KR100791859B1 - 53 53 TRANSFORMANT EXPRESSING A FUSION PROTEIN OF p53 TUMOR SUPPRESSOR PROTEIN AND A FLUORESCENCE PROTEIN AND METHOD FOR SCREENING SUBSTANCES RELATING TO THE ACTIVITY OF p53 TUMOR SUPPRESSOR PROTEIN USING SAME - Google Patents

Abstract

The present invention relates to a transformed cell line expressing a fusion protein of a p53 tumor suppressor, a first fluorescent protein and a second fluorescent protein, and a method for searching for a substance involved in the activity of the p53 tumor suppressor protein. In particular, the inactive p53 tumor suppressor protein mutant gene in which valine, which is the 143th amino acid in the 5 'to 3' direction, is substituted with alanine; A first fluorescent protein gene fused to the gene; Internal ribosome entry site (IRES) ribosomal binding sequence; And a recombinant expression vector comprising a second fluorescent protein gene, the expression of which is regulated by one promoter; A transformed cell line into which the expression vector is stably introduced; And a method for searching for a substance that increases or inhibits the activity of p53 tumor suppressor protein using the transformed cell line. The recombinant expression vector and the transformed cell line according to the present invention may be usefully used for screening anticancer agents, anticancer adjuvant agents and therapeutic agents related to degenerative diseases as biosensors to search for substances related to the activity of p53 tumor suppressor protein.

Description

Transformation cell line expressing fusion protein of ｐ53 tumor suppressor protein with fluorescent protein and method of screening for substances related to activating ｐ53 tumor suppressor protein using the same. TRAINFORMANT EXPRESSING A FUSION PROTEIN OF p53 TUMOR SUPPRESSOR PROTEIN AND A FLUORESCENCE PROTEIN AND METHOD FOR SCREENING SUBSTANCES RELATING TO THE ACTIVITY OF p53 TUMOR SUPPRESSOR PROTEIN USING SAME}

1 is a schematic diagram showing the signaling process of the p53 tumor suppressor protein,

2 shows a cleavage map of the expression vector pDsRed1-N1 / p53V143A expressing a fusion protein of the inactive p53 tumor suppressor protein and the red fluorescent protein DsRed1,

Figure 3 is a result confirmed by luciferase analysis that the inactive p53 tumor suppressor protein inserted into the expression vector pDsRed1-N1 / p53V143A is inactivated as a transcription factor,

4 shows a cleavage map of the expression vector pP53V143AdsRed1-IRES-GFP in which the green fluorescent protein GFP is inserted into the expression vector pDsRed1-N1 / p53V143A.

5 is a result of observing the expression vector pP53V143AdsRed1-IRES-GFP in a cell line transformed with fluorescence microscope stably expressing p53V143A-DsRed1 fusion protein and GPF fluorescent protein,

Figure 6 is a result of measuring by the microplate reader that the expression of p53V143A-DsRed1 fusion protein in the cell line transformed with the expression vector pP53V143AdsRed1-IRES-GFP was increased by the treatment with doxorubicin,

FIG. 7 shows the results of quantitative changes and distribution of p53V143A-DsRed1 fusion protein expressed in the transformed cell line of the present invention with a high-contents image analyzer.

The present invention relates to a transformed cell line expressing a fusion protein of a p53 tumor suppressor protein and a first fluorescent protein and a second fluorescent protein, and a method of searching for a substance involved in the activity of the p53 tumor suppressor protein.

Apoptosis of cells by gene expression in multicellular organisms is an essential process for development and homeostasis. In this process, cellular gene fragmentation and cell contraction occur, and the fragmented cell gene is wrapped in the cytoplasm and removed by phagocytic activity in the form of small cysts. Therefore, self-apoptosis does not cause an inflammatory response unlike necrosis, and thus plays an important function in normal development.

One of the typical features of tumors is that division and proliferation are not controlled at an appropriate level and progress excessively, and p53 is a representative tumor-suppressor gene that inhibits cells from exhibiting this pattern. . In many higher animals, including humans, the p53 protein, which acts as a tumor suppressor, is responsible for inhibiting abnormal proliferation of cells or inhibiting or eliminating the proliferation of damaged cells. Therefore, p53 protein is associated with important factors in cells such as ATM / ATR, Chk2, mdm2, p21 and Bax, and these factors maintain a constant interaction with p53 protein, which is called p53 signaling. In addition, p53 protein is known to be associated with tumor development, inhibition and aging, and drug cytotoxicity. The p53 protein is phosphorylated by various compounds and viruses that cause nucleic acid damage and stabilizes in the cytoplasm, thereby quantitatively increasing, and the stabilized p53 protein migrates to the cell nucleus. In the cell nucleus, the p53 protein acts as a transcription factor that specifically binds to the promoter region of the target gene, causing transcription and protein production of the target protein, resulting in cell cycle pauses to repair gene damage or permanent removal of damaged cells. Cause death (see FIG. 1 ). Therefore, securing a new compound that can raise or inhibit each step in relation to the p53 signaling process is very important in identifying the p53 signaling process and securing a new anticancer drug candidate.

About 50% of all cancers are caused by the loss of activity of the p53 protein due to mutations. As a result, the p53 protein does not block abnormal cell growth, leading to cancer. For this reason, there is a need for the development of a system that can effectively screen for a substance capable of repairing or increasing the expression of p53 gene to restore normal activity of p53 protein. On the other hand, some normal cells, such as lymphocytes, blood producing organ cells, and intestinal epithelial cells, have high expression rates of the p53 gene, which is impaired when treated with anticancer drugs that enhance the expression of the p53 gene. It can also cause side effects such as severe anemia, hair loss, and vomiting that can be involved in chemotherapy. Therefore, the development of a substance capable of appropriately suppressing the expression of the p53 gene is also urgently required.

Accordingly, the present inventors have made intensive studies to develop a system capable of effectively screening candidate substances capable of increasing or inhibiting the activity of p53 protein. As a result, fusion proteins and internal markers of p53 tumor suppressor protein and the first fluorescent protein A recombinant expression vector expressing a second fluorescent protein by a single promoter was prepared, and a transformed cell line into which the expression vector was introduced was used to confirm that a substance involved in the activity of p53 tumor suppressor protein could be effectively searched. By this, the present invention was completed.

Accordingly, an object of the present invention is to increase or inhibit the activity of p53 tumor suppressor protein by effectively analyzing the quantitative changes and distribution patterns of p53 tumor suppressor protein using fluorescent proteins to increase or inhibit the activity of p53 tumor suppressor protein. It is to provide a method for searching for a candidate substance that can be usefully used as.

It is also an object of the present invention to provide a biosensor capable of searching for candidate substances that increase or inhibit the activity of p53 tumor suppressor protein using the above screening method.

In order to achieve the above object, the present invention is an inactive p53 tumor suppressor protein (hereinafter abbreviated as 'inactive p53 protein') gene in which the 143th amino acid valine is substituted with alanine in the 5 'to 3' direction; A first fluorescent protein gene fused to the gene; Internal ribosome entry site (IRES) ribosomal binding sequence; And a second fluorescent protein gene, the expression of which is regulated by one promoter to provide a recombinant p53 protein expression vector.

The present invention also provides a transformed cell line in which the recombinant p53 protein expression vector is introduced to stably express a fusion protein and a second fluorescent protein of an inactive p53 protein and a first fluorescent protein.

In addition, the present invention provides a method for searching for a substance involved in the activity of p53 tumor suppressor protein using the transformed cell line.

Hereinafter, the present invention will be described in more detail.

The present invention provides a recombinant p53 protein expression vector that can be usefully used to detect whether compounds and genes that can be used as anticancer agents, anticancer adjuvants or therapeutic agents related to degenerative diseases affect the stabilization of p53 protein and transfer to the nucleus, and their stability. It relates to a transformed cell line expressed by.

The recombinant p53 protein expression vector of the present invention is an inactive p53 mutant gene in which the 143th amino acid valine is substituted with alanine in the p53 tumor suppressor protein, which is essential for human cancer cell death and cell cycle regulation in the 5 'to 3' direction; A first fluorescent protein gene fused to the mutant gene; IRES ribosomal binding sequence; And a second fluorescent protein gene, the expression of which is designed to be regulated by one promoter.

In the recombinant p53 protein expression vector of the present invention, the gene of the first fluorescent protein is fused to maintain an in-frame with the inactive p53 gene, so that the fusion protein and the second fusion protein of the p53 protein and the first fluorescent protein are derived from the expression vector. Fluorescent protein is expressed.

When the wild type p53 protein is excessively expressed in a cell, since the p53 protein causes cell death, cells which express this protein at all times cannot be produced. Thus, in the present invention, inactive p53 mutant genes that have lost the transcription factor activity of p53 protein are used in order to prevent apoptosis even when p53 protein is excessively expressed in cells. The inactive p53 protein encoded from the gene is a substitution of alanine for valine, the 143th amino acid, of the amino acid sequence of the wild type p53 protein, and has an amino acid sequence as set forth in SEQ ID NO: 1 . The 143th valine of the p53 protein is a key amino acid residue among the protein sites in which the p53 protein binds to the DNA of the target gene. The p53 protein mutated by this site cannot bind to the target gene, resulting in cell self-killing caused by p53 activity. This makes it easy to observe the quantitative increase of p53 protein and intracellular migration (see FIG. 3 ).

In addition, the present invention uses a fluorescent protein to track the distribution and movement of the p53 protein in the cell, and to accurately analyze the location and location of the cytoplasm of the protein expressed cells. Fluorescent proteins have a high tendency to maintain their original fluorescence even when fused with other proteins, and are expressed in cells in the form of a fusion protein fused with the target protein and applied to quantitative and qualitative analysis of the target protein. In particular, since the fusion protein thus expressed is determined by the intrinsic properties of the protein fused to the fluorescent protein, its location and mobility can be usefully used in studies regarding the intracellular location of a specific protein.

First, the first fluorescent protein expressed in the form of a fusion protein with p53 protein from the recombinant expression vector of the present invention is for tracking the distribution and movement of p53 protein in real time, and the second fluorescent protein is expressed with p53 protein. The purpose is to observe the morphology, the number of cells, the location of the cytoplasm, and the like. In the present invention, the first fluorescent protein and the second fluorescent protein preferably have different excitation wavelengths and emission wavelengths, and a green fluorescence protein (GFP) derived from a light emitting jellyfish called Aequorea Victoria Blue (CFP), yellow (YFP), red (DsRed) fluoroproteins engineered to genetically mutate and have different excitation and emission wavelengths can be used. In a preferred embodiment of the present invention, red fluorescent protein DsRed is used as the first fluorescent protein and green fluorescent protein EGFP (enhanced GFP) is used as the second fluorescent protein. The red fluorescent protein DsRed shows the highest excitation wavelength at 558 nm and the highest emission wavelength at 583 nm, and the green fluorescent protein GFP shows the highest excitation wavelength at 488 nm and the highest emission wavelength at 507 nm.

As described above, when two kinds of fluorescent proteins having different wavelengths are transcribed from different promoters, the expression level of the target fluorescent protein may be changed depending on the transcriptional activity or cell state difference in each well. Therefore, when observing intracellular activity due to modification or migration after the production of a specific protein, it is very important to match intracellular transcriptional activity using an internal indicator protein produced by the same promoter to eliminate the difference due to transcriptional activity. It is important. Thus, in the present invention, the fusion protein gene of the p53 protein and the first fluorescent protein and the second fluorescent protein gene are simultaneously transcribed into one mRNA by one promoter, so that two fluorescent proteins can be produced therefrom. An expression vector comprising two ribosomal binding sites is designed. In a preferred embodiment of the present invention, an IRES sequence is located between a red fluorescent protein DsRed1 fused to an inactive p53 protein and a GFP gene, a green fluorescent protein. In contrast, the 5'-terminal region of the mRNA is The new ribosome binds to the non-intermediate region, allowing the production of new proteins regardless of the 5'-terminal protein production.

In the recombinant p53 protein expression vector prepared as described above, two proteins, the fusion protein of the inactive p53 protein and the first fluorescent protein and the second fluorescent protein as internal markers, were simultaneously expressed on one mRNA transcribed from the same promoter. The quantitative changes and intracellular migration of p53 protein can be easily analyzed based on the expression of the intralabel protein. That is, the expression vector is characterized by expressing the second fluorescent protein in a non-specific and continuous manner as an internal standard to exclude nonspecific actions of the compounds that may occur when only the fusion protein is expressed, activity errors due to cytotoxicity, and the like. do.

In a preferred embodiment of the present invention, the gene DsRed1-N1 / p53V143A encoding a fusion protein of the inactive p53 tumor suppressor protein and the red fluorescent protein DsRed1; IRES sequence; And a recombinant p53 protein expression vector pP53V143AdsRed1-IRES-GFP comprising a gene encoding a green fluorescent protein GFP (see FIGS. 2 and 4 ).

The present invention is also a biosensor for searching for a substance involved in the activity of p53 protein using a recombinant p53 protein expression vector having the above characteristics, the expression vector is inserted into the chromosome of the host cell to stably maintain the recombinant p53 protein Provide a transgenic cell line that expresses. The recombinant p53 protein expression vector is conventional transfection methods, such as DEAE-dextran, calcium phosphate method, microinjection method, DNA-containing liposome method, lipofectamine-DNA complex method, etc., preferably in electroporation This can be introduced into host cells, preferably animal cells, more preferably BHK cells, CHO cells, COS cells and various cancer cells. Such transfection methods are known in the art (Molecular Cloning, Cold Spring Harbor, New York, Cold Spring Harbor Laboratory Press, 2001).

In a preferred embodiment of the present invention, the recombinant p53 protein expression vector pP53V143AdsRed1-IRES-GFP is introduced into the chromosome to prepare a transformed cell line U2OS / pP53V143ADsRed-IRES-GFP which stably expresses p53V143A-DsRed1 fusion protein and GFP fluorescent protein. , The transformed cell line was deposited on October 14, 2005 to the Gene Bank of Korea Biotechnology Research Institute (Accession Number: KCTC 10863BP).

Recombinant p53 protein expression vector and the transformed cell line stably expressing the same according to the present invention due to the screening of compounds and genes that can induce self-killing of various malignant tumor cells, due to the interaction between the anticancer agent and p53 protein in the treatment of cancer In order to prevent side effects, it can be very useful for screening compounds that can properly inhibit the activity of p53 protein, and for screening compounds that can be used as a therapeutic agent for degenerative related diseases, including each Huntington's disease. In addition, the transformed cell line may be applied to evaluate the apoptosis efficacy of the chemocancer drug that is currently used for therapeutic purposes in the clinic. In particular, the vector system of the present invention using mutant p53 protein genes will be useful for screening drugs and genes involved in the movement of p53 protein in a stabilized intracellular environment in which cell death by autokilling of p53 protein is inhibited. In addition, the recombinant p53 protein expression vector of the present invention may be introduced into a transformed cell line, a recombinant adenovirus, a transgenic animal, etc. and may be usefully used as a biosensor. The cell line into which the recombinant p53 protein expression vector is introduced is not only useful for screening anticancer agents, anticancer adjuvant agents and therapeutic agents for degenerative diseases, but also for transgenic animals produced using the recombinant vector to observe and evaluate p53 mobility and stabilization. It can be used as an experimental disease model animal.

Accordingly, the screening system of the present invention using a recombinant p53 protein expression vector and a transformed cell line stably expressing the same is a biosensor for searching for a substance involved in the activity of p53 protein. It will be a useful tool in the development of related therapies.

In addition, the present invention provides a method for searching for a substance involved in the activity of p53 protein using a transformed cell line stably expressing the recombinant p53 protein expression vector.

The search method

1) culturing the transformed cell line stably expressing the recombinant p53 protein expression vector of the present invention in a well plate;

2) adding and incubating a sample to be searched to each well of the plate;

3) measuring fluorescence of the first fluorescent protein and fluorescence of the second fluorescent protein expressed therefrom in the form of a fusion protein, and analyzing the distribution pattern of the fluorescence; And

4) measuring the amount of the expressed p53 protein by correcting the fluorescence of the first fluorescent protein using the fluorescence of the second fluorescent protein as an internal indicator.

In step 2), to search for a sample that increases the amount of p53 protein, only the sample to be searched in the culture medium of the transformed cell line needs to be processed. On the other hand, in order to search for a sample that inhibits the quantitative increase of the p53 protein due to the DNA damage signal, it is preferable to process the sample and to further culture the DNA damaging material 1 hour after the treatment in step 2). As the DNA damaging substance that can be used in this step, any anti-cancer agent commonly used in chemotherapy may be used, and doxorubicin is particularly preferable.

In step 3), the fluorescence from the first fluorescent protein can confirm the intracellular distribution and migration of the fusion protein expressed from the transformed cell line, and the fluorescence from the second fluorescent protein can determine the overall shape and number of cells. You can check it. In this case, the fluorescence of the second fluorescent protein is used as a standard internal control, which may be factors such as cytotoxicity of the compound, the number of cells, cell types, and variables according to differences between wells. It is an element that standardizes exogenous variables not related to p53 protein activation such as DNA damage.

General Luminescence Measurement Using Luciferase Since the bottom of the microplates used in cell-based screening systems does not require light to pass through, plates with opaque bottoms are usually used. However, when analyzing the movement of proteins in the cell with fluorescence as in the present invention, a microplate with a transparent bottom and low light scattering must be used. Accordingly, in the present invention, it is preferable to use a cell carrier 384-well microplate (CellCarrier 384 μClear bottom microplate, Evotech Co., Ltd.) having a transparent bottom and minimal light scattering. However, the present invention is not limited only to the microplate.

The search method according to the present invention can easily analyze the stability of p53 tumor suppressor protein and mobility to the nucleus using recombinant fluorescent protein, which can be usefully used in related fields such as screening of various drugs and high-speed evaluation system in the future. There will be.

Hereinafter, the present invention will be described in more detail by the following Preparation Examples and Examples.

The following Preparation Examples and Examples are only for illustrating the present invention, but the scope of the present invention is not limited thereto.

Example 1 Preparation of Transgenic Cell Line and Expression Vector Expressing Tumor Suppressor Protein p53 and Fluorescent Protein DsRed1 Fusion Protein and Green Fluorescent Protein GFP

<1-1> Preparation of Expression Vector Expressing p53-DsRed1 Fusion Protein

First, in order to amplify an inactive p53 gene which has lost the transcription factor activity to be fused with the red fluorescent protein DsRed1 gene, a gene encoding a mutant p53 protein in which the 143th amino acid valine is substituted with alanine in the amino acid sequence of the wild type p53 protein is included. PCR was performed using the pCMVp53-V143A plasmid as a template and primer pairs of SEQ ID NOs: 2 and 3 . PCR reaction solution was 10 ng of the plasmid template DNA, primer pairs of SEQ ID NO: 2 and 3 , respectively, 1 pM, 16 mM (NH) ₄ SO ₄ , 67 mM Tris-HCl (pH 8.8), Prepared by mixing 0.01% Tween 20, 1.5 mM MgCl ₂ , PCR reaction was repeated 20 times for 1 minute at 95 ℃, 1 minute at 55 ℃ and 1 minute at 72 ℃. At this time, the primer pairs of SEQ ID NO: 2 and 3 generate XhoI and HindIII restriction enzyme cleavage sites at both ends of the PCR product, respectively, and replace valine, the 143th amino acid, in the amino acid sequence of the wild type p53 protein with alanine. Designed. PCR products amplified therefrom were digested with restriction enzymes XhoI and HindIII and isolated and purified using a mega-spin gel extraction kit (Intron Biotechnology).

Meanwhile, pDsRed1-N1 (Clontech), a DsRed1 protein expression vector, was digested with restriction enzymes XhoI and HindIII and isolated and purified using a mega-spin gel extraction kit (Intron Biotechnology). The p53 gene fragment and the pDsRed1-N1 vector fragment isolated and purified as described above were mixed at a ratio of 3: 1, and then ligated for 16 hours at 16 ° C. to express the fusion protein of the p53 gene and the fluorescent protein DsRed1. The expression vector pDsRed1-N1 / p53V143A was prepared ( FIG. 2 ).

In order to confirm that the p53-DsRed1 fusion protein expressed from the expression vector pDsRed1-N1 / p53V143A is a mutant protein having no transcription factor activity, the expression vector is a reporter expression vector pGL3- (p53) 12 containing a p53 binding sequence. Co-transfection was performed on the HeLa cell line (ATCC CCL-2) using lipofectamine. In this case, the reporter expression vector pGL3- (p53) 12 is a vector in which the E1B TATA box and 12 p53 protein binding sites are cloned into the pGL3-basic plasmid (Promega), and the binding sequence to which the wild-type p53 protein can bind is 12 Canine drainage is located in the front of the fruit fly luciferase gene, so luciferase analysis can confirm whether the protein is expressed. The expression vector pDsRed1-N1 / p53V143A was transformed into HeLa cells using Lipofectamine Plus (Lipofectamine Plus, Invitrogen) reagent as follows. 20 μl of serum-free and antibiotic-free DMEM, 100 ng of pDsRed1-N1 / p53V143A vector DNA, 250 ng of pGL3- (p53) 12 vector DNA, 50 ng of internal standard vector pRL-CMV DNA, and 2 μl plus reagent (Invitrogen) It was added and mixed, and incubated at room temperature for 15 minutes. The mixture was further added to 22 μl of serum-free and antibiotic-free DMEM and 3 μl of lipofectamine mixture, followed by further incubation at room temperature for 15 minutes to prepare a complex of DNA: lipopeptamine: Plus reagent: DMEM. At this time, 100 ng of pEGFP-N1 / wt p53 DNA containing the wild-type p53 gene was used as a control to perform transfection as described above. During the formation of the complex, the HeLa cell lines were washed twice with PBS and then added to 200 μl of serum free and antibiotic free DMEM. The completed complex was added to the cell line and incubated in 37 ° C., 5% CO ₂ incubator for 3 hours. The medium was then replaced with DMEM supplemented with 10% fetal calf serum and penicillin (100 units / ml, Invitrogen) and streptomycin (100 μg / ml, Invitrogen) and incubated at 37 ° C. for 48 hours.

As described above, 48 hours after the transient transfection, the activity change of Drosophila luciferase by p53 protein expression was confirmed using a double luciferase kit (Dual Luciferase Kit, Promega). First, the remaining medium in the plate is removed, washed three times with PBS buffer, and then diluted 5 ml of 5 × Passive Lysis Buffer contained in the kit with 1 × Passive Cell Lysate. 100 μl each was added to lyse the cells. 5 μl of the lysed cell extracts were transferred to a 384-well plate, 25 μl of LARII reagent was added to each well containing the lysed cell extracts, and after about 1 minute, a microplate reader (Envision Reader, Perkin Elmer) was used. Luminescence by Drosophila luciferase determined by the transcriptional activity of the clone vector was measured for 0.1 second. After the first measurement, each well was again treated with 25 μl of Stop & Glow reagent, and after 1 minute, a microplate reader was applied to Renilla luciferase determined by the transcriptional activity of pRL-CMV. Luminous intensity was measured for 0.1 second. At this time, Renilla luciferase luminescence under the CMV promoter is used as a standard internal control, and this value is used for experiments such as the cytotoxicity of the compound, the number of cells, the degree of activation by doxorubicin, and variables such as the difference between wells. It is a factor that standardizes exogenous variables that may appear, ie, independent of hypoxia signals. Two days after transfection, the transcriptional activation of each p53 gene was analyzed by reporter analysis using a double-luciferase kit.

As a result, as shown in Figure 3 , the wild-type p53 protein binds to the p53 binding site of the pGL3- (p53) 12 reporter vector, the activity of Drosophila luciferase is high as the promoter operates, whereas the mutant p53 gene In this case, the inactive p53 protein did not bind to the promoter and thus had low activity because the promoter could not operate. From this, it was confirmed that the p53 gene of the expression vector pDsRed1-N1 / p53V143A prepared above was a mutant without transcription factor activity, and the expression vector was used as an intermediate for constructing the final expression vector including the IRES sequence and the green fluorescent protein GFP. Used as a vector.

<1-2> Preparation of expression vector expressing p53-DsRed1 fusion protein and green fluorescent protein GFP by one promoter

In order to clone the fusion protein genes p53V143A-DsRed1 contained in the expression vector pDsRed1-N1 / p53V143A prepared in Example <1-1> to the front of the IRES sequence, the expression vector was digested with restriction enzymes BglII and NotI and p53V143A. A fragment containing the -DsRed1 fusion protein gene was isolated and purified. In addition, in order to isolate fragments containing the IRES sequence, the IRES-containing vector pIRES-hrGFP-1a (Stratagene) was digested with restriction enzymes BamHI-MluI and MluI-NotI, respectively, and then BamHI-MluI fragments (vector sequence 2571). 2695 bp corresponding to ˜687) and MluI-NotI fragments (1904 bp corresponding to vector sequences 667 to 2571) were isolated and purified, respectively. After the three fragments were separated and purified, the p53V143A-DsRed1 gene was cloned into multiple cloning sites of the pIRES-hrGFP-1a vector by linking them by ligase treatment at 16 ° C. for 12 hours. In normal cloning, the BglII and BamHI restriction enzyme recognition sites disappear during the linkage process. From this, an expression vector in which expression of p53V143A-DsRed1 fusion protein and green fluorescent protein GFP is controlled by one promoter was constructed, which was named pP53V143ADsRed1-IRES-GFP ( FIG. 4 ).

<1-3> Transformation cell line stably introduced expression vector pP53V143ADsRed-IRES-GFP

Human osteosarcoma cell line U2OS (ATCC HTB-96) was transplanted into 5 × 10 ⁵ cells in a 100 mm culture dish and incubated in a 37 ° C., 5% CO ₂ incubator for 24 hours. The cultured cells were mixed with the expression vector pP53V143ADsRed-IRES-GFP prepared in Example <1-2> and pTK-Hyg (Clontech), a selection vector, using lipofectamine (Lipofectamine plus reagent, Invitrogen). Co-transfection. In general, when preparing a stable cell line using lipofectamine, the total amount of DNA to be transfected is 5 μg / 100 mm, so 4 μg of pP53V143ADsRed-IRES-GFP expression vector DNA and 1 μg of pTK-Hyg vector DNA are used. It was mixed and transfected. 350 μl of McCoy's 5A medium (McCoy's 5A, GIBCO Co., Ltd.) containing no antibiotics was added to the DNA mixture. 40 μl of Plus reagent was added thereto, mixed with a pipette, and left at room temperature for 15 minutes. After 15 minutes, 400 µl of McCoy's 5A medium (McCoy's 5A + lipofectamine) containing lipofectamine was added, mixed with a pipette, and left for 15 minutes at room temperature. After 15 minutes, 800 μl of the mixed solution was added to a 100 mm culture dish containing 3.2 ml of fresh medium and incubated for 3 hours at 37 ° C. in a 5% CO ₂ incubator. After 3 hours, the plate was washed three times with PBS buffer, and then 10 ml of medium was added thereto, followed by incubation for another 24 hours. After culturing cells reached a confluent growth state, hygromycin B was treated at a concentration of 200 μg / ml. Hygromycin B was treated until all control cells that had not been transfected with the expression vector were killed, and when all control cells were killed, the living cells, ie, transfected cells, were treated with 0.25% trypsin and removed from the culture dish. Thereafter, each well of the 96-well plate was diluted to enter a single cell (single cell) and cultured. Single cells isolated from the cultured cells were transferred back to 24-well plates and cultured until saturated growth. The cultured cells were transferred to 25T culture flasks and incubated for 48 hours, again transferred to 75T culture flasks, and incubated in large quantities for 72 hours, and then transplanted at 5 × 10 ⁵ cells per 60 mm culture dish and incubated for another 24 hours. . Cell lines transfected with the expression vector pP53V143ADsRed-IRES-GFP were treated with doxorubicin to a final concentration of 0.5 μM, followed by red fluorescence (maximum excitation wavelength = 558 nm; maximum emission wavelength = 583 nm) and green fluorescence under fluorescence microscopy. The cell morphology (maximum excitation wavelength = 488 nm; maximum emission wavelength = 507 nm) was confirmed.

As shown in Figure 5 , the overall shape and the number of cells can be confirmed by the green fluorescence of GFP, the intracellular distribution and migration of the fusion protein p53V143A-DsRed1 was confirmed by the red fluorescence of DsRed1. In particular, when treated with the anticancer drug doxorubicin, it was observed that the p53V143A-DsRed1 fusion protein was shifted to the nucleus with an increase in the amount of the p53V143A-DsRed1 fusion protein. it means. In addition, there was almost no difference in the shape and growth rate of normal cells from normal U2OS cells. This is because the p53 protein of p53V143A-DsRed1 fusion protein expressed in the host cell is an inactive mutation. It has no effect. As such, the transfected cell line of U2OS cell origin, which can easily observe the intracellular increase and migration of p53 protein, was named U2OS / pP53V143ADsRed-IRES-GFP. Was deposited (Accession Number: KCTC 10863BP).

Example 2 Screening of Substances Affecting Quantitative Increase and Intracellular Migration of p53 Using Transfected Cell Line U2OS / pP53V143ADsRed-IRES-GFP

In order to search for compounds affecting the quantitative increase and distribution of p53 protein in cells using the transfected cell line U2OS / pP53V143ADsRed-IRES-GFP prepared in Example 1, the cell lines were 24-, 96- and 384. The cells were transplanted to a well plate (CellCarrier 384 μClear bottom microplate, Evotech) at a density of about 30%. After incubation for about 20 hours at 37 ℃, 5% CO ₂ incubator medium was changed to fresh McCoy's 5A medium containing 10% FBS (McCoy's 5A / 10% FBS). This was again incubated for about 1 hour at 37 ℃, 5% CO ₂ incubator, each sample was treated with the sample to be searched. At this time, in order to search only the sample which increases the amount of p53 protein by the DNA damage signal, only the sample was treated in the culture medium of the transfected cells, and in order to search for the sample which inhibits the quantitative increase of p53 protein by the DNA damage signal. One hour after treatment, doxorubicin was treated to a final concentration of 0.5 mM and incubated for another 20 hours. After 20 hours, the red fluorescence of DsRed1 was measured at fluorescence microscope at 583 nm, and the green fluorescence of GFP at 507 nm was measured to confirm cell morphology. The green fluorescence of GFP can confirm the overall shape and the number of cells, and the red fluorescence of DsRed1 can confirm the intracellular distribution and movement of the fusion proteins p53V143A-DsRed1. In this case, green fluorescence by GFP is used as a standard internal control, which is a factor that may occur during experiments, such as cytotoxicity of the compound, the number of cells, cell type, and the difference between wells, that is, DNA damage It is an element that standardizes exogenous variables not related to the activation of p53 protein.

As shown in FIG. 5 , similarly to doxorubicin, leptomycin, a compound known to move or concentrate p53 protein to the nucleus by inhibiting the migration of the p53 protein from the nucleus to the cytoplasm, was used as a sample. Expression increased and it was confirmed that the p53 proteins expressed were concentrated in the nucleus.

In general, the increase in p53 protein can be observed by fluorescence microscopy, but screening for more than 10,000 sample compounds takes too much time to observe each day. Difficult to apply In order to solve this problem, in the present invention, as shown in Figure 6 , by using a microplate reader (Envision Reader, Perkin Elmer Co., Ltd.), which is a device that can measure the fluorescence of a specific wavelength in a very short time, actually by doxorubicin An increase in the red fluorescence of the p53V143A-DsRed1 fusion protein was confirmed.

Example 3 High Content Search Method of Substances Affecting Quantitative Increase and Migration of p53 Using Transfected Cell Line U2OS / pP53V143ADsRed-IRES-GFP

The microplate reader used in Example 2 can measure the increase or decrease of protein amount by absorption, luminescence or fluorescence, but cannot confirm the distribution and movement of the protein present in the cell. Accordingly, the present inventors have used quantitative changes of p53V143A-DsRed1 fusion protein expressed in cells using the High Content Conforcal Cell Screening System (Evotech Co., Ltd.), a device capable of analyzing various phenomena occurring in cells. As well as quantitatively measuring intracellular migration.

As a result, as shown in FIG . 7 , the p53V143A-DsRed1 fusion protein bound to the nuclear membrane was not distinguishable from the p53V143A-DsRed1 fusion protein present in the nucleus by a general fluorescence microscope, but the high content image used in the present example was used. The screening system was equipped with a confocal fluorescence microscope so that it could be easily distinguished. The left panel of Figure 7 shows that the expression of p53 protein is increased by the treatment of doxorubicin, the right panel is treated with doxorubicin after processing a sample affecting the nuclear migration of p53 protein p53 protein by DNA damage signal This indicates that movement to the nucleus after the quantitative increase of is suppressed.

From the above results, the expression vector pP53V143ADsRed-IRES-GFP and a transformed cell line comprising the same according to the present invention can be used to search for compounds, genes and proteins related to the activity of p53 protein or to study their mechanism of action. Confirmed that it can.

As described above, the recombinant p53 protein expression vector of the present invention and the transformed cell line including the same may be effectively used for developing anticancer agents, anticancer adjuvants, and therapeutic agents for degenerative diseases as biosensors related to the activity of p53 protein.

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Claims (12)

A gene encoding the amino acid sequence of SEQ ID NO: 1 in the 5 'to 3' direction; A first fluorescent protein gene fused to the gene; Internal ribosome entry site (IRES) sequence of picorna virus; And a second fluorescent protein gene having an excitation and emission wavelength different from that of the first fluorescent protein, the expression of which is regulated by one promoter. delete The method of claim 1, The first fluorescent protein and the second fluorescent protein are each selected from the group consisting of green fluorescence protein (GFP), blue fluorescent protein (CFP), yellow fluorescent protein (YFP) and red fluorescent protein (DsRed). Recombinant expression vector. The method of claim 1, A gene encoding the amino acid sequence of SEQ ID 1 ; A gene encoding red fluorescent protein DsRed1; IRES sequence of picorna virus; And a gene encoding a green fluorescent protein GFP. The method of claim 4, wherein Recombinant expression vector, characterized in that it has a cleavage map shown in FIG. Cell line transformed with the recombinant expression vector of claim 1. The method of claim 6, Transgenic cell line characterized in that the human cell line U2OS (Accession Number: KCTC 10863BP) transformed with a recombinant expression vector having a cleavage map shown in FIG. 1) culturing the transgenic cell line of claim 6 in a well plate; 2) adding and incubating a sample to be searched to each well of the plate; 3) A method for searching for a substance involved in the activity of the p53 tumor suppressor protein, comprising measuring the fluorescence of the first fluorescent protein and the fluorescence of the second fluorescent protein expressed therefrom in the form of a fusion protein. The method of claim 8, The method of claim 2, characterized in that to search for a sample to increase the amount of p53 protein by processing the sample to be searched in the culture of the transformed cell line. The method of claim 8, 1 hour after the sample was treated in step 2), the method was characterized by searching for a sample that inhibits the quantitative increase of the p53 protein by the DNA damage signal by treating the DNA damaging material and further culturing. delete delete

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