to cycle number, it is (common with any thresholding to calculate this amplification curve then, select near the mid point of index amplification region of amplification curve) circulation thresholding (ct) value of reporting to the leadship after accomplishing a task, carry out quantitative.Calculate ChM1L mRNA respectively with respect to relative expression quantity by calculating formula shown below as internal standard GAPDH.

Relative expression quantity=2-of ChM1L mRNA ^{(ct of the ct-ChM1L of GAPDH)}

＜result〉the expression of ChM1L mRNA in the mouse tendon cell, than the height (Figure 20) other cell strain that obtains from mesenchyme.Therefore, pointed out ChM1L specific expressed with tendon cell.

Embodiment 20

MS-ChM1L is to the research of the effect of tumor vessel generation

＜method〉by from the transplanting shown in the embodiment 14 behind the mouse extraction cancerous tissue of B16F10 cell, detect vascular endothelial cell by immunostaining, analyze the effect that soluble M S-ChM1L takes place tumor vessel.Just, implement by operation shown below.From the mouse of having used vehicle and soluble M S-ChM1L, extract cancerous tissue, fix with 4% Paraformaldehyde 96.By ordinary method with paraffin with its embedding after, make section.In order to detect vascular endothelial cell, use rabbit anti--the von Willebrand factor (vWF) Ab (CHEMICON system), the anti-mouse CD34 of rabbit mAb (Hycult Biotechnology system) are anti-as one-level antibody.To cut into slices after the dewaxing with series, add the streptavidin of one-level antibody, biotin labeling secondary antibody, peroxidase mark.At last, add 3-amino-9-ethyl carbazole (ethylcarbazole) (AEC) (Nichirei system), redye, typical imaging is taken pictures with phenodin.

In addition, use the painted section of vWF Ab, the blood vessel number in the cancer cells is carried out quantitatively by examining under a microscope.Just, observe section with low range, select the maximum zone of 3 blood vessel numbers after, under high magnification to the blood vessel counting number in described zone.Calculate the mean value in 3 zones of counting, data are expressed as blood vessel/hpf (high power field).

＜result〉in using the MS-ChM1L group, the vascular endothelial cell in vWF and the CD34 positive tumor has reduced (Figure 21 A, B).Therefore, think that MS-ChM1L is by blood vessel generation restraining effect, the propagation of anticancer in vivo.

Embodiment 21

By using the analysis of MS-ChM1L to the inhibited proliferation of Lewis lung cancer (LLC)

＜method〉at the back of C57BL6/J mouse (male, 6 week big) subcutaneous with 500000/only transplant LLC cell (ATCC NO.CRL-1642), measure the tumour size after 3 days and divide into groups.After dividing into groups, 1 day 1 time around the cancer cells subcutaneous with 50 μ l/ sites use the people recombinate MS-ChM1L (3mg/mL) or vehicle (25mM HEPES, 0.15M NaCl, ph8.3).Measure the size (length x width 2 * 0.52) of cancer cells every day with calipers (caliper).With DNA synthetic (cell is to the absorption of BrdU) is the proliferation assay of the LLC cell that index is carried out external (in vitro).With concentration culturing cell on 96 orifice plates in 3000/hole, and then cultivation 24 hours in the presence of serum-free (37 ℃, CO ₂Exist down).After each hole cleaning, the MS-ChM1L of various concentration exists down, uses 10%FBS irritation cell 24 hours.Be absorbed in the cell at 3 hours last BrdU.

＜result〉MS-ChM1L suppresses the propagation (Figure 22 A) of LLC cell in vivo.On the other hand, in the external propagation (Figure 22 B) that does not suppress the LLC cell.Therefore, think that MS-ChM1L suppresses the propagation of intravital LLC cell by blood vessel generation restraining effect.

Embodiment 22

The analysis of the apoptosis-inducing effect of the vascular endothelial cell of Caspase mediation

＜method〉known Caspase is the L-Cysteine HCL Anhydrous that has cysteine residues in the active centre, it is a medium crucial in the apoptosis.With caspACE FITC-VAD-FMK original position mark (promega system) the apoptosis-inducing effect of the vascular endothelial cell of Caspase mediation is analyzed.Known caspACE FITC-VAD-FMK original position mark is the Caspase inhibitor VAD-FMK (FITC-VAD-FMK) of the FITC mark of cell permeability, can inside and outside cell, freely move, by irreversibly combining, stay in the apoptotic cell with the activatory Caspase.Therefore, it is strong to observe the fluorescence intensity of FlTC of the apoptotic cell that the Caspase mediation takes place by the analysis of flow cytometer.On 6 orifice plates respectively with the concentration in 155000/hole cultivate HUVEC, cultivate MRC-5 cell (the Biological resources center NO.RCD02111 of RIKEN) with the concentration in 100000/hole, then cultivation 24 hours in the presence of serum-free (37 ℃, CO ₂Exist down).After each hole cleaning, 25? g/mL MS-ChM1L exists down, with 10ng/ml FGF-2 irritation cell.After 48 hours, with 10? the M final concentration adds FITC-VAD-FMK, cultivate 30 minutes 37 ℃, CO ₂Exist down).Peel off cell with trypsinase-EDTA, analyze by flow cytometer.

＜result〉MS-ChM1L induces the apoptosis of Caspase mediation in HUVEC.On the other hand, in the MRC-5 cell, do not observe the apoptosis (Figure 23) of Caspase mediation.Thus, think that MS-ChM1L has the apoptotic effect that vascular endothelial cell is induced the Caspase mediation specifically.

Embodiment 23

Expression in the ChM--I protein colon bacillus and purifying

＜method〉merged methionine(Met), the Histidine (His mark) of 6 residues and the proteinic cDNA (sequence number 7) of FLAG mark on the N-terminal by PCR method amplification coding people ChM-I, be cloned in the pet carrier (Novagen company) (pet-shChM-I).With pet-shChM-I transformed into escherichia coli OrigamiB (DE3) pLysS (Novagen company).In the LB substratum, cultivate intestinal bacteria and spend the night, its part is cultivated about 3 hours again after, add after IPTG induces the expression of recombinant protein with the final concentration of 1mM, cultivated again 4 hours.Make the intestinal bacteria granulating with 5000 * g centrifugation medium, with 6M guanidine, 0.1MNaH ₂PO ₄, after the 0.01M Tris hydrochloride buffer dissolving of pH8.0, remove insoluble fraction by centrifugation, last nickel nitrillotriacetic agarose (Qiagen) post.Use the 8M urea, 0.1MNaH ₂PO ₄, the 0.01M Tris hydrochloride buffer of pH8.0 cleans post, again with after slowly increasing the buffer solution for cleaning of imidazole concentration, with the buffer solution elution recombinant protein that adds the 200mM imidazoles.With PD-10 post on the elutriated fraction (Amersham Pharmacia biotech company), use 25mMHEPES, 0.15M NaCl, the buffer-exchanged of pH8.3.Remove intracellular toxin in the recombinant protein solution by the method shown below of the people's such as Aida that adopt Triton X-114 method (people Journal of Immunological Methods (Holland) such as Aida on September nineteen ninety 14 published the 132nd volume the 2nd phase p191-195) being carried out the part change.In recombinant protein solution, add Triton X-114, at 30 minutes, 37 ℃ following incubations of incubation on ice after 10 minutes, under 25 ℃ centrifugal 10 minutes, reclaim supernatant with 2000 * g with 1% ultimate density.Ultimate density with 1% in this supernatant adds Triton X-114, repeats aforesaid operations once more.Clean the PD-10 post with after removing the intracellular toxin in the post with 1% Sodium desoxycholate, be replaced into through PosidainFilter (Pole company) and remove endotoxic 25mM HEPES, 0.15M NaCl is behind the pH8.3, with recombinant C hM-I protein soln upper prop, remove residual Triton X-114.(Biowhittacker company) measures endotoxic concentration with the tachypleus amebocyte lysate assay method.Study plot uses bovine serum albumin (BSA), measures protein concn with BCA protein test agent (Pierce company).With 15% gel the recombinant C hM-I protein of purifying is carried out SDS-PAGE, with GelCodeBluestain reagent (Pierce company) dyeing.

＜result〉the recombinant C hM-I protein of purifying implemented SDS-PAGE after, be shown in (swimming lane 1: non-reducedization (2 mercaptoethanol), swimming lane 2: reducing (+2 mercaptoethanol)) among Figure 24 with the painted result of GelCode Bluestain reagent (Pierce company).The proteinic endotoxic concentration of reorganization group ChM-I of purifying is less than 5EU/mL/mg protein, and productivity is the 10-20mg/L culture.By the method identical, can obtain recombinant C hM-I protein in a large number with above-mentioned MS-ChM1L.

Brief description of drawings

That Fig. 1 represents is the result who detects soluble polypeptide by western blot method.

What Fig. 2 represented is to each fraction enforcement SDS-PAGE of the purge process of soluble polypeptide, uses the comparison (B) of the point cut-off of GelCode Bluestain reagent (Pierce company) painted result (A), ChM1L and ChM-I.

That Fig. 3 represents is the proteinic SDS-PAGE of reorganization MS-ChM1L that implements purifying, with the painted result of GelCode Bluestain reagent (Pierce company).

What Fig. 4 represented is that MS-ChM1L suppresses active for the DNA of various cells is synthetic.

What Fig. 5 represented is the capillary vessel formation that MS-ChM1L suppresses HUVEC.

What Fig. 6 represented is the migration that suppresses HUVEC.

What Fig. 7 represented is the adhesion for vitronectin (vitronectin) that MS-ChM1L suppresses HUVEC.

Fig. 8 represents is cell cycle of HUVEC to stop at the G1 phase (A:25mMHEPES, 0.15M NaCl, pH8.3, B:100 μ g/mL MS-ChM1L).

What Fig. 9 represented is the influence of MS-ChM1L for the expression of the mRNA of the matrix metallo-proteinase among the HUVEC.

What Figure 10 represented is that MS-ChM1L suppresses the interior blood vessel generation restraining effect (A:25mM HEPES, 0.15M NaCl, pH8.3, B:100 μ g/mL MS-ChM1L) of body in the FGF-2 inductive granuloma formation generation model.

Figure 11: expression be formation (the A:M-CSF 100ng/mL that MS-ChM1L and ChM--I suppress the osteoclast that obtains from the dependent scavenger cell of M-CSF, B:M-CSF 100ng/mL+RANKL 50ng/mL+Vehicle, C:M-CSF 100ng/mL+RANKL 50ng/mL+MS-ChM1L 10 μ g/mL, D:M-CSF 100ng/mL+RANKL 50ng/mL+MS-ChM1L100 μ g/mL, E:M-CSF 100ng/mL+RANKL 50ng/mL+ChM-I 10 μ g/mL, F:M-CSF 100ng/mL+RANKL 50ng/mL+ChM-I 25 μ g/mL).

What Figure 12 represented is that MS-ChM1L and ChM--I suppress to form osteoclast (A:1,25 (OH) by medullary cell ₂D ₃(10 ^-8M)+and Vehicle, B:1,25 (OH) ₂D ₃(10 ^-8M)+and MS-ChM1L 10 μ g/mL, C:1,25 (OH) ₂D ₃(10 ^-8M)+and MS-ChM1L 100 μ g/mL, D:1,25 (OH) ₂D ₃(10 ^-8M)+and ChM-I 10 μ g/mL, E:1,25 (OH) ₂D ₃(10 ^-8M)+ChM-I 25 μ g/mL).

What Figure 13 represented is that MS-ChM1L directly acts on the inhibition differentiation at the initial stage of osteoclast precursor cytodifferentiation, and this restraining effect is irreversible.

That Figure 14 represents is the result of the expression analysis of osteoclast marker gene.

Figure 15 represents is result by the analysis of using the melanomatous inhibited proliferation of B16F10 that MS-ChM1L causes.Expression by use MS-ChM1L suppress in vivo the melanomatous propagation of B16F10 (A, C).By using MS-ChM1L in the external melanomatous propagation of B16F10 (B) that do not suppress.

What Figure 16 represented is that MS-ChM1L suppresses the melanomatous lung transfer of B16F10.

What Figure 17 represented is the apoptosis of MS-ChM1L induction of vascular endothelial cell.

What Figure 18 represented is to have soluble M S-ChM1L in the tendinous tissue.

What Figure 19 represented is that ChM1L mRNA is specific expressed with tendon in living tissue.

What Figure 20 represented is that ChM1L mRNA is specific expressed with tendon cell in mesenchymal cell.

What Figure 21 represented is that MS-ChM1L suppresses the tumor vessel generation.

What Figure 22 represented is by using the analytical results of MS-ChM1L to the inhibited proliferation of LLC cell.Expression is by using the propagation that MS-ChM1L suppresses LLC in vivo, and MS-ChM1L is in the external propagation that does not suppress LLC.

Figure 23 represents is that MS-ChM1L induces the apoptosis in the vascular endothelial cell of Caspase mediation.

Figure 24: expression be after the recombinant C hM-I protein of purifying is implemented SDS-PAGE, with the painted result of GelCode Bluestain reagent (Pierce company).

Sequence table

<110>Teijin?Pharma?Limited

＜120〉novel secretory protein and its production and use

<130>SAP-715-PCT

<160>9

<170>PatenyIn?version?3.1

<210>1

<211>978

<212>DNA

＜213〉people

<220>

<221>CDS

<222>(1)..(951)

<223>

<400>1

atg?gca?aag?aat?cct?cca?gag?aat?tgt?gaa?gac?tgt?cac?att?cta?aat 48

Met?Ala?Lys?Asn?Pro?Pro?Glu?Asn?Cys?Glu?Asp?Cys?His?Ile?Leu?Asn

1 5 10 15

gca?gaa?gct?ttt?aaa?aag?aaa?ata?tgt?aaa?tca?ctt?aag?att?tgt 96

Ala?Glu?Ala?Phe?Lys?Ser?Lys?Lys?Ile?Cys?Lys?Ser?Leu?Lys?Ile?Cys

20 25 30

gga?ctg?gtg?ttt?ggt?act?ctg?gcc?act?cta?att?gtc?ctg?ttt?tgg 144

Gly?Leu?Val?Phe?Gly?Ile?Leu?Ala?Leu?Thr?Leu?Ile?Val?Leu?Phe?Trp

35 40 45

ggg?agc?aag?cac?ttc?tgg?ccg?gag?gta?ccc?aaa?aaa?gcc?tat?gac?atg 192

Gly?Ser?Lys?His?Phe?Trp?Pro?Glu?Val?Pro?Lys?Lys?Ala?Tyr?Asp?Met

50 55 60

gag?cac?act?ttc?tac?agc?aat?gga?gag?aag?aag?aag?att?tac?atg?gaa 240

Glu?His?Thr?Phe?Tyr?Ser?Asn?Gly?Glu?Lys?Lys?Lys?Ile?Tyr?Met?Glu

65 70 75 80

att?gat?cct?gtg?acc?aga?act?gaa?ata?ttc?aga?agc?gga?aat?ggc?act 288

Ile?Asp?Pro?Val?Thr?Arg?Thr?Glu?Ile?Phe?Arg?Ser?Gly?Asn?Gly?Thr

85 90 95

gat?gaa?aca?ttg?gaa?gta?cac?gac?ttt?aaa?aac?gga?tac?act?ggc?atc 336

Asp?Glu?Thr?Leu?Glu?Val?His?Asp?Phe?Lys?Asn?Gly?Tyr?Thr?Gly?Ile

100 105 110

tac?ttc?gtg?ggt?ctt?caa?aaa?tgt?ttt?atc?aaa?act?cag?att?aaa?gtg 384

Tyr?Phe?Val?Gly?Leu?Gln?Lys?Cys?Phe?Ile?Lys?Thr?Gln?Ile?Lys?Val

115 120 125

att?cct?gaa?ttt?tct?gaa?cca?gaa?gag?gaa?ata?gat?gag?aat?gaa?gaa 432

Ile?Pro?Glu?Phe?Ser?Glu?Pro?Glu?Glu?Glu?Ile?Asp?Glu?Asn?Glu?Glu

130 135 140

att?acc?aca?act?ttc?ttt?gaa?cag?tca?gtg?att?tgg?gtc?cca?gca?gaa 480

Ile?Thr?Thr?Thr?Phe?Phe?Glu?Gln?Ser?Val?Ile?Trp?Val?Pro?Ala?Glu

145 150 155 160

aag?cct?att?gaa?aac?cga?gat?ttt?ctt?aaa?aat?tcc?aaa?att?ctg?gag 528

Lys?Pro?Ile?Glu?Asn?Arg?Asp?Phe?Leu?Lys?Asn?Ser?Lys?Ile?Leu?Glu

165 170 175

att?tgt?gat?aac?gtg?acc?atg?tat?tgg?atc?aat?ccc?act?cta?ata?tca 576

Ile?Cys?Asp?Asn?Val?Thr?Met?Tyr?Trp?Ile?Asn?Pro?Thr?Leu?Ile?Ser

180 185 190

gtt?tct?gag?tta?caa?gac?ttt?gag?gag?gag?gga?gaa?gat?ctt?cac?ttt 624

Val?Ser?Glu?Leu?Gln?Asp?Phe?Glu?Glu?Glu?Gly?Glu?Asp?Leu?His?Phe

195 200 205

cct?gcc?aac?gaa?aaa?aaa?ggg?att?gaa?caa?aat?gaa?cag?tgg?gtg?gtc 672

Pro?Ala?Asn?Glu?Lys?Lys?Gly?Ile?Glu?Gln?Asn?Glu?Gln?Trp?Val?Val

210 215 220

cct?caa?gtg?aaa?gta?gag?aag?acc?cgt?cac?gcc?aga?caa?gca?agt?gag 720

Pro?Gln?Val?Lys?Val?Glu?Lys?Thr?Arg?His?Ala?Arg?Gln?Ala?Ser?Glu

225 230 235 240

gaa?gaa?ctt?cca?ata?aat?gac?tat?act?gaa?aat?gga?ata?gaa?ttt?gat 768

Glu?Glu?Leu?Pro?Ile?Asn?Asp?Tyr?Thr?Glu?Asn?Gly?Ile?Glu?Phe?Asp

245 250 255

ccc?atg?ctg?gat?gag?aga?ggt?tat?tgt?tgt?att?tac?tgc?cgt?cga?ggc 816

Pro?Met?Leu?Asp?Glu?Arg?Gly?Tyr?Cys?Cys?Ile?Tyr?Cys?Arg?Arg?Gly

260 265 270

aac?cgc?tat?tgc?cgc?cgc?gtc?tgt?gaa?cct?tta?cta?ggc?tac?tac?cca 864

Asn?Arg?Tyr?Cys?Arg?Arg?Val?Cys?Glu?Pro?Leu?Leu?Gly?Tyr?Tyr?Pro

275 280 285

tat?cca?tac?tgc?tac?caa?gga?gga?cga?gtc?atc?tgt?cgt?gtc?atc?atg 912

Tyr?Pro?Tyr?Cys?Tyr?Gln?Gly?Gly?Arg?Val?Ile?Cys?Arg?Val?Ile?Met

290 295 300

cct?tgt?aac?tgg?tgg?gtg?gcc?cgc?atg?ctg?ggg?agg?gtc?gactacaaag 961

Pro?Cys?Asn?Trp?Trp?Val?Ala?Arg?Met?Leu?Gly?Arg?Val

305 310 315

acgatgacga?caagtga 978

<210>2

<211>317

<212>PRT

＜213〉people

<400>2

Met?Ala?Lys?Asn?Pro?Pro?Glu?Asn?Cys?Glu?Asp?Cys?His?Ile?Leu?Asn

1 5 10 15

Ala?Glu?Ala?Phe?Lys?Ser?Lys?Lys?Ile?Cys?Lys?Ser?Leu?Lys?Ile?Cys

20 25 30

Gly?Leu?Val?Phe?Gly?Ile?Leu?Ala?Leu?Thr?Leu?Ile?Val?Leu?Phe?Trp

35 40 45

Gly?Ser?Lys?His?Phe?Tip?Pro?Glu?Val?Pro?Lys?Tys?Ala?Tyr?Asp?Met

50 55 60

Glu?His?Thr?Phe?Tyr?Ser?Asn?Gly?Glu?Lys?Lys?Lys?Ile?Tyr?Met?Glu

65 70 75 80

Ile?Asp?Pro?Val?Thr?Arg?Thr?Glu?Ile?Phe?Arg?Ser?Gly?Asn?Gly?Thr

85 90 95

Asp?Glu?Thr?Leu?Glu?Val?His?Asp?Phe?Lys?Asn?Gly?Tyr?Thr?Gly?Ile

100 105 110

Tyr?Phe?Val?Gly?Leu?Gln?Lys?Cys?Phe?Ile?Lys?Thr?Gln?Ile?Lys?Val

115 120 125

Ile?Pro?Glu?Phe?Ser?Glu?Pro?Glu?Glu?Glu?Ile?Asp?Glu?Asn?Glu?Glu

130 135 140

Ile?Thr?Thr?Thr?Phe?Phe?Glu?Gln?Ser?Val?Ile?Trp?Val?Pro?Ala?Glu

145 150 155 160

Lys?Pro?Ile?Glu?Asn?Arg?Asp?Phe?Leu?Lys?Asn?Ser?Lys?Ile?Leu?Glu

165 170 175

Ile?Cys?Asp?Asn?Val?Thr?Met?Tyr?Tro?Ile?Asn?Pro?Thr?Leu?Ile?Ser

180 185 190

Val?Ser?Gln?Leu?Gln?Asp?Phe?Glu?Glu?Glu?Gly?Glu?Asp?Leu?His?Phe

195 200 205

Pro?Ala?Asn?Glu?Lys?Lys?Gly?Ile?Glu?Gln?Asn?Glu?Gln?Trp?Val?Val

210 215 220

Pro?Gln?Val?Lys?Val?Glu?Lys?Thr?Arg?His?Ala?Arg?Gln?Ala?Ser?Glu

225 230 235 240

Glu?Glu?Leu?Pro?Ile?Asn?Asp?Tyr?Thr?Glu?Asn?Gly?Ile?Glu?Phe?Asp

245 250 255

Pro?Met?Leu?Asp?Glu?Arg?Gly?Tyr?Cys?Cys?Ile?Tyr?Cys?Arg?Arg?Gly

260 265 270

Asn?Arg?Tyr?Cys?Arg?Arg?Val?Cys?Glu?Pro?Leu?Leu?Gly?Tyr?Tyr?Pro

275 280 285

Tyr?Pro?Tyr?Cys?Tyr?Gln?Gly?Gly?Arg?Val?Ile?Cys?Arg?Val?Ile?Met

290 295 300

Pro?Cys?Asn?Trp?Trp?Val?Ala?Arg?Met?Leu?Gly?Arg?Val

305 310 315

<210>3

<211>246

<212>DNA

＜213〉people

<220>

<221>CDS

<222>(1)..(246)

<223>

<400>3

caa?gca?agt?gag?gaa?gaa?ctt?cca?ata?aat?gac?tat?act?gaa?aat?gga 48

Gln?Ala?Ser?Glu?Glu?Glu?Leu?Pro?Ile?Asn?Asp?Tyr?Thr?Glu?Asn?Gly

1 5 10 15

ata?gaa?ttt?gat?ccc?atg?ctg?gat?gag?aga?ggt?tat?tgt?tgt?att?tac 96

Ile?Glu?Phe?Asp?Pro?Met?Leu?Asp?Glu?Arg?Gly?Tyr?Cys?Cys?Ile?Tyr

20 25 30

tgc?cgt?cga?ggc?aac?cgc?tat?tgc?cgc?cgc?gtc?tgt?gaa?cct?tta?cta 144

Cys?Arg?Arg?Gly?Asn?Arg?Tyr?Cys?Arg?Arg?Val?Cys?Glu?Pro?Leu?Leu

35 40 45

ggc?tac?tac?cca?tat?cca?tac?tgc?tac?cca?gga?gga?cga?gtc?atc?tgt 192

Gly?Tyr?Tyr?Pro?Tyr?Pro?Tyr?Cys?Tyr?Gln?Gly?Gly?Arg?Val?Ile?Cys

50 55 60

cgt?gtc?atc?atg?cct?tgt?aac?tgg?tgg?gtg?gcc?cgc?atg?ctg?ggg?agg 240

Arg?Val?Ile?Met?Pro?Cys?Asn?Trp?Trp?Val?Ala?Arg?Met?Leu?Gly?Arg

65 70 75 80

gtc?taa 246

Val

<210>4

<211>81

<212>PRT

＜213〉people

<400>4

Gln?Ala?Ser?Glu?Glu?Glu?Leu?Pro?Ile?Asn?Asp?Tyr?Thr?Glu?Asn?Gly

1 5 10 15

Ile?Glu?Phe?Asp?Pro?Met?Leu?Asp?Glu?Arg?Gly?Tyr?Cys?Cys?Ile?Tyr

20 25 30

Cys?Arg?Arg?Gly?Asn?Arg?Tyr?Cys?Arg?Arg?Val?Cys?Glu?Pro?Leu?Leu

35 40 45

Gly?Tyr?Tyr?Pro?Tyr?Pro?Tyr?Cys?Tyr?Gln?Gly?Gly?Arg?Val?Ile?Cys

50 55 60

Arg?Val?Ile?Met?Pro?Cys?Asn?Trp?Trp?Val?Ala?Arg?Met?Leu?Gly?Arg

65 70 75 80

Val

<210>5

<211>303

<212>DNA

＜213〉people

<220>

<221>CDS

<222>(1)..(303)

<223>

<400>5

atg?cac?cat?cat?cat?cat?cat?gat?atc?gac?tac?aaa?gac?gat?gac?gac 48

Met?His?His?His?His?His?His?Asp?Ile?Asp?Tyr?Lys?Asp?Asp?Asp?Asp

1 5 10 15

aag?tcg?cga?caa?gca?agt?gag?gaa?gaa?ctt?cca?ata?aat?gac?tat?act 96

Lys?Ser?Arg?Gln?Ala?Ser?Glu?Glu?Glu?Leu?Pro?Ile?Asn?Asp?Tyr?Tyr

20 25 30

gaa?aat?gga?ata?gaa?ttt?gat?ccc?atg?ctg?gat?gag?aga?ggt?tat?tgt 144

Glu?Asn?Gly?Ile?Glu?Phe?Asp?Pro?Met?Leu?Asp?Glu?Arg?Gly?Tyr?Cys

35 40 45

tgt?att?tac?tgc?cgt?cga?ggc?aac?cgc?tat?tgc?cgc?cgc?gtc?tgt?gaa 192

Cys?Ile?Tyr?Cys?Arg?Arg?Gly?Asn?Arg?Tyr?Cys?Arg?Arg?Val?Cys?Glu

50 55 60

cct?tta?cta?ggc?tac?tac?cca?tat?cca?tac?tgc?tac?caa?gga?gga?cga 240

Pro?Leu?Leu?Gly?Tyr?Tyr?Pro?Tyr?Pro?Tyr?Cys?Tyr?Gln?Gly?Gly?Arg

65 70 75 80

gtc?atc?tgt?cgt?gtc?atc?atg?cct?tgt?aac?tgg?tgg?gtg?gcc?cgc?atg 288

Val?Ile?Cys?Arg?Val?Ile?Met?Pro?Cys?Asn?Trp?Trp?Val?Ala?Arg?Met

85 90 95

ctg?ggg?agg?gtc?taa 303

Leu?Gly?Arg?Val

100

<210>6

<211>100

<212>PRT

＜213〉people

<400>6

Met?His?His?His?His?His?His?Asp?Ile?Asp?Tyr?Lys?Asp?Asp?Asp?Asp

1 5 10 15

Lys?Ser?Arg?Gln?Ala?Ser?Glu?Glu?Glu?Leu?Pro?Ile?Asn?Asp?Tyr?Thr

20 25 30

Glu?Asn?Gly?Ile?Glu?Phe?Asp?Pro?Met?Leu?Asp?Glu?Arg?Gly?Tyr?Cys

35 40 45

Cys?Ile?Tyr?Cys?Arg?Arg?Gly?Asn?Arg?Tyr?Cys?Arg?Arg?Val?Cys?Glu

50 55 60

Pro?Leu?Leu?Gly?Tyr?Tyr?Pro?Tyr?Pro?Tyr?Cys?Tyr?Gln?Gly?Gly?Arg

65 70 75 80

Val?Ile?Cys?Arg?Val?Ile?Met?Pro?Cys?Asn?Trp?Trp?Val?Ala?Aag?Met

85 90 95

Leu?Gly?Arg?Val

100

<210>7

<211>420

<212>DNA

＜213〉people

<220>

<221>CDS

<222>(1)..(420)

<223>

<400>7

atg?cac?cat?cat?cat?cat?cat?gat?atc?gac?tac?aaa?gac?gat?gac?gac 48

Met?His?His?His?His?His?His?Asp?Ile?Asp?Tyr?Lys?Asp?Asp?Asp?Asp

1 5 10 15

aag?tcg?cga?gaa?gtg?gta?aga?aaa?att?gtt?cca?act?acc?aca?aaa?aga 96

Lys?Ser?Arg?Glu?Val?Val?Arg?Lys?Ile?Val?Pro?Thr?Thr?Thr?Lys?Arg

20 25 30

cca?cac?agt?gga?cca?cgg?agc?aac?cca?ggc?gct?gga?aga?ctg?aat?aat 144

Pro?His?Ser?Gly?Pro?Arg?Ser?Asn?Pro?Gly?Ala?Gly?Arg?Leu?Asn?Asn

35 40 45

gaa?acc?aga?ccc?agt?gtt?caa?gag?gac?tca?caa?gcc?ttc?aat?cct?gat 192

Glu?Thr?Arg?Pro?Ser?Val?Gln?Glu?Asp?Ser?Gln?Ala?Phe?Asn?Pro?Asp

50 55 60

aat?cct?tat?cat?cag?cag?gaa?ggg?gaa?agc?atg?aca?ttc?gac?cct?aga 240

Asn?Pro?Tyr?His?Gln?Gln?Glu?Gly?Glu?Ser?Met?Thr?Phe?Asp?Pro?Arg

65 70 75 80

ctg?gat?cac?gaa?gga?atc?tgt?tgt?ata?gaa?tgt?agg?cgg?agc?tac?acc 288

Leu?Asp?His?Glu?Gly?Ile?Cys?Cys?Ile?Glu?Cys?Arg?Arg?Ser?Tyr?Thr

85 90 95

cac?tgc?cag?aag?atc?tgt?gaa?ccc?ctg?ggg?ggc?tat?tac?cca?tgg?cct 336

His?Cys?Gln?Lys?Ile?Cys?Glu?Pro?Leu?Gly?Gly?Tyr?Tyr?Pro?Trp?Pro

100 105 110

tat?aat?tat?caa?ggc?tgc?cgt?tcg?gcc?tgc?aga?gtc?atc?atg?cca?tgt 384

Tyr?Asn?Tyr?Gln?Gly?Cys?Arg?Ser?Ala?Cys?Arg?Val?Ile?Met?Pro?Cys

115 120 125

agc?tgg?tgg?gtg?gcc?cgt?atc?ttg?gcc?atg?gtg?tga 420

Ser?Trp?Trp?Val?Ala?Arg?Ile?Leu?Gly?Met?Val

130 135

<210>8

<211>139

<212>PRT

＜213〉people

<400>8

Met?His?His?His?His?His?His?Asp?Ile?Asp?Tyr?Lys?Asp?Asp?Asp?Asp

1 5 10 15

Lys?Ser?Arg?Glu?Val?Val?Arg?Lys?Ile?Val?Pro?Thr?Thr?Thr?Lys?Arg

20 25 30

Pro?His?Ser?Gly?Pro?Arg?Ser?Asn?Pro?Gly?Ala?Gly?Arg?Leu?Asn?Asn

35 40 45

Glu?Thr?Arg?Pro?Ser?Val?Gln?Glu?Asp?Ser?Gln?Ala?Phe?Asn?Pro?Asp

50 55 60

Asn?Pro?Tyr?His?Gln?Gln?Glu?Gly?Glu?Ser?Met?Thr?Phe?Asp?Pro?Arg

65 70 75 80

Leu?Asp?His?Glu?Gly?Ile?Cys?Cys?Ile?Glu?Cys?Arg?Arg?Ser?Tyr?Thr

85 90 95

His?Cys?Gln?Lys?Ile?Cys?Glu?Pro?Leu?Gly?Gly?Tyr?Tyr?Pro?Trp?Pro

100 105 110

Tyr?Asn?Tyr?Gln?Gly?Cys?Arg?Ser?Ala?Cys?Arg?Val?Ile?Met?Pro?Cys

115 120 125

Ser?Trp?Trp?Val?Ala?Arg?Ile?Leu?Gly?Met?Val

130 135

<210>9

<211>80

<212>PRT

＜213〉people

<400>9

Ala?Ser?Glu?Glu?Glu?Leu?Pro?Ile?Asn?Asp?Tyr?Thr?Glu?Asn?Gly?Ile

1 5 10 15

Glu?Phe?Asp?Pro?Met?Leu?Asp?Glu?Arg?Gly?Tyr?Cys?Cys?Ile?Tyr?Cys

20 25 30

Arg?Arg?Gly?Asn?Arg?Tyr?Cys?Arg?Arg?Val?Cys?Glu?Pro?Leu?Leu?Gly

35 40 45

Tyr?Tyr?Pro?Tyr?Pro?Tyr?Cys?Gln?Gln?Gly?Gly?Arg?Val?Ile?Cys?Arg

50 55 60

Val?Ile?Met?Pro?Cys?Asn?Trp?Trp?Val?Ala?Arg?Met?Leu?Gly?Arg?Val

65 70 75 80

Claims (26)

1, that comprise the aminoacid sequence shown in the sequence number 9 or have the aminoacid sequence of at least 70% homology with the aminoacid sequence shown in the sequence number 9, and have suppress that blood vessel takes place active and/or suppress the active polypeptide of bone resorption.

2, according to the polypeptide of claim 1, its N-terminal and/or C-terminal also are added with amino-acid residue.

3, according to the polypeptide of claim 2, the amino-acid residue that its N-terminal adds is made of the initial aminoacid sequence of methionine(Met).

4, according to the polypeptide shown in claim 2 or 3, the amino-acid residue that its N-terminal and/or C-terminal add is made up of the aminoacid sequence that comprises flag sequence, and described flag sequence is by the flag sequence of a successive 6-8 Histidine and/or contain the FLAG flag sequence and constitute.

5, according to the polypeptide shown in claim 2 or 3, the amino-acid residue that this N-terminal and/or C-terminal add is fluorescence protein or its analogue from jellyfish, or the aminoacid sequence of secretor type alkaline phosphatase or its analogue constitutes.

6, according to the polypeptide of claim 1, the amino-acid residue that its N-terminal and/or C-terminal add contains modified amino-acid residue.

7, according to the polypeptide of claim 6, the amino-acid residue that its N-terminal adds is glutamine or pyroglutamyl amine residue.

8,, it is characterized in that described modified amino-acid residue has to be selected from least a modification group in ethanoyl, formyl radical, biotinyl, Boc base or the Fmoc base according to the polypeptide of claim 6.

9, the nucleic acid molecule that has the nucleotide sequence of the aminoacid sequence shown in the encoding sequence numbers 9.

10, the nucleic acid molecule of nucleotide sequence that has the 4-243 position of sequence number 3, perhaps under stringent condition with the complementary sequence hybridization of the nucleotide sequence of the 4-243 position of sequence number 3 and have the nucleic acid molecule of nucleotide sequence that coding has suppress that blood vessel takes place active and/or suppresses the active polypeptide of bone resorption.

11, the nucleic acid molecule that has the nucleotide sequence of the polypeptide of coding claim 2-6 shown in each.

12, the carrier that contains the nucleic acid molecule of claim 9-11 shown in each.

13, by the carrier transformed host cells of claim 12.

14,, it is characterized in that this method comprises the step of the transformed host cell of cultivating claim 13 and reclaims the step of the polypeptide that generates according to the preparation method of the polypeptide of claim 1-8 shown in each.

15,, it is characterized in that in the presence of protein denaturant, from transformed host cell, reclaiming the extracting solution that contains polypeptide according to the preparation method of the polypeptide of claim 14.

16, according to the preparation method of the peptide of claim 14 or 15 shown in each, it comprise handle the extracting solution that from host cell, reclaims with TritonX-114 after, carry out centrifugation and handle to remove the step of thermal source.

17, according to the preparation method of the polypeptide of claim 14-16 shown in each, it is characterized in that the institute after host cell reclaims the extracting solution that contains polypeptide in steps, the pH that will contain the solution of this polypeptide is adjusted in the scope of 8.0-8.5.

18, the method for preparing recombinant human ChM-I or recombinant human ChM1L, its be adopt can expressing human ChM-I or the recombinant host cell of people ChM1L prepare the method for recombinant human ChM-I or recombinant human ChM1L, this method is included in protein denaturant and has the step that reclaims the extracting solution that contains recombinant human ChM-I or recombinant human ChM1L down from the host cell of reorganization, and carries out centrifugation to remove the step of thermal source after with TritonX-114 this extracting solution being handled.

19, according to the preparation method of claim 18, it is characterized in that after reclaiming the step of extracting solution in steps in, the pH that will contain the solution of this polypeptide is adjusted in the scope of 8.0-8.5.

20, contain the pharmaceutical composition of claim 1 to the polypeptide of claim 8 shown in each.

21, according to the pharmaceutical composition shown in the claim 20, it is angiogenesis inhibitor and/or osteoclast activation inhibitor.

22, according to the pharmaceutical composition shown in the claim 21, it is the therapeutical agent of tendonitis, rheumatoid arthritis, osteoarthritis, malignant tumour, diabetic retinopathy, glaucoma, psoriasis, keloid or arteriosclerosis.

23, be used for measuring the composition for diagnosis of the polypeptide that is made of aminoacid sequence shown in the sequence number 9 or 4 of body fluid components, it contains antibody or its fragment of the polypeptide of at least a identification claim 1 or 7 shown in each.

24, according to the composition for diagnosis shown in the claim 23, it is used for the diagnosis of tendonitis, rheumatoid arthritis, osteoarthritis, the arbitrary illness of malignant tumour.

25, according to the composition for diagnosis shown in the claim 23, it is used for the diagnosis of diabetic retinopathy, glaucoma, psoriasis, keloid, the arbitrary illness of arteriosclerosis.

26, through the transgenic nonhuman animal of genetic manipulation, it contains the nucleic acid molecule of aforementioned claim 9-11 shown in each.

Images