KR20030094905A - Mouse oncogene, protein encoded thereby and expression vector containing same - Google Patents

Abstract

PURPOSE: A mouse oncogene, a protein encoded thereby and an expression vector containing the same are provided. The mouse oncogene has homology to human oncogene, so that it can be useful for treatment of various human cancers. CONSTITUTION: A mouse oncogene having the nucleotide sequence of SEQ ID NO: 1 or its fragments are provided. The mouse oncogene having the nucleotides positioned at 191 to 1,006 in the nucleotide sequence of SEQ ID NO: 1 or its fragments are provided. A protein encoded by the mouse oncogene has the amino acid sequence of SEQ ID NO: 2. An expression vector contains the mouse oncogene having the nucleotide sequence of SEQ ID NO: 1 or its fragments. A microorganism transformed with the expression vector containing the mouse oncogene of SEQ ID NO: 1 is provided, wherein the transformed microorganism is E. coli DH5α /MCC-1/pGEM-T(KCTC 10179BP).

Description

Mouse oncogene, protein encoded by it, and expression vector containing same {MOUSE ONCOGENE, PROTEIN ENCODED THEREBY AND EXPRESSION VECTOR CONTAINING SAME}

The present invention relates to novel mouse carcinogens and homologous proteins encoded by homologous human primary oncogenes.

Mice are considered to be the first mammals to confirm the genetic basis of more than one gene, as well as complex diseases caused by genes and their interactions with genes and the environment. Although some phenotypes differ from those in humans, the phenotypes of humans and mice are very similar in phenotype. Moreover, with the development of gene mapping and the ability to isolate and replicate single gene disorders, mice are now becoming a model system for finding genes that cause disease in humans (West, DB et. al., Med Res Rev , 20 (3): 216-230, 2000).

Transgenesis using mice (Gordon, JW et.al. Proc Natl Acad Sci USA 77: 7380-7384, 1980) and gene targeting (Peng, KW Mol Med Today 5 (10): 448- 453, 1999). Techniques have been used to determine the function and role of specific genes in diseases occurring in humans. For example, specifically engineered transgenic mice and genetically targeted mice have been developed for the development of new therapeutic agents. It is useful for searching for pathways and efficacy.

As such, mice have been useful for the study of genetic diseases in humans for the past 40 years (Beadle MA Genes Develop 11: 1-10, 1997), which is a genetic linkage due to the short generation time of mice. This is because the research can be easily performed and a genetic model can be easily produced. In mammals, mouse genome research has been actively performed after human genome research. In addition, there is considerable gene sequence and structural homology between humans and mice, and the technology for producing genetically-transformed mice and genetically-targeted mice has reached a considerable level, even though mice are smaller than normal animals. Nevertheless, it has the advantage of being able to reliably obtain various types of phenotypes. As a result, mice have become an essential model animal for the discovery of disease genes and drug development in humans.

Mouse genome research can be carried out simultaneously with human genome research, which is aimed at the discovery of genes related to human diseases and the development of therapeutic agents to prevent and treat these diseases. Discovery of disease genes, candidate targets in disease-causing processes, search for candidate pathways and characterization of phenotypes. One of the most fundamental approaches to this study is the conserved synteny where the same genomic region that is functionally involved in inducing disease phenotype in both humans and mice is conserved. This can be said to be the case. If the disease gene is located on the same chromosome, it will be easier to find the disease gene in the mouse gene than in humans. At the same time, if the same gene is involved in causing the disease in human polygenic disease, mutation-induced technology can be used to identify the site of disease-induced disease. It can be a means to check.

Therefore, the inventors of the present invention reported the human primary cancer gene HCCR-1 (human cervical cancer oncogene-1; Korean Patent Publication No. 2001-39556 and GenBank Registration No. AF283671) and HCCR-2 (Korean Patent Application No. 2000- 71202) and genetically homologous mouse genes were developed and anti-cancer drugs developed for human cancers such as breast cancer, leukemia, lymphoma, kidney cancer, ovarian cancer, liver cancer, colon cancer, gastric cancer, lung cancer and uterine cancer. As a result of approach at the gene level for development and anti-sense gene therapy, the present invention has been completed by identifying a carcinogen named mouse cervical cancer 32 (MCC-32). .

An object of the present invention is a novel mouse oncogenic gene having homology with the human proto-oncogene, which can be effectively used for anticancer drug development, gene targeting mouse development and anti-sense gene therapy, and the protein encoded by the same. To provide.

Figure 1a and 1b is a result of comparing the gene homology of the mouse carcinogenic gene MCC-32 (SEQ ID NO: 1) of the present invention and HCCR-2 human primary oncogene,

Figure 2a and 2b is a result of comparing the gene homology of the mouse carcinogenic gene MCC-32 (SEQ ID NO: 1) of the present invention and HCCR-1 human human oncogene,

Figure 3 is a result of comparing the homology of the protein sequence of the mouse carcinogen of MCC-32 (SEQ ID NO: 2) and the protein sequence of HCCR-2 human human oncogene,

4 is a result of comparing the homology of the protein sequence of the mouse carcinogen MCC-32 of the present invention (SEQ ID NO: 2) and the protein sequence of the HCRC-1 human human oncogene,

FIG. 5 shows the electrophoresis of sodium dodecyl sulfate (SDS) -polyacrylamide gel (PAGE) of the protein expressed before and after IPTG induction after transduction of MCC-32 mouse carcinogen of the present invention. Result,

6 is a result of Northern blot analysis to determine whether the MCC-32 mouse carcinogen of the present invention is expressed in human normal tissue and cancer tissue,

7A shows expression of the MCC-32 mouse carcinogen of the invention in normal human 12-row multiple tissues, ie brain, heart, skeletal muscle, colon, thymus, spleen, kidney, liver, small intestine, placenta, lung and peripheral blood leukocytes Northern blot analysis to confirm whether

FIG. 7B shows the results obtained by hybridizing the same samples as in FIG. 7A with β-actin,

FIG. 8A shows HL-60, a promyelocytic leukemia cell line, HeLa, a uterine cancer cell line, K-562, a chronic myeloid leukemia cell line, MOLT-4, a lymphocyte leukemia cell line, Raji, a Burkitt lymphoma cell line, and SW480, a colorectal cancer cell line. , Lung cancer cell line A549 and melanoma skin cancer cell line G361 shows the results of Northern blot analysis confirming the expression of MCC-32 mouse carcinogen of the present invention,

FIG. 8B shows the results obtained by hybridizing the same samples as in FIG. 8A with β-actin.

In accordance with the above object, the present invention provides a mouse oncogene or a fragment thereof having the nucleotide sequence of SEQ ID NO: 1.

The present invention also provides a recombinant vector comprising the mouse oncogene or a fragment thereof and a microorganism transformed thereby.

The invention also provides a protein or fragment thereof having the amino acid sequence of SEQ ID NO: 2.

The present invention also provides a pharmaceutical composition for treating cancer comprising the oncogene or a fragment thereof.

The present invention also provides a pharmaceutical composition for treating cancer comprising the carcinogenic protein or fragment thereof.

In addition, the present invention provides an anti-sense gene having a nucleotide sequence complementary to all or a part of the sequence of mRNA transcribed from the oncogene or fragment thereof, and binds to the mRNA to inhibit the expression of the oncogene or fragment. do.

Finally, the present invention provides a pharmaceutical composition for treating cancer comprising the anti-sense gene as an active ingredient.

Hereinafter, the present invention will be described in detail.

The mouse cervical cancer gene 32 (Mouse Cervical Cancer Oncogene 32, hereinafter abbreviated as "MCC-32 oncogene"), which is an oncogene of the present invention, has an entire base sequence of 1,085 bp in length represented by SEQ ID NO: 1.

The base sequence of the mouse MCC-32 carcinogen of the present invention set forth in SEQ ID NO: 1 was registered under the registration number AF401483 in the NIH GenBank database of the National Institutes of Health (published date: August 1, 2002). , A novel gene with little similarity with other base sequences registered in the database.

In the nucleotide sequence of SEQ ID NO: 1, the open reading frame corresponding to the nucleotide numbers 191-1,006 (1,004-1,006: end codon) is the entire protein coding region and the amino acid sequence derived therefrom is SEQ ID NO: It consists of 271 amino acids, described as 2 (“MCC-32 protein”).

However, due to the degeneracy of the codons or in view of the codons preferred in organisms that wish to express the mouse MCC-32 oncogenes, the mouse MCC-32 oncogenes of the present invention are characterized by Various modifications may be made to the coding region within a range that does not change the amino acid sequence, and various modifications or modifications may be made within a range that does not affect the expression of the gene even in portions other than the coding region. It is included in the range of. Accordingly, the present invention also encompasses polynucleotides and fragments of the genes having base sequences substantially the same as those of the mouse MCC-32 oncogene of SEQ ID NO: 1. By substantially identical polynucleotides are meant those having sequence homology of at least 80%, preferably at least 90% and most preferably at least 95%.

The protein expressed from the mouse MCC-32 oncogene of the present invention consists of 271 amino acids, has an amino acid sequence set forth in SEQ ID NO: 2, and is about 32 kd in size. However, even in the amino acid sequence of the protein, substitution, addition or deletion of amino acids may be made within a range that does not affect the function of the protein, and only a part of the protein may be used depending on the purpose. Such modified amino acid sequences are also within the scope of the present invention. Accordingly, the present invention also encompasses polypeptides and fragments thereof having substantially the same amino acid sequence as the carcinogenic protein, wherein substantially identical polypeptides are at least 80%, preferably at least 90%, most preferably at least 95% It means things with same sex.

Mouse MCC-32 oncogenes and proteins of the present invention may be isolated from tissues of mice or synthesized according to known DNA or peptide synthesis methods. In addition, the gene thus prepared may be inserted into a microorganism expression vector known in the art to prepare an expression vector, and then introduced into a suitable host cell, for example, E. coli or yeast cell. Using the transformed host as described above, the DNA of the gene of the present invention can be replicated in large quantities or a large amount of protein can be produced.

For example, in the present invention, the mouse MCC-32 oncogene of the present invention is inserted into a commercially available pGEM-T easy vector and then transfected into E. coli DH5α, and E. coli DH5α obtained therefrom. / MCC-1 / pGEM-T was deposited on February 18, 2002 to the Korea Collection for Type Cultures (KCTC) (Accession No .: KCTC 10179BP).

In the production of the vector, expression control sequences such as promoters and terminators, self-replicating sequences, and secretion signals may be appropriately selected and combined depending on the type of host cell to produce the mouse MCC-32 oncogene or protein.

The mouse MCC-32 oncogene of the present invention is HCCR-, a human primary oncogene reported to increase expression in human cancers such as breast cancer, leukemia, lymphoma, kidney cancer, ovarian cancer, liver cancer, colon cancer, gastric cancer, lung cancer, and uterine cancer. It is a mouse gene that has genetic homology with HCCR-2, and it is used to develop anticancer drugs for the cancers in mice, development of gene-targeted mice and anti-sense gene therapy. Can be used effectively.

The therapeutic agent using the mouse carcinogen includes all or part of a cancer gene and all or part of a protein expressed by the gene, and inactivates a gene or protein site directly involved in the development of the cancer through mutation induction. Sites that directly affect the cancer process can be identified. Accordingly, the present invention also provides a pharmaceutical composition for treating cancer comprising all or part of the mouse MCC-32 oncogene or protein.

Gene-targeted animals can be produced by introducing a specific site involved in cancer development of the oncogene of the present invention into a mammal, for example, a rodent animal such as a rat, and introducing the gene at least at the 8-cell stage in the fertilized egg stage. It is desirable to. The genetically targeted animals thus prepared can be usefully used for research on the mechanism of suppressing the development of specific cancers, the search for carcinogenic substances, and the search for anticancer agents.

The present invention also provides anti-sense genes that are effective for gene therapy. In the present invention, the "anti-sense gene" is a polynucleotide having a sequence complementary to some or all of the mRNA transcribed from the MCC-32 carcinogen or a portion thereof of SEQ ID NO: 1, the protein binding of the mRNA By introducing a DNA sequence having a sequence that binds to a site and destroys an open reading frame (ORF) of the oncogene into the body of a patient, cancer may be prevented or treated by expression of the oncogene.

The invention also encompasses a method for treating or preventing cancer by administering to a patient a therapeutically effective amount of an antisense gene of the invention within the scope.

In the antisense gene therapy of the present invention, the antisense gene of the present invention is administered to a patient in a conventional manner to prevent the expression of oncogenes. For example, antisense genes may be mixed with poly-L-lysine derivatives by electrostatic attraction according to the method of Kim JS et al., J. Controlled Release 53: 175-182, 1998, and the mixture may be To be administered intravenously.

The scope of the present invention also includes a pharmaceutical composition for treating cancer comprising the antisense gene of the present invention as an active ingredient together with a pharmaceutically acceptable carrier, excipient or optionally other additives. The pharmaceutical composition of the present invention is preferably formulated as an injection.

The amount of antisense gene actually administered is determined in consideration of various related factors such as the condition to be treated, the route of administration, the age and weight of the patient and the severity of the condition.

Proteins derived from MCC-32 oncogenes of the present invention and proteins produced by mutating specific sites can be used for the purpose of suppressing the occurrence of specific cancers.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Isolation and Identification of Mouse Carcinogens

(1-1) Isolation of Total RNA from Mouse Brain Tissue

Total RNA was extracted from brain tissues of 13-week-old FVB mice (Central Experimental Animal, Korea) using RNeasy total RNA kit (RNeasy total RNA kit; Qiagen Inc., CA, USA) and cleaned according to manufacturer's instructions. DNA contaminants were removed from the extracted RNA using a kit (Message clean kit, GenHunter Corp., Brookline, Mass.).

(1-2) cDNA Library Construction and Southern Blot Analysis

The above procedure was performed to search for mouse genes having the full base sequence homology with HCCR-1 (Korean Patent Application Laid-Open No. 10-2001-39556) and HCCR-2 (Korean Patent Application No. 2000-71202). CDNA libraries were prepared using mouse brain tissue RNA prepared in Example (1-1) as follows (Okayama, H. and Berg, P., Mol. Cell Biol. 2: 161-170, 1982).

The principle of Capfinder PCR cDNA Library Construction Kit (Clontech) was used to construct the cDNA library (Capfinder PCR cDNA synthesis kit (January 1996) CLONTECHniques XI (1): 2-4, 1996). First, 1 ㎍ of total RNA extracted from normal mouse brain tissue was reverse transcribed to synthesize first strand complementary DNA (cDNA), and then subjected to long distance PCR (LD, Clontech). Amplified. After attaching an adapter (Clontech) to the PCR product, T4 polynucleotide kinase 30 units (Clontech) were treated and phosphorylated, ligated to λ phage vector (Clontech) and transformed into Y1090 E. coli (Clontech). A lambda DNA packaging system (Promega) was used to connect the cDNA to the vector.

Southern blot analysis using a clone with a DNA partial sequence (nt 1810-2057, 248 bp) of the human primary oncogene HCCR-1 gene whose base sequence was identified in the cDNA library thus prepared (probe) Sambrook, J., et al., Molecular cloning : A laboratory Manual, Cold Spring Harbor Laboratory Press, 1989). From this, positive clones were selected and phage DNA and plasmid DNA were isolated.

(1-3) Cloning

It has both a T7 promoter and an SP6 promoter, and can be used to make riboprobes easily by extracellular transcription, regardless of the insertion direction of the clone, and to facilitate the restriction enzyme treatment of plasmid DNA. easy) A vector (Promega, USA) was selected and used for the experiment.

For ligation reaction, 2 µl of plasmid DNA obtained in Example (1-2), 1 µl of pGEM-T easy vector (50 ng), 1 ml of 10 × T4 DNA ligase buffer solution, T4 DNA ligase in a 0.5 ml tube. (3 weiss units / μl; T4 DNA ligase, Promega Co., Ltd.) 1 μl was added, distilled water was added until the final reaction solution was 10 μl, mixed well by pipetting, and cultured at 4 ° C. to perform a ligation reaction.

E. coli DH5α was incubated in 10 ml of LB broth (10 g of Bacterium-Tryptone, 5 g of Bacterial-Yeast extract, 5 g of NaCl) to give an OD ₆₀₀ of about 0.3 to 0.6, and then left on ice for about 10 minutes. . Thereafter, the supernatant was discarded by centrifugation at 4,000 rpm for 10 minutes at 4 ° C., and the cells were recovered. The recovered cell precipitate was exposed to 10 ml of ice cold 0.1 M CaCl ₂ for about 30 minutes to 1 hour to induce transformation of the cells. This was again centrifuged at 4,000 rpm for 10 minutes at 4,000 rpm to discard the supernatant, collect the cells, mix with 2 ml of ice-cold 0.1 M CaCl ₂ , transfer 200 μl of competent cell suspension to a new microfuge tube, and transfer the competent cells. Ready.

2 μl of the ligation reaction product was added thereto and reacted at 42 ° C. for 90 seconds in a water bath. After completion of the reaction, the cells were transferred directly to an ice bath containing ice, and the cells were cooled for 1 to 2 minutes, followed by 800 μl of SOC medium (2.0 g of bacto-tryptone, 0.5 g of bacto-yeast extract, 1 ml of 1 M NaCl, The transformation reaction was carried out by adding 0.25 ml of 1 M KCl, 97 ml of TDW, 1 ml of 2 M Mg ^{2+, and} 1 ml of 2 M glucose) and incubating in a shaking water bath at 37 ° C. for 45 minutes at 220 rpm.

20 μl of X-gal was added to an LB plate containing ampicillin pre-loaded in a 37 ° C. incubator, and then diffused into a glass rod. Then, 200 μl of transformed cells were added dropwise and diffused into the glass rod, followed by 12 hours at 37 ° C. Incubated for Three to four white colonies formed after incubation were selected to inoculate the selected clones into LB plates to which ampicillin was added. Of these, only 10 ml of colony (terrific broth; TDW 900 ml), bacto-tryptone 12 g, bacto-yeast extract 24 g, glycerol 4 ml, 0.17 M KH ₂ Each selected colony was incubated in 100 ml of PO ₄ , 0.72 MK ₂ HPO ₄ ).

The transgenic E. coli thus selected was named DH5α / MCC-1 / pGEM-T and was deposited on February 18, 2002 to the Korea Biotechnology Research Institute Gene Bank (Accession Number: KCTC 10179BP).

Example 2 DNA Sequence Analysis

(2-1) Isolation of Recombinant Plasmid DNA

Inserting the Wizard Plus Mini prep DNA purification kit from known colony; using (Wizard ^TM Plus Minipreps DNA Purification Kit Promega, USA) was isolated plasmid DNA as follows. After centrifugation of transgenic E. coli DH5α / MCC-1 / pGEM-T enriched in 10 ml of terry broth at 10,000 x g for 5 minutes, most of the supernatant was removed, and 250 μl of cell resuspension solution was added thereto. The precipitate was mixed by vortex to completely suspend and 250 μl of cell lysis solution was added thereto. Then 10 μl of alkaline protease solution was added and incubated for 5 minutes at room temperature, followed by mixing with 350 μl of neutralization solution. The bacterial lysate formed was centrifuged at 14,000 x g for 10 minutes at room temperature, and then the clarified lysate was transferred to a spin column and centrifuged at 14,000 x g for 1 minute at room temperature to remove the eluate. 750 μl of column wash solution diluted with 95% ethanol was added to the spin column and centrifuged at 14,000 × g for 1 minute at room temperature to discard the passed eluate and 250 μl of column eluate diluted with 95% ethanol. The eluate was removed by centrifugation at 14,000 x g for 2 minutes at room temperature. The spin column was then transferred to a 1.5 ml sterile tube and 100 μl of nuclease-free water was added to the spin column to centrifuge at 14,000 × g for 1 minute at room temperature to obtain passed plasmid DNA. 5 μl of the prepared 100 μl plasmid DNA was isolated, and 0.5 μl of Eco RI enzyme (Boehringer Mannheim, USA), 1 μl of 10 × Eco RI buffer, and 3.5 μl of TDW were added to digest the enzyme. Electrophoresis was performed to confirm the experimental process.

(2-2) DNA sequencing

5 μl of the plasmid DNA product obtained in Example (2-1) was added 1 μl of exonuclease I (10 units / μl) and 1 μl of shrimp alkaline phosphatase (2 units / μl), followed by mixing well. The reaction solution was incubated at 37 ° C. for 15 minutes in a PCR machine, followed by pretreatment using a sequencer PCR product sequencing kit (Amersham, USA).

7 μl of distilled water and 1 μl of T7 or SP6 primer (5-10 pmol / μl) were added to 2 μl of the pretreated DNA product, and the reaction mixture was mixed well and reacted at 100 ° C. for 2 minutes and 30 seconds immediately. The annealing reaction was performed by cooling in an ice-filled water bath for 5 minutes, centrifuging, and cooling again.

Prepare four tubes labeled A, T, G and C, remove the ddGTP termination mix, ddATP termination mixture, ddTTP termination mixture, ddCTP termination mixture (Amersham, USA) and pre-warm them It was left at room temperature for the warm) step and the termination reaction step. Each termination mixture is then placed in four tubes, 2.5 μl of the ddGTP termination mixture in the tube labeled G, 2.5 μl of the ddATP termination mixture in the tube labeled A, 2.5 μl of the ddTTP termination mixture in the tube labeled T, and C 2.5 μl of the ddCTP termination mixture was added to each tube, and the lid was closed to prevent evaporation, and four termination tubes were left in the reactor at 37 ° C. for at least 1 minute to prepare the termination mixture.

2 μl of a labeling mix prepared by diluting 5 μl of reaction buffer (USB), 1 μl of 0.1 M DTT, 5 × labeling mixture with distilled water to 10 μl of the cooled annealed DNA mixture prepared above, [ ³⁵ S] dATP (Amersham, USA) 0.5 [mu] l (5 [mu] Ci) and 2 [mu] l of the quenase DNA polymerase were added well, left to stand at room temperature for 5 minutes, and labeled.

3.5 μl of the labeling reaction solution was transferred to each termination tube containing 2 μl of the termination mixture, mixed well, and transferred to a 37 ° C. water bath and left for 5 to 10 minutes. Then, 4 μl of a reaction stop solution (USB) was added to stop the reaction. Immediately before loading on the sequencing gel, the sequence was analyzed by processing at 75 ° C. for 2 minutes and immediately loading 2-3 μl into each lane.

As a result, the mouse oncogene of the present invention showing homology with the human primary cancer genes HCCR-1 and HCCR-2 and isolated from mouse brain tissue has a nucleotide sequence set forth in SEQ ID NO: 1, wherein the gene is SEQ ID NO: Encodes a protein of about 32 kDa described as 2. In the present invention, the mouse oncogene was named as mouse cervical cancer gene 32 (MouseCervical Cancer Oncogene 32, MCC-32 cancer-related gene) and the protein encoded therefrom was named as MCC-32 protein.

Example 3 Homologous Analysis of MCC-32 Mouse Carcinogenic Genes and Human Primary Cancer Genes, HCCR-1 and HCCR-2

Nucleotide sequences of HCCR-1 and HCCR-2, which are human primary cancer genes homologous to the MCC-32 mouse carcinogen of the present invention, are available from http: //www.ncbi.nlm of the National Institute of Health, USA. .nih.gov).

As a result, as shown in Figure 1a and 1b, MCC-32 mouse oncogene of the present invention showed homology of more than 83% gene sequence with HCCR-2, a human primary oncogene.

In addition, as shown in Figure 2a and 2b, MCC-32 mouse oncogene of the present invention showed homology of more than 83% gene sequence with HCCR-1, a human primary oncogene.

In addition, as shown in Figures 3 and 4, MCC-32 mouse carcinogenic protein of the present invention showed 77% and 77% homology with the protein sequences of the human primary oncogenes HCCR-1 and HCCR-2, respectively.

Example 4 Western Blot Analysis

The entire MCC-32 oncogene of SEQ ID NO: 1 was inserted into multiple cloning sites of the pGEX-4T-3 vector (Amersham Pharmacia), and the resulting pGEX-4T-3 / MCC-32 vector was transferred to Escherichia coli BL21 (ATCC 47092). Was transfected. The expression protein glutathione S transferase (GST) is inserted at the front of the multiple cloning site of the pGEX-4T-3 vector. The transfected E. coli was shaken in LB broth, diluted to 1/100 and incubated for another 3 hours. 1 mM isopropyl β-D-thiogalactopyranoside (Isopropyl beta-D-thiogalactopyranoside, IPTG, Sigma) was added thereto to induce protein production.

E. coli cells in culture medium before and after IPTG induction were sonicated and electrophoresed on 12% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE). 5 shows the results of SDS-PAGE expression of the protein in E. coli BL21 strain transfected with the pGEX-4T-3 / MCC-32 vector. A fusion protein band of about 58 kDa was clearly observed after IPTG induction. The 58 kDa fusion protein contains about 26 kDa GST protein and about 32 kDa MCC-32 protein inserted into the pGEX-4T-3 vector.

Example 5 Northern Blot Analysis Using MCC-32 Gene 1

Northern blot analysis was performed as follows to investigate whether cancer could be detected in human tissues using the MCC-32 oncogene.

Total RNA isolation from tissues was isolated using the RNeasy total RNA kit (Qiagen Inc., CA, USA). Twenty micrograms of total RNA of two human normal cervical tissue samples and two primary cancerous cervical cancer tissue samples were denatured and electrophoresed on a 1% formaldehyde agarose gel and then transferred to a nylon membrane (Boehringer-Mannheim, Germany). I was. The nylon membrane was then hybridized overnight at 42 ° C. with a ³² P-labeled random prime probe using the MCC-32 full length cDNA obtained in Example 1. The Northern blot process was repeated twice in the same manner, one was quantified by a densitometer, and the other was hybridized with β-actin probe to confirm the total amount of mRNA.

FIG. 6 shows Northern blot analysis showing expression levels of human oncogenes homologous to MCC-32 oncogenes in normal cervical tissue and primary cervical cancer tissue. B is a hybridization of the same sample with β-actin probe. The presence of mRNA was confirmed. In FIG. 6A, lanes 1 and 2 (denoted Normal) are normal cervical tissue, and lanes 3 and 4 (denoted Cancer) are cervical cancer tissue. As shown in FIG. 6, when the MCC-32 oncogene is used as a probe, a band is detected in human cervical cancer tissues but is rarely detected in normal tissues, thereby confirming that the gene may be usefully used to detect cancer. .

Example 6 Northern Blot Analysis Using MCC-32 Gene 2

To determine whether cancer can be detected using the mouse MCC-32 oncogene in a variety of cells and tissues, 12 normal organs were extracted from normal RNA and blotted onto nylon membranes. RNAs were extracted from ten multiple tissues (Clontech) and eight types of cancer cells and commercialized human cancer cell 8-column multiple tissues (Clontech) were compared and compared for expression. Both membranes hybridized blots with ³² P-labeled random primed MCC-32 full cDNA probes prepared using the Rediprime II random prime labeling system (Amersham). Northern blot analysis was repeated twice, and the results were quantified using densitometry and normalized to β-actin.

7 shows the results of Northern blot analysis confirming the expression of human oncogenes homologous to the MCC-32 oncogene in normal human 12-row multiple tissues (Clontech), b is the same sample β-actin Hybridization with a probe confirms the presence of mRNA. As can be seen in Figure 7a, no band was detected in the Northern blot analysis using the MCC-32 gene as a probe in various normal tissues.

FIG. 8 shows the results of Northern blot analysis confirming the expression of human oncogenes homologous to the MCC-32 oncogene in human cancer cell lines (Clontech). B is a hybridization of the same sample with β-actin probe. The presence of mRNA was confirmed. As can be seen in Figure 8a, in the Northern blot analysis using the MCC-32 gene as a probe, about 1 kb in size transcripts of the promyelocytic leukemia cell line HL-60, uterine cancer cell line HeLa cells, chronic myelogenous leukemia High levels were detected in cell lines K-562, lymphocyte leukemia cell line MOLT-4, Burkitt's lymphoma cell line Raji, colon cancer cell line SW480, lung cancer cell line A549 and skin cancer melanoma cell line G361 cell lines. From the above results, the mouse MCC-32 oncogene of the present invention may be usefully used as a probe for detecting human cancer as a carcinogen having homology with human oncogenes common to various cancers.

Example 7 Locus on the Chromosome of an MCC-32 Oncogene

Mouse gene chromosome 15 corresponds to 12 for human chromosomes (Li YJ, et. Al. Genes Chromosomes Cancer 30 (1): 91-95, 2001; Xin Y., et. Al. Genomics 74 (3): 408-413, 2001). Therefore, all bases homologous to HCCR-1 (Korean Patent Application Laid-Open No. 10-2001-39556) and HCCR-2 (Korean Patent Application No. 2000-71202), which are present on the human chromosome 12, After searching for the mouse gene MCC-32 with the sequence, the chromosomal location of the gene was found at the National Institute of Health, USA at http://www.ncbi.nlm.nih.gov/genome/guide/ M_musculus.html) analysis of the website, it was confirmed that the mouse MCC-32 carcinogen of the present invention is present in the mouse chromosome 15.

As described above, the mouse carcinogen of the present invention can identify specific cancer-causing sites in the mouse gene sequence using genetic mutation induction techniques in mice or human cancers such as breast cancer, leukemia, lymphoma, kidney cancer, ovarian cancer, It can be effectively used in the development of anticancer drugs for liver cancer, colorectal cancer, gastric cancer, lung cancer and uterine cancer, gene targeting mouse development and anti-sense gene therapy.

Claims (11)

Carcinogen or fragment thereof having the nucleotide sequence of SEQ ID NO: 1. The method of claim 1, A carcinogen or fragment thereof having a nucleotide sequence corresponding to nucleotide numbers 191 to 1006 of SEQ ID NO: 1. Carcinogenic proteins or fragments thereof having the amino acid sequence of SEQ ID NO: 2. A vector comprising the oncogene or fragment thereof of claim 1. Microorganism transformed by the vector of claim 4. The method of claim 5, Microorganism that is Escherichia coli DH5α / MCC-1 / pGEM-T (Accession No .: KCTC 10179BP). A method for preparing a protein or fragment thereof having an amino acid sequence of SEQ ID NO: 2 using the microorganism of claim 5 or 6. A pharmaceutical composition for treating cancer comprising the oncogenic gene of claim 1 or a fragment thereof. A pharmaceutical composition for treating cancer comprising the protein of claim 3 or a fragment thereof. An anti-sense gene having a nucleotide sequence complementary to all or a portion of an mRNA sequence transcribed from the oncogene or fragment thereof of claim 1 or 2, which binds to the mRNA and inhibits the expression of the oncogene or fragment. A pharmaceutical composition for treating cancer containing the anti-sense gene of claim 10 as an active ingredient.