KR101032814B1 - Analysis of Bacillus anthracis Lethal Toxin - Google Patents

Abstract

The present invention relates to a method for analyzing genes whose expression is increased or decreased in macrophages by anthrax toxin specifically, in particular, in macrophages by anthrax toxin. Alternatively, the genes that are reacted by anthrax lethal toxin can be utilized as biomarkers by searching for the reduced genes using microarray technology and measuring the difference in expression.

Bacillus anthracis, anthrax toxin, cDNA microarray, mouse macrophage

Description

Analysis of macrophage response gene analysis by anthrax lethal toxin {Analysis of Bacillus anthracis Lethal Toxin}             

1 is a result of polymerase chain reaction using reverse transcriptase after treatment of lethal toxin. (a) NEK 6, (b) RPL10A, (c) JAK1, (d) ATP1B1, (e) BACE. PA was obtained by treating 100ng / ml and 16ng / ml LF.

The present invention uses a microarray technology to search for genes that are specifically increased or decreased in macrophages by anthrax toxin to determine the difference in expression and to simply analyze the infection of anthrax using DNA chips. It is about.

Anthrax is caused by anthrax and is a common infectious disease classified into pulmonary anthrax, gastrointestinal anthrax and cutaneous anthrax. Pulmonary anthrax enters lung macrophages and germinates, causing anthrax to proliferate, resulting in sepsis and shock with dyspnea due to toxins produced from anthrax. The macrophage acts as a mediator to spread anthrax to the whole body through blood vessels.

Anthrax is an aerobic and gram positive bacterium that is 5 μm × 5-8 μm in size. Anthrax is not motile and forms oxygen when there is oxygen. Anthrax toxin-constituting protein is composed of protective antigen (PA, 83 kDa), edema factor (EF, 89 kDa) and lethal factor (LF, 90 kDa). One molecule of lethal and one edema combine with seven protective antigen molecules to form lethal toxin (LeTx) and edema toxin (EdTx). Lethal toxin (LeTx) appears to play a major role in triggering apoptosis when anthrax enters macrophages and becomes toxic.

The specific activity of lethal toxin is that 20 kDa of the protective antigen is cleaved by furin-like protease when the protective antigen specifically binds to the cell membrane receptor, and 63 kDa of the remaining protective antigen is cleaved by ananthrax toxin receptor (ATR). It binds to form heptamer, and then a molecule of lethal element binds to the protective antigen heptamer and enters into cell. Lethal toxin enters and destroys macrophages. Low concentration of lethal toxin causes apoptosis, and high concentration of lethal toxin overexpresses reactive oxygen species (ROS), causing macrophage sudden death. Let's do it.

After high concentrations of lethal toxins are added to macrophages, up to 45 minutes is called the early stage and 60 minutes later is called the late stage. Lethal toxin increases the membrane permeability of Na + / K + in the early stage and consumes ATP. In the later stages, membrane permeability modifications of Ca2 +, Cr2 +, Cl-, SO42-, amino acid and uridine occur, inhibiting the synthesis of macromolecule (macromolecule), resulting in morphological changes of cells. Tyrosine kinase inhibitors and tyrosine kinase inhibitors increase the survival rate of macrophages, suggesting that tyrosine kinase may be involved in the lethal toxin mechanism.

Methods for studying gene expression include differential display PCR (DD-PCR) [Liang et al., Science, 258, 967-971, 1992], serial analysis gene expression (SAGE) [Velculescu et al., Science , 270, 484-487, expressed sequence tags (EST) database comparison [Vasmatizis et al., Proc. Natl. Acad. Sci. USA, 95, 300-304, 1998], but the biggest problem with these methods is the fact that they are time-consuming and expensive, resulting in high time and economic burden. In addition, the process is complicated and difficult to handle many genes at the same time.

In the present invention, a DNA microarray method was used as a method to compensate for the shortcomings of the above methods. DNA microarray is a technology that can simultaneously analyze the expression patterns of genes expressed in a specific cell, and can analyze a large amount of genes whose expression is specifically induced or inhibited.

The present invention provides biological analysis of gene expression patterns of macrophages using a microarray chip in which a gene whose differential expression is increased or decreased in macrophages by anthrax lethal toxin is biologically analyzed and provided as a biomarker for anthrax infection. There is a purpose.

The present invention provides a method for analyzing a response in macrophages by anthrax lethal toxin, wherein (1) a DNA chip differentially increased or decreased by anthrax lethal toxin and DNA fragments derived from the gene are immobilized: ( 2) Simple and economical anthrax infections can be easily and economically determined by measuring gene expression that is increased or decreased from anthrax toxin-containing samples using DNA chips that are spotted genes differentially expressed in macrophages by anthrax. Provide a method for analysis.

When affected by an lethal toxin of anthrax, changes in gene expression occur. DNA chip refers to a spot on a glass slide of some genes affected by anthrax among 3 to 40,000 genes. Total mRNA was isolated from macrophages (control group) without Anthrax lethal toxin and macrophages (sample group) with Anthrax lethal toxin, and two fluorescent substances (Cy-3, Only one of each of Cy-5 fluorescence is combined to prepare a probe. When hybridization of each fluorescent substance bound to the cDNA chip, probes prepared from the control group and the sample group competitively bind to one spot, and the difference in relative gene expression can be distinguished by the intensity of fluorescence. Can be.

In the present invention, a gene affected by anthrax toxin is defined as a gene whose expression is promoted or inhibited by anthrax toxin. The number of such genes may be one or more. Therefore, genes of agonists or related genes whose expression is directly or indirectly affected by anthrax toxin can be used as genes immobilized on the DNA array of the present invention.

The gene affected by the lethal toxin of anthrax can be detected as follows. DNA array refers to a support on which a gene or cDNA fragment derived from the gene is immobilized and includes, for example, a DNA chip. Supports that can be used for hybridization generally include slide glass, silicon chips, nitrocellulose or nylon membranes, and cDNA fragments derived from genes or genes affected by Anthrax lethal toxin are immobilized.

By comparing the results for macrophages treated with Anthrax lethal toxin with those for control macrophages, genes with significantly different signal intensities can be detected between the two samples. Signal intensities, such as radiation, fluorescence, and luminescence, for the hybridized arrays are measured using a special measuring instrument such as an image analyzer. Thereby, the gene whose expression was remarkably changed by the lethal toxin of anthrax can be detected based on the difference of signal intensity.

Using a multi-wavelength detection fluorescence analyzer that can detect two types of fluorescent labels simultaneously, gene expression for macrophages exposed to anthrax lethal toxin on the same DNA array can be compared with gene expression for control macrophages. have. For example, nucleic acid samples derived from cells affected by anthrax lethal toxin are fluorescently-labeled with Cy3-dUTP and control nucleic acid samples with Cy5-dUTP. The same amount of two nucleic acid samples are mixed and the mixture is hybridized with the DNA array to determine the difference in gene expression between the two nucleic acid samples by color difference, based on which a gene whose expression level has been significantly changed by anthrax toxin is detected. can do.

Genes affected by anthrax lethal toxin are selected by comparing the signal intensity measured for the sample expected to include anthrax lethal toxin as the expression level index to that for the control sample. For example, this value is calculated by dividing the fluorescence signal value for the sample containing anthrax lethal toxin by the fluorescence signal value for the control sample. Values greater than 1.00 mean that gene expression is facilitated by the treatment of the test substance. A value less than 1.00 means that gene expression was inhibited by the treatment of the test substance. A value equal to 1.00 indicates that the gene was not affected by the treatment of the test material. If expression is promoted, the value is greater than 1.5, preferably greater than 2.0. If expression is inhibited, the value appears to be less than 0.9, preferably 0.8.

In the present invention, the treatment of anthrax toxin to macrophages was measured to determine the difference in gene expression patterns.

Hereinafter, the present invention will be described in more detail with reference to the following examples. The following examples illustrate the invention, but the invention is not limited thereto.

Example 1

Mouse macrophage RAW 264.7 cells were cultured in a 37 ° C., 5% CO ₂ incubator using DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin.

MTT assay was performed to confirm the physiological activity of protective antigen and lethal factor. MTT assay is a method of measuring cell viability by reacting with dehydrogenase of mitochondria. Mouse macrophage RAW 264.7 cells were dispensed 6 × 10 ⁴ into 96-well plates and incubated overnight at 37 ° C., 5% CO ₂ incubator. Protective antigen concentrations were fixed at 100 ng / ml and lethal elements were treated by concentration (2, 4, 8, 16, 32, 64 and 100 ng / ml) plates. At hourly (0, 10, 20, 30, 60, 90, 120, 180 and 240 minutes) plates were removed from the CO ₂ incubator and washed five times with PBS pH 7.2 buffer. 100 μl of DMEM medium (10% FBS, 1 × PS) was added and 25 μl MTT (5 mg / ml in PBS) was added, followed by incubation for 3 hours in a 37 ° C., 5% CO ₂ incubator. 100 μl of the stop solution (20% SDS, 50% dimethyl formamide, pH 4.7) was added to the culture plate, and the mixture was stirred slowly at room temperature. After 3 hours, a graph was prepared by measuring with an ELISA reader at a wavelength of 570 nm.

As a result, when the lethal urea concentration of lethal toxin exceeds 8 ng / ㎖, it can be seen that the cells die at 90 minutes. In the present invention, it is intended to see the change in the gene expression for the lethal toxin in the cell death, the protective antigen concentration based on the above results was carried out with a concentration of 100 ng / ㎖, 16 ng / ㎖ of lethal urea.

Example 2

RNA isolation was performed by a method provided by GibcoBRL. Mouse macrophage RAW 264.7 cells were dispensed into 6 × 10 ⁶ cells on a plate using DMEM medium (10% FBS, 1 × PS) and incubated for 2 hours at 37 ° C. in a 5% CO ₂ incubator. After incubation, the cells were adhered to the bottom of the plate, washed twice with PBS (pH 7.2) buffer, and then 100 ng / ml of protective antigen and 16 ng / ml of lethal element were added. The plate was washed twice with PBS (pH 7.2) buffer after incubation for 30 min at 37 ° C., 5% CO ₂ incubator. After washing, 2 ml of Trizol reagent was added and the cells were destroyed by pipeting 50-70 times. The cell disrupted sample was dispensed into tubes by 1 ml and 200 μl chloroform was added. The tube lid was closed and vortexed for 15 seconds and left on ice for 15 minutes. After centrifugation at 12,000 g, 15 minutes, 4 ℃. After that, the supernatant was carefully collected, transferred to a new tube, and the same amount of isopropyl alcohol was added and left on ice for 1 hour. Thereafter, the mixture was centrifuged at 12,000 × g for 15 minutes at 4 ° C. Gel-like aggregate formation was confirmed and the supernatant was removed. After adding 1 ml of ethanol 75% (DEPC-treated distilled water), the mixture was stirred and centrifuged at 12,000 × g for 15 minutes at 4 ° C. to remove the supernatant, and again, the remaining supernatant was removed by centrifugation under the same conditions. The aggregates were dried at room temperature for 5 to 10 minutes and dissolved by adding 30 μl of DEPC treated tertiary distilled water. Samples were measured at a wavelength of 260 nm and 280 nm with a spectrophotometer to determine whether the value of A260 / A280 was at least 1.7. Electrophoresis also confirmed that the RNA was well separated.

Example 3

The labeling kit was purchased by Macrogen (Korea) and performed by the method provided by the company. Prepare an annealing reaction mixture (18 μl total RNA, 1 μl control mRNA (1 ng), 3 μl oligo dT primer) in a PCR (Polymerase chain reaction) tube with total RNA from the control group and total RNA from the experimental group, respectively. It was left for 5 minutes at ℃ and immediately the tube was transferred to ice. After dividing the new 0.2 ml PCR tube into the experimental and control reactions, the label mixture (8 μl 5X AMV RT buffer, 4 μl low dT dNTP, 1 μl Cy3, Cy5-dUTP 3 μl, RNase inhibitor (40 u / μl) 1 μl 2 μl of AMV RT (50 units) was prepared. The binding reaction mixtures were added to the labeling mixtures respectively and left at 42 ° C. for 1 hour in a PCR instrument. The reaction was then stopped by adding 5 μl of 0.5 M EDTA. 10 μl of 1 N NaOH was added to each tube and allowed to stand at 37 ° C. for 10 minutes. Then 25 μl of 1 M Tris.Hcl (pH 7.5) buffer was added to the tube. The Chromaspin column (Macrogen) was placed in a 2 ml tube and centrifuged at 1,300 xg for 3 minutes at room temperature to remove the buffer in the column. The spin column from which the buffer was removed was placed in a new tube, and the prepared control and experimental reaction samples were placed in the center of the column, and centrifuged at 1,300 × g for 5 minutes at room temperature. 300 μl of 100% ethanol and 10 μl of 3 M NaOAc were added to each sample and allowed to stand at −70 ° C. for 30 minutes. After centrifugation at 12,000 × g for 20 minutes at room temperature to remove ethanol. Then 1 ml of 70% ethanol was added and ethanol was removed by centrifugation under the same conditions. The aggregates were dried in air by blocking light. The dried aggregates were each dissolved in 45 μl of tertiary distilled water and centrifuged at 12,000 × g for 2 minutes. 5 μl of each of the mixture of Cy3 (control) and Cy5 (sample) was taken and diluted 1/20, and the absorbance (OD) value was measured with a spectrophotometer. Based on the measured value, the amount of each cDNA was calculated, and the volume of the test group and the control group was added to one tube so that the volume was equal to 80 µl. 20 μl of 5 × segmentation solution was then added to a total volume of 100 μl. Thereafter, the mixture was left at 95 ° C. for 15 minutes, transferred to ice, and the cDNA mixture, which had been segmented with a Speed-Vac, was dried to form aggregates.

cDNA microarray pre-hybridization method was performed by the method provided by Digital Genomics (Korea). Pre-hybridization buffer (25% formamide, 5X SSC, 0.1% SDS, 10 mg / ml BSA) was previously heated to 42 ° C. The slides were placed in a 50 ml tube filled with pre-hybridization buffer and left at 42 ° C. for 45 minutes. After that, the slide was removed, washed 5 times with distilled water, and centrifuged at 60 × g for 5 minutes at room temperature to remove moisture on the microarray.

Sample aggregates prepared during the probe preparation were dissolved in 15 μl hybridization buffer (25% formamide, 5 × SSC, 0.1% SDS) and heated at 95 ° C. for 5 minutes. Then centrifuged lightly for 2 minutes. The slide was transferred to the chamber, and the prepared sample was loaded onto the slide surface and the cover glass was covered. This was then left at 42 ° C. for 16-20 hours.

Wash buffer I (2X SSC, 0.1% SDS) was heated to 42 ° C., slides were inserted and shaken to remove cover glass. The slides were placed in Wash Buffer I (2 × SSC, 0.1% SDS) and left at 42 ° C. for 5 minutes. The slides were placed in Wash Buffer II (0.1 × SSC, 0.1 × SDS) and left at room temperature for 1 minute and the procedure was repeated four times. Thereafter, the microarray was dried by centrifugation at 60 xg for 5 minutes at room temperature.

I ran the Axon GenePix scanarray 4000 (Axon 4000, USA) program and slide the slides down the stage with the hybridized parts down. Values such as Scan parameter, slide, wavelength 532 nm (Cy5), and 635 nm (Cy3) were entered. Two fluorescence images (Cy3, Cy5) were scanned at wavelengths 532 nm and 635 nm, respectively, and laser intensity and PMT sensitivity at which the signal could be maximized without increasing the background value were set, respectively. Scanning was performed after examining the values of the normalization control genes of Cy3 and Cy5. The image was saved and converted to an Excel file by taking the fluorescence signal intensity and background intensity at each spot location designated as the taken image.

Comparing the signal strengths of Cy3 and Cy5, Cy3 has a higher degree of incorporation than Cy5, so it has a similar normalization. In this case, the data is compared, analyzed and normalized through several experiments. After that, the fluorescence ratio of Cy3 and Cy5 of the control gene was 1, and compared with that, the fluorescence ratio of Cy3 and Cy5 was 2 times or more, and the gene expression was inhibited.

Reverse transcription-polymerase chain reaction (PCR) was performed by a method provided by Invitrogen (USA). Total RNA from mouse macrophage RAW 264.7 cells treated with protective antigen concentration of 100 ng / ml and lethal concentration of 16 ng / ml for 30 minutes was isolated. 2 μl total RNA, 1 μl oligo dT primer, 1 μl 10 mM dNTP, and 9 μl of DEPC treated distilled water were added to the PCR tube and allowed to stand at 65 ° C. for 5 minutes in a PCR instrument. After that, 4 μl of 5 × first-strand buffer, 2 μl of 0.1M DTT, and 1 μl of recombinant ribonuclease inhibitor were added and left at 42 ° C. for 2 minutes. Then, 1 μl of SuperScript II RNase H-reverse transcriptase was added and left at 42 ° C. for 50 minutes to synthesize cDNA. After heating at 70 ℃ for 15 minutes to inactivate the residual proteins. 30 μl of tertiary distilled water was added to the tube and stored at −20 ° C. until the next use.

As described above, based on the results of the MTT assay, the conditions were set for 30 minutes at a concentration of 100 ng / ml protective antigen and 16 ng / ml lethal concentration. 6 × 10 ⁶ mouse macrophage RAW 264.7 cells were added with 100 ng / ml of protective antigen concentration and 16 ng / ml of lethal urea, and 30 minutes later, total RNA was isolated and hybridized to the cDNA microarray. Data analysis confirmed 226 gene expression changes. Of these, 32 genes were inhibited and 194 genes were induced.

As a result, the RPL10A, ARBP, RPL30, RPS3, RPL3, RPS16 and MPRS17 genes involved in protein biosynthesis and NEK6 genes that regulate cell mitosis initiation were inhibited and the results are shown in Table 1.

  List of genes whose gene expression is reduced when anthrax lethal toxin is treated in macrophages gene product Biological function RPL10A Ribosomal protein L10A Protein biosynthesis NOC4 Neighbor of Cox4 SORT1 Sortilin 1 SERPIN1 Serine (or cystein) protease
nhibitor ARBP Acidic ribosomal phosphoprotein P Protein biosynthesis MPRS17 Mitochondria ribosomal protein S1 Protein biosynthesis RPL30 Ribosomal protein L30 Protein biosynthesis RPS3 Ribosomal protein S3 Protein biosynthesis TPM3 Tropomyosin 3 gamma Muscle development CREM cAMP responsive element modulator Regulation of
ranscription TLN Talin Ube2i Ubiquitin-conjugating enzyme E21 Ubiquitin dependent
rotein catabolism MSH6 MutS homolog 6 ( E.coli ) DNA repair PTDSS2 Phosphatidylserine synthase 2 SELENBP1 Selenium binding protein 1 RPL3 Ribosomal protein L3 Protein biosynthesis GNB2-RS1 Guanine nucleotide binding
rotein β-2 ROBO1 Roundabout homolog 1 NEK6 NIMA-related expressed kinase 6 RPS16 Ribosomal protein S16 Protein biosynthesis HIST2 Histone protein 4

On the other hand, as shown in Table 2, STAT4, STAT5A, JAK1 and CNIH genes involved in signaling, BTG1 genes involved in germination (SE), SERPINB2 genes involved in tissue reconstruction / coagulation, and proteolysis are shown in Table 2. CTSW1, BACE and MCPT5 genes, GAS1 genes that inhibit the cell cycle, BIRC1E and CAP8AP2 genes involved in cell death, GLRX1, CYP7B1, AOX1 and CYP4A10 genes involved in electron transfer, AQP3, FXC1, ATP1A1, SLC16A1, APOM and KCNE4 genes, SCYB9 and PSMB9 genes involved in immune response.

 List of genes whose gene expression is increased when anthrax lethal toxin is treated in macrophages gene product Biological function JAK1 Janus kinase Intracellular signal
cascade STAT4 Signal transducer and activatior
of transcription 4 Intracellular signal
cascade BTG1 B-cell translocation gene 1,
nti-proliferative Prolierating SERPINB2 Serine (or cysteine) protease
inhibitor, clade B Tissue
remodeling / cloting STAT5A Signal transducer and activator
f transcriptor5A Intracellular signal
cascade CTSW1 Cathepsin w Proteolysis GAS1 Growth arrest specific 1 Cell cycle arrest CNIH Cornichon homolog (Drosophila) Intracellular signal
cascade GLRX1 Glutaredoxin 1 (thiol transferase) Electron transport AQP3 Aquaporin 3 Transport FXC1 Fractured callus expressed
ranscript 1 Transport CYP7B1 Cytochrome p-450,7b1 Electron transport ATP1A1 ATPase, NA + / K + transporting
eta 1 polypeptide Na + / K + transport AOX1 Aldehyde oxidase 1 Electron transport CYP4A10 Cytochrome p-450,4a10 Electron transport BIRC1E Baculoviral IAP repeat-containing
1e Apoptosis SLC16A1 Solute carrier family 16 Transport BACE Beta-site APP cleaving enzyme Proteolysis CAMK4 Calcium / calmodulin-dependent
rotein kinase IV Amino acid
hosphorylation APOM Apolipoprotein M Transport MCPT5 Mast cell protease 5 Proteolysis SCYB9 Small inducible cytokine
subfamily (cys-x-cys) Immune response KCNE4 Potassium voltage-gated channel Ion transport PSMB9 Proteasome subunit, beta type 9 Immune response CASP8AP2 Caspase 8 associated protein 2 Apoptosis

Example 4

Mouse macrophage RAW 264.7 cells were treated with 100 ng / ml of protective antigen and 16 ng / ml of lethal urea and total RNA was isolated from cells after 30 minutes. 2 μl of cDNA synthesized with total RNA, 5 μl of 10X PCR buffer, 1.5 μl of 50 mM MgCl ₂ , 1 μl of the pre-starter, 1 μl of the pre-starter, 1 μl of Taq DNA polymerase (5 U / μl, Biotool), tertiary distilled water 37.5 μl was added to a 0.2 ml PCR tube to bring the total volume to 50 μl. Thereafter, denatured at 94 ° C. for 5 minutes, denatured at 94 ° C. for 1 minute, bound for 1 minute at the melting temperature for each primer, and stretched for 30 minutes at 72 ° C. for 1 minute. It was then stretched for 10 minutes at 72 ° C. and the results were confirmed on 2% TAE agarose gel. The primer sequence of each gene used is as follows.

NEK6: 5'-TTCTGAAGGGGTGACAG-3 ', 5'-CTGTCTTCCAGATGGCT-3'

RPL10A: 5'-GCTCATCATCGGTCATC-3 ', 5'-AAGTTGGTCAAGAAGCT-3'

JAK1: 5'-GCGAGACACAGGTTTGACG-3 ', 5'-CGGACAGTGGCGTAAACAG-3'

ATP1B1: 5'-GGAAAAGCCAAGGAGGAAGG-3 ', 5'-TTGGATGGTCCCGATGAAG-3'

BACE: 5'-GAATAGGGGAAAAAGCCAG-3 ', 5'-ATTTGCCTCTGCCTGGATT-3'

The primers for genes involved according to biological function were envisioned as described above to perform RT-PCR. That is, the gene NEK6 involved in cell growth, gene RPL10A involved in protein biosynthesis, JAK1 involved in intracellular signaling, gene ATP1A1 involved in migration, and gene BACE involved in protein degradation. 1 is shown. As a result, it can be seen that by measuring the change in the genetic expression that occurs when the sudden death level of anthrax lethal toxin to the macrophages, lethal toxin further stimulates the macrophage decomposing function to further promote cell death. It was confirmed that the anthrax infection can be easily measured using this.

The present invention utilizes genes that respond to anthrax toxin toxin as a biomarker by searching for genes that are specifically increased or decreased in macrophages by anthrax toxin toxin and by measuring the difference in expression. Thus, in the presence of lethal toxin of anthrax can be analyzed simply and economically.

Claims (3)

RPL10A, NOC4, SORT1, SERPIN1, ARBP, MPRS17, RPL30, RPS3, TPM3, CREM, TLN, Ube2i, MSH6, PTDSS2, SELENBP1, RPL3, GNB2-RS1, ROBO1, NEK6, RPS16, HIST2, JAK1, STAT4 Measuring SERPINB2, STAT5A, CTSW1, GAS1, CNIH, GLRX1, AQP3, FXC1, CYP7B1, ATP1A1, AOX1, CYP4A10, BIRC1E, SLC16A1, BACE, CAMK4, APOM, MCPT5, SCYB9, KCNE4, MP9 Anthracnose lethal toxin detection DNA chip comprising the agent. The DNA chip of claim 1, wherein the agent for measuring mRNA expression level of the gene is a probe that specifically binds to the gene. RPL10A, NOC4, SORT1, SERPIN1, ARBP, MPRS17, RPL30, RPS3, TPM3, CREM, TLN, Ube2i, MSH6, PTDSS2, SELENBP1, RPL3, GNB2-RS1, ROBO1, NEK6, RPS16, HIST2, JAK1 STAT4, BTG1, SERPINB2, STAT5A, CTSW1, GAS1, CNIH, GLRX1, AQP3, FXC1, CYP7B1, ATP1A1, AOX1, CYP4A10, BIRC1E, SLC16A1, BACE, CAMK4, APNEOM, MCPT5, SCYB8, SCYB8 Measuring expression levels; Comparing the mRNA expression level of the gene with the mRNA expression level of the gene of the normal control sample; And mRNA expression levels of the RPL10A, NOC4, SORT1, SERPIN1, ARBP, MPRS17, RPL30, RPS3, TPM3, CREM, TLN, Ube2i, MSH6, PTDSS2, SELENBP1, RPL3, GNB2-RS1, ROBO1, NEK6, RPS16 and HIST2 genes Reduced and the JAK1, STAT4, BTG1, SERPINB2, STAT5A, CTSW1, GAS1, CNIH, GLRX1, AQP3, FXC1, CYP7B1, ATP1A1, AOX1, CYP4A10, BIRC1E, SLC16A1, BACE, CAMK4, APOM4, APNE4 If the amount of mRNA expression of the PSMB9 and CASP8AP2 gene is increased, the method for providing information necessary for diagnosing anthrax infection, comprising determining anthrax infection.

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