JPH0851982A - Modified gene coding human serum albumen - Google Patents

Abstract

PURPOSE:To provide a modified gene which can give a fused protein containing a cancer metastasis inhlbitory peptide or the like through the gene recombination technique because one or more restriction enzyme cleavage sites are introduced into the gene coding a natural type human albumen at desired positions. CONSTITUTION:At least one of restriction enzyme cleavage sites is introduced into a gene coding a natural type human serum albumen in at least one position, for example, of the amino terminus gene of human serum albumen containing the base sequence of formula I, the carboxyl terminus gene of human serum albumen containing the base sequence of formula II, the gene between the first and the second domains of human serum albumen containing the base sequence of formula III, a gene between the second and the third domains of human serum albumen containing the base sequence of formula IV, or in their arbitrary combination. Thus, the introduction of a gene, for example coding a peptide having cancer metastasis inhibitory activity is fasilitated on the amino terminal side whereby a modified gene coding a novel human serum albumen which can readily produce fused proteins is obtained through the gene recombination technique.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a modified human serum albumin gene which is most suitable for producing a novel fusion protein by gene recombination technology.

[0002]

2. Description of the Related Art When a peptide having physiological activity is used for the purpose of medicine or the like, when the peptide alone is administered, it often does not exhibit the desired physiological activity. This is mainly due to destabilization by degrading enzymes and adsorption to organs. to solve this problem,
Usually, a method of producing a fusion of a physiologically active peptide and a biopolymer and administering the fusion is used. Although there are many examples of biopolymers that can be used, it is preferable to use serum albumin, which is expected to have the least side effects because it is present in a large amount in the body, particularly in blood.

In order to produce a fusion product of a physiologically active peptide and serum albumin, a chemical bonding method is commonly used. For example, IIF-2, which is a peptide confirmed to have cancer metastasis inhibitory activity (Japanese Patent Laid-Open No. 3-349).
93, Isoai et al., Jpn. J. Cancer Res., 81,
909-914 (1992) and Isoai et al., Cancer Res., 5
2, 1422-1426 (1992)), the peptide and serum albumin are linked with a water-soluble carbodiimide to prepare a novel fusion protein, which is used to produce a stronger cancer cell than the peptide alone. It has been confirmed by the present inventors that they exhibit an invasion inhibitory activity and a cancer metastasis inhibitory activity (JP-A-4-254000, JP-A-4-304000).
0899, 4-300900 and Biochem.
Biophys. Res. Commun., 192, 7-14 (1993)). That is, compared to the case where the peptide is used alone, 1/50 to
A low concentration of 1/60 strongly suppressed the infiltration of human and mouse-derived highly metastatic cancer cells into the extracellular basement membrane. further,
In a so-called "experimental metastasis model" system in which cancer cells are injected into the tail vein of a mouse and metastasize to major organs such as lung and liver, a novel fusion protein consisting of the peptide and serum albumin is compared to the case where the peptide is used alone. , 1 /
At a low dose of 10 or less, the same or higher metastasis inhibitory activity was exhibited.

As in the case of the above-mentioned fusion of the cancer metastasis inhibiting peptide and serum albumin, both the bioactive peptide and the biopolymer constituting the fusion protein are both composed of a linear bond of proteinaceous amino acids. In this case, it can be easily estimated that the target fusion protein can be prepared by genetic engineering. That is, a gene encoding the desired fusion protein may be prepared and introduced into a heterologous protein production system using Escherichia coli or yeast as a host. By making it genetically,
It is possible to produce a fusion protein that cannot be produced by the method of chemically coupling. For example, it becomes easy to introduce a physiologically active peptide into a specific position of the biopolymer and to control the ratio of the number of the physiologically active peptide to the biopolymer in the fusion protein.

Therefore, when a bioactive peptide is bound using a genetic engineering technique, how to select a biopolymer used as a carrier that easily incorporates the desired bioactive peptide, or how to modify the biopolymer so that it can be easily incorporated Was a problem. At the same time, it is necessary to have a structure capable of binding any type of bioactive peptide.

[0006]

The present invention has been made in view of the above circumstances, and provides an optimum serum albumin gene as a carrier for producing a fusion protein. Then, using this, a fusion protein can be produced efficiently and in large quantities by gene recombination technology.

[0007]

[Means for Solving the Problems] The inventors of the present invention have conducted extensive studies to solve the above-mentioned problems, devised and designed a human serum albumin gene into which a bioactive peptide can be easily incorporated, and carried out gene recombination technology. It was confirmed that this fusion protein exhibits the desired physiological activity by producing the novel fusion protein actually bound with the physiologically active peptide.

That is, according to the present invention, a modified gene encoding human serum albumin, which is obtained by introducing a restriction enzyme cleavage site into at least one or more desired positions of a gene encoding natural human serum albumin. Will be provided.

Here, the restriction enzyme cleavage site is introduced at any one of the amino terminal, the carboxyl terminal, between the first and second domains, or between the second and third domains of the polypeptide chain of human serum albumin. Alternatively, the positions are preferably any combination thereof.

The present invention also provides a fusion protein prepared by introducing a gene encoding a peptide having physiological activity into the restriction enzyme cleavage site of any of the above genes.

The present invention further provides a gene encoding the above fusion protein.

The present invention will be described in detail below.

The modified human serum albumin gene of the present invention is prepared by newly introducing a restriction enzyme cleavage site into a gene encoding natural human serum albumin in order to facilitate binding with a peptide having physiological activity. To be done.

The natural human serum albumin gene used for the production of the modified human serum albumin gene is, for example, a plasmid pI derived from a human liver cDNA library.
It can be obtained by cloning using the restriction enzyme PvuII-HindIII fragment of LMALB5 (National Institute for Preventive Health, Gene Bank) as a probe. It has been reported that the human serum albumin gene has a polymorphism in which the amino acid sequences thereof are slightly different from each other, and the human serum albumin gene cloned by the above method also falls into this category. The "human serum albumin" in the present invention may include all of these polymorphisms.

Next, a fragment having a restriction enzyme cleavage site is introduced into a predetermined position of this human serum albumin gene to prepare a modified human serum albumin gene. The introduction of this restriction enzyme cleavage site is for facilitating the subsequent binding of a peptide gene having a physiological activity (for example, a cancer metastasis inhibition gene). This is for cutting at, and binding the cancer metastasis inhibitor gene to this cut.

The position at which the restriction enzyme cleavage site to be modified is introduced can be set at any position in the polypeptide chain of human serum albumin, but considering that the activity is sufficiently exhibited, It is preferable that the position is located on the surface of the protein and does not destroy the three-dimensional structure. For example, a position such as an amino terminus (N terminus) or a carboxy terminus (C terminus) that is considered not to affect the formation of the three-dimensional structure of human serum albumin is desirable. In addition, the three-dimensional structure of human serum albumin has been studied in detail by X-ray crystallography (Xia
o, M, H., and Carter, DC Nature, 358: 209-215, 1992
) Between the three domains, that is, between the first and second domains or between the second and third domains can also be candidates for the introduction site. The number of restriction enzyme cleavage sites to be introduced may be singular or plural at a single position or a plurality of positions, if necessary.

The restriction enzyme cleavage site to be introduced may be one that can be recognized by a known restriction enzyme. Desirably, a cleavage site that hardly exists in human serum albumin, and a cleavage enzyme is easily available. Particularly, those that recognize 6 bases and form sticky ends after digestion are preferable for ligation. Also,
Naturally, it is desirable not to change the natural amino acid sequence at all and, at the same time, to change the base sequence as little as possible.
In consideration of the above points, a restriction enzyme AflIII cleavage site is introduced at the amino terminus and a carboxyl terminus, a restriction enzyme HindIII cleavage site is introduced between the first and second domains, and a restriction enzyme EcoRI cleavage site is introduced between the second and third domains. Is most preferred. Although any method can be used as a method for introducing a restriction enzyme cleavage site, PC commonly used in the art is used.
A mutation introduction method using R is preferably used.

Further, in the present invention, a gene encoding a peptide having physiological activity is prepared, and this gene is linked to the restriction enzyme cleavage site of the modified human serum albumin gene to prepare a fusion protein gene having physiological activity. Then, this gene is introduced into an expression vector, the gene is expressed in a host cell using this vector, and the physiologically active fusion protein is produced by extracting and purifying from the inside of the host cell.

As the peptide having this physiological activity,
For example, a peptide having a cancer metastasis inhibitory activity represented by the amino acid sequence of SEQ ID NO: 6 (cancer metastasis inhibitory peptide; JP-A-3-34993) and the like can be mentioned. The gene encoding this cancer metastasis-inhibiting peptide can theoretically have a large number of sequences, but it is preferable to match the frequency of codon usage of the host cell used for gene recombination. It is better to design using the codons used in.

The host cell used here is not particularly limited, but it is desirable that the culture method be easy,
Microorganisms that can be cultivated at low cost are often used.
cherichia coli), various yeasts, Bacillus subtilis, filamentous fungi, etc., and host cells commonly used in the art. Transformation methods using prokaryotes as host cells are not always effective for all polypeptides, and it is not always easy to reproduce complex post-translational modifications of eukaryote-derived proteins or the same three-dimensional structures as natural bodies. Absent. Further, the presence of a specific endotoxin is not preferable because it may become a contaminant of the final product. Therefore, it is preferable to use various yeasts that do not contain endotoxin, have a well-established culture method, have been used in the fermentation and food industries, and have a well-established safety for humans. Among them, the fission yeast Schizosaccharomyces pombe, which is expected to be able to obtain a gene product closer to a natural body, is said to have characteristics similar to animal cells genetically and molecularly.
Is most preferred. Examples of the strain of Schizosaccharomyces pombe include deposit number ATCC 38399 (leu-
32h ^-) and ATCC38436 (ura4-294h ^-) American Type Culture Collection as such (ATC
The ones deposited in C) are listed and available.

Therefore, in the present invention, SEQ ID NO: 6
The gene encoding the cancer metastasis inhibitory peptide represented by
Most preferably, it is designed and synthesized using a codon most suitable for high expression in Schizosaccharomyces pombe. The optimal codon usage frequency of Schizosaccharomyces pombe is found, for example, in A. Nasim et al .: Molecular Biology oft.
You can learn from he Fission Yeast, p.263, Academic Press (1983), etc. As a result of various studies conducted by the present inventors, the inventors have concluded that the gene represented by the nucleotide sequence of SEQ ID NO: 7 is most suitable, and designed and synthesized it (however, the nucleotide sequence of SEQ ID NO: 7 is a translation initiation sequence). Signal (ATG) and translation termination signal (TAA)). In addition,
Gene production (synthesis) is performed by the triester method (Nuc. Acid.
Res. 10, p.6553, (1982)) and the phosphoamidite method (Tet
Various methods such as rahedron Letters 22, p.1859, (1981)) have already been developed, and any method may be used. In addition, since DNA synthesizers (DNA synthesizers) and the like are commercially available, they may be used.

Next, the novel cancer metastasis inhibiting fusion protein gene prepared as described above is incorporated into a vector to prepare a recombinant vector. The vector to be used is not particularly limited, but it is capable of autonomously replicating in the host cell, has an insertion site into which a cancer metastasis-inhibiting fusion protein synthetic gene (foreign gene) can be incorporated, and further, this synthetic gene Must have a region that allows it to be expressed in the host cell. As such a vector, for example, the foreign gene expression vector pTL2M (Japanese Patent Application No. 5-249310), which uses the Schizosaccharomyces pombe as a host, which has been successfully created by the present inventors, is advantageously used. The above synthetic gene can be easily incorporated into these vectors.

Next, the above recombinant vector is introduced into a host cell to obtain a transformant. The method of introducing the recombinant vector into the host cell can be carried out by a conventionally used method, such as the competent cell method, the protoplast method, the calcium phosphate coprecipitation method, the electroporation method, the microinjection method, There are various methods such as the liposome fusion method and the particle gun method, and any method can be adopted depending on the host to be used. When using Schizosaccharomyces pombe as a host,
For example, the lithium acetate method (K. Okazaki et al., Nucleic A
Cids Res., 18, 6485-6489 (1990)) and the like can be used to efficiently obtain a transformant.

By culturing the transformant thus obtained, a cancer metastasis inhibiting fusion protein is produced in the culture. The desired cancer metastasis-inhibiting fusion protein can be obtained by isolating it by a known method and optionally purifying it.

A medium for culturing the transformant is known, and a nutrient medium such as YPD medium (MD Rose et al.,
"Methods In Yeast Genetics", Cold Spring Harbor L
abolatory Press (1990) r) and minimal medium such as MB medium (K. Okazaki et al., Nucleic Acids Res., 18, 6485-
6489 (1990)) and the like can be used. Cultivation of the transformant is usually at 16 to 42 ° C, preferably 25 to 37 ° C,
It is carried out for 8 to 168 hours, preferably for 24 to 72 hours. Both shaking culture and static culture are possible, but agitation and aeration may be added if necessary.

As a method for isolating and purifying the fusion protein produced in the culture, a method utilizing a difference in solubility such as a known salting out method or a solvent precipitation method, a dialysis method, an ultrafiltration method or a gel electrophoresis method, etc. Methods that utilize differences in molecular weight, methods that utilize differences in charge such as ion exchange chromatography, methods that utilize specific affinity such as affinity chromatography, and differences in hydrophobicity such as reversed-phase high-performance liquid chromatography Examples of the method include a method of utilizing a difference in isoelectric points such as an isoelectric focusing method.

As a method for confirming the isolated and purified fusion protein, known Western blotting method, activity measuring method and the like can be mentioned. The structure of the purified fusion protein can be clarified by amino acid analysis, amino terminal analysis, primary structure analysis and the like.

[0028]

The present invention will be described more specifically by the following examples. However, the technical scope of the present invention is not limited by these examples. In addition, for each operation in the examples, except for those specifically described, methods commonly used in the art (for example, J. Sambrook et al .: Molecular
Cloning. A Laboratory Manual. 2nd ed. Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, New Y
ork, USA, 1989.).

[Example 1] Preparation of modified human serum albumin gene of SEQ ID NO: 1 Human serum albumin c cloned from human liver cDNA library onto pUC19 (Takara Shuzo Co., Ltd.)
PCR is performed using DNA as a template and the primers represented by the nucleotide sequences of SEQ ID NOs: 12 and 13,
Then, the restriction enzymes NcoI (Takara Shuzo Co., Ltd.) and Hi
Terminal adjustment (partial digestion) was performed with ndIII (Takara Shuzo Co., Ltd.). After purification by phenol extraction and ethanol precipitation, agarose gel electrophoresis was performed to obtain about 1800
The band corresponding to the base pair is cut out and DNA-PREP
It was purified by the glass bead method using (Asahi Glass) to obtain an insert fragment.

Separately from this, a Schizosaccharomyces pombe expression vector pTL2M was prepared. This vector pTL2M has been constructed by the present inventors (Japanese Patent Application No. 5-249310). The manufacturing method will be described below.

[Preparation of vector pRL2M] First, pcD4CAT prepared by a known method was cleaved with BamHI to remove the CAT gene and then ligated to obtain pcD4.
Was produced. pcD4 was partially cleaved with BamHI, blunt-ended, and ligated to prepare pcD4B (JP-A-5-15380).

This plasmid pcD4B was used as a restriction enzyme Sa
After digestion with cI, the ends were blunted with T4 DNA polymerase, further digested with a restriction enzyme BamHI, and then purified by phenol extraction and ethanol precipitation. Further, after agarose gel electrophoresis, DNA corresponding to about 4500 base pairs was purified by the glass bead method.

Separately from this, an Okayama-Berg cDNA library (pcD vector) derived from human fibroblasts was prepared by a known method. Furthermore, the already known gene sequence of human lipocortin I (Nature, 320,
77, (1986)), the gene of lipocortin I was obtained by the colony hybridization method from the above library using the gene sequence of 50 bases encoding the N-terminal amino acid sequence of the protein as a DNA probe, and the nucleotide sequence was determined. By doing so, it was confirmed that it encodes the full-length lipocortin I protein. The obtained clone was named pcDlipoI. (JP-A-5
-15380). And this human lipocortin I
The vector pcDlipoI containing the gene (cDNA) was digested with restriction enzymes XmnI and BamHI, and then purified by phenol extraction and ethanol precipitation. Further, after agarose gel electrophoresis, the DNA corresponding to about 1300 base pairs was purified by the glass bead method.

After ligating both DNAs, this was introduced into Escherichia coli DH5 strain (Toyobo Co., Ltd.) for transformation. A vector was prepared from the obtained transformant, and a transformant having the target vector pRL2L (FIG. 5) was screened. From the confirmation of the partial base sequence and the construction of the restriction enzyme map, the target vector was confirmed.

This lipocortin I expression vector pRL2
L was digested with restriction enzymes EcoRI and HindIII, extracted with phenol and precipitated with ethanol, a band corresponding to about 5000 base pairs was cut out by agarose gel electrophoresis, and purified by a glass bead method. Separately from this, a known plasmid pUC19 was added to the restriction enzyme EcoRI.
After digestion with HindIII and phenol extraction and ethanol precipitation, a band corresponding to about 60 base pairs was cut out by polyacrylamide gel electrophoresis and extracted and purified from the gel.

After ligation of these two fragments,
Target vector pR obtained by transforming Escherichia coli strain DH5
L2M (Figure 6) was screened. From the confirmation of the partial base sequence and the construction of the restriction enzyme map, the target vector was confirmed.

[Preparation of vector pTL2M] The above pRL
Oligodeoxyribonucleotide 5'- with 2M as template
TTGACTAGTTATTAATAGTA-3 'and oligodeoxyribonucleotide 5'-CTAGAATTCACATGTTTGAAAAAGTGTCTTTATC-
The target fragment was amplified by PCR using Taq polymerase with 3'as a synthetic primer. Restriction enzyme Sp
end-adjusted with eI and EcoRI, phenol extracted,
After ethanol precipitation, a band corresponding to about 600 base pairs was cut out by agarose gel electrophoresis and purified by a glass bead method.

Separately from this, pRL2M was digested with restriction enzymes SpeI and EcoRI, extracted with phenol and precipitated with ethanol. Then, a band corresponding to about 4500 base pairs was cut out by agarose gel electrophoresis and purified by a glass bead method. did. After ligation of these two fragments, Escherichia coli DH5 strain was transformed to screen the target vector pTL2M (FIG. 7). From the confirmation of the partial base sequence and the construction of the restriction enzyme map, the target vector was confirmed.

The pTL2M thus prepared was double-digested with the restriction enzymes AflIII and HindIII,
A band corresponding to about 5000 base pairs was cut out.

Then, the above-mentioned insert and this expression vector p
A total of two pieces of TL2M and the double digested product with the above restriction enzymes were ligated using a DNA ligation kit (Takara Shuzo Co., Ltd.). This was introduced into Escherichia coli DH5 strain (manufactured by Toyobo Co., Ltd.) for transformation, and the desired plasmid pTL2Bma was obtained. A large amount of pTL2Bma was prepared according to the alkali-SDS method, and it was confirmed that the plasmid had the sequence of SEQ ID NO: 1 by constructing a restriction map and determining the nucleotide sequence.

[Example 2] Preparation of modified human serum albumin gene of SEQ ID NO: 2 It is represented by the nucleotide sequences of SEQ ID NOS: 12 and 14 using human serum albumin cDNA cloned from human liver cDNA library on pUC19 as a template. PCR amplification is performed using the primers and then the restriction enzyme N
End-regulation was done with coI and HindIII. After phenol extraction and purification by ethanol precipitation,
After agarose gel electrophoresis, a band corresponding to about 550 base pairs was cut out and purified by a glass bead method using DNA-PREP to obtain an insert fragment 1.

Separately from this, PCR amplification was carried out using the same cDNA as a template and the primers represented by the nucleotide sequences of SEQ ID NOs: 15 and 13 to obtain the restriction enzyme Hi.
End-regulation was done with ndIII. After purification by phenol extraction and ethanol precipitation, agarose gel electrophoresis was performed, and a band corresponding to about 1350 base pairs was cut out and purified by a glass bead method using DNA-PREP to obtain an insert fragment 2.

Separately from this, a Schizosaccharomyces pombe expression vector pTL2M prepared in the same manner as in Example 1 was prepared, and this vector pTL2M was double-digested with restriction enzymes AflIII and HindIII to give about 5000 bases. The band corresponding to the pair was cut out.

A total of 3 of the above-mentioned two insert fragments and the double digest of the expression vector pTL2M with the above-mentioned restriction enzymes.
The book was ligated using a DNA ligation kit. This was introduced into Escherichia coli strain DH5 for transformation, and the desired plasmid pTL2Bmb was obtained. A large amount of pTL2Bmb was prepared according to the alkali-SDS method, and it was confirmed that the plasmid had the sequence of SEQ ID NO: 2 by constructing a restriction enzyme map and determining the nucleotide sequence.

[Example 3] Preparation of modified human serum albumin gene of SEQ ID NO: 3 It is represented by the nucleotide sequences of SEQ ID NOS: 12 and 16 using human serum albumin cDNA cloned from human liver cDNA library on pUC19 as a template. PCR amplification is performed using the primers and then the restriction enzyme N
The ends were adjusted with coI and EcoRI (Takara Shuzo Co., Ltd.). After purification by phenol extraction and ethanol precipitation, agarose gel electrophoresis was performed to obtain about 1100.
A band corresponding to a base pair was cut out and purified by a glass bead method using DNA-PREP to obtain an insert fragment 1.

Separately from this, PCR amplification was carried out using the same cDNA as a template and the primers represented by the nucleotide sequences of SEQ ID NOs: 17 and 13, and then the ends were regulated by the restriction enzymes EcoRI and HindIII. After purification by phenol extraction and ethanol precipitation, agarose gel electrophoresis was performed, and a band corresponding to about 700 base pairs was cut out and purified by the glass bead method using DNA-PREP to obtain insert fragment 2.

Separately from this, a Schizosaccharomyces pombe expression vector pTL2M prepared in the same manner as in Example 1 was prepared, and this vector pTL2M was double-digested with restriction enzymes AflIII and HindIII to give about 5000 bases. The band corresponding to the pair was cut out.

A total of 3 of the above-mentioned two insert fragments and the double digest of the expression vector pTL2M with the above-mentioned restriction enzymes.
The book was ligated using a DNA ligation kit. This was introduced into Escherichia coli strain DH5 for transformation, and the desired plasmid pTL2Bmc was obtained. A large amount of pTL2Bmc was prepared according to the alkali-SDS method, and it was confirmed that the plasmid had the sequence of SEQ ID NO: 3 by constructing a restriction enzyme map and determining the nucleotide sequence.

[Example 4] Preparation of modified human serum albumin gene of SEQ ID NO: 4 Using the human serum albumin cDNA cloned from human liver cDNA library on pUC19 as a template, it is represented by the nucleotide sequences of SEQ ID NOs: 12 and 18. PCR amplification is performed using the primers and then the restriction enzyme N
End-regulation was done with coI and AflIII. After phenol extraction and purification by ethanol precipitation,
After agarose gel electrophoresis, a band corresponding to about 1800 base pairs was cut out and purified by a glass bead method using DNA-PREP to obtain an insert fragment.

Separately from this, a Schizosaccharomyces pombe expression vector pTL2M prepared in the same manner as in Example 1 was prepared, and this vector pTL2M was double-digested with restriction enzymes AflIII and HindIII to give about 5000 The band corresponding to the base pair was cut out.

Then, the above-mentioned insert and this expression vector p
A total of two pieces of TL2M and a double digest with the above restriction enzymes were ligated using a DNA ligation kit. After introducing this into Escherichia coli DH5 strain and transforming it, the desired plasmid pTL2Bmd was obtained. A large amount of pTL2Bmd was prepared according to the alkali-SDS method, and it was confirmed that the plasmid had the sequence of SEQ ID NO: 4 by constructing a restriction map and determining the nucleotide sequence.

[Example 5] Preparation of modified human serum albumin gene of SEQ ID NO: 5 It is represented by the nucleotide sequences of SEQ ID NOS: 12 and 14 using human serum albumin cDNA cloned from human liver cDNA library on pUC19 as a template. PCR amplification is performed using the primers and then the restriction enzyme N
End-regulation was done with coI and HindIII. After phenol extraction and purification by ethanol precipitation,
After agarose gel electrophoresis, a band corresponding to about 550 base pairs was cut out and purified by a glass bead method using DNA-PREP to obtain an insert fragment 1.

Separately from this, PCR amplification was carried out using the same cDNA as a template and the primers represented by the nucleotide sequences of SEQ ID NOs: 15 and 16, and then the restriction enzyme H
End-regulation was done with indIII and EcoRI. After purification by phenol extraction and ethanol precipitation, agarose gel electrophoresis was performed, and a band corresponding to about 700 base pairs was cut out and purified by the glass bead method using DNA-PREP to obtain insert fragment 2.

Separately from this, PCR amplification was performed using the same cDNA as a template and the primers represented by the nucleotide sequences of SEQ ID NOs: 17 and 18, and then the ends were regulated by the restriction enzymes EcoRI and AflIII. After purification by phenol extraction and ethanol precipitation, agarose gel electrophoresis was performed, and a band corresponding to about 700 base pairs was cut out and purified by the glass bead method using DNA-PREP to obtain insert fragment 3.

Separately from this, a Schizosaccharomyces pombe expression vector pTL2M prepared in the same manner as in Example 1 was prepared, and this vector pTL2M was double-digested with restriction enzymes AflIII and HindIII to give about 5000 bases. The band corresponding to the pair was cut out.

Then, a total of four pieces of the above-mentioned insert fragment and the double digest of pTL2M with the above-mentioned restriction enzyme were ligated using a DNA ligation kit. After introducing this into Escherichia coli DH5 strain for transformation, the desired plasmid pTL2Bme was obtained. A large amount of pTL2Bme was prepared according to the alkali-SDS method, and it was confirmed that the plasmid had the sequence of SEQ ID NO: 5 by constructing a restriction enzyme map and determining the nucleotide sequence.

Example 6 Preparation of Gene Represented by Nucleotide Sequence of SEQ ID NO: 7 Encoding Cancer Metastasis Inhibitory Peptide Based on the amino acid sequence of SEQ ID NO: 6, the codon usage of Schizosaccharomyces pombe (Nasim , A. et al: Molec
ular Biology of the Fission Yeast, Academic Press,
1989, p263.) And two single-stranded oligo DNAs represented by the nucleotide sequences of SEQ ID NOs: 10 and 11
It synthesize | combined using the automatic synthesizer (Applied Biosystems). The nucleotide sequence of SEQ ID NO: 10 has an insertion site into the restriction enzyme BamHI and a start codon ATG at the 5'end,
A termination codon TAA and a restriction enzyme HindIII at the 3'end
Is the sense strand of the gene into which the insertion site was introduced, and the base sequence of SEQ ID NO: 11 is its antisense strand. After deprotection and purification, these two were annealed at 70 ° C.

On the other hand, apart from this, the plasmid pUC19
With the restriction enzymes BamHI (Takara Shuzo Co., Ltd.) and Hi.
After double digestion with ndIII, extraction with phenol, purification by ethanol precipitation, agarose gel electrophoresis,
A band corresponding to 600 base pairs was cut out and DNA-PR
It was purified by the glass bead method using EP.

Both of these fragments were ligated using a DNA ligation kit. E. coli J
After introduction into M109 strain (Takara Shuzo Co., Ltd.) and transformation, a positive clone having ampicillin resistance and displaying white colonies on X-gal plate was screened, and the desired plasmid, namely the restriction enzyme BamH.
Upon double digestion with I and HindIII, pI2A was obtained which showed a cleavage fragment of about 70 base pairs. A large amount of pI2A was prepared according to the alkali-SDS method, and it was confirmed that the plasmid had the desired sequence by constructing a restriction map and determining the nucleotide sequence.

Example 7 Construction of Expression Vector pTL2BmI Containing Cancer Metastasis Inhibitory Peptide Gene Plasmid pI2A was digested with restriction enzymes NcoI and Hind.
The ends were adjusted by double digestion of III, and a band corresponding to about 70 base pairs was cut out by acrylamide gel electrophoresis and eluted from the gel to obtain a cancer metastasis-inhibiting peptide gene insert fragment.

This gene fragment and pT prepared in Example 4
L2Bmd restriction enzyme AflIII digested product (after partial digestion, a band corresponding to about 7000 base pairs was subjected to DNA-PR
2), which were purified using EP), were ligated using a DNA ligation kit. E. coli DH5
After transforming the strain, the desired plasmid pTL2BmI shown in FIG. 1 was obtained. P according to the alkali-SDS method
A large amount of TL2BmI was prepared, and it was confirmed that it was a plasmid having the sequence of SEQ ID NO: 7 by constructing a restriction map and determining the nucleotide sequence.

[Example 8] Expression vector pTL2BmI
Leucine auxotrophic strain of Schizosaccharomyces transformation of Mrs. pombe Schizosaccharomyces pombe using, h ^-
leu 1-32 (ATCC 38399) was grown in leucine-containing minimal medium MB-leu to reach 10 ⁷ cells / ml. After centrifugation and washing with water, 10 ⁹ cells /
100 mM lithium acetate (pH 5.
0), and incubated at 30 ° C. for 60 minutes.
Thereafter, pAL7 (K. Okazaki et al .: Nucl. Acids R digested with the restriction enzyme PstI was added to 100 μl of the above suspension.
es. 18, 6485-6489 (1990)) 1 μg and 2 μg of the expression vector pTL2BmI dissolved in 10 μl of TE buffer were added, and 50% PEG4000 was added to 290 μm.
After adding 1 well and mixing well, the mixture was incubated at 30 ° C. for 60 minutes, 43 ° C. for 15 minutes, and room temperature for 10 minutes in this order.
PEG4000 was removed by centrifugation, and the suspension was suspended in 1 ml of culture medium 1 / 2YEL-Leu.

100 μl was taken from this suspension, further diluted with 900 μl of culture medium 1 / 2YEL-Leu, incubated at 32 ° C. for 30 minutes, and then 300 μl.
Was spread on the minimal agar medium MMA. After incubating at 32 ° C. for 3 days, the obtained transformant was transferred to a YEA medium containing 25 μg / ml of G418 and further cultured at 32 ° C. for 5 days, and the obtained clone was used as each target transformant.

Separately from this, transformants were prepared by the same method for the plasmids pTL2M (described above) and pTL2Bm (Japanese Patent Application No. 5-249310) which do not have a cancer metastasis inhibitory peptide gene. It was used as a negative control. The plasmid pTL2Bm was prepared as follows.

[Preparation of plasmid pTL2Bm] Oligodeoxyribonucleotide 5'-AGACCA using the vector pILMALB5 containing human serum albumin cDNA, which was provided by the National Institute of Preventive Health, Gene Bank, as a template.
The target fragment was amplified by PCR using Taq polymerase with TGGATGCACACACAAGAGTGAGGT-3 ′ and oligodeoxyribonucleotide 5′-CAGGAAACAGCTATGACCAT-3 ′ as synthetic primers. Restriction enzymes NcoI and H
End-adjusted with indIII, extracted with phenol, precipitated with ethanol, and then subjected to agarose gel electrophoresis to obtain about 1800
The band corresponding to the base pair was cut out and purified by the glass bead method.

Separately from this, pTL2M was used as a restriction enzyme Af.
After digestion with lIII and HindIII, phenol extraction and ethanol precipitation, a band corresponding to about 5000 base pairs was cut out by agarose gel electrophoresis and purified by the glass bead method.

After ligation of these two fragments,
Target vector pT obtained by transforming Escherichia coli DH5 strain
L2Bm (Figure 8) was screened. From the confirmation of the partial base sequence and the construction of the restriction enzyme map, the target vector was confirmed.

[Example 9] Cultivation of transformants and preparation of cell-free extract 50 ml of YPD medium containing the antibiotic G418 (GIBCO BRL) at a concentration of 200 µg / ml [(2% glucose (Wako Pure Chemical Industries, Ltd. )) 1% Bacto yeast extract (Di
fco), 2% bactopeptone (Difco)] in Example 8
The transformant prepared in 1. was inoculated and cultured at 32 ° C. for 5 days. After collecting 10 ⁸ cells from the culture and washing the cells,
The cells were suspended in 50 mM Tris-HCl buffer (pH 7.5) and sonicated. 10% S so that the final concentration is 1%
The DS solution was added and heated at 80 ° C. for 15 minutes. A cell-free extract (supernatant) was obtained by centrifugation.

Separately from this, a cell-free extract was prepared in the same manner for the transformants into which the above-mentioned pTL2M and pTL2Bm having no cancer metastasis-inhibiting peptide gene were introduced and used as a negative control.

[Example 10] Expression analysis of cancer metastasis inhibition fusion protein by SDS-polyacrylamide gel electrophoresis Expression analysis was performed on the cell-free extract derived from each transformant prepared in Example 9 by SDS-PAGE. It was The results are shown in Figure 2. As is clear from the figure, pT
The L2mBI transformant had a molecular weight of 6 as compared with the control pTL2Bm transformant.
The band of 9,000 (indicated by * in the figure) moves to the position of molecular weight of 71,000 (indicated by ** in the figure) due to the production of the cancer metastasis inhibiting fusion protein. I was able to detect that. When measured by a densitometer, the amount of the cancer metastasis-inhibiting fusion protein produced was about 30% of the total bacterial protein.

[Example 11] Confirmation of cancer metastasis-inhibiting fusion protein by Western blotting The cell-free extract derived from each transformant prepared in Example 9 was subjected to SDS-PAGE in the same manner as in Example 10. The obtained gel was transferred to a PVDF membrane (Bio-Rad), and an antibody (A. Isoai et al.
al .: Biochem. Biophys. Res. Commun. 192, 7-14 (199
Western blotting using 3)) and ECL
(Manufactured by Amersham Co., Ltd.). The result is shown in Figure 3.
Shown in As is clear from the figure, since a unique clear band was obtained at a position near the molecular weight of 71,000 corresponding to the sequence containing the fusion protein, an amino acid sequence specific to the fusion protein was contained. It was confirmed that the fusion protein was produced.

[Example 12] Purification of cancer metastasis-inhibiting fusion protein The transformant transformed with pTL2BmI was transformed into G
50 ml of YPD containing 418 at a concentration of 25 μg / ml
After pre-culturing in the medium at 32 ° C for 1 day, G418 was added to 200
1 x 10 ⁸ / in 1 liter of YPD medium containing μg / ml
The cells were inoculated at a ratio of ml and further cultured for 4 days. 50 mM Tris-hydrochloric acid buffer solution (pH 7.
5) [12 μM APMSF (manufactured by Wako Pure Chemical Industries, Ltd.), 2
5 μM leupeptin (manufactured by Wako Pure Chemical Industries, Ltd.), 2 mM E
It was suspended in DTA] and crushed at 0 ° C. using an equal amount of glass beads (bead beater). 12,000 rpm
The precipitate was centrifuged for 20 minutes, washed with the same buffer, and then solubilized in 50 mM Tris-HCl buffer (pH 7.5) containing 6 M guanidine hydrochloride and 10 mM dithiothreitol at 50 ° C. for 1 hour. 12,000 rpm, 2
The supernatant obtained by centrifuging for 0 minutes was treated with 0.1 M NaCl and 1 mM.
50 mM Tris-HCl buffer containing DTA, 2 mM reduced glutathione and 0.2 mM oxidized glutathione (pH
7.5) was gradually diluted 100 times (v / v) at 4 ° C. After being left overnight at 4 ° C., it was concentrated with an ultrafiltration membrane (Amicon), gel-filtered with Superose 12 column, and each fraction was analyzed by SDS-PAGE to have a molecular weight of 71,000.
Fractions showing a unique band at position 0 were collected and used as a purified cancer metastasis-inhibiting fusion protein.

Example 13 Measurement of Cancer Cell Invasion Inhibitory Activity of Purified Cancer Metastasis Inhibition Fusion Protein The cancer cell invasion inhibition effect of the cancer metastasis inhibition fusion protein purified in Example 12 was examined. Evaluation method is Albin
i et al. (Albini et al .: Cancer Res. 47, 3239-324).
5 (1987)). 10 μg of Matrigel (manufactured by Collaborative Co., Ltd.) was applied to the upper surface of the chemotaxel (manufactured by Kurabo Industries, Ltd.), which was divided into an upper layer and a lower layer by a polycarbonate filter having a pore size of 8 μm, and dried overnight at room temperature. Immediately before use, it was swollen with a culture solution and set on a 24-well culture plate. Highly metastatic clone B16FE7 derived from B16 melanoma was used as the cancer cell.

The cells were treated with 1.85 kBq / ml [ ¹²⁵ I].
The cells were cultured in the presence of IUdR (manufactured by Amersham Co., Ltd.) for 2 days. Just before use, after collecting cells with trypsin solution,
The cells were suspended in a culture medium containing 0.1% bovine serum albumin, and the radioactivity of the incorporated [ ¹²⁵ I] IUdR was measured. 20 μg / ml of human fibronectin was placed in the lower layer of chemotaxel, and 5 × 10 ⁴ cells were placed in the upper layer together with various concentrations of the cancer metastasis-inhibiting fusion protein, and cultured in a carbon dioxide incubator for 20 hours.

After the completion of the culture, the cells remaining on the upper surface of the filter were scraped off with a cotton swab, and the filter was lysed with the cells that had moved to the lower surface using a tissue solubilizer (manufactured by Amersham Co.), and the radioactivity was measured. . Fig. 4 shows the results.
Shown in As is clear from the figure, it was shown that the invasion of cancer cells was significantly inhibited by the present cancer metastasis inhibiting fusion protein.

[0076]

As described above in detail, by using the modified human serum albumin gene according to the present invention, any form of bioactive peptide can be genetically engineered at a specific position of human serum albumin. It has become possible to easily incorporate into. Therefore, it can be said that a novel physiologically active fusion protein can be easily prepared using the present invention.

[0077]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 1763 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Sequence type: cDNA to mRNA Sequence features Characteristic symbols: CDS Location: 3. ． 1763 Method of characterizing: E sequence CC ATG GAT GCA CAC AAG AGT GAG GTT GCT CAT CGG TTT AAA GAT TTG GGA 50 GAA GAA AAT TTC AAA GCC TTG GTG TTG ATT GCC TTT GCT CAG TAT CTT 98 CAG CAG TGT CCA TTT GAA GAT CAT GTA AAA TTA GTG AAT GAA GTA ACT 146 GAA TTT GCA AAA ACA TGT GTA GCT GAT GAG TCA GCT GAA AAT TGT GAC 194 AAA TCA CTT CAT ACC CTT TTT GGA GAC AAA TTA TGC ACA GTT GCA ACT 242 CTT CGT GAA ACC TAT GGT GAA ATG GCT GAC TGC TGT GCA AAA CAA GAA 290 CCT GAG AGA AAT GAA TGC TTC TTG CAA CAC AAA GAT GAC AAC CCA AAC 338 CTC CCC CGA TTG GTG AGA CCA GAG GTT GAT GTG ATG TGC ACT GCT TTT 386 CAT GAC AAT GA GAG ACA TTT TTG AAA AAA TAC TTA TAT GAA ATT GCC 434 AGA AGA CAT CCT TAC TTT TAT GCC CCG GAA CTC CTT TTC TTT GCT AAA 482 AGG TAT AAA GCT GCT TTT ACA GAA TGT TGC CAA GCT GCT GAT AAA GCT 530 GCC TGC CTG TTG CCA AAG CTC GAT GAA CTT CGG GAT GAA GGG AAG GCT 578 TCG TCT GCC AAA CAG AGA CTC AAA TGT GCC AGT CTC CAA AAA TTT GGA 626 GAA AGA GCT TTC AAA GCA TGG GCA GTG GCT CGC CTG AGC CAG AGA T TT 674 CCC AAA GCT GAG TTT GCA GAA GTT TCC AAG TTA GTG ACA GAT CTT ACC 722 AAA GTC CAC ACG GAA TGC TGC CAT GGA GAT CTG CTT GAA TGT GCT GAT 770 GAC AGG GCG GAC CTT GCC AAG TAT ATC TGT GAA AAT CAG GAT TCG ATC 818 TCC AGT AAA CTG AAG GAA TGC TGT GAA AAA CCT CTG TTG GAA AAA TCC 866 CAC TGC ATT GCC GAA GTG GAA AAT GAT GAG ATG CCT GCT GAC TTG CCT 914 TCA TTA GCT GCT GAT TTT GTT GAA AGT AAG GAT GTT TGC AAA AAC TAT 962 GCT GAG GCA AAG GAT GTC TTC CTG GGC ATG TTT TTG TAT GAA TAT GCA 1010 AGA AGG CAT CCT GAT TAC TCT GTC GTG CTG CTG CTG AGA CTT GCC AAG 1058 ACA TAT GAA ACC ACT CTA GAG AAG TGC TGT GCC GCT GCA GAT CCT CAT 1106 GAA TGC TAT GCC AAA GTG TTC GAT GAA TTT AAA CCT CTT GTG GAA GAG 1154 CCT CAG AAT TTA ATC AAA CAA AAC TGT GAG CTT TTT AAG CAG CTT GGA 1202 GAG TAC AAA TTC CAG AAT GCG CTA TTA GTT CGT TAC ACC AAG AAA GTA 1250 CCC CAA GTG TCA ACT CCA ACT CTT GTA GAG GTC TCA AGA AAC CTA GGA 1298 AAA GTG GGC AGC AAA TGT TGT AAA CAT CCT GAA GCA AAA AGA ATG CCC 1346 TGT GCA GAA GAC TAT CTA TCC GTG G TC CTG AAC CAG TTA TGT GTG TTG 1394 CAT GAG AAA ACG CCA GTA AGT GAC AGA GTC ACA AAA TGC TGC ACA GAG 1442 TCC TTG GTG AAC AGG CGA CCA TGC TTT TCA GCT CTG GAA GTC GAT GAA 1490 ACA TAC GTT CCC AAA GAG TTT AAT GCT GAA ACA TTC ACC TTC CAT GCA 1538 GAT ATA TGC ACA CTT TCT GAG AAG GAG AGA CAA ATC AAG AAA CAA ACT 1586 GCA CTT GTT GAG CTC GTG AAA CAC AAG CCC AAG GCA ACA AAA GAG CAA 1634 CTG AAA GCT GTT ATG GAT GAT TTC GCA GCT TTT GTA GAG AAG TGC TGC 1682 AAG GCT GAC GAT AAG GAG ACC TGC TTT GCC GAG GAG GGT AAA AAA CTT 1730 GTT GCT GCA AGT CAA GCT GCC TTA GGC TTA TAA 1763 SEQ ID NO: 2 Sequence length: 1761 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Sequence features Characteristic symbols: CDS Location: 1. ． 1761 Method of characterizing: E sequence ATG GAT GCA CAC AAG AGT GAG GTT GCT CAT CGG TTT AAA GAT TTG GGA 48 GAA GAA AAT TTC AAA GCC TTG GTG TTG ATT GCC TTT GCT CAG TAT CTT 96 CAG CAG TGT CCA TTT GAA GAT CAT GTA AAA TTA GTG AAT GAA GTA ACT 144 GAA TTT GCA AAA ACA TGT GTA GCT GAT GAG TCA GCT GAA AAT TGT GAC 192 AAA TCA CTT CAT ACC CTT TTT GGA GAC AAA TTA TGC ACA GTT GCA ACT 240 CTT CGT GAA ACC TAT GGT GAA ATG GCT GAC TGC TGT GCA AAA CAA GAA 288 CCT GAG AGA AAT GAA TGC TTC TTG CAA CAC AAA GAT GAC AAC CCA AAC 336 CTC CCC CGA TTG GTG AGA CCA GAG GTT GAT GTG ATG TGC ACT GCT TTT 384 CAT GAC AAT GAA GAG ACA TTT TTG AAA AAA TAC TTA TAT GAA ATT GCC 432 AGA AGA CAT CCT TAC TTT TAT GCC CCG GAA CTC CTT TTC TTT GCT AAA 480 AGG TAT AAA GCT GCT TTT ACA GAA TGT TGC CAA GCT GCT GAT AAA GCT 528 GCC TGC CTG TTG CCA AAG CTT GAT GAA CTT CGG GAT GAA GGG AAG GCT 576 TCG TCT GCC AAA CAG AGA CTC AAA TGT GCC AGT CTC CAA AAA TTT GGA 624 GAA AGA GCT TTC AAA GCA TGG GCA GTG GCT CGC CTG AGC CAG AGA TTT 672 CCC AAA GCT GAG TTT GCA GAA GTT TCC AAG TTA GTG ACA GAT CTT ACC 720 AAA GTC CAC ACG GAA TGC TGC CAT GGA GAT CTG CTT GAA TGT GCT GAT 768 GAC AGG GCG GAC CTT GCC AAG TAT ATC TGT GAA AAT CAG GAT TAT ATC 816 TCC AGT AAA CTG AAG GAA TGC TGT GAA AAA CCT CTG TTG GAA AAA TCC 864 CAC TGC ATT GCC GAA GTG GAA AAT GAT GAG ATG CCT GCT GAC TTG CCT 912 TCA TTA GCT GCT GAT TTT GTT GAA AGT AAG GAT GTT TGC AAA AAC TAT 960 GCT GAG GCA AAG GAT GTC TTC CTG GGC ATG TTT TTG TAT GAA TAT GCA 1008 AGA AGG CAT CCT GAT TAC TCT GTC GTG CTG CTG CTG AGA CTT GCC AAG 1056 ACA TAT GAA ACC ACT CTA GAG AAG TGC GGT GCC GCT GAT CCT CAT 1104 GAA TGC TAT GCC AAA GTG TTC GAT GAA TTT AAA CCT CTT GTG GAA GAG 1152 CCT CAG AAT TTA ATC AAA CAA AAC TGT GAG CTT TTT AAG CAG CTT GGA 1200 GAG TAC AAA TTC CAG AAT GCG CTA TTA GTT CGT TAC ACC AAG AAA GTA 1248 CCC CAA GTG TCA ACT CCA ACT CTT GTA GAG GTC TCA AGA AAC CTA GGA 1296 AAA GTG GGC AGC AAA TGT TGT AAA CAT CCT GAA GCA AAA AGA ATG CCC 1344 TGT GCA GAA GAC TAT CTA TCC GTG GTC CTG AAC CAG TTA TGT GTG TTG 1392 CAT GAG AAA ACG CCA GTA AGT GAC AGA GTC ACA AAA TGC TGC ACA GAG 1440 TCC TTG GTG AAC AGG CGA CCA TGC TTT TCA GCT CTG GAA GTC GAT GAA 1488 ACA TAC GTT CCC AAA GAG TTT AAT GCT GAA ACA TTC ACC TTC CAT GCA 1536 GAT ATA TGC ACA CTT TCT GAG AAG GAG AGA CAA ATC AAG AAA CAA ACT 1584 GCA CTT GTT GAG CTC GTG AAA CAC AAG CCC AAG GCA ACA AAA GAG CAA 1632 CTG AAA GCT GTT ATG GAT GAT TTC GCA GCT TTT GTA GAG AAG TGC TGC 1680 AAG GCT GAC GAT AAG GAG ACC TGC TTT GCC GAG GAG GGT AAA AAA CTT 1728 GTT GCT GCA AGT CAA GCT GCC TTA GGC TTA TAA 1761 SEQ ID NO: 3 Sequence length: 1761 Sequence Type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Sequence features Characteristic symbols: CDS Location: 1. ． 1761 Method of characterizing: E sequence ATG GAT GCA CAC AAG AGT GAG GTT GCT CAT CGG TTT AAA GAT TTG GGA 48 GAA GAA AAT TTC AAA GCC TTG GTG TTG ATT GCC TTT GCT CAG TAT CTT 96 CAG CAG TGT CCA TTT GAA GAT CAT GTA AAA TTA GTG AAT GAA GTA ACT 144 GAA TTT GCA AAA ACA TGT GTA GCT GAT GAG TCA GCT GAA AAT TGT GAC 192 AAA TCA CTT CAT ACC CTT TTT GGA GAC AAA TTA TGC ACA GTT GCA ACT 240 CTT CGT GAA ACC TAT GGT GAA ATG GCT GAC TGC TGT GCA AAA CAA GAA 288 CCT GAG AGA AAT GAA TGC TTC TTG CAA CAC AAA GAT GAC AAC CCA AAC 336 CTC CCC CGA TTG GTG AGA CCA GAG GTT GAT GTG ATG TGC ACT GCT TTT 384 CAT GAC AAT GAA GAG ACA TTT TTG AAA AAA TAC TTA TAT GAA ATT GCC 432 AGA AGA CAT CCT TAC TTT TAT GCC CCG GAA CTC CTT TTC TTT GCT AAA 480 AGG TAT AAA GCT GCT TTT ACA GAA TGT TGC CAA GCT GCT GAT AAA GCT 528 GCC TGC CTG TTG CCA AAG CTC GAT GAA CTT CGG GAT GAA GGG AAG GCT 576 TCG TCT GCC AAA CAG AGA CTC AAA TGT GCC AGT CTC CAA AAA TTT GGA 624 GAA AGA GCT TTC AAA GCA TGG GCA GTG GCT CGC CTG AGC CAG AGA TTT 672 CCC AAA GCT GAG TTT GCA GAA GTT TCC AAG TTA GTG ACA GAT CTT ACC 720 AAA GTC CAC ACG GAA TGC TGC CAT GGA GAT CTG CTT GAA TGT GCT GAT 768 GAC AGG GCG GAC CTT GCC AAG TAT ATC TGT GAA AAT CAG GAT TAT ATC 816 TCC AGT AAA CTG AAG GAA TGC TGT GAA AAA CCT CTG TTG GAA AAA TCC 864 CAC TGC ATT GCC GAA GTG GAA AAT GAT GAG ATG CCT GCT GAC TTG CCT 912 TCA TTA GCT GCT GAT TTT GTT GAA AGT AAG GAT GTT TGC AAA AAC TAT 960 GCT GAG GCA AAG GAT GTC TTC CTG GGC ATG TTT TTG TAT GAA TAT GCA 1008 AGA AGG CAT CCT GAT TAC TCT GTC GTG CTG CTG CTG AGA CTT GCC AAG 1056 ACA TAT GAA ACC ACT CTA GAG AAG TGC GGT GCC GCT GAT CCT CAT 1104 GAA TGC TAT GCC AAA GTG TTC GAT GAA TTC AAA CCT CTT GTG GAA GAG 1152 CCT CAG AAT TTA ATC AAA CAA AAC TGT GAG CTT TTT AAG CAG CTT GGA 1200 GAG TAC AAA TTC CAG AAT GCG CTA TTA GTT CGT TAC ACC AAG AAA GTA 1248 CCC CAA GTG TCA ACT CCA ACT CTT GTA GAG GTC TCA AGA AAC CTA GGA 1296 AAA GTG GGC AGC AAA TGT TGT AAA CAT CCT GAA GCA AAA AGA ATG CCC 1344 TGT GCA GAA GAC TAT CTA TCC GTG GTC CTG AAC CAG TTA TGT GTG TTG 1392 CAT GAG AAA ACG CCA GTA AGT GAC AGA GTC ACA AAA TGC TGC ACA GAG 1440 TCC TTG GTG AAC AGG CGA CCA TGC TTT TCA GCT CTG GAA GTC GAT GAA 1488 ACA TAC GTT CCC AAA GAG TTT AAT GCT GAA ACA TTC ACC TTC CAT GCA 1536 GAT ATA TGC ACA CTT TCT GAG AAG GAG AGA CAA ATC AAG AAA CAA ACT 1584 GCA CTT GTT GAG CTC GTG AAA CAC AAG CCC AAG GCA ACA AAA GAG CAA 1632 CTG AAA GCT GTT ATG GAT GAT TTC GCA GCT TTT GTA GAG AAG TGC TGC 1680 AAG GCT GAC GAT AAG GAG ACC TGC TTT GCC GAG GAG GGT AAA AAA CTT 1728 GTT GCT GCA AGT CAA GCT GCC TTA GGC TTA TAA 1761 SEQ ID NO: 4 Sequence length: 1765 Sequence Type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Sequence features Characteristic symbols: CDS Location: 1. ． 1758 Method of characterizing: E sequence ATG GAT GCA CAC AAG AGT GAG GTT GCT CAT CGG TTT AAA GAT TTG GGA 48 GAA GAA AAT TTC AAA GCC TTG GTG TTG ATT GCC TTT GCT CAG TAT CTT 96 CAG CAG TGT CCA TTT GAA GAT CAT GTA AAA TTA GTG AAT GAA GTA ACT 144 GAA TTT GCA AAA ACA TGT GTA GCT GAT GAG TCA GCT GAA AAT TGT GAC 192 AAA TCA CTT CAT ACC CTT TTT GGA GAC AAA TTA TGC ACA GTT GCA ACT 240 CTT CGT GAA ACC TAT GGT GAA ATG GCT GAC TGC TGT GCA AAA CAA GAA 288 CCT GAG AGA AAT GAA TGC TTC TTG CAA CAC AAA GAT GAC AAC CCA AAC 336 CTC CCC CGA TTG GTG AGA CCA GAG GTT GAT GTG ATG TGC ACT GCT TTT 384 CAT GAC AAT GAA GAG ACA TTT TTG AAA AAA TAC TTA TAT GAA ATT GCC 432 AGA AGA CAT CCT TAC TTT TAT GCC CCG GAA CTC CTT TTC TTT GCT AAA 480 AGG TAT AAA GCT GCT TTT ACA GAA TGT TGC CAA GCT GCT GAT AAA GCT 528 GCC TGC CTG TTG CCA AAG CTC GAT GAA CTT CGG GAT GAA GGG AAG GCT 576 TCG TCT GCC AAA CAG AGA CTC AAA TGT GCC AGT CTC CAA AAA TTT GGA 624 GAA AGA GCT TTC AAA GCA TGG GCA GTG GCT CGC CTG AGC CAG AGA TTT 672 CCC AAA GCT GAG TTT GCA GAA GTT TCC AAG TTA GTG ACA GAT CTT ACC 720 AAA GTC CAC ACG GAA TGC TGC CAT GGA GAT CTG CTT GAA TGT GCT GAT 768 GAC AGG GCG GAC CTT GCC AAG TAT ATC TGT GAA AAT CAG GAT TAT ATC 816 TCC AGT AAA CTG AAG GAA TGC TGT GAA AAA CCT CTG TTG GAA AAA TCC 864 CAC TGC ATT GCC GAA GTG GAA AAT GAT GAG ATG CCT GCT GAC TTG CCT 912 TCA TTA GCT GCT GAT TTT GTT GAA AGT AAG GAT GTT TGC AAA AAC TAT 960 GCT GAG GCA AAG GAT GTC TTC CTG GGC ATG TTT TTG TAT GAA TAT GCA 1008 AGA AGG CAT CCT GAT TAC TCT GTC GTG CTG CTG CTG AGA CTT GCC AAG 1056 ACA TAT GAA ACC ACT CTA GAG AAG TGC GGT GCC GCT GAT CCT CAT 1104 GAA TGC TAT GCC AAA GTG TTC GAT GAA TTT AAA CCT CTT GTG GAA GAG 1152 CCT CAG AAT TTA ATC AAA CAA AAC TGT GAG CTT TTT AAG CAG CTT GGA 1200 GAG TAC AAA TTC CAG AAT GCG CTA TTA GTT CGT TAC ACC AAG AAA GTA 1248 CCC CAA GTG TCA ACT CCA ACT CTT GTA GAG GTC TCA AGA AAC CTA GGA 1296 AAA GTG GGC AGC AAA TGT TGT AAA CAT CCT GAA GCA AAA AGA ATG CCC 1344 TGT GCA GAA GAC TAT CTA TCC GTG GTC CTG AAC CAG TTA TGT GTG TTG 1392 CAT GAG AAA ACG CCA GTA AGT GAC AGA GTC ACA AAA TGC TGC ACA GAG 1440 TCC TTG GTG AAC AGG CGA CCA TGC TTT TCA GCT CTG GAA GTC GAT GAA 1488 ACA TAC GTT CCC AAA GAG TTT AAT GCT GAA ACA TTC ACC TTC CAT GCA 1536 GAT ATA TGC ACA CTT TCT GAG AAG GAG AGA CAA ATC AAG AAA CAA ACT 1584 GCA CTT GTT GAG CTC GTG AAA CAC AAG CCC AAG GCA ACA AAA GAG CAA 1632 CTG AAA GCT GTT ATG GAT GAT TTC GCA GCT TTT GTA GAG AAG TGC TGC 1680 AAG GCT GAC GAT AAG GAG ACC TGC TTT GCC GAG GAG GGT AAA AAA CTT 1728 GTT GCT GCA AGT CAA GCT GCC TTA GGC TTA TAC ATG T 1765 SEQ ID NO: 5 Sequence length: 1767 Sequence type: Nucleic acid Number of strands: Double strands Topology: Linear Sequence type: cDNA to mRNA Sequence characteristics Characteristic symbols: CDS Location: 3. ． 1760 Method of characterizing: E sequence CC ATG GAT GCA CAC AAG AGT GAG GTT GCT CAT CGG TTT AAA GAT TTG GGA 50 GAA GAA AAT TTC AAA GCC TTG GTG TTG ATT GCC TTT GCT CAG TAT CTT 98 CAG CAG TGT CCA TTT GAA GAT CAT GTA AAA TTA GTG AAT GAA GTA ACT 146 GAA TTT GCA AAA ACA TGT GTA GCT GAT GAG TCA GCT GAA AAT TGT GAC 194 AAA TCA CTT CAT ACC CTT TTT GGA GAC AAA TTA TGC ACA GTT GCA ACT 242 CTT CGT GAA ACC TAT GGT GAA ATG GCT GAC TGC TGT GCA AAA CAA GAA 290 CCT GAG AGA AAT GAA TGC TTC TTG CAA CAC AAA GAT GAC AAC CCA AAC 338 CTC CCC CGA TTG GTG AGA CCA GAG GTT GAT GTG ATG TGC ACT GCT TTT 386 CAT GAC AAT GA GAG ACA TTT TTG AAA AAA TAC TTA TAT GAA ATT GCC 434 AGA AGA CAT CCT TAC TTT TAT GCC CCG GAA CTC CTT TTC TTT GCT AAA 482 AGG TAT AAA GCT GCT TTT ACA GAA TGT TGC CAA GCT GCT GAT AAA GCT 530 GCC TGC CTG TTG CCA AAG CTT GAT GAA CTT CGG GAT GAA GGG AAG GCT 578 AGC TCT GCC AAA CAG AGA CTC AAA TGT GCC AGT CTC CAA AAA TTT GGA 626 GAA AGA GCT TTC AAA GCA TGG GCA GTG GCT CGC CTG AGC CAG AGA T TT 674 CCC AAA GCT GAG TTT GCA GAA GTT TCC AAG TTA GTG ACA GAT CTT ACC 722 AAA GTC CAC ACG GAA TGC TGC CAT GGA GAT CTG CTT GAA TGT GCT GAT 770 GAC AGG GCG GAC CTT GCC AAG TAT ATC TGT GAA AAT CAG GAT TCG ATC 818 TCC AGT AAA CTG AAG GAA TGC TGT GAA AAA CCT CTG TTG GAA AAA TCC 866 CAC TGC ATT GCC GAA GTG GAA AAT GAT GAG ATG CCT GCT GAC TTG CCT 914 TCA TTA GCT GCT GAT TTT GTT GAA AGT AAG GAT GTT TGC AAA AAC TAT 962 GCT GAG GCA AAG GAT GTC TTC CTG GGC ATG TTT TTG TAT GAA TAT GCA 1010 AGA AGG CAT CCT GAT TAC TCT GTC GTG CTG CTG CTG AGA CTT GCC AAG 1058 ACA TAT GAA ACC ACT CTA GAG AAG TGC TGT GCC GCT GCA GAT CCT CAT 1106 GAA TGC TAT GCC AAA GTG TTC GAT GAA TTC AAA CCT CTT GTG GAA GAG 1154 CCT CAG AAT TTA ATC AAA CAA AAC TGT GAG CTT TTT AAG CAG CTT GGA 1202 GAG TAC AAA TTC CAG AAT GCG CTA TTA GTT CGT TAC ACC AAG AAA GTA 1250 CCC CAA GTG TCA ACT CCA ACT CTT GTA GAG GTC TCA AGA AAC CTA GGA 1298 AAA GTG GGC AGC AAA TGT TGT AAA CAT CCT GAA GCA AAA AGA ATG CCC 1346 TGT GCA GAA GAC TAT CTA TCC GTG G TC CTG AAC CAG TTA TGT GTG TTG 1394 CAT GAG AAA ACG CCA GTA AGT GAC AGA GTC ACA AAA TGC TGC ACA GAG 1442 TCC TTG GTG AAC AGG CGA CCA TGC TTT TCA GCT CTG GAA GTC GAT GAA 1490 ACA TAC GTT CCC AAA GAG TTT AAT GCT GAA ACA TTC ACC TTC CAT GCA 1538 GAT ATA TGC ACA CTT TCT GAG AAG GAG AGA CAA ATC AAG AAA CAA ACT 1586 GCA CTT GTT GAG CTC GTG AAA CAC AAG CCC AAG GCA ACA AAA GAG CAA 1634 CTG AAA GCT GTT ATG GAT GAT TTC GCA GCT TTT GTA GAG AAG TGC TGC 1682 AAG GCT GAC GAT AAG GAG ACC TGC TTT GCC GAG GAG GGT AAA AAA CTT 1730 GTT GCT GCA AGT CAA GCT GCC TTA GGC TTA TAC ATG T 1767 SEQ ID NO: 6 Sequence length : 21 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence Ala Glu Asp Gly Asp Ala Lys Thr Asp Gln Ala Glu Lys Ala Glu Gly 1 5 10 15 Ala Gly Asp Ala Lys 20 21 SEQ ID NO: 7 Sequence Length: 71 Sequence type: Nucleic acid Number of strands: Double stranded Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence CC ATG GCC GAG GAC GGT GAC GCC AAG ACC GAC CAA GCT GAG AAG GCT GAG 50 GGT GCC GGT GAC GCC AAG TAA 71 SEQ ID NO: 8 Sequence length: 609 Sequence type: Amino acid Topology: Linear Sequence type: Protein sequence Met Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly 1 5 10 15 Glu Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu 20 25 30 Gln Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr 35 40 45 Glu Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp 50 55 60 Lys Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr 65 70 75 80 Leu Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu 85 90 95 Pro Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn 100 105 110 Leu Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe 115 120 125 His Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala 130 135 140 Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys 145 150 155 160 Arg Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala 165 170 175 Ala Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala 180 185 190 Ser Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly 195 200 205 Glu Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe 210 215 220 Pro Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr 225 230 235 240 Lys Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp 245 250 255 Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile 260 265 270 Ser Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser 275 280 285 His Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro 290 295 300 Ser Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr 305 310 315 320 Ala Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala 325 330 335 Arg Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys 340 345 350 Thr Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His 355 360 365 Glu Cys Tyr Ala Lys Val Ph e Asp Glu Phe Lys Pro Leu Val Glu Glu 370 375 380 Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Lys Gln Leu Gly 385 390 395 400 Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val 405 410 415 Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly 420 425 430 Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro 435 440 445 Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu 450 455 460 His Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu 465 470 475 480 Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu 485 490 495 Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala 500 505 510 Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr 515 520 525 Ala Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln 530 535 540 Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys 545 550 555 560 Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu 565 570 575 Val Ala Ala Ser Gln Ala Al a Leu Gly Leu Tyr Met Ala Glu Asp Gly 580 585 590 Asp Ala Lys Thr Asp Gln Ala Glu Lys Ala Glu Gly Ala Gly Asp Ala 595 600 605 Lys 609 SEQ ID NO: 9 Sequence length: 1832 Sequence type: Nucleic acid chain Number: Double-stranded Topology: Linear Sequence type: cDNA to mRNA Sequence features Symbols representing features: CDS Location: 3. ． 1832 Method of characterizing: E sequence CC ATG GAT GCA CAC AAG AGT GAG GTT GCT CAT CGG TTT AAA GAT TTG GGA 50 GAA GAA AAT TTC AAA GCC TTG GTG TTG ATT GCC TTT GCT CAG TAT CTT 98 CAG CAG TGT CCA TTT GAA GAT CAT GTA AAA TTA GTG AAT GAA GTA ACT 146 GAA TTT GCA AAA ACA TGT GTA GCT GAT GAG TCA GCT GAA AAT TGT GAC 194 AAA TCA CTT CAT ACC CTT TTT GGA GAC AAA TTA TGC ACA GTT GCA ACT 242 CTT CGT GAA ACC TAT GGT GAA ATG GCT GAC TGC TGT GCA AAA CAA GAA 290 CCT GAG AGA AAT GAA TGC TTC TTG CAA CAC AAA GAT GAC AAC CCA AAC 338 CTC CCC CGA TTG GTG AGA CCA GAG GTT GAT GTG ATG TGC ACT GCT TTT 386 CAT GAC AAT GA GAG ACA TTT TTG AAA AAA TAC TTA TAT GAA ATT GCC 434 AGA AGA CAT CCT TAC TTT TAT GCC CCG GAA CTC CTT TTC TTT GCT AAA 482 AGG TAT AAA GCT GCT TTT ACA GAA TGT TGC CAA GCT GCT GAT AAA GCT 530 GCC TGC CTG TTG CCA AAG CTC GAT GAA CTT CGG GAT GAA GGG AAG GCT 578 TCG TCT GCC AAA CAG AGA CTC AAA TGT GCC AGT CTC CAA AAA TTT GGA 626 GAA AGA GCT TTC AAA GCA TGG GCA GTG GCT CGC CTG AGC CAG AGA T TT 674 CCC AAA GCT GAG TTT GCA GAA GTT TCC AAG TTA GTG ACA GAT CTT ACC 722 AAA GTC CAC ACG GAA TGC TGC CAT GGA GAT CTG CTT GAA TGT GCT GAT 770 GAC AGG GCG GAC CTT GCC AAG TAT ATC TGT GAA AAT CAG GAT TCG ATC 818 TCC AGT AAA CTG AAG GAA TGC TGT GAA AAA CCT CTG TTG GAA AAA TCC 866 CAC TGC ATT GCC GAA GTG GAA AAT GAT GAG ATG CCT GCT GAC TTG CCT 914 TCA TTA GCT GCT GAT TTT GTT GAA AGT AAG GAT GTT TGC AAA AAC TAT 962 GCT GAG GCA AAG GAT GTC TTC CTG GGC ATG TTT TTG TAT GAA TAT GCA 1010 AGA AGG CAT CCT GAT TAC TCT GTC GTG CTG CTG CTG AGA CTT GCC AAG 1058 ACA TAT GAA ACC ACT CTA GAG AAG TGC TGT GCC GCT GCA GAT CCT CAT 1106 GAA TGC TAT GCC AAA GTG TTC GAT GAA TTT AAA CCT CTT GTG GAA GAG 1154 CCT CAG AAT TTA ATC AAA CAA AAC TGT GAG CTT TTT AAG CAG CTT GGA 1202 GAG TAC AAA TTC CAG AAT GCG CTA TTA GTT CGT TAC ACC AAG AAA GTA 1250 CCC CAA GTG TCA ACT CCA ACT CTT GTA GAG GTC TCA AGA AAC CTA GGA 1298 AAA GTG GGC AGC AAA TGT TGT AAA CAT CCT GAA GCA AAA AGA ATG CCC 1346 TGT GCA GAA GAC TAT CTA TCC GTG G TC CTG AAC CAG TTA TGT GTG TTG 1394 CAT GAG AAA ACG CCA GTA AGT GAC AGA GTC ACA AAA TGC TGC ACA GAG 1442 TCC TTG GTG AAC AGG CGA CCA TGC TTT TCA GCT CTG GAA GTC GAT GAA 1490 ACA TAC GTT CCC AAA GAG TTT AAT GCT GAA ACA TTC ACC TTC CAT GCA 1538 GAT ATA TGC ACA CTT TCT GAG AAG GAG AGA CAA ATC AAG AAA CAA ACT 1586 GCA CTT GTT GAG CTT GTG AAA CAC AAG CCC AAG GCA ACA AAA GAG CAA 1634 CTG AAA GCT GTT ATG GAT GAT TTC GCA GCT TTT GTA GAG AAG TGC TGC 1682 AAG GCT GAC GAT AAG GAG ACC TGC TTT GCC GAG GAG GGT AAA AAA CTT 1730 GTT GCT GCA AGT CAA GCT GCC TTA GGC TTA TAC ATG GCC GAG GAC GGT 1778 GAC GCC AAG ACC GAG CAA GCT GAG AAG GCT GAG GGT GCC GGT GAC GCC 1826 AAG TAA 1832 SEQ ID NO: 10 Sequence length: 73 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid synthesis DNA sequence GATCC ATG GCC GAG GAC GGT GAC GCC AAG ACC GAC CAA GCT GAG AAG GCT 50 GAG GGT GCC GGT GAC GCC AAG TA 73 SEQ ID NO: 11 Sequence length: 73 Sequence Type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: Yes Sequence AGCTTA CTT GGC GTC ACC GGC ACC CTC AGC CTT CTC AGC TTG GTC GGT CTT 51 GGC GTC ACC GTC CTC GGC CAT G 73 SEQ ID NO: 12 Sequence length: 28 Sequence type: Nucleic acid Number of strands: Single-stranded Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence AGACCATGGA TGCACACAAG AGTGAGGT 28 SEQ ID NO: 13 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single-stranded Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence AATAAGCTTT TGATCTTCAT 20 SEQ ID NO: 14 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence AGCAAGCTTT GGCAACAGGC 20 SEQ ID NO: 15 Sequence length: 29 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear array type: other Acid Synthetic DNA Sequence AGCAAGCTTG ATGAACTTCG GGATGAAGG 29 SEQ ID NO: 16 Sequence Length: 24 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence AGCGAATTCA TCGAACACTT TGGC 24 Sequence No .: 17 Sequence length: 29 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence AGCGAATTCA AACCTCTTGT GGAAGAGCC 29 SEQ ID NO: 18 Sequence length: 40 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence AAGAAGCTTG AATTCACATG TATAAGCCTA AGGCAGCTTG 40

[Brief description of drawings]

FIG. 1 is a constitutional view of an expression vector pTL2BmI.

FIG. 2 is an SDS-PAGE observation view.

FIG. 3 is a Western blot observation view.

FIG. 4 is a graph showing the measurement results of cancer cell invasion inhibitory activity.

FIG. 5 is a structural diagram of expression vector pRL2L.

FIG. 6 is a structural diagram of the expression vector pRL2M.

FIG. 7 is a structural diagram of the expression vector pTL2M.

FIG. 8 is a structural diagram of expression vector pTL2Bm.

Continuation of front page (51) Int.Cl. ⁶ Identification number Reference number within the agency FI Technical indication (C12N 1/19 C12R 1: 645) (C12P 21/02 C12R 1: 645) (72) Inventor Yoko Tsukamoto Kanagawa Asahi Glass Co., Ltd. Central Research Institute, 1150, Hazawa-machi, Kanagawa-ku, Yokohama, Japan (72) Inventor Atsushi Isogo, Asahi Glass Co., Ltd. Central Research Laboratory, 1150, Hazawa-machi, Kanagawa-ku, Yokohama, Kanagawa (72) Inventor Kumagaya Hiromichi Kanagawa, Kanagawa Asahi Glass Co., Ltd. Central Research Laboratory, 1150, Hazawa-machi, Tokyo

Claims (13)

[Claims] 1. A modified gene encoding human serum albumin, which is obtained by introducing a restriction enzyme cleavage site into at least one or more desired positions of a gene encoding natural human serum albumin. 2. The restriction enzyme cleavage site is introduced at any one of the amino terminus, the carboxyl terminus, between the first and second domains, or between the second and third domains of the polypeptide chain of human serum albumin, or these. The gene according to claim 1, which is located in any combination of 3. The gene according to claim 2, which is represented by the nucleotide sequence of SEQ ID NO: 1 in which the introduction position of the restriction enzyme cleavage site is the amino terminus of the polypeptide chain of human serum albumin. 4. The gene according to claim 2, which is represented by the nucleotide sequence of SEQ ID NO: 2 in which the introduction position of the restriction enzyme cleavage site is between the first and second domains of the polypeptide chain of human serum albumin. 5. The gene according to claim 2, which is represented by the nucleotide sequence of SEQ ID NO: 3 in which the introduction position of the restriction enzyme cleavage site is between the second and third domains of the polypeptide chain of human serum albumin. 6. The gene according to claim 2, which is represented by the nucleotide sequence of SEQ ID NO: 4 in which the introduction position of the restriction enzyme cleavage site is the carboxyl terminus of the polypeptide chain of human serum albumin. 7. The nucleotide sequence of SEQ ID NO: 5 wherein the restriction enzyme cleavage site is introduced at the amino terminus, between the first and second domains, between the second and third domains, and at the carboxy terminus of the polypeptide chain of human serum albumin. The gene of claim 2 represented. 8. A fusion protein produced by introducing a gene encoding a peptide having physiological activity into the restriction enzyme cleavage site of the gene according to any one of claims 1 to 7. 9. A gene encoding the fusion protein according to claim 8. 10. The fusion protein according to claim 8, wherein the physiologically active peptide is represented by the amino acid of SEQ ID NO: 6. 11. The gene according to claim 9, wherein the gene encoding a peptide having physiological activity is represented by the nucleotide sequence of SEQ ID NO: 7. 12. The fusion protein according to claim 8, which is represented by the amino acid sequence of SEQ ID NO: 8. 13. The gene encoding the fusion protein according to claim 12, which is represented by the nucleotide sequence of SEQ ID NO: 9.