KR101583397B1 - Kit for determining infection of influenza A virus - Google Patents

Abstract

The present application relates to a kit for determining whether or not an influenza virus A infection is detected, and a method for determining whether or not an influenza virus A infection has occurred. According to the present application, it is possible to quickly and accurately determine whether an influenza virus A infection is caused by providing a protein marker capable of discriminating whether or not an influenza virus A has been infected.

Description

[0001] KIT FOR DETERMINATION OF INFLUENZA VIRUS A INFECTION [

The present application relates to a kit for determining whether or not an influenza virus A infection is detected, and a method for determining whether or not an influenza virus A infection has occurred.

Influenza virus is a genomic virus that belongs to the genome of a single strand RNA fragment of the negative, which is a major causative agent of viral respiratory disease. From the 8 RNA fragments, hemagglutinin (HA), neuraminidase (NA), nucleocapsid protein (NP), matrix proteins 1 and 2 (matrix, M1, M2) The enzyme units A, B1 and B2 (polymerase subunit A, B1 and B2, respectively PA, PB1 and PB2) and nonstructural proteins 1 and 2 (NS1 and NS2, respectively) are produced. Influenza viruses are classified as A, B, and C viruses according to the antigenicity of nucleocapsid proteins and matrix proteins, and the same A viruses are classified as either nucleocapsid proteins or matrix proteins This is the same. Among them, type A is classified as H1 type to H15 type according to the antigen characteristics of HA (hemagglutinin) protein, and N1 type to N9 type according to the nature of NA protein antigen.

Influenza virus A is spread rapidly through the respiratory tract mainly by coughing or sneezing of an infected person. Due to the influenza A (H1N1) influenza virus in 2009, only 763,752 patients were reported in Korea, of whom 2370 have died.

Therefore, there is a need for a method that can promptly and economically confirm the infection of influenza virus A in order to prevent the rapid spread of the disease.

A method for diagnosing influenza virus infection through the detection of influenza virus using a primer for a matrix gene of influenza virus, a hemagglutinin gene and the like has been reported.

However, no protein markers have yet been reported to determine whether influenza A virus is infected.

1. Korean Patent Publication No. 2012-0099816 2. Korean Patent Publication No. 2013-0081948

The present application provides a kit for determining whether or not an influenza virus A infection is detected, and a method for providing information for determining whether an influenza virus A infection has occurred.

The present application is based on the finding that the present invention relates to a method of specifically binding to one or more proteins selected from the group consisting of Rcl (protein C6orf108), OBFC (CST complex subunit STN1) and annexin 1, And a binding substance that binds to the nucleotide sequence of the influenza A virus.

The present application also provides a kit for determining whether or not an influenza virus A infection is contained, which comprises the protein array.

The present application also relates to a method of detecting influenza virus A infection in a sample of a subject, comprising the steps of: (a) selecting one of the group consisting of Rcl (protein C6orf108), OBFC (CST complex subunit STN1) and annexin 1 The present invention provides a method for measuring the expression amount of the above protein.

According to the present application, it is possible to quickly and accurately determine whether an influenza virus A infection is caused by providing a protein marker capable of discriminating whether or not an influenza virus A has been infected.

Figure 1 shows a two-dimensional electrophoresis pattern of human lung cell proteins infected with influenza virus using an IEF (isoelectric focusing) strip and a large gel system.
FIG. 2 shows a two-dimensional electrophoresis pattern of human lung cell proteins infected with influenza virus using an IEF (isoelectric focusing) strip and a large gel system.
Figure 3 shows the Rcl expression pattern of influenza virus-infected human lung cell line secretory protein by Western blot analysis.
Figure 4 shows the Rcl expression pattern of human plasma proteins infected with influenza virus by Western blot analysis.
Figure 5 shows the OBFC expression pattern of human lung cell line secretory protein infected with influenza virus by Western blot analysis.
Figure 6 shows OBFC expression patterns of human plasma proteins infected with influenza virus by Western blot analysis.
7 shows the annexin 1 expression pattern of human lung cell line secretory protein infected with influenza virus by Western blot analysis.
Figure 8 shows the annexin 1 expression pattern of human plasma protein infected with influenza virus by Western blot analysis.

The inventors of the present application studied the means for rapidly and accurately diagnosing influenza virus A infection, and as a result, discovered protein markers for influenza virus (FIGS. 1 and 2).

That is, it was confirmed that Rcl (protein C6orf108), OBFC (CST complex subunit STN 1) and annexin 1 are protein markers capable of determining whether influenza virus A is infected.

The protein marker for confirming the infection of influenza virus A of the present application means a protein showing a difference in the frequency of expression in human blood infected with influenza virus A. The difference in the expression frequency of the protease can be understood as an increase in expression in a living body infected with influenza virus A. Specifically, Rcl (protein C6orf108), OBFC (CST complex subunit STN1), and annexin 1 are proteins that are increased in expression upon influenza virus A infection.

Accordingly, the present application is directed to protein arrays for determining whether influenza virus A infection is caused by using Rcl (protein C6orf108), OBFC (CST complex subunit STN 1) and annexin 1 as protein markers for determining influenza virus A infection, A kit for determining whether or not an influenza virus A has been infected, and a method for measuring the expression level of the protein for the determination of whether influenza virus A has been infected.

The present application is based on the finding that the present invention relates to a method of specifically binding to one or more proteins selected from the group consisting of Rcl (protein C6orf108), OBFC (CST complex subunit STN1) and annexin 1, And a binding substance that binds to the nucleotide sequence of the influenza A virus.

In one embodiment, the binding material can be one or more selected from the group consisting of an oligopeptide, an antibody, a ligand, a peptide nucleic acid (PNA), an aptamer, a primer, and a probe.

In the present application, the protein arrays can include a binding agent for one marker in one protein array with Rcl (protein C6orf108), OBFC (CST complex subunit STN1) and annexin 1 as markers, respectively; The two markers may be combined to include a binding material for each of the two markers in one protein array; All three markers may be combined to include all of the binding agents for the three markers in a single protein array. Combining all three protein markers can increase the detection sensitivity and reliability of the protein arrays.

The term "array" as used herein is an array of elements (e.g., polypeptides) present on a solid support and / or in a vessel array. Arrays are most often considered physical elements with specific spatial-physical relationships, and the present invention can use a " logical knife "array that does not have a linear spatial organization. For example, a computer system may be used to track the location of one or several corresponding components that are physically present on different components or physically different components. The computer system creates a logical array by providing an "index" table of the physical location of the array members. Thus, the moving component may also be part of a logic array, as long as the members of the array are specified and the location can be ascertained. This is appropriate when the array of the present invention is present as a flow microscale system or when present in one or more microtiter trays.

Certain array formats are sometimes also referred to as "chips" or "biochips ". The array may include, for example, a low density that is an addressable location of from 2 to about 10 addresses, a medium density of about 100 or more locations, or, for example, more than 1000 high density numbers. Typically, the chip array format is a geometrically uniform shape that allows for easy assembly, handling, placement, stacking, reagent introduction, detection and storage. However, it may be irregular. As one typical format, arrays are structured in an orthographic format, with regular gaps between each position of the set of members on the array. Alternatively, the positions may be bundled, mixed or homogenized for even treatment or sampling. The array may include a plurality of addressed locations where each location is structured such that high throughput handling, automatic transfer, masking, or sampling of the reagent is spatially addressed. The array can facilitate detection or quantitation by specific means including laser irradiation, confocal or deflected light collection, CCD detection and chemiluminescence, and is not limited to the above examples. An "array" format as described herein may be an array (i.e., an array of a plurality of chips), a microchip, a microarray, a microarray assembled on a single chip, an array of macromolecules attached to the microwell plate, But not limited to, other suitable formats for use.

Protein arrays for the determination of influenza virus A infection can be produced by methods known to those skilled in the art.

Specifically, a micropipetting method using a piezoelectric method for immobilizing an antibody specifically binding to the searched protein marker on a substrate of a protein array using a probe molecule (probe) or A method using a pin-type spotter, or the like is preferably used, but the present invention is not limited thereto. The substrate of the protein array is preferably coated with an activator selected from amino-silane, poly-L-lysine, or aldehyde, but is not limited thereto . The substrate may be a slide glass, a plastic, a metal, a silicon, a nylon film, a nitrocellulose film, or the like, but is not particularly limited thereto.

As used herein, the term "oligopeptide " refers to a peptide that specifically binds to a protein marker, such as a dipeptide, a tripeptide, or the like consisting of 2 to 20 amino acids.

As used herein, the term "antibody" refers to a specific protein molecule directed against an antigenic site, which specifically binds to a protein marker.

In the present application, "PNA (peptide nucleic acid) " means a synthetic polymer similar to DNA or RNA.

As used herein, "aptamer" refers to an oligonucleotide or peptide molecule that binds to a target molecule.

As used herein, the term "primer" refers to a short nucleic acid sequence capable of forming a base pair with a complementary template with a short free 3-terminal hydroxyl group and functioning as a starting point for template strand copy. The primer can initiate DNA synthesis in the presence of reagents and four different nucleoside triphosphates for polymerization reactions (i. E., DNA polymerase or reverse transcriptase) at appropriate buffer solutions and temperatures. The primer may be a sense and antisense nucleic acid having 7 to 50 nucleotide sequences as a primer specific to each marker protein. Primers can incorporate additional features that do not alter the primer's basic properties that serve as a starting point for DNA synthesis. Methods for making primers are well known in the art and can be chemically synthesized using, for example, the phosphoramidite solid support method, or other known methods.

As used herein, " probe " refers to a linear oligomer of natural or modified monomer or linkages, including deoxyribonucleotides and ribonucleotides, which can hybridize specifically to the target nucleotide sequence, It is artificially synthesized.

The binding substance that specifically binds to the protein marker may be labeled with a labeling substance such as an enzyme, a fluorescent substance, a ligand, a luminescent material, a microparticle, a redox molecule, and a radioactive isotope. When the enzyme is used as a labeling substance, available enzymes include? -Glucuronidase,? -D-glucosidase,? -D-galactosidase, urease, peroxidase, alkaline phosphatase, acetylcholine But are not limited to, thiazolidinediones, terazo, glucose oxidase, hexokinase, GDPase, RNase, glucose oxidase, luciferase, phosphofructokinase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, ≪ / RTI > beta-lactamase, and the like. Fluorescent materials include, but are not limited to, fluorescein, isothiocyanate, rhodamine, picoeriterine, picocyanin, allophycocyanin, and fluorescamine. Ligands include biotin derivatives and the like. Emitters include, but are not limited to, acridinium ester, luciferin, luciferase, and the like. Fine particles include, but are not limited to, colloidal gold, colored latex, and the like. The redox molecule includes, but is not limited to, ferrocene, ruthenium complex, viologen, quinone, Ti ion, Cs ion, diimide, 1,4-benzoquinone, hydroquinone and the like. Radiation isotopes include, but are not limited to, 3H, 14C, 32P, 35S, 36Cl, 51Cr, 57Co, 58Co, 59Fe, 90Y, 125I, 131I and 186Re.

Rcl (C6orf108 protein) is the If Rcl (C6orf108 protein) of the known human-derived amino acid sequence is not particularly limited, and registered in the http://www.uniprot.org/ assession no. The amino acid sequence described in H0Y8X4 can be used.

OBFC (CST STN complex subunit 1) is a OBFC If the amino acid sequence of the known human-derived is not particularly limited, and registered in the http://www.uniprot.org/ assession no. The amino acid sequence described in Q9H668 can be used.

Annexin 1 protein is the Annexin If one of the known human-derived amino acid sequence is not particularly limited, and registered in the http://www.uniprot.org/ assession no. The amino acid sequence described in P04083 can be used.

In one embodiment, the influenza virus A can be HlNl or H3N2.

In one embodiment, the antibody may be a monoclonal antibody, a polyclonal antibody, or a chimeric antibody. Monoclonal antibodies, polyclonal antibodies, and chimeric antibodies can be used without limitation in antibodies produced by conventional methods, and methods for producing them are well known in the art. For example, a polyclonal antibody can be produced by a method well known in the art for obtaining an antibody-containing serum by injecting an animal with a marker protein antigen for the above-mentioned influenza virus A infection determination and collecting it from an animal . Such polyclonal antibodies can be prepared from any animal species host, such as goats, rabbits, sheep, monkeys, horses, pigs, small dogs, and the like.

Monoclonal antibodies may be obtained from the hybridoma method (see Kohler and Milstein (1976) European Jounal of Immunology 6: 511-519), or the phage antibody library (Clackson et al, Nature, 352: Biol., 222: 58, 1-597, 1991) techniques. The antibody prepared by the above method can be isolated and purified by gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, affinity chromatography, and the like.

Antibodies also include functional fragments of antibody molecules as well as complete forms with two full-length light chains and two full-length heavy chains. A functional fragment of an antibody molecule refers to a fragment having at least an antigen binding function, and includes Fab, F (ab ') 2, F (ab') 2 and Fv.

The present application also provides a kit for determining whether or not an influenza virus A infection is contained, which comprises the above protein array. In one embodiment, the kit may be, but is not limited to, a luminex assay kit, a protein microarray kit, or an ELISA kit.

The kits of the present application can include an antibody against one or more protein markers to quantify the protein of a sample to determine whether or not the influenza virus A is infected. For example, the kits of the present application can be used to analyze influenza (anti-Rcl (protein C6orf108) antibody, anti-OBFC (anti-OBTC) antibody, anti- The antigen-antibody binding reaction can be detected by an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay (RIA) assay, a sandwich assay, a Western blot on polyacrylamide gel, , An immunoblot assay, and an immunohistochemical staining method, but the present invention is not limited thereto.

The kits of the present application may include a combination of packaging material (material for packaging the array), instructional material (e.g., instructions for using the array to detect one or more reagents or samples interacting with the array), control reagents And the like), a sample, and the like.

The present application also relates to a method of detecting influenza virus A infection in a sample of a subject, comprising the steps of: (a) selecting one of the group consisting of Rcl (protein C6orf108), OBFC (CST complex subunit STN1) and annexin 1 The present invention provides a method for measuring the expression amount of the above protein. In one embodiment, the influenza virus A may be HlNl or H3N2.

In one embodiment, the amount of expression of the protein can be measured using an antigen-antibody reaction. The antigen-antibody reaction is as follows.

In one embodiment, the protein sample may be isolated from somatic cells or blood of a subject, but is not limited thereto. For example, somatic cells of the subject may be lung cells, and blood of the subject may be plasma.

Hereinafter, a method for measuring the expression level of the above-mentioned protein marker in order to determine whether or not the virus is infected with influenza virus A will be described. In order to measure the expression level of the target protein in the protein sample of the subject, a protein sample is first separated from the blood of the subject. After the separated protein sample is contacted with the antibody against the labeled protein, the degree of protein expression according to the contact is measured. Then, by comparing the degree of expression of the measured protein with the control group, it is possible to determine whether or not the virus is infected with influenza virus A. The term " subject " means a group of subjects suspected of having or not having been infected with influenza virus A, and the control group means a normal population that is not infected with influenza virus A. Methods for separating proteins from blood samples are well known in the art, and conventional protein extraction methods can be used without limitation.

Methods for confirming the expression level of the protein can be used without limitation in any method known in the art. In one embodiment, the amount of expression of the protein can be measured using an antigen-antibody reaction. As mentioned above, an antibody specifically recognizing Rcl (protein C6orf108), an antibody specifically recognizing OBFC (CST complex subunit STN1), or an antibody specifically recognizing annexin 1 are Rcl C6orf108), OBFC or annexin 1, the amount of the protein expressed can be determined by quantifying the bound antigen-antibody complex. In addition, the ionic strength obtained with the protein can be used to calculate the concentration of the target protein. The target protein is identified using a characteristic single ion pair (or several ion pairs), and the resulting ionic strength is related to the known concentration of the target protein, allowing the quantification of the corresponding target protein. Target proteins are known in advance and can be pre-annotated. Thus, the detected and quantified proteins are already tinned, allowing rapid and direct interpretation.

The mass spectrometry of the target protein can be performed using a mass spectrometer capable of MSMS analysis in order to distinguish ion species, but the mass spectrometer is not particularly limited thereto.

From the above results, the increase of the ionic strength by quantitative analysis of the protein can be compared to determine whether or not the influenza virus A has been infected.

In one embodiment, if an increase or decrease in the ionic strength of ETFB (Isoform 1 of electron transfer flavoprotein subunit beta) or ITIH-2 (Inter-alpha-trypsin inhibitor heavy chain H2) is observed in the sample, .

For example, when the expression levels of Rcl (FIGS. 3 and 4), OBFC (FIGS. 5 and 6) and annexin 1 (FIGS. 7 and 8) can do.

For example, a case in which the expression level of Rcl, OBFC and annexin 1 is normal (control group) can be determined as influenza virus A-infected group. When the three markers are discriminated from each other, the reliability and sensitivity of discrimination of influenza virus A infection can be increased.

The present application is also directed to a method of screening a cell or tissue comprising (a) one or more proteins selected from the group consisting of Rcl (protein C6orf108), OBFC (CST complex subunit STN1) and annexin 1 ; And a method for screening a substance for preventing or treating influenza virus A infection, which comprises measuring the expression level of one or more proteins or each of the polynucleotides encoding them in the step (a). In the case of infection with influenza virus A, the amount of expression of Rcl, OBFC and annexin 1 is increased. Therefore, when the sample to be analyzed reduces the expression amount of Rcl, OBFC or annexin 1, the sample is prevented or treated with influenza virus A It can be judged as a material for use.

Hereinafter, the present application will be described in detail by way of examples. The following examples are illustrative of the present application and the scope of the present application is not limited to the following examples.

EXAMPLES Example 1 Detection of Protein Index for Influenza Virus in Human Plasma Protein

For the detection of proteomic indicators for influenza viruses in human plasma proteins, the protein samples were subjected to a narrow range of immobiline dry strips (pH 4-7, pH 6-9, 24 cm) at a fixed pH One - dimensional electrophoresis was performed.

Next, the human plasma protease was isolated through 2-DE analysis (12% SDS-PAGE, 34 × 45 cm, OWL) and protein markers for influenza virus were analyzed by silver staining and image analysis (Progenesis SameSpots (Nonlinear) (Fig. 1 and Fig. 2).

Next, accurate proteomic identification was performed using EBI (European Bioinformatics Institute) IPI human database using ESI-MS / MS (LCQ Deca XP-Plus, Thermo Finnigan, USA) and Western blot. The overexpression and low expression proteins according to ESI-MS / MS analysis are shown in Table 1 below.

 [Table 1]

As shown in Table 1, the expression of Rcl (protein C6orf108), OBFC (CST complex subunit STN 1) and annexin 1 was increased in influenza virus A infected samples.

Western blot analysis showed that Rcl (protein C6orf108) (FIG. 3), CST complex subunit STN 1 (FIG. 5) and annexin 1 (FIG. 5) in human lung cell lines infected with influenza virus H1N1 or H3N2 7), and the expression of Rcl (protein C6orf108) (FIG. 4), OBFC (CST complex subunit STN 1) (FIG. 6) and annexin 1 .

Therefore, it was confirmed that Rcl (protein C6orf108), OBFC (CST complex subunit STN 1) and annexin 1 can be used as an index for determining whether or not an influenza virus A infection has occurred.

Claims (14)

A protein array for determining whether an influenza virus A infection is infected, comprising a binding substance specifically binding to Rcl (protein C6orf108) protein or binding to a polynucleotide encoding said protein.
The method according to claim 1,
A binding substance that specifically binds to one or more proteins selected from the group consisting of OBFC (CST complex subunit STN 1) and annexin 1, or binds to a polynucleotide encoding each protein, A protein array for determining whether an influenza virus A infection is included.
3. The method according to claim 1 or 2,
The binding substance is at least one selected from the group consisting of an oligopeptide, an antibody, a ligand, a peptide nucleic acid (PNA), an aptamer, a primer, and a probe.
3. The method according to claim 1 or 2,
The binding substance is labeled with a labeling substance, and the protein array for determining whether influenza virus A is infected.
5. The method of claim 4,
The labeling substance is at least one selected from the group consisting of an enzyme, a fluorescent substance, a ligand, a luminescent substance, a microparticle, a redox molecule and an isotope.
The method of claim 3,
The antibody may be a monoclonal antibody, a polyclonal antibody, or a chimeric antibody, a protein array for determining whether an influenza virus A infection is present.
A kit for determining whether or not an influenza virus A infection is infected comprising the protein array according to claim 1.
8. The method of claim 7,
Kits include Luminex assay kits, protein microarray kits, or ELISA kits for influenza virus A infection kits.
A method for providing information to determine whether or not an influenza virus A infection is detected, comprising measuring the expression level of Rcl (protein C6orf108) protein in a protein sample of a subject.
10. The method of claim 9,
A method of providing information to determine whether or not an influenza virus A infection is further included, which comprises measuring the expression level of at least one protein selected from the group consisting of OBFC (CST complex subunit STN 1) and annexin 1 .
11. The method according to claim 9 or 10,
Wherein the measurement of the amount of protein expression is carried out using an antigen-antibody reaction.
11. The method according to claim 9 or 10,
Wherein the protein sample is obtained from influenza virus A, which is isolated from somatic cells or blood of a subject.
13. The method of claim 12,
A method of providing information to determine whether a somatic cell of a subject is infected with influenza virus A which is a lung cell.
13. The method of claim 12,
A method for providing information to determine whether blood of a subject is infected with influenza virus A which is plasma.

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