KR20030013951A - Polyclon al antibody of tumor-related CMT2 gene for controlling division of the chromo some number - Google Patents

Abstract

PURPOSE: Provided is a polyclonal antibody of tumor-related CMT2 gene for controlling division of the chromosome number, thereby excellently analyzing carcinogenesis and abnormal chromosome number. CONSTITUTION: The tumor-related CMT2 gene has the nucleotide sequence of the SEQ ID NO:1. A transformant BL21 pGEX-2T/CMT2 is prepared by transformation with a recombination vector pGEX-2T/CMT2(KCTC 10000BP) including the CMT2 gene. The polyclonal antibody specifically binds to CMT2 protein having the amino acid sequence of the SEQ ID NO:2.

Description

Polyclonal antibody of tumor-related CMT2 gene for controlling division of the chromo some number

The present invention relates to a polyclonal antibody of CMT2, a tumor-related gene that regulates chromosome number separation, and more particularly, to a CMT2 polyclonal molecule that can detect CMT2 proteins involved in the mitosis phase in which intracellular chromosome separation occurs. Relates to raw antibodies.

There have been many studies to conquer cancer over the last few decades, but no breakthroughs have yet been made for their prevention or treatment. Recently, as a field of study on the mechanism of tumor development, researches to find the gene involved in the cell cycle and investigate its function have been actively conducted. This interest is because one of the characteristics of tumor cells is the growth of transformed cells through continuous cell division. That is, normal cells stop growing after proliferating for a certain period of time, but cancer cells continue to divide. One such cause is that scientists believe that a problem occurs in the cell cycle that governs cell proliferation, causing cells to continue to proliferate. Over the past decade, cell cycle research has focused primarily on the study of genes involved in the G1 / S phase. The result was a Cdk / cyclin dependent kinase complex, the engine of the cell cycle (Lee and Nurse, Nature 327: 31-35, 1987), a gene that inhibits the activity of these kinases. (P21, p27, p16, p18, etc.) were found continuously (Serrano et al., Nature 366: 704-707, 1993; Xiong et al., Genes Dev. 7: 1572-1583, 1993; Kiyokawa et al., Cell 85: 721 -732, 1996; Waldman et al., Nature 381: 713-716, 1996). Currently, researches on new drug development and gene therapy using these genes are being actively conducted at home and abroad. Research on G2 and mitosis, another axis of the cell cycle, began more than a decade ago (Kung et al., Proc Natl Acad Sci US A. 87: 9553-9557, 1990; Minshull et al., Cell 79: 475-486). , 1994; Hardwick and Murray, J Cell Biol 131: 709-720, 1995), the rate of progression was relatively slower than that of the G1 phase. In recent years, studies on higher animals about spindle checkpoints that control chromosomal segregation during mitosis have been actively conducted.

The mitosis phase in cell division is an essential time for the delivery of genetic information to daughter cells and for the preparation of the next cell cycle. In particular, the mid to late transition phase of mitosis is a very important time for the equal division of genetic information through the accurate separation of daughter chromosomes. Accurate separation of daughter chromosomes is achieved by a combination of centripetal and isotope, centromere, spindle fiber, and protein degradation. Damage to these control systems results in instability in the delivery of the genetic material. It is noted that such anuploidy of chromosome numbers is generally observed in many tumor cells, which supports the hypothesis that even separation of each chromosome may occur due to mitosis. Especially in many cancers to date, spindle checkpoint related genes have been reported (McDonald et al., Ann Med. 33: 113-122, 2001; Tsuiki et al., Oncogene 20: 420-429, 2001; Wassmann and Benezra, Curr Opin Genet Dev. 11: 83-90, 2001; Percy et al., Genes Chromosomes Cancer 29: 356-362, 2000; Jaffrey et al., Cancer Res. 60: 4349-4352, 2000), but the exact mechanism and spindle checkpoint in mammals Gene identification is not well understood.

Accordingly, the present inventors completed the present invention by confirming that tumor-associated gene CMT2, which regulates chromosome number separation, can be usefully used as a biological marker of tumor cell generation and chromosome number, while studying spindle checkpoint-related genes.

Accordingly, an object of the present invention is to provide a recombinant vector pGEX-2T / CMT2 comprising a tumor-associated gene CMT2 which regulates chromosome number separation.

Another object of the present invention to provide a polyclonal antibody of the CMT2 gene.

The object of the present invention is to search for CMT2 genes that interact with MAD2, clone them into pGEX-2T, inject CMT2 protein into rabbits to produce antibodies, and express the expression patterns of CMT2 protein according to cell type and cell cycle. Investigations were made by identifying intracellular expression sites and their interaction with Mad2.

Hereinafter, the configuration of the present invention will be described in detail.

1 is a schematic diagram of a vector pGEX-2T / CMT2 comprising the CMT2 gene of the present invention.

2 is an amino acid sequence encoded with the base sequence of the CMT2 gene of the present invention.

3 shows the CMT2 protein detected in HeLa cells.

4 is a result of examining the expression of CMT2 protein using a tumor cell line.

Figure 5 shows the results of stagnating the cells in the S phase using hydroxyurea (Hydroxyurea) and when the cells were stagnated in the mitosis step by the addition of nocodazole (A) and the double thymidine method is used. The cells were stagnated in G1 and then released (B).

Figure 6 shows the expression of CMT2 in HeLa cells.

Figure 7 shows the results of the GST-CMT2 fusion protein and GST pull down assay.

performing a yeast two hybride assay to search for a CMT2 gene that interacts with spindle checkpoint and MAD2; Cloning the CMT2 gene in pGEX-2T and inducing the expression of the protein to produce the CMT2 protein and inject it into rabbits to produce polyclonal antibodies; Investigating the expression pattern of CMT2 protein according to cell type (HeLa, U2OS, Hep3B, HepG2 D54), cell cycle; Investigation of intracellular expression site by immunofluorescence assay and GST pull down assay to confirm the interaction between CMT2 and Mad2.

The plasmid pGEX-2T used in this step was Amersham Pharmacia Biotech Ltd. It was purchased from the company and used, catalog number is 27-4587-01. pGBT8, pGAD is a Ph.D. in Albert Einstein College of Medicine Donated by Dr. Matsumoto.

As used herein, the term 'Mad2 gene' refers to a gene having a function of regulating the separation of daughter chromosomes replicated during mitosis.

Hereinafter, specific examples of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited thereto.

Example 1 performed a yeast two hybrid assay to search for genes that bind to Mad2

In order to search for a new gene source that interacts with Spindle checkpoint, Mad2, we performed a yeast two hybrid assay using Mad2 as a bait.

Mad2 inserted into pGBT8 plasmid with DNA binding domain was transformed into HF7c yeast using LiAc method (Kim et al., Science 279: 1045-1047, 1998). After transformation, the cells were cultured in Dropout medium supplied with histidine and leucine at 32 ° C. for 3 days. After the transformants were selected, the yeast was transformed for the second time using a HeLa cDNA library constructed with a pGAD vector having a DNA transactivation domain. After incubating in histidine and leucine-deficient dropout medium at 32 ° C. for 5 days, β-galactosidase analysis by filter lift was performed on the yeasts that formed colonies using replicas. Only blue colonies were selected and plasmids were extracted from yeast using glass beads, followed by DNA sequencing to confirm the nucleotide sequence of the DNA inserted into the pGAD vector, and searched in the Genebank to investigate new genes that reacted with bait.

As a result, five colonies were obtained, three of which were Mad1, one of which was a hypothetical protein with no specific function in general, and the other was a new gene. Because Mad1 is already known as a protein that already interacts with Mad2, we wanted to study the function of the new gene. The new gene was named CMT2 (SEQ ID NO: 1, SEQ ID NO: 2). The base of CMT2 was 825 bp and the expected protein size was 32 kDa (FIG. 2).

Example 2 CMT2 Antibody Production

To study the function for CMT2, the CMT2 gene was cloned into the pGEX-2T vector (FIG. 1). GST-CMT2 fusion protein was transformed into BL21 (DE3) competent cells, and GST-CMT2 fusion protein was induced with 0.1 mM IPTG. As a result, it was observed that the GST-CMT2 fusion protein was induced in the sample to which IPTG was added. The induced protein was precipitated using GSH-beads, followed by elution to purify the protein. Approximately 1 mg of GST-CMT2 fusion protein was purified and injected into rabbits to produce polyclonal antibodies, and GSH-agarose beads were used to produce affinity-purified antibodies. 3 shows the CMT2 protein detected in HeLa cells. As expected, one band was detected at 32 kDa and not in preimmune serum.

The recombinant vector pGEX-2T / CMT2 constructed by cloning the CMT2 gene into the pGEX-2T vector was deposited on July 2, 2001, with the accession number KCTC 10000BP to the Microbial Depository Center of the Korea Institute of Science and Technology.

Example 3 Investigation of Expression Pattern of CMT2 Protein by Cells

An immunoblotting assay was performed to investigate the expression of CMT2 protein using tumor cell lines that are frequently used to investigate the expression of CMT2 expressed in various cell lines (HeLa, U2OS, Hep3B, HepG2 D54).

Cell extracts were reacted with a sample loading buffer containing β-mercaptoethanol and electrophoresed using 5-20% SDS / polyacrylamide gel. Proteins in the gel were transferred to nitrocellulose membrane and subjected to western blotting. That is, the membrane was blocked with 5% bovine serum albumin, washed with PBS, and reacted with an antibody that recognizes CMT2. The membrane was washed and reacted with HRP-conjugated anti-rabbit IgG. Proteins in the membrane were analyzed using the ECL kit.

As shown in FIG. 4, the expression was decreased in Hep3B (hepatocellular carcinoma cell line) and D54 (brain cancer cell line). It was confirmed that CMT2 is expressed in the cell line. These results indicate that the CMT2 gene is involved in tumor formation.

Example 4 Investigation of Expression Pattern of CMT2 Protein by Cell Cycle

Since the function of Mad2 related protein may change in cell cycle level, the expression pattern of CMT2 protein was examined. HeLa cells expressing CMT2 were used.

HeLa cells were cultured in DMEM medium containing glucose, antibiotics (100 units / ml penicillin, 100 μg / ml streptomycin) and 10% fetal bovine serum. The following methods were used to stagnate the cells at specific stages of the cell cycle. 2 mM hydroxyurea was added for 12 hours to incubate at the G1 / S stage, and 100 ng / ml nocodazole was incubated together for approximately 16 hours to stagnate the cells in the middle of mitosis. In order to stagnate the cells by non-drug treatment, double-thymidine blocking was used at the G1 / S stage. In other words, the cells were stagnated with 2mM thymidine for 14 hours, washed, incubated for 10 hours in a thymidine-free medium, and then stagnated in a medium containing 2mM thymidine for 14 hours. I was. The cells were washed, replaced with fresh culture, and collected at predetermined time periods, and subjected to immunoblotting assay in the same manner as in Example 3.

After cloning the new gene, a stable cell line was constructed by transfection of HeLa cells with lipofectin.

First, in the case of stagnation of cells using drugs for each cell cycle, the results of stagnation of cells in the S phase using hydroxyurea (Hydroxyurea) and the stagnation of cells in the mitosis phase by the addition of nocodazole. Cases could not detect significant differences in the expression of CMT2 protein (FIG. 5A). However, these results may be effective by the drug, so the cells were stagnated and released by G1 using the double thymidine method and observed at each time point. As shown in FIG. 5B, a large difference could not be distinguished for each cell cycle stage. This means that the protein expression pattern of CMT2 does not show a difference by cell cycle. These results are similar to the expression pattern of Mad2, and further studies on the activity of CMT2 are needed.

Example 5: Immunofluorescence Assay for Observing Expression Location of CMT2 in Cells

In order to investigate the function of CMT2 with no difference in expression in each cell cycle, immunofluorescence assay was performed to observe the expression position of intracellular CMT2 by mitosis.

Cells were grown on coverslips and washed with lysis buffer containing 60 mM PIPES, 25 mM HEPES, pH 6.9, 10 mM EGTA, 4 mM MgCl ₂ . The cells were fixed with 1% paraformaldehyde and then washed. Anti-CMT2 serum was diluted 1: 100 and antitubulin serum was diluted 1:80. Primary incubation was performed at 37 ° C. for 1 hour. Cy3 or FITC conjugated secondary antibodies were diluted 1: 100. Secondary antibodies were reacted for 30 minutes at 37 ℃. The reaction was performed with 0.1 μg / ml DAPI to observe the DNA before mounting. Cells were observed with Carl Zeiss fluorescence microscopy.

Figure 6 shows the expression of CMT2 in HeLa cells. CMT2 was distributed in the cytoplasm mainly in the interphase, and it was observed that the CMT2 was particularly present near the nuclear membrane. This shows a pattern very similar to Mad2. The intracellular phases of mitosis tended to be distributed in spindle mainly in the metaphase, and in the spindle in the late phase, and in the midzone in the cytoplasmic phase. Indicated. This pattern was similar to tubulin. This tends to be very similar to previously reported p55CDC (Weinstein, J Biol Chem. 272: 28501-28511, 1997) and Mad2 (Li and Beneza, Science 274: 246-248, 1996). However, the expression of CMT2 in premetap hase has not been investigated. In other words, precisely the function at the spindle checkpoint is expected to be possible after further research.

Example 6: GST pull down assay to confirm the mutual binding of CMT2 and Mad2

In order to investigate the mutual binding of CMT2 to Mad2 by yeast two hybrid assay, GST-CMT2 fusion protein was made and subjected to GST pull down assay.

As shown in FIG. 7, CMT2 binds to Mad2 regardless of the cell cycle, and the above results suggest that the role between Mad2 and CMT2 may be maintained throughout the cell cycle stage. can do.

As is apparent from the above examples, the present invention analyzes the CMT2 gene involved in the mitosis phase in which intracellular chromosome separation occurs, and determines tumor formation and chromosome abnormality by constructing polyclonal antibodies against the CMT2 protein. It can be used as a marker that can be a very useful invention in the biopharmaceutical industry and diagnostic industry.

Claims (3)

Recombinant vector pGEX-2T / CMT2 comprising the CMT2 gene of SEQ ID NO: 1 (Accession No. KCTC 10000BP). Transformant BL21 pGEX-2T / CMT2 transformed with the recombinant vector of claim 1. Polyclonal antibody that specifically binds to the CMT2 protein of SEQ ID NO: 2.