JPH09275983A - P53 target protein gml - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new DNA that codes for a p53 target protein GML having a specific amino acid sequence, whose expression is induced by a cancer- inhibitory gene p53, and which is useful for judgment of functions of p53, diagno sis of cancer and the like from the degree of manifestation of the GML protein or the DNA. SOLUTION: This new DNA that codes for a p53 target protein GML, which contains an amino acid sequence expressed by the formula, and whose expression is induced by a cancer-inhibitory gene p53 is obtained by screening a human genomic DNA cosmid library by the use of DNA fragments that contain a p53 responsive sequence labeled with a radioisotope as a probe and by a colony hybridization method, and by amplifying exons on both sides of the obtained cosmid clone C169.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel gene whose expression is induced by the tumor suppressor gene p53 and belongs to the field of medicine.

[0002]

2. Description of the Related Art p53 was discovered in 1979 as an intracellular protein that binds to an oncovirus protein. Since a mutation in the p53 gene was found in colorectal cancer and lung cancer in 1989, and the mutation was subsequently found in various types of cancer such as breast cancer, bladder cancer, and brain tumor, it is involved in the development and progression of cancer. It is considered. Although the mechanism of action of p53 has been unclear for a long time, it is speculated that it functions as a tumor suppressor gene because it has the ability to suppress the growth of cancer cells when forced expression of normal p53 is carried out.

As a physiological function of p53, the cell cycle G
The arresting action in the 1st phase and the apoptotic action due to DNA damage are known, but the mechanism of action has been unclear for a long time. However, recently, it was revealed that p53 has an activity as a transcription factor, and it was speculated that the gene group whose expression is induced by p53 is directly involved in the physiological function of p53 that is suppression of cell growth. Subsequent studies showed that p21 /
WAF1, MDM2, GADD45, BAX, cyclin G, IGF-BP3, etc. have been reported (El-Deiry,
WS et al., Cell, 75 , 817-825,1993 .Wu, X. et a
l., Genes Dev., 7 , 1126-1132,1993 .Kastan, MB e
t al., Cell, 71 , 587-597, 1992. Miyashita, T. & Ree
d, CR Cell, 80 , 293-299, 1995. Okamoto, K. & Beac
h, D. EMBO J., 13 , 4816-4822,1994. Buckbinder, L.
et al., Nature, 377 , 646-649, 1995.). These genes were found to have p53 binding sequences in the promoter region or flanking regions, and their expression was shown to correlate with the expression of p53 protein.

In order to identify a p53 target gene, a normal p53 is introduced into a cancer cell deficient in a normal p53 and forcedly expressed, and a cDNA having an increased expression level is subtracted by a subtraction method. Screening and screening methods by differential display (differential amplification) have been used. These methods can identify genes that are expressed in a relatively large amount, but it is difficult to identify genes that are expressed in a trace amount and genes that have organ specificity.

With the progress of molecular biology, it has been considered that the cause of cancer is the stepwise accumulation of mutations in a plurality of genes involved in cell growth control in a single somatic cell. It has been desired to elucidate a novel target gene of the tumor suppressor gene p53, elucidate the mechanism of carcinogenesis, and apply it to medicine.

[0006]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a new target gene for p53.

[0007]

[Means for Solving the Problems] By the screening method using yeast (Saccharomyces cerevisiae), a large number of human genomic sequences (p53 responsive sequences) whose transcription is promoted by p53 have been identified so far (Tokino, T. et.
al., Hum. Mol. Genet., 3 , 1537-1542, 1994). Most of these sequences had two copies of 5'-PuPuPuC (A / T) (T / A) GPyPyPy-3 ', which is a consensus sequence for the p53 binding site (El-Deiry, WS et al., NatureGenet.,
1 , 45-49, 1992).

[0008] Therefore, the present inventors diligently screened a human cosmid library using the p53 responsive element as a probe to identify a gene containing the p53 responsive element and expressed in a p53-dependent manner. As a result of isolating the DNA, amplifying the exon portion, and analyzing the structure, a new target gene of p53 was successfully identified, and the present invention was completed. The amino acid sequence deduced from the DNA of the present invention was searched for homology in a database, and as a result, a glycosylphosphatidylinositol (GPI) anchor molecule (Br
akenhoff, RH et al., J. Cell Biol., 129 , 1677-16
89, 1995) and has a remarkable similarity to GML.
It was named (G PI anchor m olecular l ike protein) proteins.

That is, the present invention provides a protein containing all or part of the amino acid sequence of SEQ ID NO: 1 and a DNA encoding the protein. The present invention relates to the DNA-containing vector, transformant, method for producing the protein, DNA probe derived from the DNA, primer, method for analyzing the gene, and antibody binding to the protein. Of the amino acid sequences of the GML protein, those containing all or part of the amino acid sequence in which one or more amino acids have been added, deleted or substituted, and the DNA encoding the same are also included in the present invention.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The present inventors isolated a p53 responsive element from human genomic DNA using the transcriptional activation ability of p53 in yeast as an index (Tokino, J. et al., Human Mol. Ge.
net., 3 , 1537-1542, 1994). That is, human genome D
A DNA fragment obtained by treating NA with a restriction enzyme MboI was inserted upstream of HIS3 reporter plasmid pBM947 to prepare a reporter library, and the reporter plasmid was introduced into yeast having a plasmid expressing human p53. Using the requirement as an index, 1000 clones were isolated. It was reported that about 1/4 of these clones have a consensus sequence that promotes transcription in a p53-dependent manner.

From these clones, p53 was digested with a restriction enzyme.
The p53 responsive sequence having the consensus sequence of SEQ ID NO: 3 was labeled and used as a probe from among those obtained by excising the response sequence from the structural analysis, and the human genome cosmid library (Gene, 10 , 63, 1980) was used for colony hybridization. Tokino, T. et al., Am. J. Hum. Ge
net., 48 , 258-268, 1991), and a cosmid clone that hybridizes with the probe can be obtained. By subcloning the thus obtained cosmid clone into an appropriate plasmid vector and determining the nucleotide sequence, the genomic nucleotide sequence of the p53 responsive gene can be determined.

The cosmid clone thus obtained is digested with restriction enzymes such as Bam HI and Bgl II, the obtained DNA fragment is inserted into a trapping vector such as pSPL3, and the exon portion is amplified by the exon trapping method. , Isolation and structural analysis of exon sequences,
Exon sequences can be obtained by homology search against public databases such as GenBank. This exon sequence is then added to po prepared from human tissue, such as testis.
Using ly (A) RNA as a template, the target cD is obtained by extending both sides of the exon by the 5'- and 3'-RACE method.
NA can be obtained. The intron-exon junction is analyzed by comparing the structure-determined nucleotide sequences of these cDNAs and genomic clones.

The structure of the nucleotide sequence is the Maxam-Gilbert method (Maxam, AM and Gilbert, W., Proc. Natl. Acad. S
ci. USA, 74, 560, 1977) or dideoxy method (Messing, J et al., Nucl. Acids Res., 9, 309, 198).
1) determined by. The GML protein recombinant expression vector and its transformant can be prepared by incorporating the GML cDNA obtained by the above-mentioned method into an appropriate vector and transferring the vector into an appropriate host cell. it can. This can be cultured by a conventional method to produce a large amount of GML protein from the culture.

CDNA encoding the GML protein was GM
A recombinant expression vector can be prepared by recombining by a known method using a restriction enzyme and DNA ligase downstream of the promoter of a vector suitable for L protein expression. Vectors that can be used include E. coli-derived plasmid p
BR322, pUC18, plasmid pUB11 derived from Bacillus subtilis
0, yeast-derived plasmid pRB15, bacteriophage λgt10, λgt11 or SV40.
The vector is not particularly limited as long as it can be replicated and amplified in the host. The promoter and the terminator are not particularly limited as long as they correspond to the host used for expressing the nucleotide sequence encoding the GML protein, and an appropriate combination is also possible depending on the host.

The cDNA encoding the GML protein is GM
Any DNA may be used as long as it encodes L protein, or it may be one synthesized by chemical synthesis. Further, if the expressed protein has the physiological activity of GML protein, it is not limited to the base sequence shown in SEQ ID NO: 2, and a part of the amino acid sequence is added, deleted or substituted, and is substantially It may be a DNA encoding an equivalent protein.

[0016] The recombinant expression vector thus obtained is used in the competent cell method (J. Mol. Biol., 53, 154, 197).
0), protoplast method (Proc. Natl. Acad. Sci. USA,
75, 1929, 1978), calcium phosphate method (Science, 22
1, 551, 1983), in vitro packaging method (Porc.
Natl, Acad. Sci. USA, 72, 581, 1975), virus vector method (Cell, 37, 1053, 1984) and the like are introduced into a host to prepare a transformant. As a host, Escherichia coli, Bacillus subtilis, yeast, insect cells, animal cells and the like are used, and the obtained transformant is cultured in an appropriate medium according to the host. Cultivation is usually carried out at a temperature of 20 ° C to 45 ° C and a pH of 5 to 8, and aeration and agitation are carried out if necessary. Separation / purification of the GML protein from the culture may be carried out by appropriately combining known separation / purification methods. Examples of these known methods include salting out, solvent precipitation, dialysis gel filtration, electrophoresis, ion exchange chromatography, affinity chromatography, reverse phase high performance liquid chromatography and the like. The GML protein thus obtained is expected to exhibit an activity of suppressing the growth of the cells when injected into the cells by the microinjection method.

Whether or not the GML gene is expressed in a tissue or cell is determined by the mRNA or total R obtained from the tissue or cell.
It can be examined by analyzing NA by RT-PCR. For example, a guanidine thiocyanate method (Chirgwin, JM et al., Biochemistry, 1
8, 5294, 1979) etc. to extract RNA. If necessary, the total RNA obtained can be further purified by chromatography using an oligo (dT) cellulose column to prepare mRNA. Single-stranded cDNA is synthesized by reverse transcriptase using the obtained RNA as a template and oligo (dT) or random hexadeoxynucleotide as a primer. This single chain c
A suitable primer may be selected from GML cDNA using DNA as a template, and PCR may be performed to measure the amount of PCR product.

When measuring the amount of mRNA, the extracted m
It can also be measured by performing a gel electrophoresis on a gel, and then performing a Northern blot analysis in which the RNA is transferred to a membrane and hybridized with a labeled GML DNA probe.
The exon trapping method may follow the method of Buckler et al. (Buckler, AJ et al., Proc. Natl. Acad. Sci.
USA, 88 , 4005-4009, 1991). For example, human cosmid DNA was completely cleaved with Bam HI and Bgl II restriction enzymes, cloned into the Bam HI site of splicing vector pSPL1, transformed into E. coli DH5α, and the plasmid DNA was recovered and Cos 7 Transfection (transformation) is performed on the cells by electroporation. Then, after about 48 hours, cytosolic RNA was prepared from Cos 7 cells, RT-PCR was performed using primers designed based on the DNA sequence of the vector,
Furthermore, unknown exons can be amplified by performing 2nd-PCR with the inner primers. The obtained exon part can be cloned into a vector such as pBluescript II and the nucleotide sequence can be determined to obtain the target exon. In the RACE method, 5'-RACE that amplifies the 5'side and 3'-RACE that amplifies the 3'side of a known portion of a cDNA sequence using mRNA as a template.
Can be done by the method of Frohman et al. (Fr
ohman, MA et al., Proc. Natl. Acad. Sci. USA, 8
5, 8998-9002, 1988).

Antibodies can be prepared by inoculating a GML protein as an antigen in accordance with a conventional method, for example, subcutaneously, intramuscularly, intraperitoneally, or intravenously inoculating a plurality of animals such as mice, guinea pigs, rabbits, etc. Blood collection and serum separation from anti-GM
L polyclonal antibodies can be made. In addition,
Commercially available adjuvants can also be used. The monoclonal antibody can be produced by a known method. For example, preparing a hybridoma obtained by cell fusion of splenic cells of a mouse immunized with GML protein and commercially available mouse myeloma cells, and then preparing an anti-GML monoclonal antibody from the hybridoma culture supernatant or ascites of the hybridoma-administered mouse. You can The GML protein used as an antigen does not have to have the entire amino acid structure, and may be a peptide having a partial structure or a fusion protein with another peptide. The preparation method may be a biological method or a chemical synthesis method. These antibodies enable the identification and quantification of GML protein in human biological samples and are expected to be used as cancer diagnostic reagents. GML
The method for immunologically measuring the protein may be based on a known method, for example, a fluorescent antibody method, a passive agglutination method, an enzyme antibody method, or the like.

The above method can be implemented by a known method.

[0021]

The present invention will be described in detail and specifically with reference to the following examples, but the present invention is not limited to these examples. Embodiment 1 FIG. Cloning of exon sequences from genomic DNA and analysis of nucleotide sequences. p53 response element (Tokino, T. et al., Hum. Mol. Genet.,
3 , 1537-1542, 1994) was radiolabeled and the human genomic DNA cosmid library was screened by the colony hybridization method according to a conventional method to obtain cosmid clone c169.
The DNA of this clone was cleaved with Bam HI and Bgl II, inserted into exon trap vector pSPL3 (Gibco-BRL) and exon trapped to isolate one exon sequence.

When the obtained exon sequences were compared with sequences in public databases by homology search using the BLAST algorithm, one of GPI-anchored proteins, E48 (Brakenhoff, RH et al., J. Cell Biol.,
129 , 1677-1689, 1995) and its amino acid sequence showed remarkable homology. So this new gene
(GPI-anchored molecule-like protein).
The GML protein of the present invention is E, which is a membrane protein having a GPI anchor, as analyzed by the BLASTA program.
It was confirmed to have homology with 48 antigens.

The DNA sequencing was carried out using a double-stranded DNA as a template and an ABI377 type DNA sequencer (Applied Biosystems) using a cycle sequencing kit (using dye-terminator, Perkin Elmer) using AmpliTaq FS DNA polymerase. Confirm the DNA sequence by Sequenase Version 2.0 (Unit
edStates Biochemical) was used for manual sequencing with ³⁵ S-label.

Embodiment 2 FIG. Structure of GML gene The exon portion obtained in Example 1 was replaced by RA of Froman et al.
It was extended to both sides by the CE method. 5'- and 3'-R
ACE uses testicular poly (A) RNA (Clontech),
This was done using a Clontech kit. Furthermore, the total genomic DNA sequence contained in cosmid clone c169 was determined, and a sequence of possible exons was searched using a computer program. Search by GRAIL and 5 '
By combining the studies of -RACE and 3'-RACE, almost full-length transcript of 722 bp (Nort
In hern blot analysis, the size of the transcript was estimated to be 0.9 kb including the poly signal). First A
The TG codon was present at base number 90 and the stop codon was at base number 564 (SEQ ID NO: 2). 474 bp ORF
The 17kd translation product is encoded by (translation region). Genomic DNA sequencing revealed that a functional p53 binding site was located 19 kb upstream of the coding sequence. Up to 10 out of 11 cysteine residues possessed by the GML protein were conserved in the family of proteins having these GPI anchors. GML gene is 8q on the chromosome
A GP that maps to 24.3-q ter and includes E48 and GML
It was suggested that genes of the protein family having I-anchors might be gathered in this region.

Embodiment 3 FIG. Confirmation of expression induction of wild-type p53 of GML gene (mRNA) In order to confirm whether GML is induced by wild-type p53, a cell line expressing a wild-type or mutant p53 gene was prepared, and from the cells, RT-
Analysis by PCR was performed. In the colon cancer cell line SW480, one p53 allele is a mutant type (273 Arg to His),
It is known that the other allele is deleted and the growth of this strain is suppressed by introducing a vector expressing wild-type p53.

Therefore, first, wild type or mutant type (Kern, S. et al., Science, 256 , 827-83) was added to this SW480.
0, 1992) transfected with the p53 expression vector. 1 × 10 ⁶ cells were seeded in a 25 cm ² flask and cultured for 24 hours, followed by transfection using 25 μg of lipofectin and 5 μg of plasmid DNA. Total R from these cells using TRizol reagent (Gibco-BRL)
NA was extracted, digested with DNase I (Boeringer Mannheim), and then cDNA was synthesized using Superscript II (Gibco-BRL) and used as a template for PCR. The PCR reaction was performed using 12.5μ of cDNA generated from 200ng of total RNA.
In addition to the reaction solution of l, 94 ℃, after denaturation for 2 minutes, 94 ℃,
It was repeated for 25 to 35 cycles of 30 seconds, 55-60 ° C, 30 seconds, 72 ° C and 1 minute. PCR amplification product is 3% NuSieve
Analyzed by electrophoresis on GTG 2: 1 agarose gel. The sequences of the primers used are SEQ ID NOS: 4 and 5
It described in. These correspond to the 73-93th position of the sense code and the 247-268th position of the antisense code. At this time, the expression level of p53 target gene p21 / WAF1 was also measured, and SEQ ID NOS: 6 and 7 were used as primers. As a control gene, the expression level of GAPDH was measured, and SEQ ID NOs: 8 and 9 were used as primers.

As a result, as shown in FIG. 1, the GML mR
NA was more strongly expressed in cells transfected with the wild-type p53 expression vector as was p21 / WAF1. Embodiment 4. FIG. GML in normal tissues and esophageal cancer cell lines
Expression of mRNA. Human GMLc labeled with ³² P using poly (A) RNA Northern blot membrane (Clontech) of various human tissues.
Hybridization was performed using DNA (73-673th position DNA described in SEQ ID NO: 2, 601 bp) as a probe. Hybridization was performed as instructed by Clontech, and washing was performed with 0.1 × SSC and 0.1% SDS at 50 ° C. for 20 minutes. RT-PCR analysis of total RNA of the esophageal cancer cell line was performed by the method described in Example 3. The PCR reaction was performed at least twice to confirm the result. RNA integrity was confirmed by amplification of a positive control GAPDH gene transcript that showed similar signals in all samples. GAP for this
PCR primers for the DH gene are shown in SEQ ID NOs: 8 and 9.

As a result of the measurement, mRN from 16 kinds of normal tissues was obtained.
Approximately 0.9 kb GMLmR from Northern blot analysis of A
It was revealed that NA was expressed in the testis. Next, a panel of esophageal cancer cell lines (Nishihira, T. et al., J. C.
ancer Res. Clin. Oncol., 119 , 441-449, 1993) was used to analyze G in 11 cell lines by RT-PCR analysis.
The expression of ML was examined. As a result, as shown in FIG. 2, 4 out of 11 strains expressed GML mRNA, and 1 strain weakly expressed GML mRNA.

Example 5. Phase of anticancer drug sensitivity and GML expression
Seki. Depending on whether or not GML is expressed, resistance to anticancer drugs
I examined how sex changes. Esophageal cancer cell line used
1 x 10 per 100mm dish 24 hours before drug addition ⁶ Individual details
Plate the cells. Bleomycin for 24 hours
The viability of the cells was measured 4 days after the addition. Each
Points are averages from at least 3 independent experiments
Values, all values are the corresponding unprocessed
Shown as relative survival relative to control.
The properties of the esophageal cancer cell line used are shown in Table 1. So
As a result, as shown in FIG. 3, five kinds of GML expressing GML were expressed.
Esophageal cancer cell lines were sensitive to bleomycin.
On the other hand, if the expression of GML gene was not recognized by RT-PCR
6 cell lines were compared to the same bleomycin treatment.
It was relatively resistant. Note that similar results are obtained for CDDP.
Shown (Fig. 3).

Thus, the fact that the expression of GML, drug sensitivity and resistance were clearly correlated in esophageal cancer cell lines suggests that GML plays an important role in the p53-induced apoptosis pathway. Was done.

[0031]

[Table 1]

Example 6 Preparation of Transformant Primers GML-A and GML using the DNA (SEQ ID NO: 2) encoding the GML protein (SEQ ID NO: 1) as a substrate.
-Using B, a DNA encoding a part of the MDC protein
The fragment is amplified by PCR. The sequences of the primers used are as follows. GML-A 5'-CACAGATCTGTGAAGTGATGCTCCTCT- 3 '
(Coding strand, corresponding to nucleotide numbers 82-99 of SEQ ID NO: 2) GML-B 5'-AACAAGCT TCA TGGCAATATATTGCT-3 '(
(Antisense strand, corresponding to nucleotide numbers 548-566 of SEQ ID NO: 2, underlined indicates stop codon)
The cleavage site sequences of gl II and Hind III are added.

The PCR amplification product was separated by agarose gel electrophoresis and cleaved with BglII and HindIII. DNA encoding a part of the GML protein thus obtained
The fragment was previously digested with BamH I and Hind III p
The plasmid pMAL-GML is constructed by ligating to the MAL-c2 (New England Biolabs) vector. Similarly, in advance Bam
The plasmid pH6-GML is constructed by ligating to the pQE-13 (Diagen) vector that had been cut with HI and Hind III.

The fragment incorporated into the pMAL-c2 vector was
Since it is expressed as a fusion protein having maltose binding protein (MBP) on the N-terminal side, the fusion protein is affinity-purified by an amylose column. Also,
Since the fragment incorporated into the pQE-13 vector is expressed as a fusion protein having a peptide (His 6) consisting of 6 histidine residues on the N-terminal side, the fusion protein is affinity purified by a metal chelate column.

E. coli JM109 is transformed with each of the plasmids pMAL-GML and pH6-GML, and selected with ampicillin resistance to obtain respective transformants. Example 7 Expression and Purification of Recombinant MDC Protein Each transformant obtained in Example 6 is cultured, and recombinant MDC fusion protein is extracted and purified from the culture.

That is, each transformant was treated with 100 ml of LB.
In medium (1% polypeptone, 0.5% yeast extract, 1% NaCl)
Incubate with shaking at 37 ℃ overnight. The culture broth was diluted 10-fold with LB medium preheated to 37 ° C, and then for 30 minutes to 9 minutes.
Incubate for 0 minutes to obtain a culture in the logarithmic growth phase. IPTG (Isopropyl-beta-D-thiogalactopyr) was added to 1 liter of the culture.
anoside) to a final concentration of 1 mM to add 3-4
Cultured for hours. The cells are collected from the culture by centrifugation.

In the case of the transformant using the plasmid pMAL-GML, the cells were added with 10 ml of column buffer (20 mM TriM).
Add s-HCl pH7.4, 200mM NaCl) and disrupt by sonication. Since the recombinant GML fusion protein exists in the insoluble fraction of the disrupted solution, it is centrifuged and dissolved in a denaturing buffer (8M urea, 20 mM Tris-HCl pH8.5, 10 mM dithiothreitol). Then, this is dialyzed against a column buffer and then centrifuged to collect a supernatant soluble fraction. The dialyzed insoluble fraction is further subjected to denaturation, dialysis and centrifugation to collect a supernatant soluble fraction. The collected soluble fraction is applied to an amylose column (New England Biolabs), washed with a column buffer, and then eluted with a column buffer containing 10 mM maltose. The elution fraction is analyzed and fractionated by absorbance at 280 nm and SDS polyacrylamide gel electrophoresis (Coomassie blue staining). As a result, a fraction in which the GML fusion protein having the expected molecular weight is detected as a major band is obtained in each of the transformants using the plasmid pMAL-GML. Hereinafter, these fusion proteins will be referred to as MBP-GML.

Similarly, in the case of a transformant using the plasmid pH6-GML, 10 ml of sonication buffer (10 mM sodium phosphate pH8.0, 200 mM NaCl) is added to the cells and disrupted by sonication. Since the recombinant GML fusion protein exists in the insoluble fraction of the lysate, it was centrifuged and buffer A (6M guanidinium hydrochloride, 100 mM NaH ₂ PO ₄ , 10 mM Tr was added.
is-HCl, pH8.0) and centrifuged to collect the supernatant-soluble fraction, which was applied to a Ni-NTA column (manufactured by Diagen), buffer A, and then buffer B (8M urea, 100 mM NaH ₂
After washing with PO ₄ , 10mM Tris-HCl, pH8.0, buffer C
(8M _{Urea, 100mMNaH 2 PO 4, 10mMTris-} HCl, pH6.3), buffer D (8M _{urea, 100mM NaH 2 PO 4, 10mMTris} -HCl, p
H5.9), Buffer E (8M Urea, 100mM NaH ₂ PO ₄ , 10mMT
ris-HCl, pH4.5) and buffer F (6M guanidine hydrochloride,
Elute stepwise with 200 mM acetic acid. The elution fraction is 28
The absorbance is analyzed at 0 nm and analyzed by SDS polyacrylamide gel electrophoresis (Coomassie blue staining) to fractionate. As a result, about 34
A fraction is obtained in which the His6 fusion protein of Kd is detected as a single band. This fusion protein is hereinafter referred to as His 6-GML.

Example 8 Production of Monoclonal Antibody and Rabbit Polyclonal Antibody His 6-G, two recombinant fusion proteins obtained in Example 7
ML and MBP-GML are used as an immunogen, an antibody for antibody purification / screening, and a standard antigen for measurement, respectively. Anti-G
The ML protein-specific monoclonal antibody is prepared by immunizing a mouse with His 6 -GML. That is, His 6-GML 3M
A urea / PBS solution (500-1000 ug / ml) is mixed with a complete adjuvant at a ratio of 1: 1 and the mouse is intraperitoneally immunized with 100 ug / mouse 4 to 6 times at 2-week intervals. After completion of immunization, hybridomas of P3U1 cells and B cells are prepared using PEG1500, the antibody titer in the culture supernatant is monitored, and hybridomas producing anti-GML protein-specific antibodies are selected. The antibody titer was measured by immobilizing the MBP-GML fusion protein obtained in Example 7 (5 ug / ml).
100 ul of culture supernatant was added to the polystyrene cup to perform the first reaction, and after washing, anti-mouse IgG-HRP (Ho
rse-raddish peroxidase) is added and the second reaction is performed. After washing, enzyme substrate solution (hydrogen peroxide solution and ABTS
(2,2′-azino-bis- (3-ethylbenzothiazoline-6-sulfonic acid) mixed solution) is added, and a color reaction (third reaction) is performed and monitored.

Hybridomas are cultured in a 96-well multiplate, HAT selection is performed, and after about 2 weeks, the antibody titer in the culture supernatant is measured to select clones that react specifically with the antigen. Furthermore, the cloning operation was performed, and 0.5 ml of each hybridoma cell was preliminarily prepared in a volume of 0.5 ml approximately one week before.
Is inoculated into the BALB / c mouse which has been intraperitoneally administered, and ascites is collected 8 to 10 days later. The antibody is purified from each ascites by affinity chromatography with a protein G column.

Similarly, a rabbit is immunized with His6-GML obtained in Example 7 as an immunizing antigen to prepare an anti-GML protein polyclonal antibody. That is, like a mouse, H
is6-GML 3M urea / PBS solution (500-1000u
g / ml) is mixed with complete adjuvant at a ratio of 1: 1 for immunization. After completion of immunization, antiserum is obtained, and the antibody titer is measured using a polystyrene cup on which the MBP-GML fusion protein obtained in Example 7 is immobilized. Dilute the antiserum appropriately,
100 ul thereof is added to the well, and the antibody titer is examined using goat anti-rabbit IgG-HRP. Further, this antiserum is purified by affinity chromatography using a protein G column and a Sepharose column on which an MBP-MDC (dC1) fusion protein is immobilized.

E using the purified monoclonal antibody and the purified rabbit polyclonal antibody thus obtained
A method for quantifying GML protein by the LISA method is established. That is, a purified monoclonal antibody derived from a hybridoma was immobilized on a 96-well plate, and BSA (Bovine ser
um albumin) and then purified MBP-GML solution as a test solution in the range of 0.156 to 5.00 ug / ml, 100 ul /
Add wells and incubate for 1 hour at room temperature. After washing the wells, add purified rabbit polyclonal antibody solution (5ug / ml)
Add 100 ul / well each and incubate for 1 hour at room temperature.
After washing the wells, add anti-rabbit IgG-HRP (5ug / ml)
Add 100 ul / well each and incubate for 1 hour at room temperature.
After completion of the reaction, 2 mM sodium azide is added at 100 ul / well, and the absorbance values at 405 nm and 490 nm are measured.

[0043]

EFFECTS OF THE INVENTION Mutations in the P53 gene are found in about 50% of cancers, and the detection of such changes has extremely important significance in the development, diagnosis, treatment and the like of cancer. However, P5 in cancer tissues
Since the mutation sites of the 3 genes are not constant and many kinds of mutations have been found, it took a lot of labor and time to detect and identify the p53 gene transition. Therefore, by measuring the expression of the novel target protein GML gene of the present invention or the protein itself instead of measuring the P53 mutation, it becomes possible to determine the P53 mutation and inactivation, and it is expected as a diagnostic tool for cancer. To be done. In addition, application to gene therapy for expressing GML gene in cancer lesions, P53
It is expected to be applied to a screening method of a drug that expresses GML without depending on the function, and conversely, to a drug that suppresses the expression of GML and an antisense drug.

[0044]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 158 Sequence type: Amino acid Sequence type: Protein Topology-: Linear origin Biological name: Homo sapiens Direct origin Library-Name: Human DNA cosmid library-Sequence Met Leu Leu Phe Ala Leu Leu Leu Ala Met Glu Leu Pro Leu Val Ala 1 5 10 15 Ala Ser Ala Thr Met Arg Ala Gln Trp Thr Tyr Ser Leu Arg Cys His 20 25 30 Asp Cys Ala Val Ile Asn Asp Phe Asn Cys Pro Asn Ile Arg Val Cys 35 40 45 Pro Tyr His Ile Arg Arg Cys Met Thr Ile Ser Ile Arg Ile Asn Ser 50 55 60 Arg Glu Leu Leu Val Tyr Lys Asn Cys Thr Asn Asn Cys Thr Phe Val 65 70 75 80 Tyr Ala Ala Glu Gln Pro Pro Glu Ala Pro Gly Lys Ile Phe Lys Thr 85 90 95 Asn Ser Phe Tyr Trp Val Cys Cys Cys Asn Ser Met Val Cys Asn Ala 100 105 110 Gly Gly Pro Thr Asn Leu Glu Arg Asp Met Leu Pro Asp Glu Val Thr 115 120 125 Glu Glu Glu Leu Pro Glu Gly Thr Val Arg Leu Gly Val Ser Lys Leu 130 135 140 Leu Leu Ser Phe Ala Ser Ile Ile Val Ser Asn Ile Leu Pro 145 150 155

SEQ ID NO: 2 Sequence length: 722 Sequence type: Nucleic acid Number of strands: Double strand Topology :: Linear Sequence type: cDNA to mRNA Origin organism name: Homo sapiens Direct origin Library name : Human DNA cosmid library-sequence GTCGGCGCGG GAGGCTCAAA TGGCTGGAGG CGGCGGGCAC GCACACTGCG GGCTCCGAGG 60 GGCACAGGTC CAGGCTCCTG CGTGAAGTG ATG CTC CTC TTT GCC TTA CTC CTA 113 Met Leu Leu GTG ATC CTC CTA 113 Met Leu CCA GTC GCT CAG 161 Ala Met Glu Leu Pro Leu Val Ala Ala Ser Ala Thr Met Arg Ala Gln 10 15 20 TGG ACT TAC AGT TTG AGA TGC CAT GAC TGT GCG GTC ATA AAT GAC TTC 209 Trp Thr Tyr Ser Leu Arg Cys His Asp Cys Ala Val Ile Asn Asp Phe 25 30 35 40 AAC TGT CCC AAC ATT AGA GTA TGT CCG TAT CAT ATT AGG CGC TGT ATG 257 Asn Cys Pro Asn Ile Arg Val Cys Pro Tyr His Ile Arg Arg Cys Met 45 50 55 ACA ATC TCC ATT CGC ATA AAT TCT CGT GAA CTA CTT GTT TAT AAG AAC 305 Thr Ile Ser Ile Arg Ile Asn Ser Arg Glu Leu Leu Val Tyr Lys Asn 60 65 70 TGT ACA AAC AAC TGC ACA TTT GTA TAT GCA GCT GAA CAG CCT CCT GAA 353 Cys Thr Asn Asn Cys Thr Phe Val Tyr Ala Ala Glu Gln Pro Pro Glu 75 80 85 GCC CCA GGA AAA ATC TTC AAA ACT AAT AGC TTC TAC TGG GTT TGT TGT 401 Ala Pro Gly Lys Ile Phe Lys Thr Asn Ser Phe Tyr Trp Val Cys Cys 90 95 100 TGT AAT AGC ATG GTT TGC AAT GCA GGA GGA CCT ACT AAT CTT GAA AGG 449 Cys Asn Ser Met Val Cys Asn Ala Gly Gly Pro Thr Asn Leu Glu Arg 105 110 115 120 GAC ATG TTA CCC GAT GAA GTA ACT GAG GAG GAG CTT CCA GAA GGA ACT 497 Asp Met Leu Pro Asp Glu Val Thr Glu Glu Glu Leu Pro Glu Gly Thr 125 130 135 GTG AGG CTG GGG GTA TCA AAA CTG TTG CTG AGT TTT GCC TCT ATC ATA 545 Val Arg Leu Gly Val Ser Lys Leu Leu Leu Ser Phe Ala Ser Ile Ile 140 145 150 GTC AGC AAT ATA TTG CCA TGAGGACCCC ACCTTGGAGG GTCTGACCAT 593 Val Ser Asn Ile Leu Pro 155 CTTCACCTGT TCCGCAGAGA AATGTTGCTC TCCATTATTC CCTTCTAAGC CAGAGACCCT 653 TATCCACTGC TCCTCTAGGT GGCCCATTTA TGGTTTGTTG TAAGAGAAAA ATTAAAAAAA 713 TATTGTTTA 722

SEQ ID NO: 3 Sequence length: 30 Sequence type: Nucleic acid Number of strands: Single strand Topology :: Linear Sequence type: Other nucleic acid (synthetic DNA) sequence ATGCTTGCCC AGGCATGTCC AGGCTGGTCC

SEQ ID NO: 4 Sequence length: 21 Sequence type: Nucleic acid Number of strands: Single strand Topology :: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence GGCTCCTGCG TGAAGTGATG C

SEQ ID NO: 5 Sequence length: 22 Sequence type: Nucleic acid Number of strands: Single strand Topology :: Linear Sequence type: Other nucleic acid (synthetic DNA) sequence ATGGAGATTG TCATACAGCG CC

SEQ ID NO: 6 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology-: Linear Sequence type: Other nucleic acid (synthetic DNA) sequence GTTCCTTGTG GAGCCGGAGC

SEQ ID NO: 7 Sequence length: 21 Sequence type: Nucleic acid Number of strands: Single strand Topology :: Linear Sequence type: Other nucleic acid (synthetic DNA) sequence GGTACAAGAC AGTAGCAGGT C

SEQ ID NO: 8 Sequence length: 22 Sequence type: Nucleic acid Number of strands: Single strand Topology :: Linear Sequence type: Other nucleic acid (synthetic DNA) sequence CAACTACATG GTTTACATGT TC

SEQ ID NO: 9 Sequence length: 18 Sequence type: Nucleic acid Number of strands: Single strand Topology :: Linear Sequence type: Other nucleic acid (synthetic DNA) sequence GCCAGTGGAC TCCACGAC

[0053]

[Brief description of drawings]

FIG. 1: Expression of GML gene by p53 in SW480 cell line. W: wild type p53, M: mutant p53

FIG. 2. Expression of GML gene in esophageal cancer cell line.
W: wild type p53, M: mutant p53

[Fig. 3] Anti-cancer drug (Bleomycinn, C in esophageal cancer cell line
Correlation between DDP) sensitivity and GML expression.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical location C12P 21/02 C12P 21/08 21/08 7823-4B C12Q 1/68 A C12Q 1/68 C12N 5 / 00 B // (C12N 15/09 ZNA C12R 1:91) (C12N 5/10 C12R 1:91) (C12P 21/02 C12R 1:91) (C12P 21/08 C12R 1:91)

Claims (12)

[Claims] 1. A DNA encoding a protein containing the amino acid sequence of SEQ ID NO: 1. 2. The DNA according to claim 1, which is the DNA according to SEQ ID NO: 2. 3. One of the amino acid sequences of SEQ ID NO: 1
Alternatively, a DNA encoding a protein containing an amino acid sequence in which a plurality of amino acids are added, deleted or substituted. 4. A vector containing the DNA according to claim 1, 2 or 3. 5. A transformant carrying the vector according to claim 4. 6. A protein containing the amino acid sequence of SEQ ID NO: 1. 7. A protein comprising an amino acid sequence in which one or more amino acids of the amino acid sequence of SEQ ID NO: 1 has been added, deleted or substituted. 8. The method for producing the protein according to claim 6, which comprises culturing the transformant according to claim 5 and recovering the expression product. 9. A DNA probe comprising a single-stranded DNA containing all or part of the nucleotide sequence of SEQ ID NO: 2 or its complementary sequence. 10. A DNA primer comprising a single-stranded DNA containing a part of the nucleotide sequence of SEQ ID NO: 2 or its complementary sequence. 11. A polyclonal antibody or a monoclonal antibody which binds to the protein according to claim 6. 12. The method for analyzing DNA according to claim 2, wherein the probe or primer according to claim 9 or 10 is used.