JPH0751065A - Glycoprotein 39 gene - Google Patents

Abstract

PURPOSE:To provide a new gene useful for the production of a human-originated mucin glycoprotein 39 useful as a tumor marker, immune abnormality marker and marker for various inflammatory diseases. CONSTITUTION:This glycoprotein 39 gene is cDNA clone pKP39 coding glycoprotein 39 and having restriction map and a base-sequence determination method shown in the drawing. The glycoprotein 39 gene can be produced by separating whole RNA from a cell expressing glycoprotein 39 (e.g. gastric cancer cell strain KATO-III), purifying mRNA therefrom, synthesizing cDNA by conventional method, constructing a library by integrating into an expression vector and selecting a clone having glycoprotein 39 gene by using an anti-glycoprotein 39 antibody.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel mucin glycoprotein 39 gene, and more specifically, codes for a human-derived mucin glycoprotein 39 core protein useful as a tumor marker, an immune abnormality marker or various inflammatory disease markers. The present invention relates to a gene containing a nucleotide sequence that

[0002]

2. Description of the Related Art Generally, it is known that glycoproteins such as glycoproteins and glycolipids different from those of normal cells are present in the cell membrane of cancerous cells. Further, in diagnosing cancer, a method of measuring a protein antigen or a sugar chain antigen specifically produced in a cancer patient is performed. Examples include carcinoembryonic antigen (CEA), α-fetoprotein, CA19
Diagnosis of digestive system cancer by measurement of -9 etc. is known [Takashi Muramatsu, Japan Clinic, 44 , p.337-344 (1986); Reiji Kanaki, Clinicopathology, 35 , p.1247-1264. (1986); History of Medicine, Vol. 106, No. 5, 5th Saturday, 235-250 (1978)
Year)〕.

[0003]

However, the conventional cancer diagnostic methods using various types of cancer antigen measurement are relatively limited in the types of cancer that can be applied, and cross-react with other diseases such as healthy people and hepatitis. Therefore, a diagnostic method applicable to a wider range of cancers or a highly specific diagnostic method is desired. Further, an accurate diagnostic method is desired for diseases caused by various immune abnormal responses and various inflammatory diseases. Then, the development of new glycoproteins and genes encoding their core proteins, which can be used as tumor markers, immunological abnormality markers or various inflammatory disease markers that can be used for diagnosis of such diseases, has been earnestly desired.

On the other hand, in recent years, structural analysis of sugar chains and core proteins of mucin expressed in various cancer tissues has progressed, and its relevance to various diseases including cancer has been attracting attention [Bhavanandan, VP, Glycobiology, 1 ,
493-503 (1991)].

[0005]

[Means for Solving the Problems] Therefore, the present inventors have studied for the purpose of solving the above problems by focusing on glycoproteins expressed on the surface of human gastric cancer cells. A gene encoding a core protein of a novel mucin glycoprotein 39, which is expected to be applied to the diagnosis of diseases, was found, and this gene was found in breast cancer and pancreatic cancer, and was found to be polymorphic epithelial mucin (P
The present invention has been completed by revealing that it is a new mucin that clearly shows a high homology with EM).

That is, the present invention provides a glycoprotein 39 gene encoding a novel human-derived mucin core protein.

The gene of the present invention includes, for example, a nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 1 and a nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 2, a nucleotide sequence complementary to these amino acid sequences, or a sequence thereof. Both are included. In the sequence listing, the lower part of the base sequence is the amino acid sequence deduced from the upper base sequence.

The glycoprotein 39 gene of the present invention is prepared, for example, as follows. That is, first, total RNA is isolated from cells expressing glycoprotein 39, mRNA is purified from this, cDNA is synthesized by a conventional method, and then this is incorporated into an expression vector to construct a library.
Next, a clone having the glycoprotein 39 gene is selected from this cDNA library using the anti-glycoprotein 39 antibody to obtain the glycoprotein 39 gene of the present invention. Next, the method for producing the gene of the present invention will be described in detail.

[1] Construction of cDNA Library As tissue cells used for extraction of total RNA, human gastric cancer tissue or cell lines already established as cell lines, for example, gastric cancer cell line KATO-III [Sekiguchi M., Sakakibara K . an
d Fujii G. (1978). Jpn. J. Exp. Med., 48 , p.61-6
8].

RNA is extracted by a guanidine-isothiocyanate mixture or a suitable detergent such as SDS, NP-4.
0, Triton X-100, deoxycholic acid, etc., or by partially or completely disrupting and solubilizing the cells by a homogenizer method or a physical method such as freeze-thawing, the chromosomal DNA is treated with polytron, etc. This is carried out by using a mixer or a syringe described in 1. above, shearing to some extent, and then separating the protein and nucleic acid fractions. For this operation, in particular, the CsCl overlay method using phenol / chloroform extraction or ultracentrifugation [Chirgwin, JM, et al., Bioch
emistry, 18 , p. 5294 (1979)] is generally used.

In each of the above methods, RNase
In order to prevent RNA degradation, it is advisable to add an RNase inhibitor such as heparin, polyvinylsulfate, diethylpyrocarbonate, a vanadium complex, bentonite and macaloid.

From RNA obtained according to the above extraction procedure
Separation and purification of mRNA can be carried out by a method using an extract using an adsorption column such as oligo dT-cellulose (Colaborative Research Inc.) or poly U-Sepharose (Pharmacia), or by a batch method.

The purified mRNA obtained as described above is usually unstable, is replaced by a stable complementary DNA (cDNA) type, and is ligated to a microorganism-derived vector in order to enable amplification of the target gene. The above-mentioned conversion of mRNA into cDNA, that is, synthesis of cDNA in vitro can be generally performed as follows.

That is, first, using oligo dT as a primer (this primer may be either free oligo dT or oligo dT already added to the vector primer), mR
Single-stranded cDNA complementary to mRNA using reverse transcriptase in the presence of dNTP (dATP, dGTP, dCTP or dTTP) using NA as a template
To synthesize. The next steps differ as follows depending on whether the free oligo dT or the oligo dT added to the vector primer was used in the above.

In the former case, the mRNA used as a template is decomposed and removed by alkali treatment or the like, and then the single-stranded DNA is used as a template to form a double-stranded DNA using reverse transcriptase or DNA polymerase.
Create A. Next, treat both ends of the resulting double-stranded DNA with exonuclease,
Multiple bases of DNA or an annealable combination are added, and this is incorporated into an appropriate vector. This is a known method depending on the vector used, such as the method of Young et al. [Youn
g, RA et al., in "DNA Cloning, Vol. 1", p.49 (1
985)], or the method of Gubler and Hoffman [Gubler,
U. and Hoffman, BJ Gene, 25 , p.263 (1983)]. Further, the above-mentioned cDNA can be easily synthesized by using a commercially available cDNA synthesis kit.

The vector is not particularly limited, but λgt
A system such as a phage vector or a plasmid vector can be appropriately selected depending on the host or used in combination. Examples of the vector used here include λgt10 and λgt11, and the method of using λgt10 and λgt11 as a vector can be performed according to the method of Young et al.

CDNA integrated into a λgt-based phage vector
The recombinant becomes a cDNA recombinant phage by reacting with an in vitro packaging solution, and becomes λgt10 or λgt10.
A gt11 cDNA library is constructed. The above-mentioned λgt-based phage library was prepared using commercially available λgt10 or λgt.
It can be easily performed by using the 11 cDNA cloning kit.

In the latter case, the open circular plasmid to which the same linker as above is added while leaving the mRNA as a template and the linker DNA (often containing a region capable of autonomous replication in animal cells and a transcription promoter region of mRNA) Can be used as a DNA fragment) to form a closed circle, and then RNase and DNA polymerase are allowed to coexist in the presence of dTNP to substitute the mRNA for the DNA chain to form a complete plasmid DNA.

The cDNA recombinant plasmid obtained as described above is introduced into a host microorganism to transform the microorganism. As a host microorganism, Escherichia coli
However, the Bacillus subtilis ( Bacillus subtilis ), yeast ( Saccharomyces cer
visiae ) etc. can also be used.

As a method for introducing DNA into a host microorganism and transforming the same by a generally used method,
For example, a method in which cells in the logarithmic growth phase are mainly collected and treated with CaCl ₂ so that DNA can be easily taken up naturally and the plasmid is taken up can be adopted. In the above method, MgCl ₂ and RbCl can be further coexisted in order to further improve the efficiency of transformation, as is commonly known. Alternatively, a method in which microbial cells are transformed into spheroplasts or protoplasts and then transformed can also be employed.

[2] Selection of Glycoprotein 39 Gene Clone As a method for selecting a strain containing a cDNA encoding the core protein of the glycoprotein 39 of the present invention from the transformants obtained as described above, for example, the following various types are selected. The method can be adopted.

(1) Method of selecting using lectin-binding glycoprotein containing glycoprotein 39 of the present invention using an antibody against the core protein: cDNA is previously incorporated into a vector capable of expressing the protein in the transformant, And a second antibody against the core protein of the lectin-binding glycoprotein containing the glycoprotein 39 of the present invention and a second antibody against the antibody to produce a polypeptide producing strain of the lectin-binding glycoprotein containing the glycoprotein 39 of the present invention. Detect and obtain the target strain.

(2) Method of Screening by Producing Polypeptide of Glycoprotein 39 of the Present Invention in Animal Cell A transformed strain is cultured, a gene is grown, and the gene is transfected into an animal cell (in this case, It may be either a plasmid that is replicable and contains an mRNA transcription promoter region or a plasmid that integrates into the chromosome of an animal cell), produces a protein encoded by a gene, and forms a core protein of a lectin-binding glycoprotein containing the glycoprotein 39 of the present invention. By detecting the polypeptide of the lectin-binding glycoprotein containing the glycoprotein 39 of the present invention using an antibody against the above, a strain having a cDNA encoding the polypeptide portion of the target glycoprotein 39 of the present invention from the original transformant strain can be obtained. elect.

(3) Method Using Selective Hybridization Translation System cDNA obtained from the transformant strain is blotted on a nitrocellulose filter or the like to produce a polypeptide of a lectin-binding glycoprotein containing the glycoprotein 39 of the present invention. After the mRNA from the cells is hybridized, the mRNA corresponding to the cDNA is recovered. A lectin-binding glycoprotein containing the glycoprotein 39 of the present invention containing the glycoprotein 39 of the present invention obtained by injecting the recovered mRNA into a protein translation system, for example, injecting it into oocytes of Xenopus laevis or translating it into a protein in a cell-free system such as rabbit reticulocyte lysate or wheat germ. The target strain is obtained by detection using an antibody against the core protein of.

The antibody to the core protein of the lectin-binding glycoprotein containing the glycoprotein 39 of the present invention used in the above method can be prepared by a known method.

That is, first, the cell membrane of a tissue cell expressing the glycoprotein 39 of the present invention is solubilized using a surfactant, and this is adsorbed on a lectin-binding agarose column to which the glycoprotein 39 can bind to give a lectin. Prepare the conjugated glycoprotein.

As a means for separating cell membrane solubilized fractions of various tissue cells, for example, human cancer tissue and human cells are crushed in an appropriate buffer and subjected to high-speed centrifugation at 100,000 × g, and the residue is triton-based. Dissolve in surfactant and re-add 100,00
A method of subjecting to 0 × g high-speed centrifugation and collecting the supernatant can be mentioned.

The lectin used for separating the lectin-binding glycoprotein containing the glycoprotein 39 of the present invention from the obtained cell membrane solubilized fraction includes, for example, peanut bean lectin (Peanut agglutinin, PNA),
As such a lectin, a commercially available one may be used, or, for example, one obtained by extracting and separating from peanut beans by a method known per se may be used. Commercially available lectin-bound agarose may be used, or it may be obtained by coupling it to an agarose gel by a conventional means.

To elute the lectin-binding glycoprotein, a hapten sugar such as lactose solution is used. The concentration of the eluate used here is preferably 0.05 to 0.2M.

Next, the lectin-binding glycoprotein containing the glycoprotein 39 of the present invention is treated with trifluoromethanesulfonic acid (TFMS) or hydrogen fluoride to remove the sugar chain, and this is used together with a complete adjuvant in small animals such as rabbits. Antiserum is collected after immunization and several immunizations with incomplete adjuvant at appropriate intervals. Next, the bacterial cell component obtained by heat-treating E. coli and centrifuging and the antiserum obtained above are mixed at 4 ° C. and then centrifuging to obtain the desired polyclonal antibody.

The gene clone of the present invention obtained above can be subcloned into various plasmids by a conventional method. For example, a cDNA fragment containing the gene of the present invention purified by digestion with EcoRI was similarly digested with EcoRI to obtain pUC18 [Yanisch-Perron, C., et al., Gene, 8
3 , p.103-119 (1985)] and the like into the cleavage site of the cloning vector. As a result, the desired recombinant plasmid can be obtained. In addition, the introduction of the obtained recombinant plasmid into a host, and the amplification and individualization of the recombinant plasmid thereby, can be performed by various commonly used methods, for example, by collecting cells mainly in logarithmic growth phase and
₂ Treatment may be carried out by a method such that the DNA is naturally incorporated into the DNA easily, and the vector is incorporated into the DNA.

Various operations adopted in the above, for example, chemical synthesis of a part of DNA, cleavage and deletion of DNA chain,
Enzymatic treatment for the purpose of addition or binding, DNA isolation, purification, replication, selection, etc. can all follow conventional methods.
More specifically, the isolation and purification of the above DNA can be performed by agarose gel electrophoresis or the like.

The nucleotide sequence of the gene of the present invention obtained above can be determined by digesting DNA with an appropriate restriction enzyme and then using the dideoxy method [Sanger, et al., Proc. Natl. Acad. Sci.
USA, 74 , p.5463 (1977)] and the Maxam-Gilbert method (AM Maxam and W. Gilbert, Methods in Enzymolog
y, 65 , p. 499 (1980)] and the like. Furthermore, the above-mentioned nucleotide sequence can be easily determined by using a commercially available sequence kit or the like.

The glycoprotein 39 of the present invention thus obtained
A part of the base sequence of the gene and the corresponding amino acid sequence are shown in SEQ ID NO: 1 and SEQ ID NO: 2. The base number is 1 at the 5'end and is assigned in the direction from the 5'end to the 3'end. The amino acid residues are numbered from the N-terminal to the C-terminal, and the first encoded amino acid is 1. SEQ ID NO: 1 is a translation region having a length of 180 bases located at the most 5 ′ end in the translation region of the glycoprotein 39 gene whose sequence can be determined, and corresponds to a protein portion of 60 amino acids. This sequence is similar to the PEM gene in 60 bases (2
Repeated sequence (tandem with 0 amino acid residues) as one unit
It is considered to be a repeats) region, and there seems to be individual differences in the number of repeats. The translation region of the glycoprotein 39 gene shown in SEQ ID NO: 2 has a length of 981 bases and corresponds to a protein portion of 327 amino acids. The sequence of SEQ ID NO: 1 is further connected to a repeating sequence on the 3'side (the C-terminal side of the amino acid sequence) in the base sequence, to which the sequence number 2 is connected.

The obtained gene of the present invention can be used by a conventional publicly known general gene recombination technique [Science, 2
24 , p.1431 (1984); Biochem. Biophys. Res. Comm., 13
0 , p.692 (1985); Proc. Natl. Acad. Sci., USA, 80 ,
p.5990 (1983); see EP Patent Publication No. 187991, etc.),
The core protein of glycoprotein 39 can be easily produced and obtained in large quantities. Further, by using the core protein of glycoprotein 39 thus obtained, an antibody specific to the core protein of glycoprotein 39 can be prepared. Antibodies are produced according to the usual methods for producing polyclonal antibodies and monoclonal antibodies. The polyclonal antibody against the core protein complex of glycoprotein 39 is analyzed by the method of Weinberger et al.
ence, 228 , p.740-742 (1985)], it is also possible to obtain an epitope-specific antibody. Antibody is glycoprotein 39
And its core protein are used for purification, measurement, identification and the like.

The glycoprotein 39 core protein obtained as described above includes a polypeptide in which methionine is linked to the N-terminus of the amino acid sequence shown in the sequence listing, and a glycoprotein 39 at the N-terminus of the above amino acid sequence. Also included are intermediates in which part or all of the signal peptide for binding or deficiency is bound. Such a mutation can be obtained naturally, for example, by post-translational modification, or in a genetic engineering technique, a gene obtained from nature is modified by a method such as cytospecific mutagenesis, or a phosphite triester method or the like. Genes can be synthesized by synthesizing mutated DNA by a chemical synthesis method or by combining both. By utilizing these genes, incorporating them into a microbial vector, and producing them from a transformed microorganism, a component having a mutation can be obtained. In addition, these proteins can be partially substituted with amino acids or modified in sequence by natural or artificial mutation while maintaining their functions. Therefore, the glycoprotein 39 gene of the present invention also includes genes encoding proteins having the above-mentioned various mutations. Stop codons such as TAG and TAA can be added to the end of the genetic code. The genetic code is not limited to the codons exemplified in SEQ ID NOS: 1 and 2 above, and any codon can be selected for each amino acid without changing the amino acid sequence. For example, consider the frequency of codon usage of the host used for gene recombination. You can follow the usual method [Nucl. Acids. Res., 9 , p.43-74
(1981)].

[0037]

EFFECT OF THE INVENTION By using the glycoprotein 39 gene of the present invention, the core protein of the glycoprotein 39 can be easily produced in a large amount. Glycoprotein 39 of the present invention
Is expressed in human cancer tissues, particularly gastric cancer, colon cancer, pancreatic cancer, liver cancer, esophageal cancer, lung cancer, etc., while it is expressed in secretory normal tissues such as stomach, colon, lung, etc. It is expected to be applied as an abnormal marker or various inflammatory disease markers.

[0038]

The present invention will be described in more detail with reference to the following examples.

Example 1 Preparation of glycoprotein 39 of the present invention: Gastric cancer cell KATO-III 2
g was minced in PBS [PBS (+)] supplemented with CaCl ₂ and MgCl ₂ under ice cooling and homogenized with a Potter-Elvehjem type homogenizer.

This disrupted liquid was centrifuged at 4 ° C. for 1 hour under high speed (10
5,000 × g), and the supernatant was removed, and the pellet was added to 2% Triton X-100, 0.15M NaCl, 0.01M Tris-HCl (pH 7.6).
And 60 ml of a solution containing PMSF (phenylmethylsulfonylfluoride) (Sigma), which is a protease inhibitor of 50 μg / ml, were homogenized, and the mixture was allowed to stand at 4 ° C. for 30 minutes to solubilize the cell membrane. This at 4 ℃
High-speed centrifugation (105,000 × g) for 1 hour to obtain the supernatant.

The supernatant was added to a commercially available PNA-binding agarose column (manufactured by EY Laboratories) to adsorb the PNA-binding glycoprotein.

The column was loaded with 0.1% Triton X-100, 0.15MN.
It was washed with 200 ml of a washing solution (pH 7.6) containing aCl and 0.01 M Tris-HCl. Then, the PNA-bound glycoprotein was eluted with 50 ml of 0.05 M lactose solution.

The protein concentration of PNA-binding glycoprotein in the eluate was measured by the Lowry method to obtain a total amount of 3 mg (protein content) of PNA-binding glycoprotein.

Example 2 (1) Removal of sugar chain from PNA-bound glycoprotein: 2 mg of PNA-bound glycoprotein was lyophilized and then dissolved by adding 1 ml of trifluoromethanesulfonic acid (TFMS) -anisole (2: 1) solution. Nitrogen gas was bubbled through the reaction solution to replace it.
The mixture was stirred at 0 ° C for 5 hours to decompose sugar chains. After completion of the reaction, a double amount of diethyl ether was added and mixed, and then the mixture was allowed to stand at -80 ° C for 1 hour. Next, an equal volume of ice-cooled 50% pyridine solution was added and stirred with a vortex mixer, and then the ether layer was removed. Ether was further added and the mixture was extracted twice with ether in the same manner, and then dialyzed against 4 l of 2 mM pyridine-acetic acid buffer (pH 5.5).

(2) Preparation of Polyclonal Antibody against Core Protein of PNA-Binding Glycoprotein: 0.5 ml of PBS (-) solution (protein concentration 800 μg / ml) of sugar chain-eliminating PNA-binding glycoprotein prepared in (1) and Freund's The suspension prepared by mixing 0.5 ml of complete adjuvant was subcutaneously inoculated into the foot pad of a New Zealand White male rabbit. Thereafter, the suspension of Freund's incomplete adjuvant and the PNA-binding glycoprotein was subcutaneously inoculated into the foot pad or the back three times every two weeks for immunization. On the 10th day after the final immunization, blood was collected from the ear vein of the rabbit, and after complete coagulation, high-speed centrifugation (150,000 rpm) was repeated twice at 4 ° C. for 20 minutes twice, and the supernatant was collected to obtain antiserum.

(3) Absorption treatment of antibody: The antibody used in the screening for the isolation of the recombinant phage clone encoding the core protein of the glycoprotein 39 of the present invention shown in Example 3 (2) below is an E. coli cell. It is desired that it not cross-react with the components. Therefore, the antibody used for screening is reacted with E. coli Y1090 bacterial cell components in advance,
Antibodies that crossed this were removed.

E. coli Y1090 strain was treated with LB medium [Molecular C
loning (A Laboratory Manual); T. Maniatis, EF Fr
itsch, J. Sambrook; Cold Spring Harbor Laboratory
(1982), p.68] Incubate in 500 ml at 37 ℃ overnight,
The cells were collected by centrifugation at pm for 10 minutes. This was suspended in 20 ml of distilled water and heat-treated at 100 ° C for 5-10 minutes. Furthermore,
After centrifugation at 10,000 rpm for 10 minutes, the supernatant was separated. Next, 1 ml of this supernatant was added to 100 ml of a solution prepared by diluting the antiserum prepared in Example 2 (2) 50 times with PBS (-), and mixed.
After leaving it at 4 ° C. for 2 hours, it was centrifuged at 10,000 rpm for 15 minutes, and the supernatant was separated to obtain an antibody against the core protein of glycoprotein 39 of the present invention.

Example 3 (1) Preparation of cDNA library of gastric cancer cell line KATO-III: 5% of gastric cancer cell line KATO-III was added to RPMI-1640 medium at a ratio of 10% fetal calf serum. Subculture was performed at 37 ° C. under aeration of CO ₂ gas. From the obtained gastric cancer cell line KATO-III 1 g, the guanidinium isothiocyanate method [Molecular Cloning
(A Laboratory Manual); T. Maniatis, EF Fritsch,
J. Sambrook; Cold Spring Harbor Laboratory (198
2), p.196], 3 mg of total RNA was extracted, and this was extracted with an oligo (dT) cellulose column (Colaborative Research In
c., column volume 1 ml) was used to obtain 200 μg of poly (A) ⁺ RNA. Then, double-stranded cDNA was synthesized according to the protocol of the cDNA synthesis system of Amersham. That is, the applicable poly
Reverse transcriptase (Amersham) was allowed to act on 5 μg of (A) ⁺ RNA to synthesize the first DNA strand. Next, E. coli ribonuclease H (RNase H) and E. coli DNA polymerase I (both Amersham) are allowed to act, and the second DNA chain is synthesized using the first DNA chain as a template while digesting RNA, and the exonuclease activity of T4 DNA polymerase is A double-stranded cDNA (ds-cDNA) having a blunt end was synthesized by utilizing the method.

The ds-cDNA obtained above was further cloned into the expression vector λgt11 using the cDNA cloning system λgt11 of Amersham. That is, ds-cDNA
EcoRI methylase (Amersham) is allowed to act on ds-c
The recognition site for the restriction enzyme EcoRI inside the DNA is protected with a methyl group, and then a synthetic EcoRI linker (Amersham) is connected to both ends with T4 DNA ligase (Amersham), and finally the restriction enzyme EcoRI (Amersham) is connected to this. Company) to make both ends sticky ends.

This ds-cDNA and λgt11 arm (Amersham) were ligated with T4 DNA ligase (Amersham) to prepare a recombinant DNA. An in vitro packaging solution (Amersham) was allowed to act on this to prepare a cDNA library.

(2) Isolation of recombinant phage clone encoding the glycoprotein 39 of the present invention: λgt1 obtained in (1)
1 cDNA library and E. coli Y1090 were incubated at 37 ℃ for 20 minutes and recombinant phage was used as host strain Y109.
After being adsorbed at 0, it was mixed with the dissolved upper soft agar and spread on agar plates. After solidifying the upper agar plate, place the agar plate at 42 ° C.
The cells were cultured for 4 to 8 hours to form plaques. Then 10
Saturated with isopropyl-1-thio-β-D-galactoside (IPTG) and dried nitrocellulose filter was placed on the surface of agar plate and incubated at 37 ° C for 2 hours,
The β-galactosidase fusion protein was expressed.

Thereafter, this was cooled at 4 ° C. for 1 hour or more, and then the filter was removed. Apply this filter at room temperature for 1 hour in blocking solution (2% horse hemoglobin, 0.1% Twe).
The glycoprotein 39 of the present invention, which was subjected to the absorption treatment in Example 2 (3) in the blocking solution after being soaked in en20, PBS (-))
Was reacted with 50 μg / ml of an antibody against the core protein of and was incubated at room temperature for 2 hours. Turn the filter to 0.
After washing 5 times with PBS (-) containing 1% Tween20, this filter was washed with horseradish peroxidase (HRP) -labeled anti-rabbit IgG antibody (Cappel) blocking solution (2
The reaction was carried out for 2 hours at room temperature in a (00-fold diluted solution), and after completion of the reaction, the above-mentioned washing solution was washed 5 times. Then, a clone expressing a fusion protein corresponding to the core protein of the glycoprotein 39 of the present invention was selected by coloring with a 4-chloro-1-naphthol solution containing hydrogen peroxide. After separating a single plaque of the obtained clone, it was grown using Y1090 as a host, suspended in SM buffer, and stored at 4 ° C. The clone was named λKP39.

(3) Preparation of lysogen of recombinant phage encoding glycoprotein 39 of the present invention: Huynh, TV, Young,
RA, Davis, RW: DNA Coloning Vol.1 A Practic
al Approach, (ed.) Glover, DM, IRL Press (1985)
A lysogen was prepared by lysogenizing λKP39 into E. coli BNN103 according to the method described on p.49-78.

(4) Isolation of Recombinant Phage DNA Encoding the Glycoprotein 39 of the Present Invention: The recombinant phage clone (λKP39) encoding the glycoprotein 39 of the present invention obtained in (2) was transformed into E. coli Y1090. After being grown as a host, (Molecular Cloning (A Laboratory Manual); T.
Maniatis, EF Fritsch, J. Sambrook; ColdSpring H
Arbor Laboratory (1982) p.371-372], the recombinant phage DNA of the present invention (λKP39 DNA) was prepared.

(5) Construction of plasmid pKP39 transformant:
λKP39 DNA was digested with restriction enzyme EcoRI (manufactured by Nippon Gene Co., Ltd.) to obtain a DNA fragment of about 1900 base pairs.

On the other hand, the plasmid vector pBluescript II
After KS (Stratagene) was similarly digested with EcoRI, both fragments were ligated with T4 DNA ligase (Takara Shuzo) to obtain a recombinant plasmid pKP39 encoding the polypeptide chain of glycoprotein 39 of the present invention. .

The obtained recombinant plasmid pKP39 was transformed with E. c.
oli JM83 competent cells were transduced.

(6) Construction of restriction enzyme map: p obtained in (5)
KP39 (Molecular Cloning (A Laboratory Manual);
T. Maniatis, EF Fritsch, J. Sambrook; Cold Spr
ing Harbor Laboratory (1982) p.104-106], and a restriction enzyme map of the pKP39 clone encoding the glycoprotein 39 of the present invention is prepared according to the method of the above-mentioned documents p.374-p.381. (Fig. 1).

(7) Determination of nucleotide sequence of pKP39 clone: pKP3
9 The nucleotide sequence of the clones was determined by the method of Sanger et al. [Sanger F., Nicklen S. & Coulson AR, Proc.
Natl. Acad. Sci. USA, 74 , p.5463-5467 (1977)].

Glycoprotein 39 obtained from the above results
The gene sequence is composed of about 1900 bases in total including the translated region and the 3'-untranslated region. Of these, about 600 bases from the 5'end is a repeating sequence region having 60 bases as one unit. It was found that the translation region including this region is about 1560 bases long and encodes a protein portion of about 553 amino acids. However, the sequence of the repetitive sequence region could be determined 180 bases (which can encode 60 amino acid residues) (SEQ ID NO: 1), but other repetitive sequences have not been determined. The sequence of 1320 bases underlined from the repeated sequence was determined and is shown in SEQ ID NO: 2.

Example 4 (1) Preparation of total RNA and poly (A) ⁺ RNA Total RNA was extracted from gastric cancer cell line KATO-III according to the guanidinium isothiocyanate method described in Example 3- (1),
In addition, a commercially available oligo (dT) cellulose column (Colaborati
Ve Research Inc.) was used to prepare poly (A) ⁺ RNA (see Molecular Cloning p. 196-198 above).

(2) 20 μg of total RNA prepared in Northern blotting (1) or 10 μg of poly (A) ⁺ RNA was added to the above M
After denaturing by heating at 50 ° C for 1 hour in the presence of glyoxal according to the method of olecular Cloning (p.200-201), it was applied to a 1% agarose gel containing 10 mM sodium phosphate solution at 90V for 3-4. Electrophoresis was performed for a period of time. Next, the separated RNA was transferred to a nitrocellulose filter (Schlier and Shell) in 20 × SSC for 15 hours. After RNA transfer, dry the nitrocellulose filter at room temperature, bake at 80 ℃ for 2 hours to fix it, and then fix it at 20 mM.
Glyoxal was removed by heating in Tris-HCl buffer (pH 8.0) at 100 ° C. for 5 minutes. This filter was shaken in the prehybridization solution described in Example 3- (7) at 42 ° C. for 3 hours, and then the hybridization solution containing the α- ³² P-dCTP-labeled probe (composition other than the probe was used. It was transferred to the same as the hybridization solution) and shaken at 42 ° C. for 20 hours. The probe is pKP39
A fragment obtained by cleaving the cDNA of the clone with the restriction enzyme EcoRI was labeled with α- ³² P-dCTP using a multiprime DNA labeling system (Amersham) to give 0.5 to 1 × 10 ⁷ cp.
Used at a concentration of m / ml. After hybridization, transfer the filter to 2 x SSC-0.1% SDS solution and wash 3 times at room temperature for 10 minutes each, and further wash 0.1 x SSC-0.1% SDS solution at 60 ° C for 30 minutes 3 times each. After that, it was dried at room temperature. Put the filter on the filter paper, put it in the X-ray film cassette, and stack the X-ray film (Konica XAR-5)-
It was exposed to light at 70 ° C. for 1 to 3 days.

The obtained northern blotting results are shown in FIG.

28S and 18S ribosomal RNAs were used as RNA molecular weight markers. As a result, two mRNAs having a length of 4400 bases and a length of 6800 bases were detected. It is considered that this is caused by alternative splicing as well as known PEM mRNA.

[0065]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 180 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Origin Biological name: Homo sapiens Cell type: Gastric signet ring cell carcinoma Cell Line: KATO-III Direct origin Library name: λgt11 KATO-III cDNA library Clone name: λKP39 Sequence characteristics Characteristic symbol: mat peptide Location: 1..180 Method of determining characteristic: S Characteristic symbol : Repeat region Location: 1..180 Characteristic determination method: S Characteristic symbol: repeat unit Location: 1..60 Characteristic determination method: S Sequence GGC TCC ACC GCC CCC CCA GCC CAC GGT GTC ACC TCG GCC CCG GAG AGC 48 Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Glu Ser 1 5 10 15 AGG CCG GCC CCG GGC TCC ACC GCG CCC GCA GCC CAC GGT GTC ACC TCG 96 Arg Pro Ala Pro Gly Ser Thr Ala Pro Ala Ala His Gly Val Thr Ser 20 25 30 GCC CCG GAG AGC AGG CCG GCC CCG GGC TCC ACC GCG CCC GCA GCC CAC 144 Ala Pro Glu Ser Arg Pro Ala Pro Gly Ser Thr Ala Pro Ala Ala His 35 40 45 GGT GTC ACC TCG GCC CCG GAC ACC AGG CCG GCC CCG 180 Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro 50 55 60

SEQ ID NO: 2 Sequence length: 1320 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Origin organism name: Homo sapiens Cell type: Gastric signet ring Cellular cancer Cell line: KATO-III Direct origin Library name: λgt11 KATO-III cDNA library Clone name: λKP39 Sequence features Characteristic symbols: mat peptide Location: 1..981 Method of determining features: S features Symbol for expressing: polyA signal Location: 1267..1272 Method for determining feature: S Feature for expressing: polyA site Location: 1293..1320 Method for determining: S sequence GGC TCC ACC GCC CCC CCA GCC CAC GGT GTC ACC TCG GCC CCG GAC ACC 48 Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr 5 10 15 AGG CCC GCC TTG GGC TCC ACC GCG CCT CCA GTC CAC AAT GTC ACC TCG 96 Arg Pro Ala Leu Gly Ser Thr Ala Pro Pro Val His Asn Val Thr Ser 20 25 30 GCC TCA GGC TCT GCA TCA GGC TCA GCT TCT ACT CTG GTG CA C AAC GGC 144 Ala Ser Gly Ser Ala Ser Gly Ser Ala Ser Thr Leu Val His Asn Gly 35 40 45 ACC TCT GCC AGG GCT ACC ACA ACC CCA GCC AGC AAG AGC ACT CCA TTC 192 Thr Ser Ala Arg Ala Thr Thr Thr Pro Ala Ser Lys Ser Thr Pro Phe 50 55 60 TCA ATT CCC AGC CAC CAC TCT GAT ACT CCT ACC ACC CTT GCC AGC CAT 240 Ser Ile Pro Ser His His Ser Asp Thr Pro Thr Thr Leu Ala Ser His 65 70 75 80 AGC ACC AAG ACT GAT GCC AGT AGC ACT CAC CAT AGC ACG GTA CCT CCT 288 Ser Thr Lys Thr Asp Ala Ser Ser Thr His His Ser Thr Val Pro Pro 85 90 95 CTC ACC TCC TCC AAT CAC AGC ACT TCT CCC CAG TTG TCT ACT GGG GTC 336 Leu Thr Ser Ser Asn His Ser Thr Ser Pro Gln Leu Ser Thr Gly Val 100 105 110 TCT TTC TTT TTC CTG TCT TTT CAC ATT TCA AAC CTC CAG TTT AAT TCC 384 Ser Phe Phe Phe Leu Ser Phe His Ile Ser Asn Leu Gln Phe Asn Ser 115 120 125 TCT CTG GAA GAT CCC AGC ACC GAC TAC TAC CAA GAG CTG CAG AGA GAC 432 Ser Leu Glu Asp Pro Ser Thr Asp Tyr Tyr Gln Glu Leu Gln Arg Asp 130 135 140 ATT TCT GAA ATG TTT TTG CAG ATT TAT AAA CAA GGG GGT TTT CTG GGC 480 Ile Ser Glu Met Phe Leu Gln Ile Tyr Lys Gln Gly Gly Phe Leu Gly 145 150 155 160 CTC TCC AAT ATT AAG TTC AGG CCA GGA TCT GTG GTG GTA CAA TTG ACT 528 Leu Ser Asn Ile Lys Phe Arg Pro Gly Ser Val Val Val Gln Leu Thr 165 170 175 CTG GCC TTC CGA GAA GGT ACC ATC AAT GTC CAC GAC GTG GAG ACA CAG 576 Leu Ala Phe Arg Glu Gly Thr Ile Asn Val His Asp Val Glu Thr Gln 180 185 190 TTC AAT CAG TAT AAA ACG GAA GCA GCC TCT CGA TAT AAC CTG ACG ATC 624 Phe Asn Gln Tyr Lys Thr Glu Ala Ala Ser Arg Tyr Asn Leu Thr Ile 195 200 205 TCA GAC GTC AGC GTG AGT GAT GTG CCA TTT CCT TTC TCT GCC CAG TCT 672 Ser Asp Val Ser Val Ser Asp Val Pro Phe Pro Phe Ser Ala Gln Ser 210 215 220 GGG GCT GGG GTG CCA GGC TGG GGC ATC GCG CTG CTG GTG CTG GTC TGT 720 Gly Ala Gly Val Pro Gly Trp Gly Ile Ala Leu Leu Val Leu Val Cys 225 230 235 240 GTT CTG GTT GCG CTG GCC ATT GTC TAT CTC ATT GCC TTG GCT GTC TGT 768 Val Leu Val Ala Leu Ala Ile Val Tyr Leu Ile Ala Leu Ala Val Cys 245 250 255 CAG TGC CGC CGA AAG AAC TAC GGG CAG CT G GAC ATC TTT CCA GCC CGG 816 Gln Cys Arg Arg Lys Asn Tyr Gly Gln Leu Asp Ile Phe Pro Ala Arg 260 265 270 GAT ACC TAC TAC CAT CCT ATG AGC GAG TAC CCC ACC TAC CAC ACC CAT GGG 864 Asp Thr Tyr His Pro Met Ser Glu Tyr Pro Thr Tyr His Thr His Gly 275 280 285 CGC TAT GTG CCC CCT AGC AGT ACC GAT CGT AGC CCC TAT GAG AAG GTT 912 Arg Tyr Val Pro Pro Ser Ser Thr Asp Arg Ser Pro Tyr Glu Lys Val 290 295 300 TCT GCA GGT AAT GGT GGC AGC AGC CTC TCT TAC ACA AAC CCA GCA GTG 960 Ser Ala Gly Asn Gly Gly Ser Ser Leu Ser Tyr Thr Asn Pro Ala Val 305 310 315 320 GCA GCC ACT TCT GCC AAC TTG TAGGGGCACG TCGCCCGCTG AGCTGAGTGG 1011 Ala Ala Ser Ala Asn Leu 325 327 CCAGCCAGTG CCATTCCACT CCACTCAGGT TCTTCAGGGC CAGAGCCCCT GCACCCTGTT 1071 TGGGCTGGTG AGCTGGGAGT TCAGGTGGGC TGCTCACACC GTCCTTCAGA GGCCCCACCA 1131 ATTTCTCGGA CACTTCTCAG TGTGTGGAAG CTCATGTGGG CCCCTGAGGC TCATGCCTGG 1191 GAAGTGTTGT GGTGGGGGCT CCCAGGAGGA CTGGCCCAGA GAGCCCTGAG ATAGCGGGGA 1251 TCCTGAACTG GACTGAATAA AACGTGGTCT CCCACTGCGC CAAAAAAAAA AAAAAAAAAA 1311 A AAAAAAAA 1320

[Brief description of drawings]

FIG. 1 shows a restriction enzyme map of a cDNA encoding a core protein of glycoprotein 39 of the present invention and a method for determining a nucleotide sequence. In Figure 1, the scale shown at the top is the length of the nucleotide based on the first base of the cDNA (kilobase).
Is. The lower row shows a cDNA clone pKP39 encoding the glycoprotein 39 of the present invention. The broken line on the left side of the line indicates a region encoding a repetitive sequence with 20 amino acid residues as one unit, and the thick black line in the center indicates the subsequent coding region. Arrows indicate the direction and length of the base sequence determined for each DNA fragment.

FIG. 2 is a drawing showing Northern blotting of mRNA encoding the protein of the present invention in Example 4. 44
There are two mRNAs of 00 base length and 6800 base length.

Claims (3)

[Claims] 1. A glycoprotein 39 gene. 2. The gene according to claim 1, which comprises a base sequence encoding the amino acid sequence represented by SEQ ID NO: 1 and a base sequence encoding the amino acid sequence represented by SEQ ID NO: 2. 3. The gene according to claim 1, which contains the base sequence of the 5'end portion shown in SEQ ID NO: 1 and the base sequence of the 3'end portion shown in SEQ ID NO: 2.