KR101876504B1 - Recombinant vector for stimultaneously expressing dual reporter gene - Google Patents

Abstract

The present invention relates to a recombinant vector for simultaneous expression of a dual reporter gene, a recombinant cell line, a pharmaceutical screening composition, a kit and a manufacturing method thereof. Since the recombinant vector of the present invention contains a gene encoding a luminescent enzyme linked to two different promoters in a single vector, the cell death effect and the expression of sodium iodide cotransporter can be confirmed at the same time, Since the cell line into which the recombinant vector is transfected is used, there is an advantage that the possibility of cell transformation that can occur during the establishment of two cell lines can be minimized. In addition, the present invention minimizes the influence between both promoters by reversing the directions of two promoters in the vector, and has an excellent effect of early selection of a substance with a high possibility of drug and drastically reducing drug development cost. Therefore, the recombinant vector according to the present invention can be used to easily evaluate candidate therapeutic agents for thyroid cancer, breast cancer, stomach cancer, teratoma, or salivary gland cancer using noninvasive molecular imaging techniques.

Description

Recombinant vector for stimulated expression of dual reporter gene < RTI ID = 0.0 >

The present invention relates to a recombinant vector for simultaneous expression of a dual reporter gene, a recombinant cell line using the same, a composition for screening a drug, a kit and a manufacturing method thereof.

Molecular imaging is used in the in vitro imaging of cell biology or molecular biology, in ex vivo imaging in organ or tissue culture, and in real-time in vivo imaging in animal experiments , The demand is increasing radically. In molecular imaging, the technique of labeling proteins or cells is very important, and many reporter genes have been developed for labeling.

Reporter genes are genes that biotechnology researchers attach to cell cultures or to regulatory sequences of genes of interest in plants or animals. The reporter gene is selected as a reporter because it easily distinguishes the characteristics given to the organism expressing it and becomes a measurement or selectable markers. The reporter gene is often used to determine whether a particular gene has entered or expressed in a cell or organ. Repeated, noninvasive reporter genes are luciferases.

Sodium iodide symporter (NIS) is a glycoprotein that mediates the active capture of one iodide ion and two sodium ions across the membrane of thyroid follicular cells (Dai, G. et al., Nature, 379: 458-460, 1996; Carrasco, M. et al., Biochim Biophys Acta, 1154: 65-82, 1993). Recently, cloning and functional studies on NIS genes have shown that NIS can be targeted for tumor therapy (Spizweg, C. et al., Gene Therapy, 8: 1524-1531, 2001; Nakamoto, Y. et al. (Mandell, RB et al., J Nucl Med, 41: 1898-1904, 2000; Dingli, D. et al., Blood, 102: 489-496, 2003) , Cancer Res, 59: 661-668, 1999; Spizweg, C. et al., Cancer Res, 60: 6526-6530, 2000).

KR registration number 10-0888343

Therefore, the inventors of the present invention prepared a recombinant vector expressing two reporter genes simultaneously expressing two different kinds of luminescent enzymes by using CMV promoter and NIS promoter in one vector, and using this recombinant vector, The present invention has been accomplished by confirming that a drug which can be used for prevention or treatment of diseases related to sodium iodide cotransporter can be screened.

Accordingly, an object of the present invention is to provide a gene encoding a CMV promoter (pCMV; CMV promoter) and a first luminescent enzyme operably linked to the promoter; And a gene encoding a sodium iodo symporter promoter (pNIS) and a second luminescent enzyme operably linked to the promoter.

It is also an object of the present invention to provide a recombinant cell line transformed with said vector.

Also, the object of the present invention is to provide a composition or kit for screening a drug for prevention or treatment of one or more diseases selected from the group consisting of thyroid cancer, breast cancer, stomach cancer, teratoma and salivary cancer, will be.

Further, the object of the present invention is (1) a method for producing a cell line, comprising the steps of: And (2) measuring the expression of the first luminescent enzyme and the second luminescent enzyme in the cell line; A method for screening a drug for at least one disease selected from the group consisting of thyroid cancer, breast cancer, stomach cancer, teratoma, and salivary gland cancer.

In order to accomplish the above object, the present invention provides a gene encoding a CMV promoter (pCMV; CMV promoter) and a first luminescent enzyme operably linked to the promoter; And a gene encoding a sodium lordotic symporter promoter (pNIS) and a second luminescent enzyme operably linked to the promoter.

In addition, the present invention provides a recombinant cell line transformed with said vector.

The present invention also provides a composition for screening a medicament for preventing or treating at least one disease selected from the group consisting of thyroid cancer, breast cancer, stomach cancer, teratoma and salivary gland cancer.

The present invention also provides a kit for screening a drug for preventing or treating one or more diseases selected from the group consisting of thyroid cancer, breast cancer, stomach cancer, teratoma, and salivary gland cancer, comprising the cell line.

(1) treating and culturing the cell line with a candidate substance; And (2) measuring the expression of the first luminescent enzyme and the second luminescent enzyme in the cell line; The present invention provides a method for screening a drug for at least one disease selected from the group consisting of thyroid cancer, breast cancer, stomach cancer, teratoma, and salivary gland cancer.

Since the recombinant vector of the present invention contains a gene encoding a luminescent enzyme linked to two different promoters in a single vector, the cell death effect and the expression of sodium iodide cotransporter can be confirmed at the same time, Since the cell line into which the recombinant vector is transfected is used, there is an advantage that the possibility of cell transformation that can occur during the establishment of two cell lines can be minimized. In addition, the present invention minimizes the influence between both promoters by reversing the directions of two promoters in the vector, and has an excellent effect of early selection of a substance with a high possibility of drug and drastically reducing drug development cost. Therefore, the recombinant vector according to the present invention can be used to easily evaluate candidate therapeutic agents for thyroid cancer, breast cancer, stomach cancer, teratoma, or salivary gland cancer using noninvasive molecular imaging techniques.

1 is a schematic diagram showing a dual reporter gene system.
2 is a diagram showing a schematic diagram of a dual reporter gene vector used in an experiment; pNIS is a promoter of sodium iodine cotransporter, FL2 is firefly luciferase which is a second luminescence enzyme, Turbo FP635 is a red fluorescent protein, pCMV is a CMV promoter, Rluc is a first luminescence enzyme, Renilla Luciferase.
3A shows results of confirming the operation of a dual reporter gene system in a human kidney cell line transfected with a recombinant vector of the present invention using optical imaging equipment.
FIG. 3B is a diagram showing the results of confirming the operation of the dual reporter gene system in a human kidney cell line transfected with the recombinant vector of the present invention using a fluorescence microscope.
FIG. 4A is a graph showing the results of confirming the operation of a dual reporter gene system in a thyroid cancer cell line transformed with the recombinant vector of the present invention using optical imaging equipment.
FIG. 4B is a graph showing the results of confirming the operation of a dual reporter gene system in a thyroid cancer cell line transfected with a recombinant vector of the present invention using a fluorescence microscope.
5A is a graph showing the results of confirming the operation of a dual reporter gene system by a candidate drug in a thyroid cancer cell line transfected with the recombinant vector of the present invention.
FIG. 5B is a graph showing the results of confirming the effect of the drug on the luminescence of the luminescent enzyme, in relation to the drug confirming the operation of the dual reporter gene system in FIG. 5A.
FIG. 6 is a graph showing the results of confirming the effect of screening of a cell line transformed with the recombinant vector of the present invention using Western blotting.

The present invention provides a recombinant vector for simultaneous expression of a dual reporter gene, a recombinant cell line using the same, a composition for screening a drug, a kit, and a manufacturing method.

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

The present invention provides a gene encoding a CMV promoter (pCMV; CMV promoter) and a first luminescent enzyme operably linked to the promoter; And a gene encoding a sodium lordotic symporter promoter (pNIS) and a second luminescent enzyme operably linked to the promoter.

As used herein, the term "promoter" means the minimum sequence sufficient to direct transcription.

As used herein, the term " CMV promoter (pCMV) " is a promoter that can be strongly transcribed in a host using an RNA polymerase used in almost all eukaryotic cells, and is always expressed in cells and in vivo Therefore, it can be used to evaluate the degree of cell proliferation and death, and the toxicity of the drug can be confirmed during drug screening.

As used herein, the term " sodium / iodide symporter promoter (pNIS) "refers to a transcription product of sodium iodide symporter promoter, which is a passage through which iodine is taken in a specific organ such as thyroid hormone synthesis, , And the expression of sodium iodide is decreased in thyroid cancer cells, breast cancer cells, stomach cancer cells, teratoma cells or salivary cancer cells as compared with normal cells. Therefore, the therapeutic agent for thyroid cancer, breast cancer, gastric cancer, teratoma or salivary gland cancer can be screened by measuring the activity of pNIS using the properties of sodium iodide transporter as described above. More specifically, a recombinant cell line transfected with a vector comprising a gene encoding a pNIS and a lysozyme operably linked to the promoter is used to identify a candidate compound for the expression of a lysozyme, a breast cancer, a gastric cancer , Teratoma, or salivary gland cancer.

The terms used in reference to the expression level may be referred to as having a differential level or differential value compared to a "normal" expression level or value, or expressed differentially, as well as quantitative differences in expression, Includes both of the differences.

As used herein, the term "vector" refers to a gene construct comprising a base sequence of a gene operably linked to a suitable regulatory sequence so as to be capable of expressing the gene of interest in a suitable host, A promoter that may be present, any operator sequences for modulating such transcription, and sequences that regulate the termination of transcription and translation. The vector of the present invention is not particularly limited as long as it is replicable in cells, and any vector known in the art may be used, and may be, for example, a plasmid, a cosmid, a phage particle, or a viral vector.

In the present specification, the "recombinant vector" means an expression vector of a desired polypeptide capable of expressing the desired polypeptide at a high efficiency in an appropriate host cell when the coding gene of the polypeptide of interest to be expressed is operatively linked , And the recombinant vector can be expressed in host cells. The host cell may preferably be a eukaryotic cell. Depending on the type of the host cell, an expression control sequence such as a promoter, a terminator, an inhancer, etc., a sequence for membrane targeting or secretion, And can be variously combined according to the purpose.

As used herein, "operably linked" refers to a functional linkage between a nucleic acid expression control sequence and a nucleic acid sequence encoding a desired protein to perform a general function. For example, a nucleic acid sequence encoding a promoter and a protein or RNA may be operably linked to affect the expression of the coding sequence. The operative linkage with the recombinant vector can be produced using genetic recombination techniques well known in the art, and site-specific DNA cleavage and linkage are made using enzymes generally known in the art.

In the present invention, the CMV promoter may be represented by SEQ ID NO: 1, but is not limited thereto.

In the present invention, the sodium iodo-transporter promoter may be represented by SEQ ID NO: 2, but is not limited thereto.

Variants of the nucleotide sequence represented by SEQ ID NO: 1 or 2 are also included within the scope of the present invention. The CMV promoter and the sodium iodo-cotransporter promoter nucleic acid molecule of the present invention may be used for the deletion, substitution, and / or deletion of functional bases of nucleic acid molecules constituting the CMV promoter and sodium iodide cotransporter promoter, for example, Or variants that are functionally equivalent to a CMV promoter and a sodium iodide cotransporter promoter nucleic acid molecule, although modified by insertion or insertion of the promoter. Specifically, the CMV promoter and the sodium iodide cotransporter promoter respectively have a sequence identity of 70% or more, more preferably 80% or more, still more preferably 90% or more, and most preferably 95% or more, Or more of the sequence homology. "% Of sequence homology to polynucleotides" is ascertained by comparing the comparison region with two optimally aligned sequences, and a portion of the polynucleotide sequence in the comparison region is the reference sequence for the optimal alignment of the two sequences (I. E., A gap) relative to the < / RTI >

In the present invention, a gene encoding the CMV promoter (pCMV; CMV promoter) and a first luminescent enzyme operably linked to the promoter; And a gene encoding a sodium lordotic iodide symporter promoter (pNIS) and a second luminescent enzyme operably linked to the promoter are arranged in opposite directions. Since the promoter is opposite in direction, So that the expression of the luminescence enzyme by the two promoters located in "one vector" is made independent from each other. Since the promoters that operate independently of each other are present in one vector, accuracy and efficiency in drug screening are increased as compared with the conventional invention.

Since the present invention has two promoters in one vector and utilizes the transfusion technique for the cell line, the effect of improving the disease associated with the cell killing effect and the expression of sodium iodide cotransporter is reduced for a shorter time And the possibility of cell transformation that can occur during the establishment of two cell lines can be minimized. In addition, the dual reporter system according to the present invention is advantageous in that the expression of two genes is independently expressed by the respective promoters, so that the activity of the CMV promoter and the sodium iodophosphate transporter promoter can be independently checked.

In the present specification, a reporter gene can be used without limitation as long as it is a gene capable of expressing the gene according to its upstream promoter activity and confirming its expression in the cell. In the present invention, 2 luminescent enzyme is a reporter gene, which contains all the reporter genes that can express color in addition to the luminescent enzyme.

The first luminescent enzyme and the second luminescent enzyme are each independently selected from the group consisting of firfly luciferase, renilla luciferase, Turbo FP635, Photinus pyralis luciferase, May be copepod gaussia luciferase or metridia longa luciferase and the luminescent enzyme operably linked to each promoter may be one in which the above luminescent enzyme is bound, The invention is not limited thereto.

The luciferase is an enzyme which promotes the oxidation of luciferin to convert chemical energy into light energy and emits light. It is an enzyme obtained directly from an insect such as a firefly or a glow-worm Or by expression from a microorganism comprising a recombinant DNA fragment encoding such an enzyme.

In one embodiment of the present invention, a Renilla luminescent enzyme is used as the first luminescent enzyme, and the Renilla luminescent enzyme is more preferably a luminescent enzyme encoded by the nucleotide sequence of SEQ ID NO: 3. In addition, the second luminescent enzyme may be a firefly luciferase, and the firefly luciferase may be encoded by the nucleotide sequence of SEQ ID NO: 4. However, the nucleotide sequence is merely an example, and the present invention is not limited thereto.

In the present invention, the recombinant vector has a cleavage map as shown in the following figures, but is not limited thereto, so long as it is a vector capable of achieving the dual reporter gene expression of the present invention.

The vector may further comprise an enhancer, a transcriptional regulatory factor, a replication origin, an additional reporter gene, an additional promoter, etc., and may encode the CMV promoter (pCMV; CMV promoter) and the first luciferase operably linked to the promoter gene; And a gene encoding a second luminescence enzyme operatively linked to a sodium iodide symporter promoter (pNIS) and a promoter of the sodium iodide, and which can be helpful in drug screening You can add more.

The present invention also provides a method for producing a gene encoding a CMV promoter (pCMV; CMV promoter) and a gene encoding a first luminescent enzyme operably linked to the promoter; And a gene encoding a sodium lordotic iodide symporter promoter (pNIS) and a second luminescence enzyme operably linked to the promoter, are transfected into a recombinant cell line transformed with the recombinant vector cell line.

As used herein, the term "cell line" refers to each individual of the cell line when cells are separated and cultured and subcultured, wherein the cell line can be distinguished from other cell lines by genetic traits, It is said to be maintained. In the present invention, the cell line can be used regardless of the type of cell line in which the expression of the sodium iodine transporter varies depending on the state of the cell line (cancer development, cancer treatment, etc.). Preferably, the cell line is a thyroid cancer cell line, a breast cancer cell line, , Salivary gland cancer cell line, gastric cancer cell line, and teratoma cell line, and the present invention is not limited thereto.

As used herein, the term "transduction" refers to a technique of transplanting a given DNA into a cell, that is, injecting DNA, which is a kind of nucleic acid extracted from cells of any line of the organism (excluding viruses), into living eukaryotic cells of other lines The DNA enters the cell and the genetic trait changes. That is, "transfection" means that a specific gene can be introduced into a host cell and expressed in the host cell.

The recombinant vector of the present invention may be transformed into a cell line by a method known in the art, for example, calcium phosphate coprecipitation, DEAE-textur treatment, electroporation, redistribution, and the like And the like. Preferably, the transfection can be performed by calcium phosphate co-precipitation, and the present invention is not limited thereto.

The present invention also provides a method for producing a gene encoding a CMV promoter (pCMV; CMV promoter) and a gene encoding a first luminescent enzyme operably linked to the promoter; And a recombinant vector having a recombinant vector transfected with a recombinant vector comprising a sodium iodide symporter promoter (pNIS) and a gene encoding a second luminescence enzyme operably linked to the promoter, and a thyroid cancer, breast cancer, A composition for screening a drug for at least one disease selected from the group consisting of gastric cancer, teratoma, and salivary gland cancer.

The present composition can be used for the treatment of thyroid cancer, breast cancer, stomach cancer, teratoma or saline cancer by measuring the level of the luminescence enzyme by pCMV and the pNIS using the vector of the present invention. Screening can be performed.

The present invention also provides a method for producing a gene encoding a CMV promoter (pCMV; CMV promoter) and a gene encoding a first luminescent enzyme operably linked to the promoter; And a recombinant vector having a recombinant vector transfected with a recombinant vector comprising a sodium iodide symporter promoter (pNIS) and a gene encoding a second luminescence enzyme operably linked to the promoter, and a thyroid cancer, breast cancer, There is provided a kit for drug screening for preventing or treating at least one disease selected from the group consisting of gastric cancer, teratoma, and salivary gland cancer.

The screening kit may be prepared using a method commonly used in the art, and is generally used in the art for the screening of drugs for the prevention or treatment of thyroid cancer, breast cancer, gastric cancer, teratoma or salivary gland cancer Tools, reagents, and the like. Such tools or reagents include, but are not limited to, suitable carriers, labeling substances capable of producing a detectable signal, solubilizers, detergents, buffers, stabilizers, and the like.

The screening kit may further include an instruction manual describing an optimum reaction performing condition. The instructions include instructions on the surface of the package, including the brochure or leaflet in the form of a brochure, a label attached to the kit, and a kit. In addition, the brochure may include information that is disclosed or provided through an electronic medium, such as the Internet.

The present invention also provides a method for producing a gene encoding a CMV promoter (pCMV; CMV promoter) and a gene encoding a first luminescent enzyme operably linked to the promoter; And a gene encoding a second luminescent enzyme operatively linked to a sodium iodide symporter promoter (pNIS) and a promoter, and a recombinant vector cell line transformed with the recombinant vector, ; (2) a method for preventing or treating one or more diseases selected from the group consisting of thyroid cancer, breast cancer, stomach cancer, teratoma and salivary cancer, which comprises measuring the expression of the first luminescent enzyme and the second luminescent enzyme in the cell line A method for screening pharmaceuticals is provided.

In the step (1), the culturing can be carried out in accordance with a culturing method suitable for culturing eukaryotic cells, and the culturing can be carried out in a batch, infusion or continuous manner, though not limited thereto.

The step of measuring the expression of the luminescent enzyme in step (2) may be carried out using any one or more of immunofluorescence staining, fluorescence resonance energy transfer, biomedical imaging, nuclear medicine imaging, luminescence imaging, or fluorescence imaging Preferably, the luminescence image confirmation method or the fluorescence image confirmation method can be used to measure the luminescence enzyme, but the present invention is not limited thereto.

In the present invention, when the expression of the first luminescent enzyme is maintained by the candidate substance and the expression of the second luminescent enzyme is increased as compared with the control group, the candidate screening method can be applied to the thyroid cancer, breast cancer, stomach cancer, And judging the drug as a drug for one or more diseases selected from the group consisting of More specifically, the expression of the first luminescent enzyme is maintained at a level of 50% to 100%, more preferably 60% to 70%, relative to the control, and the expression ratio of the second luminescent enzyme is about 2 times or more , The candidate substance may be judged to be a drug for one or more diseases selected from the group consisting of thyroid cancer, breast cancer, stomach cancer, teratoma and salivary gland cancer. The degree of apoptosis by the drug was measured by the first luminescent enzyme and the increase of the expression of sodium iodide cotransporter was measured through the second luminescent enzyme to measure the degree of apoptosis of thyroid cancer, breast cancer, stomach cancer, Drugs for teratoma or salivary gland cancer can be efficiently screened. In particular, since the degree of expression of the sodium iodide cotransporter promoter can be corrected by the CMV promoter expression rate, a drug having excellent therapeutic effect against thyroid cancer, breast cancer, stomach cancer, teratoma or salivary gland cancer can be more efficiently found.

The control group refers to a group which does not apply any operation or condition to judge whether the experimental result is properly derived or not. Unlike the experiment group which is set to achieve the direct purpose of the experiment, the control group makes the test result more assured . The control group in the present invention preferably refers to a group in which the candidate substance is not treated in the recombinant cell line transfected with the recombinant vector.

In the screening method of the present invention, the substance to be tested is presumed to have the possibility of preventing or treating a disease related to sodium iodine cotransporter including thyroid cancer, breast cancer, gastric cancer, teratoma or salivary gland cancer according to a conventional selection method, Randomly selected individual nucleic acids, proteins, other extracts or natural products, compounds, and the like.

The substance obtained by such a screening method acts as a leading compound in the process of developing a preventive or therapeutic agent for a disease related to sodium iodine airborne transplant including the following thyroid cancer, breast cancer, gastric cancer, teratoma or salivary gland cancer, By modifying and optimizing the material, it is possible to develop a preventive or therapeutic agent for diseases related to sodium iodide cotransporter including new thyroid cancer, breast cancer, stomach cancer, teratoma or salivary gland cancer and the like.

The redundant contents are taken into consideration in the complexity of the present specification, and terms not otherwise defined herein have the meanings commonly used in the art to which the present invention belongs.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention and are not intended to limit the scope of the present invention.

Example  One. CMV  Promoter and sodium iodine Airstream  Using a promoter Dual Reporter  Genetically recombinant plasmid production

To simultaneously express dual reporter genes using the CMV promoter (pCMV) and the sodium iodide symporter promoter (pNIS), recombinant plasmids were constructed to construct a dual reporter gene system.

First, the R-luciferase gene was inserted into the pcDNA3.1-Hyg plasmid and the CMV primer represented by SEQ. ID. NO: 1 and pCMV-Rluc, a DNA encoding the Renilla luciferase encoded by the nucleotide sequence shown in SEQ ID NO: 3, Respectively. Then, the DNA was treated with HindIII and XbaI restriction enzymes, and the pNIS-FL2-Turbo FP635 plasmid containing the sodium iodine cotransporter primer shown in SEQ ID NO: 2 and the firefly luciferase encoded with the nucleotide sequence shown in SEQ ID NO: (Obtained from Dr. Abhijit de of Molecular functional imaging laboratory (Navi Mumbai, india)) was treated with MluI restriction enzyme and left at 37 ° C for 3 hours. The DNA and the plasmid thus treated were ligated to a plasmid having a dual reporter gene system in which a CMV promoter cassette and a Renilla Luciferase were inserted in the opposite direction to the sodium iodine cotransporter promoter using a gene cloning technique common in the art . A schematic diagram of a plasmid having a dual reporter gene system prepared by the above method is shown in FIG. 1 and FIG.

Example  2. In the cell line transfected with the recombinant plasmid Dual  Identification of reporter gene expression

2-1. In the kidney cell line Dual  Identification of reporter gene expression

Experiments were carried out to confirm whether the expression of sodium iodide cotransporter and apoptosis can be visually measured by the plasmid having the dual reporter gene system prepared in Example 1 above. First, 4 × 10 ⁵ human HEK293T cells were cultured in 6-well plates for one day, and 5 μg of DNA was transfected with calcium phosphate to transform the recombinant plasmids prepared in Example 1. Then, the cell line was further cultured for one day. To promote the enzyme-substrate reaction activity, D-luciferin was added to a firefly luminescence enzyme (first luminescent enzyme) Coelenterazine was added. The luminescence of the luminescence enzyme was confirmed by an optical microscope (IVIS, Perkin Elmer), and the results are shown in Fig. In addition, the luminescence of Turbo FP635 was confirmed by fluorescence microscopy, and the results are shown in Fig. 3b.

As shown in FIG. 3, the recombinant human kidney cell line transfected with the plasmid having the dual reporter gene system prepared in Example 1 showed that both of the firefly luciferase and the Renilla luminescence were normally emitted.

2-2. In thyroid cancer cell line Dual  Identification of reporter gene expression

Experiments were carried out to confirm whether the expression of sodium iodide cotransporter and apoptosis can be visually measured by the plasmid having the dual reporter gene system prepared in Example 1 above. First, ⁴ thyroid cancer cell line 8505C (1.25x10) was cultured on a 96-well plate, and then a cell line transformed with the combined vector was constructed in the same manner as in Example 2-1. Thereafter, the luminescence of the luminescence enzyme was confirmed by an optical microscope (IVIS, Perkin Elmer). The results are shown in FIG. 4A. In addition, the luminescence of Turbo FP635 was confirmed by fluorescence microscopy, and the result is shown in FIG. 4B.

As shown in FIG. 4, the recombinant thyroid cancer cell line transfected with the plasmid having the dual reporter gene system prepared in Example 1 was found to normally emit both the firefly luciferase and the Renilla luminescence enzyme.

Example  3. Dual  Identification of drug screening effect through reporter gene system

3-1. Identify visual drug screening effects

The optical imaging equipment was used to confirm whether the plasmid having the dual reporter gene system prepared in Example 1 could be used for drug screening. First, a recombinant thyroid carcinoma cell line transfected in the above Example 2-2 was cultured on a plate storing 3200 species provided by the Korean Chemical Bank of Korea Research Institute of Chemical Technology. After 24 hours, the luminescence of the luminescence enzyme was confirmed by optical image equipment (IVIS, Perkin Elmer). The results are shown in Fig. 5A.

After the luminescence was confirmed, only the plate in which the expression of the first luminescence enzyme was maintained at 70% or more and at the same time, the luminescence of the second luminescence enzyme became more remarkable was separately cultured and the luminescence was confirmed by separating the plate separately. 5b.

3-2. Identification of drug screening effect at protein level

In order to confirm whether the expression of the sodium iodide cotransporter actually increased in the plate to which the luminescence was confirmed in Example 3-1, the expression of the first luminescence enzyme in the above-mentioned drug was maintained at 70% or more While K905-0266 (Chemdiv., Inc.), a substance that significantly increased the luminescence of the second luminescent enzyme, was selected. Western blotting was performed on the cell line of the plate treated with the concentrations of 6.25 μM and 12.5 μM. Western blotting was performed using methods commonly used in the art, and the results are shown in Fig. The control shown in the figure represents a recombinant cell line without drug treatment, and actin was used as a reference for confirming the operation of Western blot.

As shown in FIG. 6, the increase in the expression of sodium iodophosphate was remarkably observed in the cell line of the plate treated with 12.5 μM of the drug, and the drug (K905-0266) was found to be effective as a therapeutic agent for thyroid cancer Can be used. This indicates that the recombinant cell line transformed with the plasmid having the dual reporter gene system according to the present invention can be used for the screening of medicines for the prevention or treatment of thyroid cancer, breast cancer, gastric cancer, teratoma and salivary gland cancer.

In general, the recombinant vector according to the present invention has a gene encoding a luminescent enzyme linked to two different promoters in a single vector, and the cell line into which the recombinant vector is transduced is used. Therefore, It can be confirmed that the cell killing effect and the expression of sodium iodide cotransporter can be confirmed at the same time as compared with the inventions, and at the same time, the possibility of cell transformation that can occur in the establishment of two cell lines can be minimized. In addition, since the effect of both promoters is minimized by reversing the directions of two promoters in the vector, the present invention is advantageous in early selection of a substance with a high possibility of drug use and drastically reducing drug development cost have. Therefore, it can be seen that the candidate agent for the prophylactic or therapeutic treatment of thyroid cancer, breast cancer, stomach cancer, teratoma or salivary cancer can be simply evaluated by the noninvasive molecular imaging technique using the recombinant vector according to the present invention .

Although the present invention has been described in terms of the preferred embodiments mentioned above, it is possible to make various modifications and variations without departing from the spirit and scope of the invention. It is also to be understood that the appended claims are intended to cover such modifications and changes as fall within the scope of the invention.

<110> Kyungpook National University Industry-Academic Cooperation Foundation <120> Recombinant vector for stimultaneously expressing dual reporter          gene <130> p-1287 <160> 4 <170> KoPatentin 3.0 <210> 1 <211> 204 <212> DNA <213> Artificial Sequence <220> <223> cytomegalovirus promoter <400> 1 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt 60 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac 120 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg 180 tgggaggtct atataagcag agct 204 <210> 2 <211> 1245 <212> DNA <213> Artificial Sequence <220> <223> sodium / iodide symporter promoter <400> 2 ttgtattttt catacagaca agatctcact atgttgctca gggtagtctc gaattctggg 60 actcaaatga tcctcccact tcagcctccc aaagtgctgg gattacaggc ataagccatc 120 atgcccggcc tctgacgctg tttctttcaa cccccaggat ttcagattcc accagcttat 180 ggagaaggga accaagtttg agatgcgtga ttgcccagaa agttggaggc tgagctgaga 240 cttgaaccca gagaccagaa cctccagagg tcaaagtcct cctgggtccc ccagagaagg 300 gccctgagat gacagctcgt tggtcctcat ggaagcgtga cccccccagt agactttctc 360 ccacacccaa ccttggtttc ctcatctata tgatagggac aagccagact ctacctccct 420 ggtggtcatg gtctccgctt attcgggttc ataaccttaa aggcccctcg caccacctca 480 gtgagccatt tatgcctggc acagggccaa ctctcagtgc atatctgcaa aggagccaat 540 gaatgaatga atgaagtgac aaatgaataa aggaataaat gaatgaggca cttatcatgt 600 accaggcttt cgttaccacg tcccatttat tcctctgagg cagggtctat tttatccttg 660 ttacagatgg ggaaactaag gcccagggag gagcaaagtc ttccccaagt atgtacccac 720 tcagaacttg agctctgaat gtctcccacc cagcttagcc caagagcggg gttcagtgat 780 gcccaccccc taaggctcta gagaaagggg gtaggcccac atgccagttt gggggtggta 840 aagccaggta agttttcttt atgggtcccc tgaaaccctg aaagtgaacc ccagtcctgc 900 atgaaagtga gctccccata gctcaaggta ttcaagcaca atacggcttt gagtgctgaa 960 gcaggctgtg caggcttgga tagtgacatg ccctctctga gcctcaattt ccccacctgt 1020 caacagcaga cagtgacagc tgtgatcagg ggatcacagt gcatggggat gggtgggtgc 1080 atggggatgg aggggcattt gggagccctc cccgatacca ccccctgcag ccacccagat 1140 agcctgtcct ggcctgtctg tcccagtcca gggctgaaag ggtgcgggtc ctgcccgccc 1200 ctaggtctgg aggcggagtc gcggtgaccc gggagcccaa intro 1245 <210> 3 <211> 1650 <212> DNA <213> Artificial Sequence <220> <223> firefly luciferase <400> 3 atggaagatg ccaaaaacat taagaagggc ccagcgccat tctacccact cgaagacggg 60 accgccggcg agcagctgca caaagccatg aagcgctacg ccctggtgcc cggcaccatc 120 gcctttaccg acgcacatat cgaggtggac attacctacg ccgagtactt cgagatgagc 180 gttcggctgg cagaagctat gaagcgctat gggctgaata caaaccatcg gatcgtggtg 240 tgcagcgaga atagcttgca gttcttcatg cccgtgttgg gtgccctgtt catcggtgtg 300 gctgtggccc cagctaacga catctacaac gagcgcgagc tgctgaacag catgggcatc 360 agccagccca ccgtcgtatt cgtgagcaag aaagggctgc aaaagatcct caacgtgcaa 420 aagaagctac cgatcataca aaagatcatc atcatggata gcaagaccga ctaccagggc 480 ttccaaagca tgtacacctt cgtgacttcc catttgccac ccggcttcaa cgagtacgac 540 ttcgtgcccg agagcttcga ccgggacaaa accatcgccc tgatcatgaa cagtagtggc 600 agtaccggat tgcccaaggg cgtagcccta ccgcaccgca ccgcttgtgt ccgattcagt 660 catgcccgcg accccatctt cggcaaccag atcatccccg acaccgctat cctcagcgtg 720 gtgccatttc accacggctt cggcatgttc accacgctgg gctacttgat ctgcggcttt 780 cgggtcgtgc tcatgtaccg cttcgaggag gagctattct tgcgcagctt gcaagactat 840 aagattcaat ctgccctgct ggtgcccaca ctatttagct tcttcgctaa gagcactctc 900 atcgacaagt acgacctaag caacttgcac gagatcgcca gcggcggggc gccgctcagc 960 aaggaggtag gtgaggccgt ggccaaacgc ttccacctac caggcatccg ccagggctac 1020 ggcctgacag aaacaaccag cgccattctg atcacccccg aaggggacga caagcctggc 1080 gcagtaggca aggtggtgcc cttcttcgag gctaaggtgg tggacttgga caccggtaag 1140 acactgggtg tgaaccagcg cggcgagctg tgcgtccgtg gccccatgat catgagcggc 1200 tacgttaaca accccgaggc tacaaacgct ctcatcgaca aggacggctg gctgcacagc 1260 ggcgacatcg cctactggga cgaggacgag cacttcttca tcgtggaccg gctgaagagc 1320 ctgatcaaat acaagggcta ccaggtagcc ccagccgaac tggagagcat cctgctgcaa 1380 caccccaaca tcttcgacgc cggggtcgcc ggcctgcccg acgacgatgc cggcgagctg 1440 cccgccgcag tcgtcgtgct ggaacacggt aaaaccatga ccgagaagga gatcgtggac 1500 tatgtggcca gccaggttac aaccgccaag aagctgcgcg gtggtgttgt gttcgtggac 1560 gaggtgccta aaggactgac cggcaagttg gacgcccgca agatccgcga gattctcatt 1620 aaggccaaga agggcggcaa gatcgccgtg 1650 <210> 4 <211> 2240 <212> DNA <213> Artificial Sequence <220> <223> renilla luciferase <400> 4 taccacattt gtagaggttt tacttgcttt aaaaaacctc ccacacctcc ccctgaacct 60 gaaacataaa atgaatgcaa ttgttgttgt taacttgttt attgcagctt ataatggtta 120 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag 180 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgctcgaagc ggccgctcta 240 gaattattgt tcatttttga gaactcgctc aacgaacgat ttgatatatt ttcccatttc 300 atcaggtgca tcttcttgcg aaaaatgaag accttttact ttgacaaatt cagtattagg 360 aaacttcttg gcaccttcaa caatagcatt ggaaaagaat cctgggtccg attcaataaa 420 catttttggt aaatcatcac ttgcacgtag ataagcatta taattcctaa caatttgtac 480 aacgtcaggt ttaccacctt ttactaacgg gatttcacga ggccatgata atgttggacg 540 acgaacttca cctttctctt tgaatggttc aagatatgct gcaaattctt ctggttctaa 600 ctttctcatg atttttgatg gcaacatggt ttccacgaag aagttattct ccaaaaccat 660 tttttctcct tcttcagatt tgatcaacgc aatatcttct tcaatatcag gccattcatc 720 ccatgattca atcacatcta ctacactttc agcgtgaact attgctttga tcttatcttg 780 atgctcatag ctataatgaa atgccaaaca agcaccccaa tcatggccga caaaaatgat 840 cttctttggt aaattaagaa gttcaaacca tgcagtaaga tatttgtaat gatcaagtaa 900 cctataagaa ccattaccag atttgcctga tttgcccata ccaataaggt ctggtataat 960 acaccgcgct actggctcaa tatgtggcac aacatgtcgc cataaataag aagaggccgc 1020 gttaccatgt aaaaaaataa cagcattttc tgcatgtttt tctgaatcat aataattaat 1080 aaatgaatca agaacattca tttgtttaca tctggcccac cactgcggac cagttatcat 1140 ccgtttcctt tgttctggat cataaacttt cgaagtcatg gtggctagcc tatagtgagt 1200 cgtattaagt actctagcct taagagctgt aattgaactg ggagtggaca cctgtggaga 1260 gaaaggcaaa gtggatgtca gtaagaccaa taggtgccta tcagaaacgc aagagtcttc 1320 tctgtctcga caagcccagt ttctattggt ctccttaaac ctgtcttgta accttgatac 1380 ttacctgccc agtgcctcac gaccaacttc tgcagcttaa gttcgagact gttgtgtcag 1440 aagcactgac tgcgttagca atttaactgt gataaactac cgcaataaag cttctagtga 1500 tctgacggtt cactaaacga gctctgctta tatagacctc ccaccgtaca cgcctaccgc 1560 ccatttgcgt caacggggcg gggttattac gacattttgg aaagtcccgt tgattttggt 1620 gccaaaacaa actcccattg acgtcaatgg ggtggagact tggaaatccc cgtgagtcaa 1680 accgctatcc acgcccattg gtgtactgcc aaaaccgcat caccatggta atagcgatga 1740 ctaatacgta gatgtactgc caagtaggaa agtcccgtaa ggtcatgtac tgggcataat 1800 gccaggcggg ccatttaccg tcattgacgt caataggggg cggacttggc atatgataca 1860 cttgatgtac tgccaagtgg gcagtttacc gtaaatactc cacccattga cgtcaatgga 1920 aagtccctat tggcgttact atgggaacat acgtcattat tgacgtcaat gggcgggggt 1980 cgttgggcgg tcagccaggc gggccattta ccgtaagtta tgtaacgcgg aactccatat 2040 atgggctatg aactaatgac cccgtaattg attactatta ataactagtc aataatcaat 2100 gccaacatgg cggtcatatt ggacatgagc caatataaat gtacatatta tgatatagat 2160 acaacgtatg caatggccaa tagccaatat tgatttatgc tatataacca atgaataata 2220 tggctaatgg ccaatattga 2240

Claims (11)

A gene encoding a CMV promoter (pCMV; CMV promoter) and a first luminescent enzyme operably linked to the promoter; And a gene encoding a sodium iodide symporter promoter (pNIS) and a second luminescent enzyme operably linked to the promoter,
A gene encoding said CMV promoter (pCMV; CMV promoter) and a first luminescent enzyme operably linked to said promoter; And a gene encoding a sodium iodide symporter promoter (pNIS) and a second luminescence enzyme operably linked to the promoter, wherein the gene encoding the recombinant vector comprises a recombinant vector arranged in the opposite direction in the vector,
Thyroid cancer, breast cancer, stomach cancer, teratoma, and salivary gland cancer.
2. The composition for drug screening according to claim 1, wherein the CMV promoter is represented by SEQ.
2. The composition for screening pharmaceuticals according to claim 1, wherein the sodium iodoammono-transporter promoter is represented by SEQ ID NO: 2.
delete The method according to claim 1,
The first luminescent enzyme and the second luminescent enzyme are each independently selected from the group consisting of firfly luciferase, renilla luciferase, Turbo FP635, Photinus pyralis luciferase, Wherein the composition is at least one selected from the group consisting of copepod gaussia luciferase and metridia longa luciferase.
The pharmaceutical screening composition according to claim 1, wherein the recombinant vector has a cleavage map as described in the following.
[Figure 1]

A gene encoding a CMV promoter (pCMV; CMV promoter) and a first luminescent enzyme operably linked to the promoter; And a gene encoding a sodium iodide symporter promoter (pNIS) and a second luminescent enzyme operably linked to the promoter,
A gene encoding said CMV promoter (pCMV; CMV promoter) and a first luminescent enzyme operably linked to said promoter; And a sodium iodide symporter promoter (pNIS) and a gene encoding a second luminescence enzyme operably linked to the promoter, are transfected with a recombinant vector arranged in the opposite direction in the vector.
A composition for screening a drug for prevention or treatment of at least one disease selected from the group consisting of thyroid cancer, breast cancer, stomach cancer, teratoma, and salivary gland cancer, comprising the cell line of claim 7.
A pharmaceutical screening kit for preventing or treating at least one disease selected from the group consisting of thyroid cancer, breast cancer, stomach cancer, teratoma and salivary gland cancer comprising the composition of claim 8.
(1) treating and culturing the cell line of claim 7; And
(2) measuring the expression of the first luminescent enzyme and the second luminescent enzyme in the cell line; Wherein the disease is selected from the group consisting of thyroid cancer, breast cancer, stomach cancer, teratoma, and salivary gland cancer.
11. The method of claim 10,
(3) When the expression of the first luminescent enzyme is maintained at a level of 50% to 100% by the candidate substance and the expression of the second luminescent enzyme is increased by the candidate substance compared to the control, the candidate substance is preferably used as a candidate compound for the treatment of thyroid cancer, breast cancer, teratoma, and salivary gland cancer. The method for screening a pharmaceutical according to claim 1,

Images