KR101884935B1 - Aptamer binding to influenza virus and uses thereof - Google Patents

Abstract

More particularly, the present invention relates to an RNA tamtamer or a peptide specifically binding to an influenza virus, a method for detecting influenza virus containing the RNA tamtamer or a peptide as an active ingredient, A kit for detecting influenza virus containing the same, and a method for detecting influenza virus. The RNA plasmid or peptide according to the present invention can be used for the diagnosis of influenza virus infection because it is smaller in size than the antibody, has high stability, can be produced in a short time at a low cost, and can be used for a long time. There will be.

Description

Aptamer binding to influenza virus and uses thereof < RTI ID = 0.0 >

The present invention relates to an abatumer binding to influenza virus and uses thereof.

Every year, seasonal influenza is U.S. It alone causes 300,000 hospitalizations and 36,000 deaths. Influenza (influenza) is a respiratory disease caused by influenza virus. Influenza virus, the causative agent, is a member of the family Orthomyxoviridae. Influenza virus particles include a segmented negative-sense-RNA genome that encodes the following proteins: Hemagglutinin (HA), neuraminidase (NA), Matrix (M1)), a proton ion-channel protein (M2), a nucleoprotein (NP), a polymerase base protein 1 (PB1), a polymerase base protein 2 (PB2), a polymerase acid protein 2 (NS2). HA, NA, M1 and M2 are associated with membranes, whereas NP, PB1, PB2, PA and NS2 are proteins associated with nucleocapsids. The M1 protein is the most abundant protein in influenza particles. The HA and NA proteins are envelope glycoproteins responsible for viral attachment and infiltration of viral particles into the cell, the source of the major immunodominant determinants for viral neutralization and immunity protection. Both HA and NA proteins are considered to be the most important components for preventive influenza.

Influenza viruses are spherical (80-120 nm in diameter) viruses that are divided into several subtypes depending on the type of hemagglutinin and neuraminidase proteins that are major antigens on the surface. In the case of influenza A, a total of 16 HA and 9 NA are known, and a total of 144 subtypes are theoretically possible by these combinations.

Current diagnosis of influenza infection is mainly based on the detection of virus and the immune response of the patient to the virus. Therefore, diagnostic tests can be roughly classified into 4 kinds such as influenza virus isolation (culture), detection of virus antigen (rapid antigen test, immunofluorescence), viral nucleic acid confirmation (RT-PCR) and serological test. The virus culture method has a disadvantage of 2 ~ 10 days time required, skilled expert is needed and false negative is likely to occur, and the PCR method has a disadvantage that a primer suitable for each subtype is required. Immunological methods using antibodies can diagnose diseases with high accuracy. At the time of the first development of the immunoassay method, enzymes were used as markers, but organic minerals are now commonly used as a result of technology development. However, in the case of organic minerals, there is a problem that the sensitivity of the sensor is deteriorated because the brightness of the fluorescent material due to photobleaching sharply drops as well as the sensitivity to the pretreatment conditions of the sample. Therefore, development of a method capable of more effectively detecting influenza virus is required.

In order to overcome the limitations of the prior art, methods using an Aptamer have been studied. Aptamer is a single-stranded nucleic acid such as DNA or RNA having a stable tertiary structure and capable of binding to a target molecule with high affinity and specificity. Since 1990, many abundant plasmids have been unearthed that can bind to a variety of target molecules, including small molecule organisms, peptides, and membrane proteins. The aptamer has a characteristic of being able to selectively bind to a target molecule with high binding force and specificity, so that it can be used for distinguishing a specific substance.

Korean Patent Registration No. 1384178 discloses a platamer which specifically binds to the NS1 protein of influenza virus and a pharmaceutical composition containing the same, Korean Patent Laid-Open Publication No. 2015-0112573 discloses a pharmaceutical composition comprising ' Binding nucleic acid platamer and its use 'have been disclosed. However, the platamer binding to the influenza virus of the present invention and its use have not been disclosed.

The present invention has been made in view of the above-mentioned needs, and it is an object of the present invention to provide a novel immunoassay kit comprising an RNA uterum-europium complex or a peptide-europium complex according to the present invention to produce influenza virus subtypes H1N1, H7N7, H9N2 and H5N3 Type virus antigens can be detected, thereby completing the present invention.

In order to achieve the above object, the present invention provides an oligonucleotide primer consisting of the nucleotide sequence of SEQ ID NO: 1 which specifically binds to influenza virus.

The present invention also provides a composition for detecting influenza virus comprising the oligonucleotide squid polymer or a peptide consisting of the amino acid sequence of SEQ ID NO: 2 as an active ingredient.

The present invention also provides a kit for detecting influenza virus comprising the composition for detecting influenza virus as an active ingredient.

The RNA plasmids or peptides according to the present invention can be used for the rapid diagnosis of influenza virus infection because they are small in size, high in stability, can be produced in a short time at a low cost, and can be used for a long time.

1 is a graph showing the results obtained by applying various influenza A virus samples (H5N3, H9N2, H7N7 or H1N1 virus) to a fluorescent immunoassay kit in which an anti-influenza A antibody containing the RNA complex of the present invention is placed, TL) was confirmed by UV detection.
FIG. 2 shows the results obtained by applying various influenza A virus samples (H5N3, H9N2, H7N7 or H1N1 virus) to the fluorescent immunoassay kit in which the anti-influenza A antibody containing the RNA complex of the present invention is dispensed, TL) of the fluorescence intensity.
FIG. 3 is a graph showing the results obtained by applying various influenza A virus samples (H5N3, H9N2, H7N7 or H1N1 virus) to a fluorescent immunoassay kit in which an anti-influenza H5 specific antibody containing the RNA complex of the present invention is placed, (TL) fluorescence detection on UV.
FIG. 4 is a graph showing the results obtained by applying various influenza A virus samples (H5N3, H9N2, H7N7, or H1N1 virus) to a fluorescent immunoassay kit in which an anti-influenza H5 specific antibody comprising the RNA- (TL).
FIG. 5 shows the results obtained by applying various influenza A virus samples (H5N3, H9N2, H7N7 or H1N1 virus) to the fluorescence immunoassay kit in which the anti-influenza A antibody containing the peptide conjugate of the present invention was placed, TL) of the fluorescence intensity.
FIG. 6 is a graph showing the results obtained by applying various influenza A virus samples (H5N3, H9N2, H7N7, or H1N1 virus) to a fluorescent immunoassay kit loaded with an anti-influenza H5 specific antibody comprising the peptide conjugate of the present invention, (TL).

In order to achieve the object of the present invention, the present invention provides an oligonucleotide primer comprising the nucleotide sequence of SEQ ID NO: 1 which specifically binds to influenza virus.

'Aptamer' refers to single stranded DNA (ssDNA) or RNA with high specificity and affinity for a specific substance. Since aptamers can be synthesized in a relatively simple and highly stable manner with a high affinity for a specific substance, various modifications are possible in order to increase binding force, and even cells, proteins, and small organic substances can be target substances, Its specificity and stability are very high compared to antibodies that have already been developed.

The aptamer of the present invention is characterized by an RNA polymerase comprising the nucleotide sequence of SEQ ID NO: The aptamer according to the present invention may comprise, but is not limited to, the nucleotide sequence of SEQ ID NO: 1. In addition, homologues of the nucleotide sequences are included within the scope of the present invention. Specifically, the gene has a nucleotide sequence having a sequence homology of 70% or more, more preferably 80% or more, still more preferably 90% or more, and most preferably 95% or more, with the nucleotide sequence of SEQ ID NO: 1 . "% 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 >

The length of the plumper of the present invention is not particularly limited and can be generally about 15 to 200 nucleotides, but is, for example, about 100 nucleotides or less, preferably 90 nucleotides or less, more preferably about 60 nucleotides or less And most preferably about 45 nucleotides or less.

Each nucleotide contained in the platamer of the present invention may be the same or different and is a nucleotide including a hydroxyl group at the 2 'position of the ribose (i.e., a nucleotide which is a nucleotide), or a hydroxyl group at the 2 & , Any atom or a nucleotide substituted with a functional group. Examples of such an arbitrary atom or functional group include a hydrogen atom, a fluorine atom or an -O-alkyl group (e.g., -O-Me (methoxyl group)), -O-acyl group (e.g., -O- -NH2). ≪ / RTI > The aptamers of the present invention may also be modified so that at least one (e.g., 1, 2, 3 or 4 kinds) of nucleotides may contain a hydroxyl group or any of the above-mentioned atoms or functional groups such as a hydrogen atom, (E.g., 2, 3 or 4) functional groups selected from the group consisting of a hydroxyl group and a methoxyl group. In addition, the aptamer of the present invention has the same functional group selected from the group consisting of a hydroxyl group or any of the above-mentioned atoms or functional groups such as a hydrogen atom, a fluorine atom, a hydroxyl group and a methoxyl group at the 2'position of the ribose of all the nucleotides , And preferably, the hydroxyl group at the 2 'position of the ribose of all the nucleotides may be substituted with a methoxyl group, but is not limited thereto.

The aptamer of the present invention can be combined with a label that generates a detectable signal, which can be a detecting moiety that can be easily detected by a detection method known in the art. For example, the label generating the detectable signal may be a chemical label (e.g., biotin), a fluorescent label, a radioactive label (e.g., ¹²⁵ I and ¹⁴ C), a luminescent label, a chemiluminescent label, and a fluorescence resonance energy transfer labels, but are not limited thereto.

In an embodiment of the present invention, the aptamer of the present invention is characterized in that an amine linker is added to the 5 'end region to bind with the fluorescent substance.

The fluorescent substance that can be used in the present invention can be any of those known in the art. In one embodiment of the present invention, the aptamer of the present invention may be labeled with europium, but is not limited thereto.

The oligonucleotide aptamer is preferably used as an antibody substitute against influenza virus, but is not limited thereto. Since the oligonucleotide molecule is much smaller in size than the antibody and binds to the target molecule with a high binding force, it has antibody-like target affinity and is not sensitive to temperature change. Even if the oligonucleotide molecule is denatured, There are advantages.

In addition, the oligonucleotide molecule of the present invention can be used as a sensor. For example, the oligonucleotide molecule of the present invention can be used as an electrochemical sensor that responds to the degree of electron transfer appearing before and after binding between an abatumer and a target molecule, Optical sensors, and mass spectrometric sensors that measure and analyze the difference in mass before and after bonding with the target material.

The influenza virus is preferably an influenza A virus, more preferably an influenza virus of H1N1 type, H5N3 type, H7N7 type or H9N2 type, but is not particularly limited thereto.

The present invention also provides a composition for detecting influenza virus comprising the oligonucleotide squid polymer or a peptide consisting of the amino acid sequence of SEQ ID NO: 2 as an active ingredient.

The oligonucleotide aptamer which is an effective component of the composition for detecting influenza virus of the present invention is as described above.

The peptide as an active ingredient of the composition for detecting influenza virus of the present invention comprises a peptide consisting of the amino acid sequence shown in SEQ ID NO: 2 and a functional equivalent of the peptide. As a result of addition, substitution or deletion of an amino acid, at least 60% or more, preferably 70% or more, more preferably 80% or more, still more preferably Quot; refers to a peptide having substantially the same physiological activity as a peptide represented by the amino acid sequence shown in SEQ ID NO: 2, having 90% or more of sequence homology.

The peptide of the present invention consists of the amino acid sequence of SEQ ID NO: 2 and may be a peptide in which a fluorescent substance is directly bound to lysine at the N-terminal.

The present invention also provides a kit for detecting influenza virus comprising the composition for detecting influenza virus as an active ingredient.

The kit for detecting influenza virus of the present invention comprises a conjugate in which the above-mentioned platemater or peptide and a fluorescent substance are combined. In one embodiment of the present invention, the specific antibody of influenza virus in the detection kit of the present invention may be provided in a state adsorbed on the substrate. As the substrate, a PVDF film, a plate, and a slide may be used, but the present invention is not limited thereto. In addition, the detection kit may further comprise a composition, a solution or an apparatus having one or more other components suitable for the analysis method.

The kit for detecting influenza virus according to the present invention is not particularly limited as long as it comprises a conjugate in which the platemaker or peptide and the fluorescent substance are bound and can immunologically detect a desired antigen. Preferably, the kit includes an ELISA kit , More preferably a sandwich ELISA kit, and most preferably a sandwich FICT (fluorescent immunochromatographic test kit) kit in the form of a strip.

The influenza virus detection kit comprises: a sample injection unit for injecting a sample; An influenza virus binding unit comprising a composition for detecting influenza virus that binds to an influenza virus antigen in a sample located at a position spaced apart from the sample injecting unit by a predetermined distance; A test line in which a specific antibody of influenza virus is immobilized at a position spaced apart from the binding portion at a predetermined interval; And a control line to which the anti-mouse IgG is immobilized are sequentially provided. However, the present invention is not limited thereto.

For example, the kit for detecting influenza virus provided by the present invention is a kit for detecting influenza virus, which comprises a sample in which a glass fiber, a cotton or a cellulose material pad is bonded to a nitrocellulose membrane in a strip form, A binding portion of an influenza virus antigen including the platemaker-fluorescent substance conjugate or a peptide-fluorescent substance conjugate, and an antigen identical to the platemaker or the peptide can be detected while maintaining a predetermined distance from the sample injecting portion Or an immunostrip or a fluorescent immunochromatographic test kit (FICT), in which a test line to which another antibody with a different antibody is immobilized and a control line to which a secondary antibody capable of detecting the antibody contained in the composition is immobilized are sequentially provided.

In addition, the present invention is characterized in that when a specimen suspected of containing an influenza virus is put into a kit for detecting influenza virus and a fluorescent light band appears on an examination line of the test strip, the influenza virus infection is judged to be positive, Is not present, the influenza virus infection is determined to be negative.

The present invention provides a method for providing information for diagnosis of influenza virus comprising the step of adding a separated sample to the fluorescence immunoassay kit for detecting influenza virus, .

Specifically, the method comprises the steps of: (a) obtaining a sample isolated from an animal suspected of having an influenza virus infection; And (b) detecting the influenza virus from the obtained sample using the kit.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited thereto.

Materials and methods

1. Synthesis of RNA Abutamer and Peptide

As a result of screening nucleic acid that specifically binds to influenza virus in the synthetic nucleic acid library, 90mer of RNA (SEQ ID NO: 1: GGG UUC ACU GCA GAC UAG ACG AAG CUU ACA AAC AAG AGC AAA AAG GGA GUU GAC GUA GAC UGU GCG GAA UGG AUC CAC AUC UAC GAA UUC) were selected. The above-mentioned RNA aptamer was ordered so as to be able to stabilize and bind the fluorescent substance to order from Bio-Ina (Korea). 2'-O-methyl (methoxyl group) was transformed to all ribose (rA, rC, rG, rU) to stabilize the RNA sequence and an amine linker was added at the 5 'end for fluorescent substance binding.

As a result of screening for a synthetic peptide specifically binding to influenza virus, a peptide consisting of the amino acid sequence 'KPNDAINF' of SEQ ID NO: 2 was selected and the peptide was prepared according to a conventional peptide synthesis method known in the art.

2. Preparation of europium conjugate

10 μl The bangs lab (FC02F) (stock: 0.3 μM) was mixed with 920 μl of 0.1 M MES pH 6.0, 20 μl of 0.2 M EDC and 50 μl of 0.2 M NHS, followed by sonication 20s, pulse 01: 1, amp 25%). The reaction was then allowed to react slowly at room temperature for 1 hour. After centrifugation (17,000 rpm / 5 minutes), 490 μl of 0.1 M sodium phoshate (pH 8.0) was added to the harvested sediment and dispersed by sonication (20 s, pulse 01:01, amp 25%). 10 μl of RNA (10 μM) or peptide (10 μM) was added and reacted slowly at room temperature for 2 hours. After sonication (20 s, pulse 01: 1, amp 25%) was performed, 120 μl of 1 M Tris-HCl, 30 mM Glycine, pH 8 was further added and reacted by slowly turning it at room temperature for 25 minutes. (17,000 rpm / 5 minutes). The precipitate was subjected to sonication (16s, pulse 01: 1, amp 25%) with 1 ml of 2 mM Borax, pH 9.0, followed by centrifugation (17,000 rpm / 5 min). After the sonication (20s, pulse 01: 1, amp 25%) was carried out by adding 100 μl of 2 mM Borax, pH 9.0 and 1% BSA, the final prepared europium + RNA fluorescent conjugate and europium + ≪ / RTI >

3. Production of a rapid fluorescence immunoassay kit for diagnosis of influenza infection

3-1. Production of rapid fluorescence diagnostic kit with anti-influenza A antibody

A rapid fluorescence diagnostic kit for the diagnosis of influenza infection was prepared, including the prepared conjugate. In order to detect the NP antigen of influenza A virus contained in the sample, a fluorescentimmunochromatographic test (FICT) in which a sample pad, a conjugate, an inspection line and a control line are sequentially arranged on a strip type nitrocellulose membrane, Respectively.

Specifically, a sample injecting unit capable of injecting a sample by bonding a pad made of glass fiber, cotton or cellulose to the end of a strip-shaped nitrocellulose membrane was provided. To the test line (TL), 2.5 μg of an antibody against NP of influenza A virus (7307, Medix Biochemica) was added and fixed. 2.5 μg of antibody against mouse IgG was added to the control line (CL) and fixed. 2 [mu] l of the fluorescent conjugate prepared on the binding pad was dropped on the binding pad. After that, 75 μl of 1 × 10 ⁵ PFU / mL of H5N3, H1N1, H7N7 and H9N2 were added, respectively. Subsequently, 75 μl of elution buffer (Tris HCl 50 mM pH 7.5, NaCl 0.2 M, MgCl 2 5 mM, sodium azide 0.02%, Triton X-100 0.5%) was dropped into the sample inlet. After inducing lateral flow for 15 minutes, the fluorescence of the strip was measured by exposure to UV. Or fluorescence values of TL and CL were measured by a fluorescent reader (aQcard TRF, Medisensor, Korea).

3-2. Production of Rapid Fluorescence Diagnostic Kit Dosing Anti-Influenza H5 Specific Antibody

A rapid fluorescence diagnostic kit for the diagnosis of influenza infection was prepared, including the prepared conjugate. A fluorescentimmunochromatographic test (FICT) was performed in which a sample pad, a conjugate, an inspection line and a control line were sequentially arranged on a strip-shaped nitrocellulose membrane so as to detect the virus antigen of the influenza contained in the sample Respectively.

Specifically, a sample injecting unit capable of injecting a sample by bonding a pad made of glass fiber, cotton or cellulose to the end of a strip-shaped nitrocellulose membrane was provided. 2.5 μg of influenza H5 subtype specific antibody (Genbody Inc.) was added to the test line (TL). 2.5 μg of antibody against mouse IgG was added to the control line (CL) and fixed. 2 [mu] l of the fluorescent conjugate prepared on the binding pad was dropped on the binding pad. Then, 75 의 of 1x10 ⁵ PFU / ㎖ L of H5N3, H1N1, H7N7, and H9N2 were added, respectively. Subsequently, 75 μl of elution buffer (Tris HCl 50 mM pH 7.5, NaCl 0.2 M, MgCl 2 5 mM, sodium azide 0.02%, Triton X-100 0.5%) was dropped into the sample inlet. After inducing lateral flow for 15 minutes, the fluorescence of the strip was measured by exposure to UV. Or fluorescence values of TL and CL were measured by a fluorescent reader (aQcard TRF, Medisensor, Korea).

Example  1. Detection of influenza virus using RNA complex

(1) Influenza A virus rapid fluorescence immunoassay using RNA complex

H5N3, H1N1, H7N7 and H9N2 viruses were detected in the test line (TL) by fluorescence microscopy for the results of influenza A rapid fluorescence immunoassay (stripping with anti-influenza A) Image was detected and confirmed to be reactive. CL was not a conjugate using an antibody and thus did not react (Fig. 1).

As a result of fluorescent immunoassay using the kit for detecting influenza virus, fluorescent images were detected in all of H5N3, H1N1, H7N7 and H9N2 viruses on the inspection line (TL) . In CL, the reaction value was very low since it was not a conjugate using an antibody (Fig. 2).

(2) Influenza H5 subtype specific virus rapid fluorescence immunoassay using RNA complex

H5N3, H1N1, H7N7, and H9N2 viruses were detected in the test line (TL) as a result of placing the strip under UV for the purpose of reading the results according to the influenza H5 subspecies specific rapid fluorescence immunoassay (a strip dotted with anti-influenza H5 specific antibody) Fluorescent images were detected for all of the H5N3 viruses in all, confirming the H5 subtype specific reactivity. CL was not a conjugate using an antibody and thus was not reacted (Fig. 3).

As a result of fluorescent immunoassay using the kit for detecting influenza virus, fluorescent images were detected in all of H5N3, H1N1, H7N7 and H9N2 viruses on the inspection line (TL) . CL was not a conjugate using an antibody and thus was not reacted (Fig. 4).

Example 2. Detection of influenza virus using peptide conjugate

(1) Rapid fluorescence immunoassay for influenza A virus using a peptide conjugate

H5N3, H1N1, H7N7, and H9N2 viruses were tested in the test line (TL) as a result of measuring the strip with a fluorescent reader to read out the results according to the influenza A rapid fluorescent immunoassay (stripping with anti-influenza A) As a result, the fluorescence value was high for all of the applied viruses, and it was confirmed that there was reactivity to the antigen. In CL, the reaction value was very low since it was not a conjugate using an antibody (Fig. 5).

(2) Rapid fluorescence immunoassay for influenza H5 subspecies specific virus using peptide conjugate

H5N3, H1N1, H7N7, and H9N2 viruses were detected in the test line (TL) for the purpose of reading the results according to the influenza H5 subspecies specific rapid fluorescence immunoassay (strips dotted with anti-influenza H5 specific antibodies) , It was confirmed that not only the fluorescence image but also the fluorescence value of the H5N3 virus was increased and thus it was useful for the H5 subtype specific rapid fluorescence immunoassay. CL was not a conjugate using an antibody and thus did not react (Fig. 6).

<110> Wonkwang University Center for Industry-Academy Cooperation <120> Aptamer binding to influenza virus and uses thereof <130> PN16245 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 90 <212> RNA <213> Artificial Sequence <220> <223> RNA aptamer <400> 1 ggguucacug cagacuugac gaagcuuaca aacaagagca aaaagggagu ugacguagac 60 ugugcggaau ggauccacau cuacgaauuc 90 <210> 2 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> peptide <400> 2 Lys Pro Asn Asp Ala Ile Asn Phe   1 5

Claims (9)

delete delete delete delete A composition for detecting H1N1, H5N3, H7N7, and H9N2 influenza viruses comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 2 as an active ingredient. The composition for detecting influenza virus according to claim 5, wherein the peptide is a peptide conjugated with a fluorescent substance. delete A kit for detecting H1N1, H5N3, H7N7 and H9N2 influenza viruses comprising the composition for detecting influenza virus according to claim 5 as an active ingredient. 9. The influenza virus detection kit according to claim 8, wherein the influenza virus detection kit comprises: a sample injection unit for injecting a sample; H1N1, H5N3, H7N7 and H9N2 influenza viruses containing the peptide consisting of the amino acid sequence of SEQ ID NO: 2, which binds to the influenza virus antigen in the sample, A binding portion of an influenza virus antigen comprising the composition; A test line in which a specific antibody of influenza virus is immobilized at a position spaced apart from the binding portion at a predetermined interval; And a control line on which an anti-mouse IgG is immobilized are sequentially provided on a test strip. The kit for detecting H1N1, H5N3, H7N7 and H9N2 influenza viruses in test strip form.

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