JPH0787976A - New polypeptide, its production, dna coding for the polypeptide, vector composed of the dna, host cell transformed by the vector, antibody of the polypeptide and pharmaceutical composition containing the polypeptide or antibody - Google Patents

Abstract

PURPOSE:To obtain a new polypeptide having a specific amino acid sequence, which is produced by and excreted from a human glioblastoma cell line, useful for e.g. an agent for prophylaxis and/or treatment of dysgenesis or abnormal growth of cell, hypergasia or hyperergasia, tumor, etc. CONSTITUTION:A human glioblastoma cell line T98G is stimulated with human interleukin-1beta and then the entire RNAs are recovered from the stimulated cell line by an ordinary method. The recovered RNAs are fractionated through an oligo-dT- cellulose column to collect mRNAs. cDNAs are synthesized by a reverse transcriptase using the obtained mRNAs as templates and a cDNA l-ibrary is produced using the cDNAs. Subsequently, this library is cloned to select positive clones. Plasmids are recovered from the cells obtained by culturing the positive clones and the plasmids are bound to vectors after the restriction enzyme treatment. The plasmid-bound vectors are introduced to host cells to perform transformation and these transformed cells are cultured. The objective new polypeptide consisting of the amino acid sequence expressed by the formula, its homolog, its fragments, etc., are obtained from the cultured mixture.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel polypeptide produced by a certain glioblastoma cell line, a method for producing the same, DNA encoding the polypeptide, and DNA thereof.
And a host cell transformed with the vector, an antibody of the polypeptide, and a pharmaceutical composition containing the peptide or antibody.

[0002]

BACKGROUND OF THE INVENTION Cells constituting the brain are roughly classified into nerve cells and glial cells. Nerve cells play a major role in information transmission and processing in the brain. On the other hand, glial cells support the function of nerve cells. Specifically, it protects and supports nerve cells, forms a myelin sheath, forms a blood-brain barrier, supplies nutrients to nerve cells, metabolism of neurotransmitters, and further functions of nerve cell proliferation and differentiation during brain development. Control etc. are considered. Glial cells include a wide variety of cells. In the central nervous system there are astrocytes, oligodendrocytes, microglia, etc., and in the peripheral nervous system there are Schwann cells, mantle cells, etc.,
Ependymal cells present in the endothelium of the ventricles are also glial cells.

[0003]

2. Description of the Related Art Recently, many humoral factors involved in the differentiation, proliferation and growth of nerve cells and glial cells have been found, but most of them are glial cells or cancerous glial cells (hereinafter, Called glial cells, etc.). For example, nerve growth factor (NGF, molecular weight of about 13.5 kd (human)), which is known to act only on nerve cells, and brain-derived neurotrophic factor (BDN
F, molecular weight of about 13.5 kd (human), neuroblastoma growth inhibitory factor (NGIF, molecular weight of about 5 kd (human)) and glial-derived nerve growth factor (molecular weight of about 500 kd and about 110 kd (both are available) Rat)), and glial cell-derived glial cell growth inhibitory factor that acts only on glial cells (GdGGIF, molecular weight of about 100 kd or more (rat)) and platelet-derived growth factor (PDGF, molecular weight of about 32 kd (human)), and neurites. Glial-derived neurite outgrowth factor (GdNTF, molecular weight about 43 kd (rat)), which has both extension-promoting action and glial cell proliferation-promoting action, and glia cell growth factor that promotes neurite extension-promoting action and glioblast proliferation and differentiation GMF, molecular weight about 16.5 kd (bovine), ganglion cell survival maintenance, sympathetic ganglion cell growth suppression And ciliary nerve growth factor (CNTF, molecular weight of about 23 kd (rabbit and rat)) having a differentiation promoting action and astrocyte differentiation promoting action, and fibroblast growth having a proliferation promoting activity on nerve cells and astrocytes All the factors (FGF, molecular weight about 16 to 17 kd (human)) are produced from astrocytes. Furthermore, Schwann-derived nerve growth factor (SchNTF, molecular weight 8 kd or more (rat)) that acts on nerve cells is produced by Schwann cells [For details, see Protein Nucleic Acid Enzymes, 36 (No.7), 260 (1991). ]]. In addition, until now, it has been thought that cells of the immune system mainly secrete interleukins (IL-
1, IL-6, etc.) have become known to be secreted also from glial cells. Most of the amino acid sequences of these factors have already been elucidated, and at present, they are being researched not only as research subjects but also as applications to medicines.

[0004]

As described above, from glial cells and the like,
It can be seen that many humoral factors related to differentiation, proliferation and growth of nerve cells and glial cells and humoral factors related to immunity are produced. These facts suggest that other than the above, some factors having similar effects may be produced from glial cells and the like. The present inventors have paid attention to this point and have conducted diligent studies to find out a novel factor (polypeptide) produced by glial cells and the like. Conventionally, in order to obtain a specific polypeptide or a DNA encoding the same, a desired biological activity is confirmed in a tissue or cell culture medium, and then a gene is cloned through isolation and purification of the polypeptide. Method
Alternatively, a method of expressing and cloning a gene using its biological activity as an index has been generally used.

However, in vivo physiologically active polypeptides often have various biological activities, and as a result of cloning a gene using one activity as an index, it is the same as a known polypeptide. Increasing numbers of cases are being discovered later. In addition, all the factors produced by glial cells are very small, which makes isolation, purification and confirmation of biological activity difficult. On the other hand, cDNA production technology and sequencing technology have been rapidly developed, and a large amount of cDNA can be sequenced rapidly. In addition, reverse genetics (Re
The development of various methods of (verse Genetics) is remarkable.

Therefore, the present inventors decided to find a novel polypeptide using this method. That is, mRNA was isolated from glial cells and the like, cDNA was obtained using this as a starting material, its base sequence was determined, and the amino acid sequence was deduced. As a result, they succeeded in finding a completely novel polypeptide and DNA encoding the same, and completed the present invention. Swiss Plot (Swiss Prot Release
The amino acid sequence of the known polypeptide registered in 2.0) was investigated, but none of them had a sequence having the same or high homology with that of the polypeptide of the present invention. In addition, GeneBank (GenBank Release 70.0)
The nucleotide sequence registered in the above was also investigated, but none of them had a sequence having the same or high homology with the cDNA encoding the polypeptide of the present invention. Therefore, it was confirmed that the polypeptide of the present invention is completely novel.

[0007]

The present invention relates to a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 in a substantially pure form,
It relates to its homologues, fragments of its sequences and its homologues. The invention further relates to DNA encoding those polypeptides. More specifically, it relates to a DNA having the base sequence represented by SEQ ID NO: 2 or 3, and a DNA having a fragment selectively hybridizing to the base sequence represented by SEQ ID NO: 2 or 3.

In particular, the present invention relates to (1) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1, (2) a DNA encoding the polypeptide described in (1) above,
(3) DNA having the base sequence shown in SEQ ID NO: 2,
And (4) D having the nucleotide sequence shown in SEQ ID NO: 3
Regarding NA.

A polypeptide having the amino acid sequence shown in SEQ ID NO: 1 in substantially pure form is generally
90% or more of the as-produced polypeptide, eg 95,9
It means that 8 or 99% is a polypeptide having the amino acid sequence shown by SEQ ID NO: 1.

The homologue of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 generally means at least 2
0, preferably at least 30, for example 40, 60
Or a homology of at least 70%, preferably at least 80 or 90%, more preferably 95% or more in a region of 100 contiguous amino acids, and such homologues are hereinafter described as the polypeptide of the present invention. To be done. Furthermore, a fragment of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1, or a homologue thereof, means at least 10 amino acids, preferably at least 15 amino acids, for example 20,
Means 25, 30, 40, 50 or 60 amino acid moieties.

The DNA which selectively hybridizes to the DNA having the base sequence shown in SEQ ID NO: 2 or 3 generally means at least 20, preferably at least 30, for example 40, 60 or 100 consecutive bases. At least 70%, preferably at least 80 or 90%, and more preferably 95% or more homology in the sequence region, and such DNA is hereinafter described as the DNA of the present invention. The fragment of DNA having the base sequence shown in SEQ ID NO: 2 or 3 means at least 10 bases, preferably at least 15 bases, for example, 20, 25, 30 or 40 base portions, and such fragments also include the present invention. Included in DNA.

Further, the present invention includes a replication or expression vector comprising the DNA of the present invention. Examples of the vector include a plasmid, virus or phage vector comprising an ori region and, if necessary, a promoter for expressing the above-mentioned DNA, a promoter regulatory factor and the like. The vector may include one or more selectable marker genes, such as the ampicillin resistance gene. The vector is in vit
ro), for example, production of RNA corresponding to DNA,
It can be used to transform host cells. Further, in the present invention, D having a nucleotide sequence shown by SEQ ID NO: 2 or 3 or an open reading frame thereof.
Also included are host cells transformed with the vector for replicating or expressing the DNA of the present invention containing NA. Cells include, for example, bacteria, yeast, insect cells or mammalian cells.

Further, the present invention also includes a method for producing the polypeptide of the present invention, which comprises culturing the host cell of the present invention under conditions for expressing the polypeptide of the present invention. Culturing is preferably carried out under conditions in which the polypeptide of the present invention is expressed and produced by a host cell. Antisense RNA can also be produced by inserting the DNA of the present invention into the antisense region of the above vector. Such antisense RNA is
It can also be used to control the levels of the polypeptides of the invention in cells.

The present invention also includes monoclonal or polyclonal antibodies of the polypeptides of the present invention. Furthermore, it also includes a method for producing a monoclonal or polyclonal antibody of the polypeptide of the present invention. A monoclonal antibody can be produced by a usual hybridoma technique using the peptide of the present invention or a fragment thereof as an antigen. The polyclonal antibody can be produced by a usual method in which a host animal (for example, rat or rabbit) is inoculated with the polypeptide of the present invention and immune serum is collected. The present invention also includes a pharmaceutical composition containing the polypeptide of the present invention, its antibody, and a pharmaceutically acceptable excipient and / or carrier. As the polypeptide of the present invention, in addition to the polypeptide having the amino acid sequence shown by SEQ ID NO: 1, a part thereof is deleted (for example, a polypeptide consisting of only a part essential for the expression of biological activity in SEQ ID NO: 1). Peptide), a part of which is replaced with another amino acid (for example, a part of which is substituted with an amino acid having similar physical properties),
And those in which other amino acids are added or inserted in part thereof.

As is well known, 1 to 6 types of codons encoding one amino acid (for example, 1 type for Met and 6 types for Leu) are known. Therefore, the base sequence of DNA can be changed without changing the amino acid sequence of the polypeptide. The DNA of the present invention specified in (2) includes all the base sequence groups encoding the polypeptide shown in SEQ ID NO: 1 in (1). The productivity of the polypeptide may be improved by changing the base sequence. The DNA specified in (3) is
It is an embodiment of the DNA shown in (2) and represents a native sequence. The DNA shown in (4) shows the sequence obtained by adding the natural untranslated portion to the DNA specified in (3). The DNA having the nucleotide sequence represented by SEQ ID NO: 3 is prepared according to the following method.

That is, (I) cells producing the polypeptide of the present invention, for example, human glioblastoma cell line
RNA is separated, and (ii) the first strand (single-stranded DNA) and then the second strand (2) are separated from the mRNA.
Double-stranded DNA) (synthesis of cDNA), and (iii) the c
The DNA is incorporated into an appropriate plasmid vector, and (iv) the host cell is transformed with the obtained recombinant vector (cD
(Preparation of NA library), (v) A large amount of the cloned cDNA library was sequenced and an average of 300 bases was sequenced from the 5'side, and (vi) a novel clone with a nucleotide sequence was sequenced. It can be manufactured by

More specifically, step (i) comprises the step of stimulating a human glioblastoma cell line with a suitable stimulant (eg IL-
1), followed by the method of Okayama, H. et al. [Methods
in Enzymology, 154 , 3 (1987)]. The cells producing the present polypeptide preferably include human glioblastoma cell line T98G (ATCC strain number, CRL-1690). The steps (ii), (iii) and (iv) are steps for preparing a cDNA library, and the modified Gubler & Hoffman method [Gene, 25 , 263] is used.
(1983)].

The plasmid vector used in the step (iii) is one that functions in E. coli (for example, pBR).
322) or those that function in Bacillus subtilis (eg, pUB1
Although 10) is known in large numbers, pVfCS-1 (preferably described in the Examples below) preferably made from pGEM-3Zf (+) [3199 bp, sold by Promega] that functions in E. coli. ) Is used. Many host cells are already known for use in the step (iv), and any of them may be used, but preferably they are prepared by the method described in DH5 competent cell [Gene, 96 , 23 (1990)]. To be done. ].

The cloning in step (v) is performed by a known method. The sequencing is performed by the Maxam-Gilbert method or the dideoxy terminator method. Step (vi) is based on Molecular Cl
oning [Sambrook, J., Fritsch, EF and Maniati
s, T., published by Cold Spring Harbor Laboratory Press in 1989].

The DNA thus obtained is (I) D
Converting the NA sequence into an amino acid sequence in a possible frame, (II) preparing a hydrophobicity profile from the resulting amino acid sequence, and just after the start codon (ATG) (secretory protein at its N-terminal It is necessary to confirm the presence of the highly hydrophobic region (having the signal peptide of the highly hydrophobic region), and (III) confirm that the DNA is full length or almost full length. These confirmations may be performed after step (vi), but it is more efficient to perform these confirmations between step (v) and step (vi). During the above examination, confirmation of (III) is performed by Northern analysis.

Once the base sequences shown in SEQ ID NOS: 2 and 3 have been determined, they are then hybridized by chemical synthesis, PCR (Polymerase Chain Reaction) method, or a fragment of the base sequence as a probe. Thus, the DNA of the present invention can be obtained. Furthermore, the desired amount of the desired DNA of the present invention can be obtained by introducing the vector DNA containing the present DNA into a suitable host and allowing it to grow.

The method of obtaining the polypeptide of the present invention includes (1) a method of purifying and isolating from a living body or a cultured cell, (2) a method of synthesizing a peptide, or (3) a method of producing using a gene recombination technique. , And the like, but the method described in (3) is industrially preferable.

Examples of the expression system (host-vector system) for producing a polypeptide using the gene recombination technique include expression systems of bacteria, yeast, insect cells and mammalian cells. For example, in the case of expression in E. coli, D encoding the nucleotide sequence represented by SEQ ID NO: 3
A start codon (ATG) is added to the 5'end of NA, and the obtained DNA is connected downstream of an appropriate promoter (for example, trp promoter, lac promoter, λPL promoter, T7 promoter, etc.) to function in E. coli. Vector (eg, pBR322, pUC1)
8, pUC19 etc.) to prepare an expression vector. Next, E. coli transformed with this expression vector (for example, E. Coli DH1, E. Coli JM109, E. Coli H
The desired polypeptide can be obtained from the bacterial cells by culturing B101 strain or the like) in an appropriate medium. In addition, by using a bacterial signal peptide (for example, pelB signal peptide), the target polypeptide can be secreted into the periplasm. In addition, fusion proteins with other polypeptides (fusion proteins)
protein) can also be produced.

When expressed in mammalian cells, for example, a DNA encoding the nucleotide sequence shown in Sequence Listing 3 is used as a suitable vector (eg, retrovirus vector, papillomavirus vector, vaccinia virus vector, SV40 system). An expression vector is prepared by inserting it into the downstream of a suitable promoter (eg, SV40 promoter, LTR promoter, metallothionein promoter, etc.) in the vector). Then, using the obtained expression vector, suitable mammalian cells (for example, monkey COS-7) can be obtained.
Cells, Chinese hamster CHO cells, mouse L cells, etc.), and the transformant is cultured in an appropriate medium to produce the desired polypeptide in the cells. The polypeptide obtained as described above can be isolated and purified by a general biochemical method.

[0025]

EFFECTS OF THE INVENTION Since the polypeptide of the present invention is produced and secreted from human glioblastoma cell line, it is associated with biological activity and immune function related to differentiation, proliferation, growth or survival maintenance of glia and neurons. It is expected to have the above-mentioned biological activity or a biological activity related to tumor growth and growth. Therefore, the polypeptide of the present invention can be used by itself as a prophylactic or therapeutic agent for glial and neuronal dysgenesis or abnormal proliferation, decreased or enhanced immune function, or tumor. Further, using a polyclonal antibody or a monoclonal antibody of the polypeptide, it is possible to quantify the polypeptide in a living body,
As a result, it can be used for research on the relationship between the polypeptide and a disease or diagnosis of a disease. Polyclonal antibodies and monoclonal antibodies can be prepared by a conventional method using the polypeptide or a fragment thereof as an antigen.

The DNA of the present invention not only serves as an important and essential template for producing the polypeptide of the present invention, which is expected to have great utility, but also can be used for diagnosis and treatment of genetic diseases (treatment of gene deficiency). Alternatively, it can be used for the treatment by stopping the expression of the polypeptide by antisense DNA (RNA). Moreover, genomic DNA can be separated using the DNA of the present invention as a probe. Similarly, it is also possible to isolate a gene of a human related polypeptide having high homology with the DNA of the present invention or a gene of a polypeptide highly homologous to the polypeptide of the present invention in an organism other than human.

[0027]

[Application to medicine] The object of the present invention, or glial cells,
The polypeptide of the present invention is usually used systemically or locally for the treatment of growth failure or abnormal growth of nerve cells, enhancement or reduction of immune function, or tumor, diabetes, diseases caused by abnormal glucose metabolism, etc. Is administered orally or parenterally. Oral administration, intravenous administration and intracerebroventricular administration are preferred.

Dosage depends on age, body weight, symptoms, therapeutic effect,
Depending on the administration method, treatment time, etc., it is usually administered once per adult in the range of 100 μg to 100 mg once or several times a day orally, or from 10 μg per adult per administration. It is parenterally administered in the range of 100 mg once to several times a day. Of course, as described above, since the dose varies depending on various conditions, a dose smaller than the above dose may be sufficient in some cases, or a dose exceeding the range may be necessary.

When the compound of the present invention is administered, it is used as a solid composition, a liquid composition and other compositions for oral administration, an injection for parenteral administration, an external preparation, a suppository and the like. Solid compositions for oral administration include tablets,
Pills, capsules, powders, granules and the like are included. Capsules include soft capsules and hard capsules.

In such solid compositions, the one or more active substances comprise at least one inert diluent (eg lactose, mannitol, glucose, hydroxypropylcellulose, microcrystalline cellulose, starch, polyvinyl). Pyrrolidone, magnesium aluminometasilicate, etc.). The composition may be an additive other than an inert diluent, for example, a lubricant (magnesium stearate, etc.), a disintegrating agent (calcium fibrin glycolate, etc.), a stabilizer (human serum albumin, lactose, etc.) according to a conventional method. ), And a solubilizing agent (arginine, aspartic acid, etc.) may be contained.

The tablets or pills may be coated with a gastric or enteric film of sucrose, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose phthalate or the like, if necessary, or with two or more layers. Good. Also included are capsules of absorbable material such as gelatin.

Liquid compositions for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, elixirs and the like, and generally used inert diluents (eg, Purified water, ethanol, etc.) may be contained. Such a composition comprises a wetting agent, in addition to an inert diluent,
An auxiliary agent such as a suspension agent, a sweetening agent, a flavoring agent, an aromatic agent, and a preservative may be contained.

Other compositions for oral administration include spray formulations which contain one or more active substances and are formulated in a manner known per se. In addition to an inert diluent, this composition contains a stabilizer such as sodium bisulfite and a stabilizer that imparts isotonicity, an isotonic agent such as sodium chloride, sodium citrate or citric acid. May be. The method for producing the spray agent is described in, for example, U.S. Patent Nos. 2,868,691 and 3,095,
It is described in detail in No. 355.

The injections for parenteral administration according to the present invention include sterile aqueous or non-aqueous solutions, suspensions and emulsions. As aqueous or non-aqueous solutions or suspensions, one or more active substances are mixed with at least one inert diluent. Examples of the aqueous diluent include distilled water for injection and physiological saline. Examples of non-aqueous diluents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, alcohols such as ethanol, and Polysorbate 80 (registered trademark).

Such a composition may further contain a preservative, a wetting agent, an emulsifying agent, a dispersing agent, a stabilizing agent (eg, human serum albumin, lactose, etc.), a solubilizing agent (eg, arginine, aspartic acid, etc.). The auxiliary agent may be included. These are sterilized by filtration through a bacteria-retaining filter, blending of a bactericide, or irradiation. They can also be used by preparing a sterile solid composition (for example, by a freeze-drying method) and dissolving it in sterile distilled water for injection or other solvent before use. Other compositions for parenteral administration include one or more active substances, which are formulated according to a conventional method such as external liquid preparation, soft ko, liniment, suppository and pessary for rectal administration. Is included.

[0036]

EXAMPLES The present invention will be described in more detail with reference to examples below, but these do not limit the scope of the present invention.

Example 1: Construction of vector for preparing cDNA library Plasmid vector pGEM-3Zf (+) [3199b
Sold by Promega Corp.]
After cutting with III, Klenow treatment was performed to re-circularize. Escherichia coli was transformed with this plasmid and the plasmid was recovered.
Next, the AatII-NdeI fragment of the recovered plasmid was cut out, and the ends of the linear plasmid were made blunt by using T4 polymerase. A HindIII linker was ligated to this end, which was cleaved with HindIII, circularized again, and transformed into Escherichia coli to recover a plasmid. The portion of SacI to PstI in the polylinker of the obtained plasmid was replaced with the following synthetic polylinker:

[0038]

[Chemical 1]

Was replaced with The plasmid vector thus constructed (shown in FIG. 1) was designated as pVfCS-1. pVfCS-1 has the following characteristics as a multipurpose plasmid vector. 1. Okayama-Berg method and Gubler-Hoffman method can be applied. 2. High plasmid yield per culture. 3. Single-stranded DNA can also be prepared. 4. It is easy to cut out the cDNA insert. 5. Delation Mutant for Sequence
(Deletion mutant) can be easily produced. 6. In vitro transcription is possible.

Example 2: Isolation and purification of mRNA Human glioblastoma cell line T98G (ATCC strain number,
CRL-1690) 3 x 10 ⁷ 100 units / m
After stimulation with 1 human IL-1β for 4 hours, Okayama, H. et al.
MRNA was separated according to the method described in [Methods in Enzymology, 154 , 3 (1987)]. Ie 5.5 M
GTC solution (5.5 M guanidine thiocyanate, 25
After solubilizing the cells with mM sodium citrate and 0.5% sodium lauryl sarcosine, cell lysate was placed on a cushion of cesium trifluoroacetate (CsTFA) solution having a density of 1.51 and subjected to ultracentrifugation ( (120,000 × g, 20 hours) to recover 1.26 mg of total RNA during the precipitation. this,
Pass through oligo dT cellulose column twice to get 46 μg p
The oly (A) ⁺ RNA was purified and collected.

Example 3: Preparation of cDNA library The cDNA library was prepared by the Gubler & Hoffman method [Gen
e, 25 , 263 (1983)]. First strand was synthesized from poly (A) ⁺ RNA (5 μg) prepared in Example 2 by reverse transcriptase using an oligo dT primer having a NotI site. Subsequently, second strand was synthesized, ligated with SalI adapter and digested with NotI, and then subjected to gel filtration column chromatography [Sephacryl
An S-500HR (sold by Pharmacia) column was used. ], The adapter and the primer were removed, and 820 ng of cDNA fraction was recovered. The above cDNA synthesis steps are performed by the Super Script system (Super Scri
ptSystem, sold by BRL).

On the other hand, the vector was prepared by completely digesting pVfCS-1 prepared in Example 1 with NotI, further digesting it with SalI, and cutting out the band by 0.8% agarose electrophoresis, followed by a kit for glass powder method. [GE
NECLEAN II (sold by BIO 101)] was used for purification. After ligating each of the prepared cDNAs and the vector, they were transformed into DH5 competent cells prepared by the method of Inoue, H. et al. [Described in Gene, 96 , 23 (1990)]. As a result, the average length is 1.5
kb, c with 6 × 10 ⁵ independent clones
A DNA library was obtained.

Example 4: Cloning and Sequencing From the cDNA library prepared in Example 3, a diameter of 1
Clones were plated on a 0 cm LB plate at a density of about 300 colonies / plate, and randomly picked from the colony group for cloning. After culturing one colony in 3 ml of LB culture solution overnight, culture solution 200 μ
7% dimethylsulfochioside (DMSO) was added to 1 and stored at -80 ° C. Bacteria were separated from the remaining culture solution, and the plasmid was separated and purified by a conventional method. Since the plasmid has the structure shown in Fig. 2, when T7 was used as a primer for sequencing, the cloned c
The nucleotide sequence can be read from the 5'side of the DNA. DNA sequencing is based on the dideoxy terminator method of Sanger, F. et al.
ed Biosystems Inc.) using a fluorescent dye-terminator. A DNA sequencer (Model 373A) manufactured by ABI was used to read the sequence. Thus, a nucleotide sequence having an average length of 300 bases was obtained from the 5'side of each clone.

Example 5: Analysis of partial sequence data The nucleotide sequence obtained in Example 4 was Lipm.
Using the FASTA program of an, DJ and Pearson, WR, known databases (GenBank and EMB
A homology search was performed on all nucleotide sequences contained in L). As a result, clones having an unknown sequence were identified from the sequenced clone group. The nucleotide sequence of the identified clone was converted into an amino acid sequence in three possible frames. However, all of the cloned cDNA clones
It does not necessarily cover the entire length of mRNA. If it is not full-length, it is unlikely to include the N-terminal amino acid sequence portion. Therefore, Northern analysis was performed to determine whether the obtained TG0227 clone was full length. That is, poly (A) ⁺ RNA extracted and purified from a glioblastoma cell line was electrophoresed and then blotted on a nylon membrane. CDNA of TG0227
When the insert is hybridized as a probe,
One band was observed at a position of about 1900 bp. TG0
Since the insert size of clone 227 was about 1600 bp, it was confirmed that TG0227 was almost full-length cDNA.

Example 6: Sequence of full-length cDNA and determination of open reading frame The sequence of full-length cDNA was Molecular Cloning
[Sambrook, J., Fritsch, EF and Maniatis, T.
Published by Cold Spring Harbor Laboratory Press in 1989] and determined by random sequencing.

That is, the plasmid was recovered from the TG0227 clone, and the cDNA insert was separated and purified.
Ligate and fragment this,
Blunt the ends of the DNA fragment with T4 polymerase,
A DNA fragment having a length of about 400 bp was recovered using agarose electrophoresis. The obtained DNA fragment is a plasmid vector, BLUESCRIPT II (sold by Stratagene)
After cloning into the SmaI site of E. coli
li). Twenty colonies were picked up at random to prepare plasmid DNA.
Plasmids (all of these are TG0227 cDNA
It has the fragment of A as an insert. ) DNA sequencing was performed. DNA sequencing and sequence reading were performed by the method described in Example 4. The sequence data of the TG0227 cDNA fragment was edited into a continuous sequence using the DNA sequence ligation program of DNASIS to obtain the nucleotide sequence shown in SEQ ID NO: 3. An open reading frame was determined from this cDNA full-length sequence data and further translated into an amino acid sequence to obtain the sequence shown in SEQ ID NO: 1. Sequence Listing 4 shows the entire nucleotide sequence of TG0227 cDNA and the amino acid primary sequence of TG0227 protein encoded by it.

[0047]

[Sequence Listing] SEQ ID NO: 1 Sequence length: 239 Sequence type: Amino acid Topology: Linear Sequence type: Protein sequence Met Val Pro Leu Val Ala Val Val Ser Gly Pro Arg Ala Gln Leu Phe 1 5 10 15 Ala Cys Leu Leu Arg Leu Gly Thr Gln Gln Val Gly Pro Leu Gln Leu 20 25 30 His Thr Gly Ala Ser His Ala Ala Arg Asn His Tyr Glu Val Leu Val 35 40 45 Leu Gly Gly Gly Ser Gly Gly Ile Thr Met Ala Ala Arg Met Lys Arg 50 55 60 Lys Val Gly Ala Glu Asn Val Ala Ile Val Glu Pro Ser Glu Arg His 65 70 75 80 Phe Tyr Gln Pro Ile Trp Thr Leu Val Gly Ala Gly Ala Lys Gln Leu 85 90 95 Ser Ser Ser Gly Arg Pro Thr Ala Ser Val Ile Pro Ser Gly Val Glu 100 105 110 Trp Ile Lys Ala Arg Val Thr Glu Leu Asn Pro Asp Lys Asn Cys Ile 115 120 125 His Thr Asp Asp Asp Glu Lys Ile Ser Tyr Arg Tyr Leu Ile Ile Ala 130 135 140 Leu Gly Ile Gln Leu Asp Tyr Glu Lys Ile Lys Gly Leu Pro Glu Gly 145 150 155 160 Phe Ala His Pro Lys Ile Gly Ser Asn Tyr Ser Val Lys Thr Val Glu 165 170 175 Lys Thr Trp Ly s Ala Leu Gln Asp Phe Lys Glu Gly Asn Ala Ile Phe 180 185 190 Thr Phe Pro Asn Thr Pro Val Lys Cys Ala Gly Ala Pro Gln Lys Ile 195 200 205 Met Val Leu Ile Arg Ser Leu Leu Gln Glu Asp Arg Glu Ala Ile Gln 210 215 220 Gly Gln Tyr His Phe Gln His Phe Ser Trp Ser His Phe Arg Gly 225 230 235

SEQ ID NO: 2 Sequence length: 720 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: cDNA to mRNA Sequence ATGGTGCCAC TGGTGGCTGT GGTATCAGGG CCCCGTGCCC AGCTCTTTGC CTGCCTGCTC 60 AGGCTGGGCA CTCAGCAGGT CGGCCCCCGG CAGCCAGG CCATGCGGCC 120 AGGAACCATT ATGAGGTGCT GGTGCTGGGT GGGGGCAGTG GCGGAATCAC CATGGCTGCC 180 CGCATGAAGA GGAAAGTGGG TGCAGAGAAT GTGGCCATTG TTGAGCCCAG TGAGAGACAT 240 TTCTACCAGC CAATCTGGAC ACTGGTGGGT GCTGGTGCCA AACAATTGTC CTCATCTGGT 300 CGTCCCACGG CAAGTGTGAT TCCATCTGGT GTAGAATGGA TCAAAGCTAG AGTGACTGAG 360 TTGAACCCAG ACAAGAACTG CATTCACACA GATGACGACG AGAAGATCTC CTACCGATAT 420 CTTATTATTG CTCTCGGAAT CCAGCTGGAC TATGAGAAGA TTAAAGGCCT ACCTGAAGGT 480 TTCGCTCATC CCAAAATAGG GTCGAATTAT TCAGTTAAGA CTGTAGAGAA GACATGGAAA 540 GCTCTGCAGG ACTTCAAAGA GGGCAATGCC ATCTTCACCT TCCCAAATAC TCCAGTGAAG 600 TGTGCTGGAG CCCCTCAGAA GATCATGGTA CTTATCAGAA GCCTACTTCA GGAAGACAGG 660 GAAGCGATCC AAGGCCAATA TCATTTTCAA CACTTCTCTT GGAGCCATTTTT TCGGGGTTAA 720

SEQ ID NO: 3 Sequence length: 822 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: cDNA to mRNA Sequence AAGGTTTTTN CTGCGCCAAC GCAGTGACCG AAGGCTCCGC TCACGCCCGG CCTGATCCTG 60 CCTGAAGATG GTGCCACTGG TGGCCCTGTGTGCACAGC TCTTTGCCTG 120 CCTGCTCAGG CTGGGCACTC AGCAGGTCGG CCCCCTTCAG CTGCACACCG GGGCCAGCCA 180 TGCGGCCAGG AACCATTATG AGGTGCTGGT GCTGGGTGGG GGCAGTGGCG GAATCACCAT 240 GGCTGCCCGC ATGAAGAGGA AAGTGGGTGC AGAGAATGTG GCCATTGTTG AGCCCAGTGA 300 GAGACATTTC TACCAGCCAA TCTGGACACT GGTGGGTGCT GGTGCCAAAC AATTGTCCTC 360 ATCTGGTCGT CCCACGGCAA GTGTGATTCC ATCTGGTGTA GAATGGATCA AAGCTAGAGT 420 GACTGAGTTG AACCCAGACA AGAACTGCAT TCACACAGAT GACGACGAGA AGATCTCCTA 480 CCGATATCTT ATTATTGCTC TCGGAATCCA GCTGGACTAT GAGAAGATTA AAGGCCTACC 540 TGAAGGTTTC GCTCATCCCA AAATAGGGTC GAATTATTCA GTTAAGACTG TAGAGAAGAC 600 ATGGAAAGCT CTGCAGGACT TCAAAGAGGG CAATGCCATC TTCACCTTCC CAAATACTCC 660 AGTGAAGTGT GCTGGAGCCC CTCAGAAGAT CATGGTACTT ATCAGAAGCC T ACTTCAGGA 720 AGACAGGGAA GCGATCCAAG GCCAATATCA TTTTCAACAC TTCTCTTGGA GCCATTTTCG 780 GGGTTAAGAA GTATGCAGAT GCCCTGCAGG AGATCATCCA GG 822

SEQ ID NO: 4 Sequence length: 822 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: cDNA to mRNA Origin organism name: Homo sapiens Cell line: T98G Sequence features Characteristic symbol: CDS Location: 68..787 Method for determining characteristic: P sequence AAGGTTTTTN CTGCGCCAAC GCAGTGACCG AAGGCTCCGC TCACGCCCGG CCTGATC 57 CTGCCTGAAG ATG GTG CCA CTG GTG GCT GTG GTA TCA GGG CCC CGT GCC 106 Ala Val Pro Leu Gly Pro Arg Pro Arg Ala 1 5 10 CAG CTC TTT GCC TGC CTG CTC AGG CTG GGC ACT CAG CAG GTC GGC CCC 154 Gln Leu Phe Ala Cys Leu Leu Arg Leu Gly Thr Gln Gln Val Gly Pro 15 20 25 CTT CAG CTG CAC ACC GGG GCC AGC CAT GCG GCC AGG AAC CAT TAT GAG 202 Leu Gln Leu His Thr Gly Ala Ser His Ala Ala Arg Asn His Tyr Glu 30 35 40 45 GTG CTG GTG CTG GGT GGG GGC AGT GGC GGA ATC ACC ATG GCT GCC CGC 250 Val Leu Val Leu Gly Gly Gly Ser Gly Gly Ile Thr Met Ala Ala Arg 50 55 60 ATG AAG AGG AAA GTG GGT GCA GAG AAT GTG GCC ATT GTT GAG CCC AGT 298 Met Lys Arg Lys Val Gly Ala Glu Asn Val Ala Ile Val Glu Pro Ser 65 70 75 GAG AGA CAT TTC TAC CAG CCA ATC TGG ACA CTG GTG GGT GCT GGT GCC 346 Glu Arg His Phe Tyr Gln Pro Ile Trp Thr Leu Val Gly Ala Gly Ala 80 85 90 AAA CAA TTG TCC TCA TCT GGT CGT CCC ACG GCA AGT GTG ATT CCA TCT 394 Lys Gln Leu Ser Ser Ser Gly Arg Pro Thr Ala Ser Val Ile Pro Ser 95 100 105 GGT GTA GAA TGG ATC AAA GCT AGA GTG ACT GAG TTG AAC CCA GAC AAG 442 Gly Val Glu Trp Ile Lys Ala Arg Val Thr Glu Leu Asn Pro Asp Lys 110 115 120 125 AAC TGC ATT CAC ACA GAT GAC GAC GAG AAG ATC TCC TAC CGA TAT CTT 490 Asn Cys Ile His Thr Asp Asp Asp Glu Lys Ile Ser Tyr Arg Tyr Leu 130 135 140 ATT ATT GCT CTC GGA ATC CAG CTG GAC TAT GAG AAG ATT AAA GGC CTA 538 Ile Ile Ala Leu Gly Ile Gln Leu Asp Tyr Glu Lys Ile Lys Gly Leu 145 150 155 CCT GAA GGT TTC GCT CAT CCC AAA ATA GGG TCG AAT TAT TCA GTT AAG 586 Pro Glu Gly Phe Ala His Pro Lys Ile Gly Ser Asn Tyr Ser Val Lys 160 165 170 ACT GTA GAG AAG ACA TGG AAA GCT CTG CAG GAC T TC AAA GAG GGC AAT 634 Thr Val Glu Lys Thr Trp Lys Ala Leu Gln Asp Phe Lys Glu Gly Asn 175 180 185 GCC ATC TTC ACC TTC CCA AAT ACT CCA GTG AAG TGT GCT GGA GCC CCT 682 Ala Ile Phe Thr Phe Pro Asn Thr Pro Val Lys Cys Ala Gly Ala Pro 190 195 200 205 CAG AAG ATC ATG GTA CTT ATC AGA AGC CTA CTT CAG GAA GAC AGG GAA 730 Gln Lys Ile Met Val Leu Ile Arg Ser Leu Leu Gln Glu Asp Arg Glu 210 215 220 GCG ATC CAA GGC CAA TAT CAT TTT CAA CAC TTC TCT TGG AGC CAT TTT 778 Ala Ile Gln Gly Gln Tyr His Phe Gln His Phe Ser Trp Ser His Phe 225 230 235 CGG GGT TAA GAAGTATGCA GATGCCCTGC AGGAGATCAT CCAGG 822 Arg Gly

[Brief description of drawings]

FIG. 1 is a construction diagram of a plasmid vector pVfCS-1.

FIG. 2c derived from human glioblastoma cell line T98G
It is a construction drawing of the recombinant DNA in which DNA was inserted.

Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI Technical display location A61K 38/00 ADU 39/395 ABA D ADP T C07K 14/475 ZNA 8318-4H // C12P 21/02 C 9282 -4B (C12P 21/02 C12R 1:19) A61K 37/02 ADS ADU (54) [Title of the Invention] Novel polypeptide, method for producing the same, DNA encoding the polypeptide, vector comprising the DNA, Host cells transformed with the vector, antibody of the polypeptide, and pharmaceutical composition containing the polypeptide or antibody

Claims (10)

[Claims] 1. A polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1, which is in a substantially pure form, a homologue thereof, a fragment thereof or a homologue of a fragment thereof. 2. The polypeptide according to claim 1, which consists of the amino acid sequence represented by SEQ ID NO: 1. 3. A DNA encoding the polypeptide according to claim 1. 4. The DNA according to claim 3, which has the base sequence represented by SEQ ID NO: 2, or a fragment which selectively hybridizes to the sequence. 5. The DNA according to claim 3, which has the nucleotide sequence represented by SEQ ID NO: 3, or a fragment which selectively hybridizes to the sequence. 6. A replication or expression vector comprising the DNA according to any one of claims 3 to 5. 7. A host cell transformed with the replication or expression vector of claim 6. 8. A method for producing the polypeptide according to claim 1, which comprises culturing the host cell according to claim 7 under conditions for expressing the polypeptide according to claim 1 or 2. 9. A monoclonal or polyclonal antibody of the polypeptide according to claim 1 or 2. 10. A pharmaceutical composition comprising the polypeptide according to claim 1 or 2 or the antibody according to claim 9 and a pharmaceutically acceptable excipient and / or carrier.