KR20070011417A - Dna chip for screening of peroxisome proliferator - Google Patents

Abstract

A DNA chip and a kit for screening substances showing activity as a peroxisome proliferator are provided to be able to rapidly and accurately screen the peroxisome proliferator inducing abnormal cell reaction in vivo, thereby being widely applied to safety test of various materials. The DNA chip for screening of peroxisome proliferator comprises: a probe consisting of 121 to 587 nucleotides, capable of binding specifically to a gene whose expression is induced by a peroxisome proliferators; a linker including oligo (dT)15, -(CH2)6- and amine group in sequence, whose 3'-terminal of oligo (dT)15 is linked to 5'-terminal of the probe; and a solid substrate, whose surface is coated with aldehyde group, connecting with amine group of the linker via Schiff's base reaction. In the DNA chip, the probe is at least one gene selected from SEQ ID Nos: 1 to 12. The kit for screening of peroxisome proliferator comprises the DNA chip and a fluorescence-labeled deoxynucleotide to be used for labeling cDNA synthesized from RNA isolated from a sample. The method for screening peroxisome proliferator using the kit comprises the steps of: (a) administering a peroxisome proliferator candidate chemical to an animal; (b) isolating RNA from the animal; (c) synthesizing fluorescence-labeled cDNA from the isolated RNA using fluorescence-labeled deoxynucleotide in the kit; (d) applying the synthesized cDNA to a DNA chip for screening peroxisome proliferator in the kit to allow hybridization; and (e) after washing the hybridized DNA chip, examining the hybridized probe in the DNA chip by using a scanner.

Description

DNA chip for screening peroxysomal proliferators {DNA Chip for Screening of Peroxisome Proliferator}

The present invention relates to a DNA chip capable of selecting a peroxisome proliferator. More specifically, the present invention provides a DNA chip comprising a gene probe capable of specifically binding to a gene expressed upon administration of a peroxysome proliferative agent, a method of preparing an electrical DNA chip, and a peroxy including an electrical DNA chip. The present invention relates to a method for screening peroxysomal proliferative agents using a kit for screening a multiplier and an electric kit.

Peroxysome proliferators, which exhibit peroxysome proliferative activity, activate not only peroxisome proliferator activated receptors (PPARs), which promote metabolism that breaks down triglycerides in the body, but also generate excess free radicals in the body, resulting in abnormal cellular responses. It is known to play a role of inducing. When the peroxysome proliferative agent is administered to the body of the animal, excess free radicals are generated in the body, and the peroxysomes are abnormally proliferated in the cell to remove the excess free radicals generated. Peroxysomes, which are electroproliferative, absorb and remove most of the free radicals present in the cell, but not all free radicals are removed through the peroxysome, and a small amount of free radicals remain in the cells. Free radicals, which are not removed and remain in the cell, can carry out abnormal oxidation in the cell, even in small amounts. For this reason, it is considered very important from the viewpoint of safety to confirm whether or not the substance to be administered in the body of the animal exhibits peroxysome proliferation activity.

Until now, in order to confirm whether the substance to be administered in the animal shows peroxysome proliferative activity, the electrical substance is administered to the experimental animal, the liver tissue is extracted from the experimental animal to which the electrical substance is administered, and extracted. The tissue sample for optical microscopy was obtained using the prepared liver tissue, and then the obtained tissue sample was observed under an optical microscope, and compared with the size of the peroxysome originally existing in the electrical tissue sample. A method of checking the presence of moth has been used. However, such a method takes a long time, and in the case of a substance having a weak activity as a peroxysomal proliferative agent, it has a disadvantage in that accurate identification is difficult. That is, it takes more than one day to obtain a tissue sample using liver tissue extracted from an animal to which the electric substance has been administered, and relatively low activity when a substance having weak activity as a peroxysome proliferative agent is administered to the animal. In order to remove a small amount of free radicals and to remove a relatively small amount of free radicals, the peroxysome may not be excessively proliferated, in which case the size and peroxysomal proliferation of the peroxysome originally present in the tissue sample. It has been pointed out that due to the administration of the agent the size of the propoxysomes propagated may not be clearly distinguished. In order to solve these drawbacks, various studies have been conducted, but the situation has not been much so far.

Therefore, there is a constant need to develop a method capable of quickly and accurately searching for a substance exhibiting activity as a peroxysomal proliferative agent.

Summary of the Invention

Therefore, the present inventors have made intensive studies to develop a method for searching for a substance showing activity as a peroxysomal proliferative agent, and as a result, a gene probe capable of specifically binding to a gene expressed upon administration of a peroxysomal proliferative agent When using a peroxysomal proliferation agent selection kit comprising a fluorescently labeled deoxy nucleotides to label the synthesized cDNA from the DNA chip and DNA collected from the sample, the peroxysomal proliferation agent is selected quickly and accurately It was confirmed that this can be done, and this invention was completed.

After all, the first object of the present invention is to provide a DNA chip for peroxysomal proliferation agent selection comprising a gene probe capable of specifically binding to the gene expressed upon administration of the peroxysomal proliferative agent.

A second object of the present invention is to provide a method for producing an electrical DNA chip.

It is a third object of the present invention to provide a kit for selecting a peroxysomal proliferation agent comprising an electric DNA chip.

A fourth object of the present invention is to provide a method for selecting a peroxysomal proliferative agent using an electric assay kit.

The DNA chip for selecting a peroxysomal proliferative agent of the present invention comprises a gene probe which is composed of 121 to 587 nucleotides and which can specifically bind to a gene expressed by the peroxysomal proliferative agent; A linker comprising oligo (dT) ₁₅ ,-(CH ₂ ) ₆ -and an amine group sequentially, wherein the 5 ′ end of the electric gene probe is connected to the 3 ′ end of oligo (dT) ₁₅ ; And a solid substrate to which an aldehyde is attached to a surface, and an amine group of an electric linker is connected through a reaction of an aldehyde group and a Schiff's base on the surface: wherein the gene probe is not particularly limited thereto. It is preferred to use at least one gene selected from 1-12 genes as the probe.

Method for producing a DNA chip for the selection of the peroxysomal proliferation agent comprises a step of synthesizing a gene probe consisting of 121 to 587 nucleotides, which can specifically bind to a gene expressed by the peroxysome proliferative agent; Synthesizing a linker sequentially comprising an oligo (dT) ₁₅ ,-(CH ₂ ) ₆ -and an amine group; Binding the 5 'end of the electrosynthesized gene probe to the 3' end of the oligo (dT) ₁₅ of the electrosynthesized linker; Linking a gene probe-linked linker to a surface of an aldehyde-attached solid substrate through a Schiff's base reaction; And a step of reducing unreacted aldehyde, wherein the solid is not particularly limited thereto but is preferably glass, and the reducing conditions of the aldehyde are not particularly limited thereto, but a reducing agent such as NaBH ₄ may be used. It is preferable to use.

On the other hand, using the DNA chip for screening peroxysomal growth agent of the present invention, a kit for selecting a peroxysomal growth agent can be produced. That is, the peroxysomal proliferation agent selection kit of the present invention comprises an electric peroxysomal proliferation agent selection DNA chip and fluorescently labeled deoxy nucleotides to label the synthesized cDNA from RNA collected from a sample: Fluorescently labeled deoxy nucleotides include, but are not limited to, Texas Red dATP, cyanine 3 dCTP, cyanine 5 dGTP or fluorescein-12 dUTP. It is preferable to use.

The peroxysome proliferative agent can be easily selected using the above-described kit for screening peroxysomal proliferative agent of the present invention. That is, administering a peroxysomal proliferative candidate to the experimental animal; Harvesting RNA from the experimental animal to which the candidate is administered; Synthesizing the fluorescently labeled cDNA from the electrosampled RNA using the fluorescently labeled deoxy nucleotides included in the electrical kit; Hybridizing the electrosynthesized cDNA to a DNA chip for selecting a peroxysomal growth agent included in an electric kit; And peroxysomes by washing the electrohybridized DNA chip and inspecting a probe site hybridized to the DNA chip using a scanner to determine whether the administered candidate substance exhibits activity as a peroxysomal proliferative agent. Proliferative agents can be selected.

The peroxysomal proliferation agent selection kit of the present invention can easily select a peroxysomal proliferation agent that induces an abnormal cellular response in the body, and thus may be widely used for safety testing of various substances.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example 1 Screening for a Gene Sequence (EST, Expressed Sequence Tag) That Can Specificly Binding to a Gene Expressed Upon Administration of a Peroxysome Proliferative Agent

Experimental group 1 administered 6 week old rats (Sprague-Dawley VAF + albino) by intraperitoneal injection of clofibrate, a hyperlipidemia therapeutic known to have peroxisomal proliferation activity, in an amount of 0.2 mg / kg per day for 2 weeks; Experimental group 2, which was administered by intraperitoneal injection of gemfibrozil, a drug for treating hyperlipidemia known to have peroxisomal proliferation, in an amount of 0.2 mg / kg per day for 2 weeks; Experimental group 3, which was administered by intraperitoneal injection of phenytoin, which is known to have no peroxisomal proliferation activity, in an amount of 0.2 mg / kg per day for 2 weeks; And control groups to which no drug was administered, respectively, and liver tissues were extracted from each control group and the experimental group.

Using an RNA extraction kit (Micro-to-Midi Tatal RNA purification system, Life Technologies, Inc., USA), mRNA was isolated from each of the electroextracted liver tissues, and a reverse transcription kit (Platinum Biochip Reagent Kit, Genocheck, Korea) And cyanine 5 (cyanine 5, Amersham, GB) using a dGTP synthesized fluorescently labeled cDNA from mRNA was isolated.

Meanwhile, a plasmid containing each gene is extracted from a clone library (Clone-Library, Reserch Genetics, USA) containing various ESTs, amplified by PCR method, and spotted on a glass substrate for DNA chip. A DNA chip containing the electric amplified gene was constructed.

After the electrosynthesized fluorescently labeled cDNA was added to the constructed DNA chip, the hybridization reaction was performed for 12 hours, and the hybridization terminated DNA chip was scanned with a plate scanner (GenePix Scanner 4000A, Axon Inc., USA). The scan results were analyzed by an image analysis system (GenePix Pro 4.0, Axon Instrument Inc., USA) to select genes showing a relatively high degree of fluorescence in each experimental group (see Table 1).

TABLE 1

Hybridization of Genes Specific to Genes Expressed in Each Chip and Genes in DNA Chips

Table 1 shows a selection of genes showing relatively high hybridization reactivity with cDNA of each experimental group. After reacting with each experimental group, 12 kinds of EST (relative to the high fluorescence generated from cyanine 5) dGTP were detected. SEQ ID NOS: 1 to 12) and their relative fluorescence levels. At this time, as a reference for the relative fluorescence degree, the cDNA of the control group and the DNA chip were reacted, and the fluorescence degree (1.0) generated from cyanine 5 dCTP was used.

As shown in Table 1, EST performing relatively high hybridization with Experimental Group 1 is SEQ ID NO: 1 to 11, EST performing relatively high hybridization with Experimental Group 2 is SEQ ID NO: 12, Experimental Group 3 is relatively low hybridization The degree of response was shown. In particular, the experimental groups 1 and 2 administered with a substance known to have peroxysome proliferative activity showed a similar hybridization pattern, and the experimental group 3 administered to a substance known to have no peroxysome proliferative activity was completely different from the experimental groups 1 and 2. Since different hybridization patterns were shown, it was predicted that, if the 12 selected ESTs were used, substances with peroxysome proliferation activity could be selected.

Example 2 Preparation of DNA Chips

12 kinds of ESTs selected in Example 1 were synthesized for use as probes, oligo (dT) ₁₅ ,-(CH ₂ ) ₆ -and linkers with amine groups were sequentially constructed, and then electrosynthesized. The 5 'end of EST was linked to the thymine site of the linker.

Subsequently, the probes connected to the electric linker were dissolved in 50 μM each in a buffer solution (350 mM sodium bicarbonate, pH 9.0), and the arrayer (MicroGrid) was mounted on the surface of the aldehyde attached slide (CS-100, CEL, USA). II, BioRobotics, USA), and then dripped to 150 μm in size and 400 μm in interval, and then carried out a base salt reaction. Subsequently, the mixture was washed sequentially with 0.2% (w / v) SDS solution and tertiary distilled water, and immersed in NaBH ₄ solution (0.1 g NaBH ₄ , 30 ml PBS, 10 ml ethanol) for 15 minutes to bind an amine. The non-aldehyde residue was reduced, washed with tertiary distilled water and dried to prepare a DNA chip.

Example 3 Screening of Peroxysome Proliferative Agents Using DNA Chips

Using the DNA chip prepared in Example 2, it was confirmed whether the compound showing the peroxysome proliferation activity can be selected.

In other words, 1-methyl-4-piperidyl-bis (p-chlorophenoxy) acetate (1-methyl-4-piperidyl-bis (p-chlorophenoxy) acetate), which is known to exhibit peroxysome proliferative activity, is used daily. Experimental group 4 administered intraperitoneally for 2 weeks in an amount of 0.05 mg / kg; Wy-14,643 (4-chloro-6-[(2,3-dimethylphenyl) amino] -2-pyrimidinyl) thioacetic acid, A.G.Scientific. Inc., USA) experimental group 5 administered intraperitoneal injection for 2 weeks in an amount of 0.05 mg / kg per day; Experimental group which showed peroxysome proliferative activity and received di- (2-ethylhexyl) phthalate (di- (2-ethylhexyl) phthalate), known as an environmental hormone, intraperitoneally for 2 weeks in an amount of 0.05 mg / kg per day 6; And 2-hydroxy-3-propyl-4- [6- (tetrazol-5-yl) hexyloxylacetophenone (2-), which exhibits peroxysome proliferation activity and is known as an inhibitor of oxidation of fatty acids performed in the liver. Except for the use of experimental group 7, each of which was administered hydroxy-3-propyl-4- [6- (tetrazol-5-yl) hexyloxy] acetophenone) intraperitoneally for 2 weeks in an amount of 0.05 mg / kg per day. Using the same method as in Example 1, fluorescently labeled cDNA of each experimental group was synthesized. Subsequently, the fluorescently labeled cDNA of each experimental group synthesized on the DNA chip prepared in Example 2 was applied, hybridization was performed for 12 hours, and the hybridization was terminated in the same manner as in Example 1. Was analyzed and the results were analyzed to measure the relative degree of fluorescence generated in each experimental group, thereby measuring the degree of hybridization of cDNA and DNA chip of the electric experimental group (see Table 2).

TABLE 2

The degree of hybridization of genes and DNA chips that are specifically expressed when each substance is administered

As shown in Table 2, the cDNA obtained in the experimental groups 4 to 7 exhibited a high degree of hybridization with each probe provided in the electrical DNA chip, and thus showed similar results to the experimental groups 1 and 2 disclosed in Example 1 above. It can be seen that the results are different from 3.

Therefore, it was confirmed that by using the DNA chip of the present invention, a substance showing peroxysome proliferation activity can be selected quickly and accurately.

As described and demonstrated in detail above, the present invention includes a DNA chip comprising a gene probe capable of specifically binding to a gene expressed upon administration of a peroxysome proliferative agent, a method of preparing an electric DNA chip, and an electric DNA chip. It provides a method for screening peroxysome growth agent using a peroxysomal growth agent screening kit and an electric kit. The peroxysomal proliferation agent selection kit of the present invention can easily select a peroxysomal proliferation agent that induces an abnormal cellular response in the body, and thus may be widely used for safety testing of various substances.

As described above in detail the specific parts of the present invention, for those skilled in the art, these specific descriptions are only preferred embodiments, which are not intended to limit the scope of the present invention. Will be obvious. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (8)

(Iii) a gene probe consisting of 121 to 587 nucleotides and capable of specifically binding to a gene expressed by a peroxysome growth agent; (Ii) a linker comprising oligo (dT) ₁₅ ,-(CH ₂ ) ₆ -and amine groups sequentially, wherein the 5 'end of the electric gene probe is linked to the 3' end of oligo (dT) ₁₅ ; And, (Iii) An aldehyde is attached to a surface, and a DNA chip for peroxysome growth agent selection, comprising a solid substrate having an amine group of an electric linker connected through a reaction of an aldehyde group on the surface with a Schiff's base. The method of claim 1, Gene probe is characterized in that at least one gene selected from SEQ ID NOs: 1 to 12 DNA chip for peroxisomal growth agent selection. (Iii) synthesizing a gene probe consisting of 121 to 587 nucleotides and capable of specifically binding to a gene expressed by a peroxysome growth agent; (Ii) synthesizing a linker sequentially comprising an oligo (dT) ₁₅ ,-(CH ₂ ) ₆ -and an amine group; (Iii) binding the 5 'end of the electrosynthesized gene probe to the 3' end of the oligo (dT) ₁₅ of the electrosynthesized linker; (Iii) binding the linker to which the gene probe is bound to a surface of an aldehyde-attached solid substrate through a Schiff's base reaction; And, (Iii) A method for producing a DNA chip for peroxysome growth agent selection, comprising the step of reducing aldehyde remaining without reacting. The method of claim 3, wherein Gene probe is characterized in that at least one gene selected from SEQ ID NOs: 1 to 12 Method for producing a DNA chip for peroxysome growth agent selection. A peroxysomal proliferation agent selection kit comprising a fluorescently labeled deoxy nucleotide to label cDNA synthesized from a DNA chip for peroxysome proliferation agent selection of claim 1 and RNA collected from a sample. The method of claim 5, Fluorescently labeled deoxy nucleotides are Texas Red dATP, cyanine 3 dCTP, cyanine 5 dGTP or fluorescein-12 dUTP Peroxysome growth agent screening kit. (Iii) administering the peroxysomal proliferative candidate to the experimental animal; (Ii) extracting RNA from the experimental animal to which the candidate was administered; (Iii) synthesizing the fluorescently labeled cDNA from the electrosampled RNA using the fluorescently labeled deoxy nucleotides included in the kit of claim 5; (Iii) hybridizing the electrosynthesized cDNA to a DNA chip for selecting a peroxy species multiplier included in the electric kit; And, (Iii) washing the electrohybridized DNA chip and inspecting a probe region hybridized to the DNA chip using a scanner, wherein the peroxysome proliferative agent is selected using the kit of claim 5. The method of claim 7, wherein Fluorescently labeled deoxy nucleotides are characterized by Texas Red dATP, cyanine 3 dCTP, cyanine 5 dGTP or fluorescein-12 dUTP. A method for selecting a peroxysomal proliferative agent using the kit of claim 5.