KR20030046896A - Transformant having a recombinant reporter gene whose expression being regulated by glucocorticoid and method for screening analogue and inhibitor of glucocorticoid using same - Google Patents

Abstract

PURPOSE: A transformant having a recombinant reporter gene of which expression is regulated by glucocorticoid and a method for screening the analogue and inhibitor of glucocorticoid using the same are provided, thereby cheaply and rapidly screening the analogue and inhibitor of glucocorticoid. CONSTITUTION: A method for screening the analogue and inhibitor of glucocorticoid comprises the steps of: (1) preparing a recombinant reporter gene construct of which expression is regulated by glucocorticoid and a transformant containing the same; (2) treating the transformant with glucocorticoid and a testing compound and culturing the transformant; (3) separating expressed proteins from the cultured medium; (4) adding a substrate of the reporter gene to induce the enzyme-substrate reaction; and (5) measuring the luminescence level produced by the enzyme-substrate reaction, wherein the gene construct is prepared by fusion of glucocorticoid response element(GRE) and the reporter gene; the GRE has the nucleotide sequence of SEQ ID NO: 1; the reporter gene is selected from the group consisting of luciferase, alkaline phosphatase, Chloramphenicol acetyl transferase and galactosidase; the transformant is GRE luciferase-transformed mouse glioma cell; and the substrate is luciferin.

Description

TRANSFORMANT HAVING A RECOMBINANT REPORTER GENE WHOSE EXPRESSION BEING REGULATED BY GLUCOCORTICOID AND METHOD FOR SCREENING ANALOGUE AND INHIBI OF GLUCOCORTICOID USING SAME}

The present invention relates to a transformed cell line having a recombinant reporter gene whose expression is regulated by glucocorticoids, and a biological detection method of glucocorticoid-like substances and inhibitors using the same, and specifically, expression is regulated by glucocorticoids. The present invention relates to a synthetic reporter gene construct, a transformed cell line thereof, and a biological screening method for selecting analogs and inhibitors for glucocorticoids using the same.

Glucocorticoids are steroidal hormones produced and secreted by the adrenal corticotropic hormone (ACTH) secreted by the pituitary gland to produce carbohydrates from fats and proteins, as well as to produce carbohydrates from the lipids and proteins. It is involved in the proliferation of blast cells. It is also known to be secreted during physical stress and responsible for stress adaptation (Laugero KD, J. Neuroendocrinol. 13 (9): 827-835, 2001). However, excessive presence of glucocorticoids has adverse effects on the nervous system, particularly the hippocampus, disrupting synaptic flexibility, atrophy of the dendritic spine, reducing neuronal reproduction, and even neuronal death (Tome ME et al., Cell Death Differ. 8 (9): 953-961, 2001).

In most species, including humans, the physiological glucocorticoid is cortisol (hydrocortisone). As mentioned above, glucocorticoids are secreted in response to ACTH (corticotropin), which shows both changes and elevations in time-period rhythm in response to stress and food. Cortisol levels respond in minutes to many physical and psychological stresses, including trauma, surgical operations, exercise, worry, and depression. Cortisol is a steroid and works by binding to the glucocorticoid receptor (GR) in the cell. In humans, the glucocorticoid receptor is present in two forms: ligand-binding GR-alpha of 777 amino acids and different GR-beta isoforms only in the last 15 amino acids. Both types of GR have high affinity for their specific ligands and are thought to act through the same delivery pathway.

Therefore, glucocorticoids are involved in carbohydrate metabolism, development, resistance to stress, and the like, so that a similar substance can be used as a useful substance for controlling these physiological phenomena. On the other hand, if the glucocorticoid is excessive, it may show various types of toxicity in the nervous system, ranging from neurotransmission abnormalities to cell death. Therefore, identifying environmental pollutants exhibiting similar actions may help to understand the toxicity and mechanism of action of these substances. Will be In addition, the present invention may be utilized to suppress pathologies caused by excess glucocorticoids by searching for inhibitors capable of inhibiting glucocorticoid action.

The inventors have found that dioxins, which are highly toxic environmental pollutants, and estrogen-like substances that cause various endocrine disorders, are transformed into various environmental samples using a transformed cell line comprising a dioxin reactive sequence and an estrogen reactive sequence to which a reporter gene is bound. Biological screening methods have been developed to analyze existing dioxins and estrogen analogues (Korean Patent Application Publication Nos. 10-2001-46920 and 10-2001-81766).

Accordingly, the present inventors can search for biological substances capable of searching for analogues that induce gene expression in cells with a mechanism similar to glucocorticoids or inhibitors for inhibiting gene expression by glucocorticoids and endocrine disruptors (aka environmental hormones). As a result of diligent research efforts to develop a screening method, a luciferase gene is coupled to a glucocorticoid receptor to produce a recombinant reporter gene construct that can easily measure the amount of the expressed protein while its expression is regulated by the glucocorticoid. After treating various compounds in the transformed cell line, the expression of luciferase induced by glucocorticoids was measured through enzymatic activity, thereby confirming that the analogues and inhibitors for glucocorticoids can be easily selected. Completed.

SUMMARY OF THE INVENTION An object of the present invention is to provide a biological screening method that enables simple and economical screening of analogs and inhibitors for glucocorticoids involved in various physiological phenomena.

1 illustrates a process of searching for a glucocorticoid-like substance and an inhibitor by using a transformed cell line having a recombinant reporter gene whose expression is controlled by the glucocorticoid of the present invention,

Figure 2 shows a genetic map of the plasmid pGRE-Luc. Comprising a recombinant reporter gene construct produced in the present invention,

FIG. 3 shows the luciferase expression according to the concentration of dexamethasone in a transformed cell line having a recombinant reporter gene whose expression is controlled by the glucocorticoid of the present invention.

4 is a result of searching for a glucocorticoid-like substance and an inhibitor using a transformed cell line having a recombinant reporter gene whose expression is controlled by the glucocorticoid of the present invention.

In order to achieve the above object, the present invention provides a recombinant reporter gene construct in which expression is regulated by glucocorticoids.

The present invention also provides a transformed cell line comprising the recombinant reporter gene construct.

In addition, the present invention provides a biological screening method for selecting analogs and inhibitors for glucocorticoids using the transformed cell line.

Hereinafter, the present invention will be described in detail.

The present invention provides a recombinant reporter gene construct in which expression is regulated by glucocorticoids.

First, in the present invention, a glucocorticoid response element (GRE) present in a regulatory region of a protein whose expression is controlled by the glucocorticoid is fused with a reporter gene which is easy to measure the expression level, and the recombinant reporter gene construct To produce.

Glucocorticoids bind to glucocorticoid receptors to recognize and bind to specific sequences of DNA, or GRE (Ou XM et al., J. Biol. Chem . 276 (17): 14299-14307, 2001 ). The combination of these genes and proteins increases the transcription of specific proteins and induces cellular responses by glucocorticoids. Therefore, the glucocorticoid analogue and inhibitor can be searched by measuring the expression level of the gene regulated by GRE. That is, the glucocorticoid analogues will increase the expression level of the gene and the inhibitor will reduce the gene expression level by the glucocorticoid, thereby indirectly predicting the effect on these organisms.

Therefore, in the present invention, GRE, a DNA specific nucleotide sequence recognized after the glucocorticoid binds to the intracellular receptor, was selected as a gene whose expression can be controlled by the glucocorticoid. The GRE has the nucleotide sequence set forth in SEQ ID NO: 1 and is located about 2,500 bp above the transcription initiation base of the relevant gene and acts as an enhancer rather than a promoter.

Reporter genes that can be used in the recombinant reporter gene construct of the present invention include luciferase, alkaline phosphatase, CAT (Chloramphenicol acetyl transferase) or galactosidase (galactosidase), and the like. In a preferred embodiment of the GRE gene by combining the luciferase gene to prepare a GRE-luciferase recombinant gene construct (see Figure 2 ).

The present invention also provides a transformed cell line comprising the recombinant reporter gene construct.

The transformed cell line of the present invention contains a recombinant reporter gene construct, preferably a GRE-luciferase gene construct, whose expression is regulated by glucocorticoids, which is useful for the selection of analogs and inhibitors of glucocorticoids. Can be used.

That is, when a variety of compounds are treated and cultured in the transgenic cell line, and a substrate of luciferase is added to the protein isolated therefrom, a luminescence reaction is generated by an enzyme-substrate reaction in proportion to the expression level of luciferase. The increase and decrease of can detect whether the target compound is an analog or inhibitor of the glucocorticoid.

Since the glucocorticoid analogs and inhibitors introduced into the organism act on the glucocorticoid receptors and exhibit their effects, the host cell line to which the recombinant reporter gene construct of the present invention is to be transformed is used as a cell line expressing an excessive amount of the glucocorticoid receptor. Glioma cells (Be (2) C), Mouse Mammary Epithelial cells or monkey kidney cells (COS-1 cells, CV-1 cells) and the like are used, and mouse glioma cells are particularly preferred.

Specifically, the transgenic cell line comprises a plasmid containing the GRE-luciferase binding recombinant reporter gene construct of the present invention and a plasmid containing an antibiotic resistance gene that facilitates cell line selection by electroporation. After co-transformation into glial cells, the plasmids were screened for antibiotic resistance confirming their entry into the cells. The transformed cell line of the present invention thus selected was named C6 / pGRE-LUC and was deposited with the Korea Biotechnology Research Institute Genetic Bank on November 8, 2001 (Accession Number: KCTC 10109BP).

In addition, the present invention provides a biological screening method for selecting analogs and inhibitors for glucocorticoids using the transformed cell line.

The present invention is a specific compound by measuring the expression level of the protein product of the reporter gene when the glucocorticoid is treated using a transformed cell line comprising a recombinant reporter gene whose expression is controlled by the glucocorticoid produced by the above process It can be searched whether it acts as an analog or inhibitor of glucocorticoid.

Specifically, the search method of the present invention

1) preparing a recombinant reporter gene construct whose expression is regulated by glucocorticoids and a transformed cell line comprising the same;

2) culturing the transformed cell line by treating the glucocorticoid and the compound of interest;

3) separating the protein from the culture;

4) inducing an enzyme-substrate reaction by adding a substrate of a reporter gene to the isolated protein; And

6) measuring the degree of luminescence generated by the enzyme-substrate reaction (see FIG. 1 ).

Since the glucocorticoid analogs or inhibitors introduced into the organism will act on the glucocorticoid receptor and exert its effect, it is preferable to use a transgenic cell line engineered to overexpress the glucocorticoid receptor in step 1). In the present invention, a recombinant reporter gene construct is prepared by fusing the luciferase reporter gene to GRE, which is a gene sequence recognized by the glucocorticoid receptor using a gene recombination technology, and transforming it into mouse glioma cells, which is specific for glucocorticoids. The transforming cell line is selected by selectively propagating the cell group which reacts selectively.

The selected transformed cell lines were treated with the searched material and cultured, and then the luciferase expression was investigated by measuring the intensity of light generated by adding the substrate of luciferase to the proteins isolated from these cells. Search for corticosteroid analogs or inhibitors

In the present invention, as a preferred example, the search method is verified using dexamethasone, a synthetic glucocorticoid. Dexamethasone dissolved in DMSO (dimethylsulfoxide) is diluted in a culture solution, treated with the transformed cell line for 24 hours, and then centrifuged to recover the cells, lysing the cell membrane with a surfactant and homogenizing to separate only the protein. Luciferase, a substrate of luciferase, was added to the isolated protein, and the light produced by the enzyme-substrate reaction was measured by a light luminometer to confirm that the expression level of luciferase increased with the treatment concentration of dexamethasone.

As described above, the method for retrieving glucocorticoid-like substances and inhibitors using the transformed cell line of the present invention is simple, simple, and low in search cost. In particular, since hundreds to tens of thousands of substances can be searched at a time, it is suitable for a wide range of substances and has the advantage of simultaneously predicting the effects on organisms by utilizing the intracellular mechanism of action of glucocorticoid-like substances and inhibitors.

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 Construction of Recombinant Reporter Gene Construct

First, in order to obtain the GRE gene fragment, which is the glucocorticoid reaction sequence, both ends of the minimal promoter including GRE from the promoter of mouse mammary tumor virus (MMTV) are restricted to the restriction enzyme BAL 31 exonuclease. After cleavage, blunt ends were made from the Klenow fragment of DNA polymerase I. The luciferase gene was treated with the pGL2-promoter vector (Promega), the restriction enzyme SmaI, to cut the luciferase gene fragment and calf intestinal alkaline phosphatase to make both ends smooth. . The two genes were ligated to make a recombinant reporter gene construct pGRE-Luc. Comprising a GRE-luciferase fusion gene, which was confirmed by digestion analysis using restriction enzyme II ( FIG. 2A ).

Example 2 Preparation of Transgenic Cell Line

(2-1) Culture of Mouse Glioma Cells

Cultured mouse glioma C6 cells (ATCC CCL-107) in Dulbecco's Modified Eagle Medium (DMEM) containing 10% bovine calf serum (Biowhittaker) in a cell incubator at 37 ° C and 5% CO _2. It was. The culture solution was changed every two days, and when the cells were dispensed, 0.25% trypsin-EDTA was added and removed from the culture dish, and then divided into two to four.

(2-2) Production of Transgenic Cell Line with pGRE-Luc.

Mouse glioma C6 cells 1 × 10 ⁷ cells cultured as in Example (2-1) were prepared in Example 1 pGRE-Luc. Calcium phosphate coprecipitation, Hofman K., et al., Mol Cell Endocrinol. 25; 168 (1-2): 21-9 with 30 μg and 3 μg of plasmid pcDNA 3.1 (+) (Invitrogen) , 2000). At this time, the plasmid pcDNA 3.1 (+) contains a neomycin resistant gene to facilitate the selection of the transformed cell line. After incubation for 48 hours at 37 ° C., 5% CO ₂ , 750 μg / ml of G418 (neomycin) (Calbiochem) was treated to remove cells that were not transfected with the recombinant gene. The cells into which the recombinant gene is inserted survive the neomycin resistance gene in pcDNA 3.1 (+).

Single cell populations were obtained, transferred to 96 well plates, and expanded, and when the number of cells increased, each cell group was divided into two new wells, and DMSO (0.1%) and 1 μM dexamethasone (Sigma) were added for 24 hours. After treatment, cell proteins were obtained, and luciferase enzyme activity was measured to determine pGRE-Luc. Only cell populations in which genes enter the cell and function are selected.

The transformed cell line containing the recombinant reporter gene thus selected was named C6 / pGRE-LUC and was deposited with the Korea Biotechnology Research Institute Gene Bank on November 8, 2001 (Accession Number: KCTC 10109BP).

Example 3 Screening of Glucocorticoid Similar Substances and Inhibitors

In order to check whether the glucocorticoid analogs and inhibitors can be detected using the transformed cell line of the present invention, the following experiment was performed.

(3-1) Changes in Luciferase Expression by Dexamethasone Treatment

1 × 10 ⁴ cells / ml of the transformed cell lines selected in Example 2 were dispensed into each well of a 96 well plate and incubated for 24 hours, followed by 0.01, 0.1, 1, 10, 100, and DMSO (0.1%), respectively. Dexamethasone, a synthetic glucocorticoid dissolved at 1000 nM concentration, was treated for 24 hours. After treatment, the culture was aspirated off and washed twice with DPBS (Dulbecco's Phosphate Buffered Saline). 50 μl of buffer solution (nonionic surfactant, 1% Triton X-100, 20 mM Tris pH 8.0, 150 mM NaCl, 2 mM DTT) was added to lyse the cells. 10 μl of this was taken in a 12 × 75 borosilicate tube and mixed with 50 μl of luciferin substrate Luciferin (Pharmacia Co., Ltd.) to cause an enzyme-substrate reaction, and the light produced was measured with a photometer.

As a result, as shown in Figure 3 , the transformed cell line of the present invention comprising a recombinant reporter gene it can be seen that the expression level of luciferase is increased in a concentration-dependent manner by the treatment of dexamethasone, a glucocorticoid-like substance.

(3-2) Screening of Glucocorticoid Analogues and Inhibitors

In order to detect the glucocorticoid analogs and inhibitors using the transformed cell line of the present invention, DMSO, dexamethasone, bisphenol A, tributyltin, styrene, naphthalene, 2 , 4-dichlorophenol (2,4-dichlorophenol), fluoranthene, β-naphthoflavone, α-naphthoflavone, 6-coumarin , Anisaldehyde (anisaldehyde) and the like to be dissolved in a solvent DMSO at a concentration of 10 μM, each treated for 24 hours, the culture solution was aspirated and washed twice with DPBS. After this process was carried out in the same manner as in Example (3-1).

As a result, as shown in FIG . 4 , β-naphthoflavone showed the same amount of luciferase expression as dexamethasone, a synthetic glucocorticoid, while DMSO, bisphenol A, tributyltin, styrene, naphthalene, 2,4-dichlorophenol, fluoranthene, α-naphthoflavone, 6-coumarin, annisaldehyde and the like showed much lower levels of luciferase expression. From this, it can be seen that β-naphthoflavones act as analogues of glucocorticoids and the other substances act as inhibitors.

As described above, the biological screening method of the glucocorticoid-like substance and the inhibitor using the transformed cell line of the present invention 1) simple operation and does not require expensive equipment, only basic cell culture technology and enzyme activity measurement system Easy to use, 2) search for a large number of substances at a low cost at a time, 3) search for both glucocorticoid analogues and inhibitors, and 4) environmental pollutants that exhibit similar effects with glucocorticoids. Cytotoxicity can be confirmed, and 5) it has very high sensitivity.

Claims (10)

1) preparing a recombinant reporter gene construct whose expression is regulated by glucocorticoids and a transformed cell line comprising the same; 2) culturing the transformed cell line by treating the glucocorticoid and the compound of interest; 3) separating the protein from the culture; 4) inducing an enzyme-substrate reaction by adding a substrate of a reporter gene to the isolated protein; And 6) biological screening method of the glucocorticoid analogues and inhibitors, comprising measuring the degree of luminescence generated by the enzyme-substrate reaction. The method of claim 1, The gene construct of step 1) is a recombinant reporter gene construct in which the glucocorticoid response sequence (GRE) and the reporter gene are fused to regulate expression by the glucocorticoid. The method of claim 2, Search method characterized in that the GRE has a nucleotide sequence described in SEQ ID NO: 1 . The method of claim 2, The reporter gene is selected from the group consisting of luciferase, alkaline phosphatase, CAT (Chloramphenicol acetyl transferase) and galactosidase. The method of claim 2, And wherein the gene construct is GRE-luciferase with the gene map described in FIG. 2A . The method of claim 1, The cell line of step 1) is characterized in that the mouse glioma cell line transformed with GRE-luciferase. The method of claim 1, Search method characterized in that the substrate of step 4) is luciferin for luciferase. The method of claim 1, Step 5) is a screening method for measuring the enzymatic activity of luciferase that is increased or decreased by a glucocorticoid analog or inhibitor. Recombinant plasmid pGRE-Luc. Comprising a GRE-luciferase recombinant reporter gene and having the genetic map of FIG. 2A . Mouse glioma cell line C6 / pGRE-Luc transformed with plasmid pGRE-Luc. Comprising the GRE-luciferase recombinant reporter gene (Accession Number: KCTC 10109BP).