JPH09299088A - Dna fragment and production of human chondromodulin-i protein using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for stably mass-producing a human chondromodulin-I protein and simplify a purifying and isolating step due to the precipitation of the resultant human chondromodulin-I protein with ammonium sulfate and adsorption thereof on a heparin Sepharose (R), etc. SOLUTION: An expression vector containing a DNA fragment in which 3 bases on the upstream side counted from adenine of a base sequence capable of coding translation starting methionine as an origin in the base sequence of a gene capable of coding a human chondromodulin-I protein are converted is prepared. A transformant transformed with the resultant expression vector is then cultured to thereby produce the human chondromoduin-I protein.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel DNA fragment and a method for producing a human chondromodulin-I protein using the same, and more specifically, a nucleotide sequence of a gene encoding the human chondromodulin-I protein. A DNA fragment obtained by converting 3 bases upstream from adenine in the nucleotide sequence encoding the translation initiation methionine in
The present invention relates to an expression vector containing an NA fragment, a transformant transformed with the vector, and a method for producing a human chondromodulin-I protein by culturing the transformant.

[0002]

BACKGROUND OF THE INVENTION Human chondromodulin-I protein is a water-soluble protein having a molecular weight of about 26 KD as determined by SDS-polyacrylamide gel electrophoresis. It is a protein composed of one type of polypeptide having activity to grow in the presence of factors. Suzuki et al. Have been investigating to produce human chondromodulin-I protein in large quantities by recombinant DNA technology.
Novel human chondromodulin-I useful for such purpose
For the first time, a human gene encoding a protein was isolated and obtained, and this gene was further incorporated into an expression vector to obtain a transformant, which succeeded in producing a recombinant human chondromodulin-I protein (special feature: (See Kaihei 7-138295).

However, the results of expression studies using animal cells and yeast by the conventional method such as JP-A-7-138295 show that the expression level of human chondromodulin-I protein was large and inexpensive. Was not enough. Further, most of the human chondromodulin-I proteins produced by these methods did not adsorb by heparin sepharose chromatography, and the process of purification and isolation was complicated.

[0004]

[Means for Solving the Problems] As a result of intensive studies aimed at producing a recombinant human chondromodulin-I protein in large quantities and at low cost, the present inventors have focused on the nucleotide sequence around the translation initiation point. . And the method of kozak (Cell, 4
4 , 283-292 (1986)), the base sequence of the region immediately before the base sequence ATG encoding the translation initiation methionine was converted.

That is, when the ribosome of a eukaryotic cell makes a protein from messenger RNA, the base at the position (-3) upstream from the adenine of the nucleotide sequence encoding the translation initiation methionine as a starting point is a purine (adenine or Guanine) is particularly important, and the sequence of 1 to 3 bases (-1 to -3) upstream greatly affects the translation efficiency. However, it was found that in the conventional expression vector for human chondromodulin-I protein, the upstream third base (-3) is a pyrimidine (thymine), and this is the reason why the translation efficiency into protein is extremely low. .

[0006] Then, an expression vector is newly constructed by converting the nucleotide sequence in the region immediately before the nucleotide sequence ATG encoding the translation initiation methionine of the conventional expression vector, and the transformant is used to produce a recombinant human chondromodo. The inventors succeeded in producing a large amount of phosphorus-I protein at low cost, and found that the obtained protein is adsorbed by heparin sepharose chromatography, so that the process of purification and isolation can be simplified, thus completing the present invention. It was

[0007] That is, the gist of the present invention is to include the base of the third base upstream from the adenine of the base sequence encoding the translation initiation methionine in the base sequence of the gene encoding the human chondromodulin-I protein, and
A DNA fragment characterized in that the base of the third base is adenine or guanine, the DNA fragment characterized in that the base of the third base is adenine, and further upstream 2
The above D characterized in that the base of the base order is cytosine
NA fragment, wherein the upstream first base is cytosine, and the DNA fragment has a restriction enzyme at the 5'end
The above D characterized in that it contains a Hind III cleavage site.
NA fragment, the DNA fragment characterized by being used as a primer, the DNA fragment characterized by being represented by SEQ ID NO: 2 in the sequence listing, and the DN according to any one of the above.
A DNA fragment containing a gene encoding a human chondromodulin-I protein, which is obtained by a PCR method using the A fragment as a primer, which is represented by SEQ ID NO: 1 in the sequence listing. An expression vector comprising a DNA fragment containing the gene encoding the human chondromodulin-I protein or the DNA fragment represented by SEQ ID NO: 1 in the sequence listing, and transforming a host cell with the expression vector And a transformant having a gene encoding the human chondromodulin-I protein introduced into the chromosome of the host cell, and a human chondromodulin-I protein characterized by culturing the transformant. Exists in the manufacturing method of.

[0008]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail. The DNA fragment in the present invention is the third base upstream from the adenine of the nucleotide sequence encoding the translation initiation methionine in the nucleotide sequence of the gene encoding human chondromodulin-I protein (see Japanese Patent Laid-Open No. 138295/1995). And the third base is adenine or guanine. The D
In the NA fragment, among the above, it is preferable that the base at the upstream third base is adenine, and further that the base at the upstream second base is cytosine and the base at the upstream first base is cytosine Is more preferable. Also,
It is preferred that the 5 'end contain restriction enzyme Hin dIII cleavage site. Particularly preferred in the present invention is the one represented by the base sequence of SEQ ID NO: 2 in the sequence listing. The above DNA fragment can be easily obtained by known chemical synthesis.

In the present invention, the above-obtained DNA fragment and a DNA fragment encoding a nucleotide sequence corresponding to the carboxyl terminal region of human chondromodulin-I protein obtained by separately known chemical synthesis are used as primers. Conventionally used human chondromodulin-I as described in JP-A-7-138295.
A DNA fragment in which the upstream portion of the human chondromodulin-I protein gene has been modified can be obtained by performing a PCR reaction using an expression vector containing a gene encoding the protein as a template (SEQ ID NO: 1 in the sequence listing). ). The DNA fragment of the present invention is not limited to those shown in the sequence listing, and may include those having a modified base sequence as long as the gist of the present invention is not exceeded.

The expression vector of the present invention can be obtained by reinserting this DNA fragment at a predetermined position of a known expression vector. The obtained expression vector is a known lipopolyamine method in a known cell such as an animal cell host described below.
The transformant of the present invention can be obtained by introducing it by a method known per se such as the calcium phosphate method and the electroporation method.

The transformant of the present invention can be obtained, for example, as follows. The host used for transformation should be one that allows the recombinant DNA to grow stably and autonomously. Preferable examples include animal cells used in Examples described below, but the present invention is not particularly limited to animal cells, and other microorganisms (for example, Escherichia coli JM109 and HB) can be used.
101 (all commercially available from Toyobo Co., Ltd. and the like) and yeast, insect cells, etc.
A host cell as described in Japanese Patent No. 8295 can be used.

Examples of animal cells include CHO cells and C
Examples thereof include OS cells, mouse L cells, mouse C127 cells, mouse FM3A cells, osteoblasts, chondrocytes and the like. These animal cell lines are commercially available and are easily available. Further, when these cells are used as hosts, there is an advantage that the human chondromodulin-I protein in mature form is secreted and produced by introducing the human chondromodulin-I protein gene in the form of a precursor protein. There is expected.

The expression plasmid in the case of using the above cells as a host preferably has an SV40 promoter or a cytomegalovirus (hereinafter sometimes abbreviated as CMV) promoter as a promoter. Further, in order to increase the production amount of human chondromodulin-I protein, a product in which a plurality of DNA fragments having a promoter such as CMV are connected to the 5′-terminal side of each human chondromodulin-I protein gene may be inserted. . A polyadenylation site is contained downstream of the human chondromodulin-I protein gene. Examples of the polyadenylation site include SV40 DNA and β-globin gene. Specific examples of such a plasmid include a CMV promoter and pcDNA having a polyadenylation site from the rabbit β-globin gene.
3 (Invitrogen) and the like.

This expression vector is transformed into an animal cell such as a CHO cell together with a vector carrying a drug resistance gene and cultured in a medium containing the drug to obtain a drug resistant cell line. By controlling the ratio of the drug resistance gene to the expression vector during the transformation, a cell line expressing the human chondromodulin-I protein can be efficiently obtained. Further, for the purpose of further increasing the expression level of human chondromodulin-I protein, the selection marker was changed for cells in which the expression of human chondromodulin-I protein was confirmed by this double transformation method. Double transformation may be repeated and repeated.

In selecting the obtained drug-resistant strain, if there is no suitable antibody, total RNA is prepared for each cell,
A strain with a high expression level of the messenger RNA can be selected by Northern hybridization or the like using the introduced partial DNA fragment of the human chondromodulin-I protein gene as a probe. The culture of the transformed animal cells may be appropriately changed. In the case of normal CHO cells, eRDF medium (Dulbecco's modified Eagle) supplemented with 10% fetal calf serum (FCS) is usually used.
le's: Ham's Nutrient Mixtu
re F-12: RPMI1640 = 1: 1: 2) or the like is used. At the time of mass production, the FCS concentration can be lowered to, for example, 2% within a range where the productivity is not significantly reduced.

Animal cells expressing the human chondromodulin-I protein secrete the human chondromodulin-I protein into the culture supernatant thereof. It is possible to separate and purify the -I protein. The culture supernatant containing the produced human chondromodulin-I protein can be purified using various chromatography methods such as heparin sepharose, blue sepharose, gel filtration or ammonium sulfate precipitation. The recombinant human chondromodulin-I protein produced by the present invention can be adsorbed to a heparin sepharose column to obtain a molecular species that behaves similarly to the chondromodulin-I protein obtained from bovine cartilage. It is a feature.

[0017]

EXAMPLES The present invention will be described in more detail with reference to examples below, but the present invention is not limited to the following examples as long as the gist thereof is not exceeded. Example 1 Preparation of DNA fragment and acquisition of expression gene by PCR method Human chondromodulin-I shown in SEQ ID NO: 2 in the sequence listing
The translation initiation methionine region of the protein and the sequence of the immediately preceding 3 bases are referred to as ACCATG, and the restriction enzyme Hin is added to the 5'end.
Synthetic D consisting of a total of 25 bases including the dIII cleavage site
NA was prepared and used as primer 1. On the other hand, it contains the carboxyl-terminal region of human chondromodulin-I protein shown in SEQ ID NO: 3 in the sequence listing, and contains restriction enzyme X at the 3'end.
Synthetic DN consisting of a total of 25 bases including a ba I cleavage site
A was prepared and used as a primer 2.

PCR is Perkin Elmer Cetus
The procedure was carried out using a DNA thermal cycler according to the instruction manual. That is, as substrate DNA
10 ng of an expression vector containing the human chondromodulin-I protein gene obtained by the method described in JP-A-138295 is used as a template, and a 10-fold concentration of reaction buffer [500 mM KCl, 100 mM T
ris-HCl (pH 8.3), 15 mM MgC
l ₂ , 0.1% (w / v) gelatin] 10 μl, 1.2
16 μl of 5 mM 4dNTP, 5 μl each of 50 μM of primer 1 and 50 μM of primer 2, TaqDN
0.5 μl of A polymerase was added to make a 100 μl system. The reaction was pretreated at 94 ° C. for 10 minutes, followed by 35 cycles of incubation at 94 ° C. for 1 minute (denaturation step), 55 ° C. for 1.5 minutes (annealing step), and 72 ° C. for 2 minutes (extension step). Finally, the reaction was completed by incubating at 72 ° C. for 7 minutes.

[0019] After the obtained PCR product was subjected to reaction treatment with a restriction enzyme Hin dIII Oyo <br/> beauty Xba I, a conventional method ( "Molecular Cloning" 2nd edition, Cold
Recombinant modified pcDNA containing the DNA fragment shown in SEQ ID NO: 1 of the Sequence Listing, inserted and ligated into the cloning site of commercially available pcDNA3 (Invitrogen) according to Spring Harbor Laboratory, 1989).
3hChM1 was obtained.

The obtained modified pcDNA3h
The sequence of ChM1 is the fluorescence sequencer G manufactured by DuPont.
Determined using the ENESIS 2000 system. Example 2 C of human chondromodulin-I protein
Expression in HO cells modified pcDNA3 obtained in Example 1
The plasmid pSV2bsr (Funakoshi) carrying the hChM1 and blasticidin S resistance genes was prepared by the lipopolyamine method (Loeffler et al., J. Neur) using commercially available TRANSFECTAM (Bokusui Brown).
ochem. , 54 , 1812-1815 (199
0)) and co-transfected into CHO cells according to the instructions.

That is, 4 cells are added to 5 × 10 ⁵ CHO cells.
μg of modified pcDNA3hChM1, 0.
4 μg pSV2bsr and 25 μg TRAN
SFECTAM was co-introduced and cultured for 6 hours. 10%
FCS-containing eRDF medium was added and the cells were further cultured for 48 hours. Then, the cells were treated with 0.5 mg / ml trypsin-ED.
Peel off the TA solution, suspend in blasticidin S (Funakoshi) in a selective medium containing 3 μg / ml, and seed on a 96-well plate to give 500 to 1000 cells / well. A blasticidin S-resistant strain was obtained by using a selective medium containing blasticidin S for the subsequent culture. The above cell culture was performed at 37 ° C. in the presence of 5% CO ₂ .

Some of the obtained resistant strains were treated with 5 × 10 ⁴ c
After seeding at a density of ells / cm ² and culturing for 24 hours, commercially available RNAzol (BIOTECX LABORATOR
Total RNA was prepared using IES) according to the instruction manual, and Northern hybridization was performed. The human chondromodulin-I protein-specific probe used in the Northern hybridization method was prepared as follows. First, using the DNAs shown in SEQ ID NO: 4 of the Sequence Listing and SEQ ID NO: 5 of the Sequence Listing as primers,
Example 1 using fiedpcDNA3hChM1 as a template
PCR was performed by the method shown in. Next, the PCR reaction solution was analyzed by agarose gel electrophoresis, and 0.4 kb
The DNA fragment was purified by agarose gel according to a conventional method. This was labeled with ³² P by the random oligo DNA labeling method using clenow fragment and used.

Northern hybridization was performed as follows. 1 μg of total RNA obtained from each cell group
Glyoxal method (McMaster et al., P
rc. Natl. Acad. Sci. USA, 74 ,
4835-4838 (1977)) and independently electrophoresed on a 1% agarose gel. After electrophoresis, RN
A was transferred to a nylon membrane and cross-linked with UV light. This nylon membrane was hybridized with the above ³² P-labeled probe at 42 ° C. The hybridization solution was 50% formamide, 50 mM sodium phosphate buffer solution (pH 7.0), 5 × SSC, 250.
μg / ml herring tests DNA,
And those containing 10% dextran sulfate and 0.02% each of Ficoll, polyvinylpyrrolidone and BSA.

The membrane after hybridization was set to 0.1
After washing with a 1 × SSC solution containing% SDS at room temperature, it was further washed twice with a 0.1 × SSC solution containing 0.1% SDS at 50 ° C., and exposed to an X-ray film together with an intensifying screen overnight. . As a result, a human chondromodulin-I protein mRNA having the expected size (1.6 kb) from the nucleotide sequence of the introduced expression vector was confirmed. Further m
The intensity of the band existing at the position of RNA was compared, and the cell group that expressed the most was selected. The cell group was redistributed to an appropriate cell concentration and selection was repeated by the Northern hybridization method to finally obtain a single producing cell group.

Example 3 Precipitation of Human Chondromodulin-I Protein with 45% Saturated Ammonium Sulfate 1 × 10 ⁷ cell lines obtained in Example 2 were cultured in a 9 cm dish in an eRDF medium containing 2% FCS for 72 hours. To 10 ml of the collected culture supernatant, 2.8 g of ammonium sulfate powder was added under ice cooling and dissolved. After standing for 1 hour, the mixture was centrifuged at 15,000 rpm for 5 minutes, the obtained precipitate was dissolved in 0.2 ml of distilled water, and dialyzed against distilled water for 24 hours.

20 μl of the sample thus obtained (corresponding to 1 ml of culture solution) was added to 0.1% SDS-12.5% polyacrylamide gel. A Tris buffer solution (25 mM Tris-HCl pH 8.3, 192 mM glycine, 0.1% SDS) was used as an electrode solution, and a constant current of 20 mA was applied to the electrode.
After the electrophoresis for a minute, the gel was subjected to Immobilon P by a conventional method.
Overlaid with a VDF membrane (Millipore), the gel side was the cathode and the membrane side was the anode.
Transfer was performed by applying for 0 minutes. Next, this membrane was mixed with PBS buffer containing 5% skim milk (potassium chloride 0.2
g, sodium chloride 8g, potassium dihydrogen phosphate
0.2 g, disodium hydrogen phosphate dodecahydrate 2.9
g was dissolved in distilled water so as to be 1 liter: hereinafter simply referred to as PBS) and immersed for 12 hours. 20 μl
Buffer solution containing rabbit anti-human chondromodulin-I protein peptide antibody (antibody against synthetic peptide in which Cys is added to the amino terminus of Thr from 9th Thr to 33rd Pro of mature human chondromodulin-I protein) It was immersed in (PBS with 0.05% Tween 20 added) and left at 37 ° C. for 3 hours. This is P
The mixture was allowed to react with a peroxidase-labeled goat anti-rabbit antibody (Organon Technica) diluted 1000 times with BST buffer at room temperature for 1 hour. Next, this membrane was colored using Konica Immunostain HRP-1000 (manufactured by Konica) according to the instruction manual. As a result, it was revealed that most of human chondromodulin-I protein obtained from the supernatant was recovered in the precipitated fraction by 45% saturated ammonium sulfate. From this result, it can be said that the culture supernatant containing the human chondromodulin-I protein obtained by the method of the present invention can be purified using ammonium sulfate precipitation.

Example 4 Heparinto Yopard Column Chromatography of Human Chondromodulin-I Protein 1 × 10 ⁷ cell lines obtained in Example 2 were cultured in a 9 cm Petri dish for 72 hours in an eRDF medium containing 2% FCS. did. 10 ml of the collected culture supernatant was previously used in PBS.
It was applied to a heparint yopearl (manufactured by Tosoh Corporation) column (diameter 8 mm × 20 mm) equilibrated with. After washing with 5 column volumes of PBS, elution was performed with PBS containing 1.2 M NaCl. The non-adsorbed fraction and the adsorbed fraction were each concentrated and desalted with Centricon-10 (manufactured by Amicon).
Then, Western blotting was performed according to the method described above.

As a result, it was revealed that about 5% of the human chondromodulin-I protein contained in the culture supernatant was adsorbed on the heparint yopearl column.
From this result, it can be said that the culture supernatant containing the human chondromodulin-I protein obtained by the method of the present application can be purified using heparin sepharose.

Example 5 Expression of human chondromodulin-I protein-antibody constant region (Fc region) fusion protein using COS cells A fusion protein of human chondromodulin-I protein and antibody Fc region was produced. . First, a sequence translated from the nucleotide sequence of the gene encoding the human chondromodulin-I protein shown in SEQ ID NO: 1 in the sequence listing, which is recognized and cleaved by Factor Xa immediately after the carboxyl-terminal amino acid Val Ligation followed by rat immunoglobulin G (IgG)
A gene encoding the amino acid sequence in which the Fc region of 2A was linked was prepared. That is, the synthetic DNAs of SEQ ID NO: 2 and SEQ ID NO: 6 in the sequence listing were used as primers according to a conventional method (“Molecular Cloning”, Second Edition, Cold Spring Harbor Laboratory, 1989),
PCR reaction was performed using the modified pcDNA3hChM1 as a template. Separately, synthetic DNA of SEQ ID NO: 7 in the sequence listing and rat IgG2 shown in SEQ ID NO: 8 of the sequence listing
PCR was performed using a synthetic DNA corresponding to the 3'end side of the Fc region of A as a primer and rat IgG2A as a template. 10 ng of each of these two PCR products was mixed, and synthetic DNAs of SEQ ID NO: 2 and SEQ ID NO: 8 in the sequence listing were obtained.
Was used as a primer to perform a PCR reaction to prepare a modified gene encoding a human chondromodulin-I protein-antibody Fc region fusion protein. This gene uses the synthetic DNA of SEQ ID NO: 2 in the Sequence Listing as a primer,
Since the modified pcDNA3hChM1 is used as the template, it is expected that the productivity is increased.

On the other hand, as a control, a human chondromodulin-I protein-antibody Fc region fusion protein gene in which the vicinity of the translation initiation point of the nucleotide sequence of the gene encoding the human chondromodulin-I protein was not modified was prepared. First, PCR was carried out using the synthetic DNAs of SEQ ID NO: 9 and SEQ ID NO: 6 in the sequence listing as primers and a conventional expression vector as a template. This is mixed with a PCR product obtained by using the synthetic DNAs of SEQ ID NO: 7 and SEQ ID NO: 8 in the sequence listing as a primer and using rat IgG2A as a template, and performing a PCR reaction using SEQ ID NO: 9 and SEQ ID NO: 8 in the sequence listing as primers. , Human chondromodulin-I in which the translation initiation methionine region is not modified
A modified gene corresponding to the protein-antibody Fc region fusion protein was prepared.

Was introduced DNA fragment of an antibody Fc region fusion gene respectively Hin dIII at both ends thereof, the Xba I - [0031] These translation initiation unmodified to those modified point two human chondromodulin -I protein So
The enzyme treatment was carried out by each conventional manner Hin dIII and Xba I. Then, according to the usual method, the above-mentioned pc
It was inserted into the cloning site of DNA3.

COS7 cells were transfected with the two kinds of expression vectors thus prepared. That is, 1 × 10 ⁷ COS7 cells are 9 cm
Spread it on a petri dish and use the above-mentioned method for TRANSFECTAM.
Was used for gene transfer. After culturing for 6 hours, 10% F
CS-containing eRDF medium was added and the cells were further cultured for 76 hours. A part of the culture supernatant was collected 48 hours after the start of the culture.

The concentration of human chondromodulin-I protein-antibody Fc region fusion protein in the recovered culture supernatant was quantified by the EIA method using an anti-Fc antibody. That is, according to a conventional method, a 96-well plate was coated with an antibody that recognizes rat antibody Fc, a culture supernatant was added, and a rat antibody Fc recognition antibody-HRP conjugate was added. The human chondromodulin-I protein-antibody Fc region fusion gene having an improved translation initiation point as a result of color development using ortho-phenylenediamine was compared with a fusion protein gene in which the amino acid sequence at the translation initiation point remained unchanged. The expression level of human chondromodulin-I protein was increased about 3- to 8-fold (see the table below).

[0034]

[Table 1]

[0035]

INDUSTRIAL APPLICABILITY According to the present invention, human chondromodulin-I protein can be stably produced in large quantities. Further, since human chondromodulin-I protein produced by the present invention is adsorbed on ammonium sulfate precipitation, heparin sepharose and the like, purification and isolation using these can be performed, and the purification and isolation process can be simplified as compared with the conventional method. Is possible.

[0036]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 1031 Sequence type: Nucleic acid Number of strands: Double-stranded topology: Linear sequence AAGCTTCGGC ACC ATG ACA GAG AAC TCC GAC AAA GTT CCC ATT GCC CTG 49 Met Thr Glu Asn Ser Asp Lys Val Pro Ile Ala Leu 1 5 10 GTG GGA CCT GAT GAC GTG GAA TTC TGC AGC CCC CCG GCG TAC GCT ACG 97 Val Gly Pro Asp Asp Val Glu Phe Cys Ser Pro Pro Ala Tyr Ala Thr 15 20 25 CTG ACG GTG AAG CCC TCC AGC CCC GCG CGG CTG CTC AAG GTG GGA GCC 145 Leu Thr Val Lys Pro Ser Ser Pro Ala Arg Leu Leu Lys Val Gly Ala 30 35 40 GTG GTC CTC ATT TCG GGA GCT GTG CTG CTG CTC TTT GGG GCC ATC GGG 193 Val Val Leu Ile Ser Gly Ala Val Leu Leu Leu Phe Gly Ala Ile Gly 45 50 55 60 GCC TTC TAC TTC TGG AAG GGG AGC GAC AGT CAC ATT TAC TAC AAT GTC CAT 241 Ala Phe Tyr Phe Trp Lys Gly Ser Asp Ser His Ile Tyr Asn Val His 65 70 75 TAC ACC ATG AGT ATC AAT GGG AAA TTA CAA GAT GGG TCA ATG GAA ATA 289 Tyr Thr Met Ser Ile Asn Gly Lys Leu Gln Asp Gly Ser Met Glu Ile 80 85 90 GAC GCT GGG AAC AAC TTG GAG ACC TTT AAA ATG GGA AGT GGA GCT GAA 337 Asp Ala Gly Asn Asn Leu Glu Thr Phe Lys Met Gly Ser Gly Ala Glu 95 100 105 GAA GCA ATT GCA GTT AAT GAT TTC CAG AAT GGC ATC ACA GGA ATT CGT 385 Glu Ala Ile Ala Val Asn Asp Phe Gln Asn Gly Ile Thr Gly Ile Arg 110 115 120 TTT GCT GGA GGA GAG AAG TGC TAC ATT AAA GCG CAA GTG AAG GCT CGT 433 Phe Ala Gly Gly Glu Lys Cys Tyr Ile Lys Ala Gln Val Lys Ala Arg 125 130 135 140 ATT CCT GAG GTG GGC GCC GTG ACC AAA CAG AGC ATC TCC TCC AAA CTG 481 Ile Pro Glu Val Gly Ala Val Thr Lys Gln Ser Ile Ser Ser Lys Leu 145 150 155 GAA GGC AAG ATC ATG CCA GTC AAA TAT GAA GAA AAT TCT CTT ATC TGG 529 Glu Gly Lys Ile Met Pro Val Lys Tyr Glu Glu Asn Ser Leu Ile Trp 160 165 170 GTG GCT GTA GAT CAG CCT GTG AAG GAC AAC AGC TTC TTG AGT TCT AAG 577 Val Ala Val Asp Gln Pro Val Lys Asp Asn Ser Phe Leu Ser Ser Lys 175 180 185 GTG TTA GAA CTC TGC GGT GAC CTT CCT ATT TTC TGG CTT AAA CCA ACC 625 Val Leu Glu Leu Cys Gly Asp Leu Pro Ile Phe Trp Leu Lys Pro Thr 190 195 200 TAT CCA AAA GAA ATC CAG AGG GAAAGA AGA GAA GTG GTA AGA AAA ATT 673 Tyr Pro Lys Glu Ile Gln Arg Glu Arg Arg Glu Val Val Arg Lys Ile 205 210 215 220 GTT CCA ACT ACC ACA AAA AGA CCA CAC AGT GGA CCA CGG AGC AAC CCA 721 Val Pro Thr Thr Thr Lys Arg Pro His Ser Gly Pro Arg Ser Asn Pro 225 230 235 GGC GCT GGA AGA CTG AAT AAT GAA ACC AGA CCC AGT GTT CAA GAG GAC 769 Gly Ala Gly Arg Leu Asn Asn Glu Thr Arg Pro Ser Val Gln Glu Asp 240 245 250 TCA CAA GCC TTC AAT CCT GAT AAT CCT TAT CAT CAG CAG GAA GGG GAA 817 Ser Gln Ala Phe Asn Pro Asp Asn Pro Tyr His Gln Gln Glu Gly Glu 255 260 265 AGC ATG ACA TTC GAC CCT AGA CTG GAT CAC GAA GGA ATC TGT TGT ATA 865 Ser Met Thr Phe Asp Pro Arg Leu Asp His Glu Gly Ile Cys Cys Ile 270 275 280 GAA TGT AGG CGG AGC TAC ACC CAC TGC CAG AAG ATC TGT GAA CCC CTG 913 Glu Cys Arg Arg Ser Tyr Thr His Cys Gln Lys Ile Cys Glu Pro Leu 285 290 295 300 GGG GGC TAT TAC CCA TGG CCT TAT AAT TAT CAA GGC TGC CGT TCG GCC 961 Gly Gly Tyr Tyr Pro Trp Pro Tyr Asn Tyr Gln Gly Cys Arg Ser Ala 305 310 315 TGC AGA GTC ATCATG CCA TGT AGC TGG TGG GTG GCC CGC ATC CTG GGC 1009 Cys Arg Val Ile Met Pro Cys Ser Trp Trp Val Ala Arg Ile Leu Gly 320 325 330 ATG GTG TAA CTCGAGTTCT AGA 1031 Met Val Stop 335

SEQ ID NO: 2 Sequence length: 25 Sequence type: Nucleic acid Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence AAGCTTCGGC ACCATGACAG AGAAC 25

SEQ ID NO: 3 Sequence length: 25 Sequence type: Nucleic acid Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence GTTCTAGAAC TCGAGTTACA CCATG 25

SEQ ID NO: 4 Sequence length: 20 Sequence type: Nucleic acid Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence GGTGACCTTC CTATTTTCTG 20

SEQ ID NO: 5 Sequence length: 19 Sequence type: Nucleic acid Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence ACCATGCCCA AGATACGGG 19

SEQ ID NO: 6 Sequence length: 41 Sequence type: Nucleic acid Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence TTCAGCACGA CCTTCGATCA CCATGCCCAG GATGCGGGCC A 41

SEQ ID NO: 7 Sequence length: 42 Sequence type: Nucleic acid Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence ATGGTGATCG AAGGTCGTGC TGAAACAACA GCCCCATCTG TC 42

SEQ ID NO: 8 Sequence length: 33 Sequence type: Nucleic acid Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence CCCTCTAGAT CATTTACCAG GAGAGTGGGA GAG 33

SEQ ID NO: 9 Sequence length: 25 Sequence type: Nucleic acid Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence AAGCTTCGGC TTCATGACAG AGAAC 25

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location // (C12N 5/10 C12R 1:91) (C12P 21/02 C12R 1:91) (72) Inventor Midori Nakajima 1000 Kamoshida-cho, Aoba-ku, Yokohama-shi, Kanagawa Mitsubishi Chemical Co., Ltd.Yokohama Research Institute (72) Inventor Kazushi Takahashi 1000 Kamoshida-cho, Aoba-ku, Yokohama-shi, Kanagawa Mitsubishi Chemical Corporation Yokohama Research Institute (72 ) Inventor Atsushi Kondo 1000 Kamoshida-cho, Aoba-ku, Yokohama, Kanagawa Prefecture, Yokohama Research Institute, Mitsubishi Chemical Co., Ltd. (72) Sadayo Iijima 1000, Kamoshida-cho, Aoba-ku, Yokohama City, Kanagawa Mitsubishi Chemical Corporation, Yokohama Research Institute

Claims (12)

[Claims] 1. A base sequence of a gene encoding a human chondromodulin-I protein, which contains a base at the third base upstream from adenine in the base sequence encoding methionine for translation initiation, and comprises the base at the third base. A DNA fragment whose base is adenine or guanine. 2. The DNA fragment according to claim 1, wherein the third base is adenine. 3. The DNA fragment according to claim 1, wherein the second upstream base is cytosine. 4. The DNA fragment according to any one of claims 1 to 3, wherein the first upstream base is cytosine. 5. The DNA fragment according to claim 1, which contains a restriction enzyme Hin dIII cleavage site at the 5 ′ end. 6. The DNA fragment according to any one of claims 1 to 5, which is used as a primer. 7. The DNA fragment according to any one of claims 1 to 6, which is represented by the nucleotide sequence of SEQ ID NO: 2 in the sequence listing. 8. The DN according to any one of claims 1 to 7.
A DNA fragment containing a gene encoding a human chondromodulin-I protein, which is obtained by a PCR method using the A fragment as a primer. 9. A DNA fragment represented by SEQ ID NO: 1 in the sequence listing. 10. An expression vector containing the DNA fragment according to claim 8 or 9. 11. A transformant obtained by transforming a host cell with the expression vector according to claim 10, and introducing a gene encoding a human chondromodulin-I protein into the chromosome of the host cell. 12. A method for producing human chondromodulin-I protein, which comprises culturing the transformant according to claim 11.