KR102554330B1 - Peptide specifically binding to H1N1 subtype of influenza A virus and uses thereof - Google Patents

Abstract

The present invention relates to a peptide that specifically binds to influenza A H1N1 virus and uses thereof, and more particularly, to a peptide that specifically binds to hemagglutinin or neuraminidase of influenza A H1N1 virus and a method for detecting influenza A H1N1 virus using the same.
Since the peptide provided by the present invention binds to the influenza A H1N1 virus with very high specificity and affinity, it can be used very usefully in the development of a composition and detection kit for detecting influenza A H1N1 virus, and can be used to detect influenza A H1N1 virus infection. Influenza A H1N1 virus can be rapidly and accurately detected in a sample provided from a patient.

Description

Peptide specifically binding to H1N1 subtype of influenza A virus and uses thereof {Peptide specifically binding to H1N1 subtype of influenza A virus and uses thereof}

The present invention relates to a peptide that specifically binds to influenza A H1N1 virus and uses thereof, and more particularly, to peptides specific to hemagglutinin (HA) or neuraminidase (NA) of influenza A H1N1 virus. It relates to a peptide that binds to and a method for detecting influenza A H1N1 virus using the same.

Influenza virus belongs to the genus Orthomyxoviridae influenza virus as an RNA virus, and is classified as A, B, C, classified as type D. Of these, type A is classified into subtypes H1 to H18 depending on the antigenic characteristics of the HA protein and N1 to N11 types according to the antigenic characteristics of the NA protein. These virus particles have an outer envelope and are usually polymorphic spheres with a diameter of about 80 to 120 nm. Viruses immediately after isolation from fertilized eggs and susceptible cells sometimes have a thread-like filament shape with a length of 400 nm. There are specific glycoproteins in the outer envelope of the virus. Representative envelope membrane glycoproteins present in type A viruses are hemagglutinin (HA) and neuraminidase (NA), which protrude on the surface of the virus It plays an important role in the antibody response.

In general, among influenza viruses, type A virus is the most representative in terms of infectivity and pathogenicity. This virus can infect a variety of species, including birds and humans. Its natural host is known to be a wild migratory bird, and it is assumed that transmission from birds to mammals occurs through interspecies infection. The epidemic cycle of type A virus due to antigenic shift is estimated to be about 10 to 40 years, and it has been the cause of at least three influenza virus pandemics in the past.

The influenza A H1N1 subtype to be detected in the present invention is a virus that caused a pandemic called 'Spanish flu' in 1918, and was historically recorded as causing many deaths in a short period of time. Even after that, it was called 'Soviet flu' in 1977, and an epidemic occurred among young people who did not have immunity to H1N1. This resulted in an influenza virus with a new genetic combination originating from pigs that are susceptible to both human and avian influenza viruses, and caused a pandemic, especially among people without immunity. Infected patients suffered mainly lung disease because the virus caused a cytokine storm in lung tissue, stimulating the immune system.

The incubation period of H1N1 influenza is estimated to be 1 to 7 days, and in most cases, symptoms begin within 1 to 4 days after contact with an infected person (droplets of an infected person). Infectious symptoms include fever (94%), cough (92%), sore throat (66%), runny nose/stuffy nose, fatigue, and headache, which appear in the form of acute febrile respiratory disease and are similar to infections caused by common seasonal influenza or other viruses. It is difficult to distinguish. However, gastrointestinal symptoms such as diarrhea and vomiting, which do not appear in seasonal influenza, occur in 10-25% of patients, making them distinct. Most of the patients are cured spontaneously without complications, and 3-9% are hospitalized. According to a report in the United States, pneumonia and dehydration are the main causes of hospitalization. Although related complications have not yet been precisely identified, it is estimated to be respiratory complications and exacerbation of underlying diseases similar to seasonal influenza. During the H1N1 influenza outbreak in the United States, 70-80% of hospitalized patients had one or more severe diseases or high-risk factors for complications. Among hospitalized patients, asthma was the most common underlying disease, and other risk factors included pregnant women, infants under 2 years of age, diabetes, immunodeficiency disease, and cardiovascular disease. In particular, pregnant women need to be careful because they are at high risk of premature birth and death when infected with the virus.

Respiratory diseases caused by influenza virus infection are not unusual, so differential diagnosis from respiratory syncytial virus (RSV), parainfluenza virus, adenovirus, rhinovirus, mycoplasma pneumoniae, and respiratory infections caused by bacteria need this To this end, several diagnostic methods for influenza virus infection have been developed, and the first among them is a culture test.

To this end, several diagnostic methods for influenza virus infection have been developed, and the first among them is a culture test. It is cultured after treating samples (influenza virus) collected from animal cells and fertilized eggs with antibiotics, observing the cytopathic effect (CPE), detecting antigens by hemagglutination test, reverse transcription polymerase chain reaction method (RT-PCR) and real-time reverse transcription polymerase chain reaction (Real-time RT-RCR) to identify specific genes of influenza virus. The second is a gene detection test, which is a method of analyzing the type and subtype of influenza virus by amplifying influenza virus-specific genes (HA or NA region). For type and subtype analysis, HA (hemagglutination assay) and HI (hemagglutination inhibition assay) methods are being implemented. In addition, RT-PCR (reverse transcription-polymerase chain reaction), real-time RT-RCR, and EIA (enzyme linked immunoassay) methods are used to determine types A and B and subtypes, and sequencing is performed to determine HA or HA of influenza virus isolates. There is a diagnostic method that analyzes the nucleotide sequence for the NA gene and compares it with vaccine strains and foreign isolates. Third, the serological diagnosis of influenza virus can be diagnosed when the serum IgG influenza-specific antibody increases significantly by 4-fold or more during the acute phase (within 1 week of onset) and convalescent phase (2 to 4 weeks of onset). However, these diagnostic methods are complicated, lengthy, and require expert help. Lastly, Rapid influenza diagnostic tests (RIDTs) are a method of detecting influenza virus antigens. Recently, new diagnostic reagents have been developed to quickly determine whether antiviral drugs are administered by taking a sample from a patient in the clinic and diagnosing it within 30 minutes. can decide However, the sensitivity is lower than that of PCR, the viral antigen characterization is not possible, and the price is high. In addition, even if RIDTs are negative, influenza infection cannot be completely ruled out, so there is a limitation in that cell culture or genetic testing is required if necessary.

Accordingly, the present inventors conducted research to develop a diagnostic method capable of quickly and accurately detecting influenza virus A H1N1. ), a new peptide having high sensitivity and specificity was discovered, and a system capable of early diagnosis of influenza virus A H1N1 was developed using this, thereby completing the present invention.

Accordingly, an object of the present invention is to provide a peptide that specifically binds to influenza A H1N1 virus, having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 12.

Another object of the present invention is to provide a polynucleotide encoding the above peptide.

Another object of the present invention is to provide a recombinant expression vector comprising a polynucleotide.

Another object of the present invention is to provide a host cell transformed with the recombinant expression vector.

Another object of the present invention is to provide a composition for detecting influenza A H1N1 virus comprising the peptide as an active ingredient.

Another object of the present invention is to provide a kit for detecting influenza A H1N1 virus comprising the peptide as an active ingredient.

Another object of the present invention is to provide a method for detecting influenza A H1N1 virus comprising the following steps: (a) contacting a sample to be detected with the peptide according to claim 1; and (b) determining whether the peptide is bound to the influenza A H1N1 virus contained in the sample to be detected.

Another object of the present invention is to provide a composition for diagnosing influenza comprising the peptide as an active ingredient.

In order to achieve the above object of the present invention, the present invention provides a peptide that specifically binds to influenza A H1N1 virus, having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 12.

In order to achieve another object of the present invention, the present invention provides a polynucleotide encoding the peptide.

In order to achieve another object of the present invention, the present invention provides a recombinant expression vector containing a polynucleotide.

In order to achieve another object of the present invention, the present invention provides a host cell transformed with the recombinant expression vector.

In order to achieve another object of the present invention, the present invention provides a composition for detecting influenza A H1N1 virus comprising the peptide as an active ingredient.

In order to achieve another object of the present invention, the present invention provides a kit for detecting influenza A H1N1 virus comprising the peptide as an active ingredient.

In order to achieve another object of the present invention, the present invention provides a method for detecting influenza A H1N1 virus comprising the following steps: (a) contacting the peptide according to claim 1 with a sample to be detected; and (b) determining whether the peptide is bound to the influenza A H1N1 virus contained in the sample to be detected.

In order to achieve another object of the present invention, the present invention provides a composition for diagnosing influenza comprising the peptide as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention provides a peptide that specifically binds to influenza A H1N1 virus, having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 12.

The range of peptides specifically binding to influenza virus according to the present invention includes peptides having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 12 and functional equivalents of the peptides. "Functional equivalent" means at least 60% or more, preferably 70% or more, more preferably 80% or more, still more preferably, the amino acid sequence represented by SEQ ID NO: 3 as a result of addition, substitution or deletion of amino acids. refers to a peptide having a sequence homology of 90% or more and exhibiting substantially the same physiological activity as a peptide represented by an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 12.

In a preferred aspect of the present invention, the peptide may be a peptide having an amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, and most preferably a peptide having an amino acid sequence of SEQ ID NO: 3 or 4 .

The peptide according to the present invention is characterized in that it specifically binds to hemagglutinin or neuraminase of influenza A H1N1 virus, and exhibits high specificity, not binding to other subtypes of influenza A virus.

The peptides of the present invention can be prepared by chemical synthesis known in the art. Representative methods include, but are not necessarily limited to, liquid or solid phase synthesis, fragment condensation, F-MOC or T-BOC chemistries. In addition, the peptides of the present invention can be prepared by genetic engineering methods. First, a DNA sequence encoding the peptide is prepared according to a conventional method. DNA sequences can be prepared by PCR amplification using appropriate primers. Alternatively, DNA sequences may be synthesized by standard methods known in the art, such as using an automated DNA synthesizer (eg, sold by Biosearch or Applied Biosystems). The constructed DNA sequence is a vector containing one or more expression control sequences (e.g., promoter, enhancer, etc.) operatively linked to the DNA sequence to control the expression of the DNA sequence. and the host cell is transformed with the recombinant expression vector formed therefrom. The resulting transformants are cultured under appropriate media and conditions to allow expression of the DNA sequence, and a substantially pure peptide encoded by the DNA sequence is recovered from the culture. The recovery may be performed using a method known in the art (eg, chromatography). As used herein, 'substantially pure peptide' means that the peptide according to the present invention does not substantially contain any other proteins derived from the host. For the genetic engineering method for synthesizing the peptide of the present invention, reference may be made to the following documents: Maniatis et al., Molecular Cloning;

A laboratory Manual, Cold Spring Harbor laboratory, 1982.

The present invention also provides a polynucleotide encoding the peptide.

As used herein, the terms "DNA", "polynucleotide" and "nucleic acid" refer to DNA molecules that have been isolated from the total genomic DNA of a particular species. Thus, a segment of DNA encoding a polypeptide is one or more coding sequences that have been substantially isolated or purified from the total genomic DNA of the species from which the DNA segment can be obtained. refers to a piece of DNA that is made up of The terms 'DNA fragment' and 'polynucleotide' include DNA fragments and smaller fragments of such fragments, and also include recombinant vectors (eg, plasmids, cosmids, phagemids, bactericidal viruses, viruses, etc.) include

As will be appreciated by those skilled in the art, the polynucleotide sequence of the present invention expresses, or is adapted to express, a protein, polypeptide, peptide, etc., genomic sequence, extra-genomic sequence, encoding in a plasmid modified sequences and smaller engineered genetic fragments. These pieces can be isolated naturally or synthetically modified by human hands.

As recognized by those skilled in the art, polynucleotides can be single-stranded (coding sequences or antisense sequences) or double-stranded, and can be DNA molecules (genomic, cDNA or synthetic) or RNA molecules. Additional coding or non-coding sequences may be present in the polynucleotides of the present invention. Polynucleotides may also be linked to other molecules and/or supporting materials.

The polynucleotide of the present invention, regardless of the length of the coding sequence itself, can be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments (parts, segments), etc. As a result, their overall length can vary considerably. Therefore, it is contemplated that polynucleotide fragments of almost any length may be applied, and the overall length may preferably be limited by ease of manufacture and use in the intended recombinant DNA protocol.

Moreover, as a result of the degeneracy of the genetic code, it will be readily apparent to those skilled in the art that there are many nucleotide sequences encoding the polypeptides described herein. Some of these polynucleotides have minimal homology to the nucleotide sequence of any native gene. Nevertheless, other polynucleotides due to differences in codon usage (eg polynucleotides optimized for human and/or primate codon selection) are specifically contemplated by the present invention.

The present invention also provides a recombinant expression vector containing the polynucleotide and a host cell containing the expression vector.

To express a desired polypeptide, a nucleotide sequence encoding the polypeptide or functional equivalent can be inserted into an appropriate expression vector (i.e., a vector containing the necessary elements for transcription and translation of the inserted coding sequence). An expression vector containing a sequence encoding a polypeptide of interest, and appropriate transcription control elements (elements) and translation control elements (elements) can be constructed by a method well known to those skilled in the art. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. These methods are described in Sambrook et al., Molecular Cloning, A Laboratory Manual (1989) and Ausubel et al., Current Protocols in Molecular Biology (1989).

A variety of expression vector/host systems are known and can be used to contain and express polynucleotide sequences. The expression vector/host system includes, but is not limited to, bacteria transformed with, for example, recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; Yeast transformed with a yeast expression vector; insect cell lines infected with viral expression vectors (eg, baculovirus); plant cell lines transformed with viral expression vectors (eg cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or bacterial expression vectors (eg Ti or pBR322 plasmids); or microorganisms such as animal cell lines.

"Control elements" or "regulatory sequences" present in expression vectors are untranslated regions (enhancers, promoters, 5' and 3' untranslated regions) that interact with host cell proteins to effect transcription and translation. . These elements (elements) may have different strengths and specificities. Depending on the vector system and host used, any number of suitable transcription and translation elements (including constitutive and inducible promoters) may be used.

For mammalian host cells, a number of viral-based expression systems are generally available. For example, when an adenovirus is used as an expression vector, a sequence encoding a polypeptide of interest can be ligated into an adenovirus transcription/translation complex consisting of a late promoter and a tripartite leader sequence. there is. Insertion into the non-essential E1 or E3 regions of the viral genome can be used to obtain viable viruses capable of expressing the polypeptide in infected host cells. (Logan and Shenk, Proc. Natl. Acad. Sci. U.S.A.81: 3655-3659 (1984)). Transcriptional enhancers (eg Rous Sarcoma Virus (RSV) enhancers) can also be used to enhance expression in mammalian host cells.

A host cell may be selected for its ability to regulate expression of the inserted sequence or to process (process) the expressed protein in a desired manner. Such alterations of polypeptides include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation. Post-translational processing, which cleaves the 'prepro' form of a protein, can be used to facilitate correct insertion, folding and/or function. Other host cells (e.g. CHO, HeLa, MDCK, HEK293 and W138, which have specific cellular machinery and characteristic mechanisms for such post-translational activity) require precise modification of foreign proteins and It can be selected to ensure processing.

In the long term, for high-yield production of peptides, stable expression is generally desirable. For example, a cell line stably expressing a desired polynucleotide can be transformed using an expression vector, and the expression vector includes a viral origin of replication and/or an endogenous expression element (element), and the same vector or a separate vector. A selectable marker gene may be included in the phase.

After introduction of the vector, cells can be grown for 1-2 days in enriched media, and then converted to selective media. The purpose of the selectable marker is to confer resistance to selection, and its presence permits the growth and recovery of cells in which the introduced array is being successfully expressed. Resistant clones of stably transformed cells can be propagated using tissue culture techniques appropriate for the cell type.

The present invention also provides a composition for detecting influenza A H1N1 virus comprising a peptide as an active ingredient.

In order to easily identify, detect and quantify whether the peptide of the present invention forms a bond with the influenza A H1N1 virus, the peptide of the present invention may be provided in a labeled state. That is, it may be provided by linking (eg, covalent bonding or cross-linking) to a detectable label. The link may be formed directly between the peptide of the present invention and the detectable label, but may also be formed through a link. Any linker known in the art may be used, and a suitable peptide linker sequence may be selected by considering the following factors: (a) ability to be applied to flexible extended conformations; (b) the ability not to generate secondary structures that interact with the epitope; and (c) absence of hydrophobic residues or charged residues capable of reacting with the epitope.

In one aspect of the present invention, the peptide of the present invention may be labeled with one selected from the group consisting of a chromogenic enzyme, a radioactive isotope, a chromopore, a luminescent material, and a fluorescent material.

The label is specifically, for example, peroxidase corresponding to a coloring enzyme, alkaline phosphatase); 124I, 125I, 111In, 99mTc, 32P, 35S corresponding to radioactive isotopes; chromophores; It may be FITC, RITC, rhodamine, Texas Red, fluorescein, phycoerythrin, or quantum dots corresponding to a luminescent material or a fluorescent material, and is limited thereto It doesn't work.

Similarly, the detectable label may be an antibody epitope, substrate, cofactor, inhibitor or affinity ligand. This labeling may be performed during the process of synthesizing the peptide of the present invention, or may be additionally performed on already synthesized peptides.

A peptide containing the detectable label can be detected using a conventionally known detection method for each label. Specifically, for example, when using the fluorescence signal FITC as a label, the fluorescence intensity for each peptide concentration at the maximum wavelength (Excitation: 495 nm, Emission: 520 nm) of FITC linked to the peptide using a fluorescence photometer measurement methods are available.

The peptide according to the present invention has an improved manufacturing yield and a simple manufacturing process compared to detection methods using existing antibodies, aptamers, PNAs, carbohydrates, enzymes, etc. Existing influenza viruses can be detected very simply, efficiently, with high sensitivity and with high specificity.

The present invention also provides a kit for detecting influenza A H1N1 virus comprising the peptide as an active ingredient.

The influenza A H1N1 virus detection kit of the present invention may include the peptides of SEQ ID NOs: 1 to 6 or polynucleotides encoding them, and the peptides or polynucleotides may be obtained by chemical or genetic engineering methods as described above. can The peptide is the same as described above for description of other aspects of the present invention. The kit of the present invention may further include a buffer or reaction solution for stably maintaining the structure or physiological activity of the peptide. In addition, in order to maintain stability, it may be provided in a powdered state or dissolved in an appropriate buffer or maintained at 4°C.

The influenza A H1N1 virus detection kit of the present invention enables on-site diagnosis faster and more conveniently than conventional PCR methods, and can be provided in the form of an influenza fluorescence detection kit containing influenza A H1N1 virus. A type of system capable of increasing the sensitivity to influenza by linking the peptide of the present invention to other known probes, specifically detecting only food poisoning bacteria, and detecting influenza by imaging it through a fluorescence microscope can be provided as

In order to facilitate detection of influenza A H1N1, peptides included in the detection kit of the present invention may be provided in a labeled state as described above. On the other hand, when the peptide of the present invention is provided unlabeled, the influenza A H1N1 detection kit of the present invention detects whether or not the peptide of the present invention binds to the influenza A H1N1 virus included in the detection target in vitro or its location Ingredients may be further included. The component is a known compound for labeling the peptide of the present invention, or an antibody against a specific receptor binding to the peptide of the present invention or a peptide of the present invention for a search through an antigen-antibody reaction, or a secondary antibody against them, and It may be a reagent for its detection. Specifically, the diagnosis of the present invention may be performed by modifying a conventional immunoassay method or protocol. For example, the diagnosis of the present invention can be performed by using the peptide of the present invention instead of the antibody in the conventional immunoassay and using the same process. For example, an enzyme-linked immunosorbant assay (ELISA) is performed with a slight modification to an enzyme-linked oligonucleotide assay (ELONA).

In addition, the peptide of the present invention may be provided in the form of a coating on the surface of a plate. In this case, influenza A H1N1 virus can be detected by inoculating the target sample directly on the plate, reacting under appropriate conditions, and observing the binding between the influenza A H1N1 virus and the peptide contained in the target sample on the surface of the plate.

The influenza A H1N1 virus detection kit of the present invention may take the form of a bottle, tub, sachet, envelope, tube, ampoule, etc., which may be partially or wholly made of plastic or glass. , can be formed from paper, foil, wax, and the like. The container may be equipped with a fully or partially removable closure that is part of it or can be attached to the container by mechanical, adhesive or other means. The container may also be equipped with a stopper that allows access to the contents by means of an injection needle.

The present invention also provides a method for detecting influenza A H1N1 virus comprising the following steps:

(a) contacting the peptide according to claim 1 with a sample to be detected; and

(b) confirming whether the peptide is bound to the influenza A H1N1 virus contained in the sample to be detected.

The "detection target sample" of the present invention means a target sample in which influenza A H1N1 virus is likely to be present, and the presence or absence needs to be confirmed. Specifically, for example, it may be blood, urine, tears, sweat, saliva, lymph, and cerebrospinal fluid, but is not limited thereto.

Confirmation of binding in step (b) of the present invention may refer to the above description of the composition for detecting influenza A H1N1 virus, which is another aspect of the present invention, and is omitted to avoid excessive complexity of the description herein.

The present invention also provides a composition for diagnosing influenza comprising the peptide as an active ingredient.

In one aspect of the present invention, the influenza may be characterized as being caused by influenza A H1N1 virus infection.

In addition to the peptide of the present invention, the diagnostic composition of the present invention may include other subcomponents that can be used for detecting influenza A H1N1 virus, and the above description may be applied in the same manner.

Since the peptide provided by the present invention binds to the influenza A H1N1 virus with very high specificity and affinity, it can be used very usefully in the development of a composition and detection kit for detecting influenza A H1N1 virus, and can be used to detect influenza A H1N1 virus infection. Influenza A H1N1 virus can be rapidly and accurately detected in a sample provided from a patient.

1 is an experimental result confirming the highly specific binding of the peptide used in the present invention to A/H1N1/Beijing/22808/2009/HA using enzyme immunosorbent assay.
2 is an experimental result confirming the highly specific binding of the peptide used in the present invention to A/H1N1/Michigan/45/2015/NA using enzyme immunoassay.
Figure 3 shows the results of an enzyme-linked immunosorbent assay for binding to A/H1N1/Beijing/22808/2009/HA according to the concentration of the peptide used in the present invention.
Figure 4 is the result of testing the binding ability of the peptide used in the present invention to the concentration change of A / H1N1 / Beijing / 22808 / 2009 / HA by enzyme immunoassay.
5 is A / H1N1 / Beijing / 22808 / 2009 / HA, A / H1N1 / Michigan / 45 / 2015 / NA, A / H5N1 / Anhui / 1/2005 / This is the result of comparing the binding ability with HA and A / H5N1 / Hubei / 1/2011 / NA by enzyme immunoassay.

Hereinafter, the present invention will be described in detail by the following examples. However, the following examples are only for illustrating the present invention, and the present invention is not limited thereto.

Example 1: Discovery of novel peptides capable of highly specific and highly selective binding to A/H1N1/Beijing/22808/2009/HA or A/H1N1/Michigan/45/2015/NA proteins using M13 bacteriophage display

The target proteins used in the present invention were A/H1N1/Beijing/22808/2009/HA and A/H1N1/Michigan/45/2015/NA, which were purchased from Sino biological and used by Thermo scientific EZ-Link Sulfo-NHS- After undergoing the biotinylation process using the Biotinylation Kit, the concentration was calculated by BCA assay and stored in a -80°C freezer until use.

The bacteriophage random peptide library (Phage display peptide library) used in the present invention is Ph.D.-12 (New England BioLab) and Ph.D.-C7C, which are a kind of coat protein in the genome of M13 bacteriophage After artificially inserting gene sequences so that peptides of 12 or 7 random amino acid sequences are expressed at the end of the gene producing phosphorus pIII, E. coli is infected and recombinant expression of hundreds of millions of different peptides obtained It is composed of bacteriophages.

First, panning was performed a total of 4 times to discover receptors for A/H1N1/Beijing/22808/2009/HA, and a plate coated with streptavidin (Streptavidin High Binding Capacity Coated 96- Well Plates (Thermo scientific, Cat. No. 15501) were used.

After washing the streptavidin-coated plate with PBS (phosphate buffered saline) three times for 5 minutes, 0.5 μM of A/H1N1/Beijing/22808/2009/HA protein with biotin was added in a volume of 100 μL for 1 hour. while fixed at room temperature. Thereafter, the portion of streptavidin that is not bound to the target protein is blocked with 0.1 mM biotin, and 100 μL of 1 × 10 ¹¹ PFU/mL of M13 bacteriophage with a 12-mer peptide is added to the target protein at room temperature for 1 hour. reacted with After this process, the non-binding bacteriophages were repeatedly washed 10 times with 0.1% Tween-20 in 200 μL of PBS (phosphate buffered saline), and finally, those that specifically bind to A/H1N1/Beijing/22808/2009/HA Phages were eluted in 100 μL of 0.2M Glycin-HCl (pH 2.2) solution, and 15 μL of Tris-HCl (pH 9.0) was added for pH neutralization. Through a total of 4 repetitions of the above process, the amino acid sequences of bacteriophages binding to A/H1N1/Beijing/22808/2009/HA were confirmed through DNA sequencing, and among them, 5 bacteriophage candidates that bind repeatedly were selected as primary was selected (Table 1).

Next, a total of four rounds of panning were performed to discover receptors for A/H1N1/Michigan/45/2015/NA, and the overall process and concentration were the same as above. One difference is that, unlike the above, M13 bacteriophages with C7C-mer peptides were used, and 7 phage candidates that repeatedly bind to A/H1N1/Michigan/45/2015/NA were initially screened (Table 2).

Example 3: Including a novel peptide probe capable of highly specific and highly selective binding to A/H1N1/Beijing/22808/2009/HA and A/H1N1/Michigan/45/2015/NA using enzyme-linked immunosorbent assay (ELISA) Measurement of binding force of bacteriophage candidates

Amplification of each phage was carried out in order to use the 5 and 7 bacteriophage candidates, respectively, selected through bacteriophage display 4 times for the two target proteins for ELISA experiments. The phage stock was added to 20 mL LB medium together with previously cultured E.coli ER2738, and then cultured at 37°C and 190 rpm for 4-5 hours. The cultured bacteriophage candidates were centrifuged at 12,000 g for 10 minutes to recover the supernatant, and then 20% PEG/2.5 M NaCl was added to 1/6 of the supernatant capacity (3.3 mL) and left in a refrigerator at 4°C for 24 hours. did The precipitate obtained by centrifuging again at 12,000 g for 15 minutes was dissolved in 1 mL PBS (phosphate buffered saline), centrifuged again at 14,000 rpm for 5 minutes, and the supernatant was transferred to a new 1.5 mL-micro tube. One-sixth of the volume (about 166.7 μL) of 20% PEG/2.5 M NaCl was added and left on ice for 1 hour. Then, the precipitate obtained by centrifugation at 14,000 rpm for 10 minutes was dissolved in 200 μL PBS (phosphate buffered saline), and phage titration was performed to calculate the concentration of phage (PFU/mL: Plaque forming units/mL).

In order to compare the binding ability of the selected phage and the two target proteins, ELISA was performed as follows. First, biotinylated target protein (HA concentration: 250 nM, NA concentration: 500 nM) and biotinylated BSA (Bovine serum albumin) 100 μL (concentration: 250, 500 nM) were placed on a streptavidin-coated plate at room temperature for 1 hour. It was fixed and blocked with 5% BSA. After that, wash 6 times with 0.1% Tween-20 in PBS (Phosphate buffered saline), put 100 μL of phage candidates (concentration: 1×10 ¹² PFU/mL) on a protein-fixed plate, and stir at 150 rpm at room temperature. and reacted to bind for 1 hour. After the protein and phage reaction, wash 6 times with 0.1% Tween-20 in PBS (phosphate buffered saline), add anti M13-HRP antibody: 5% BSA in NaHCO ₃ in 200 μL of a 1:5000 diluted solution, and incubate at room temperature for 1 stirred for an hour. After this process, after washing 6 times with 0.1% Tween-20 in PBS (Phosphate buffered saline), the absorbance (OD) value was measured at 405 nm through a color development process using ABTS.

As a result, all of the peptides of SEQ ID NOs: 1 to 5 showed specific binding ability to A/H1N1/Beijing/22808/2009/HA, and among them, Beijing R4#1 phage showed relatively low binding ability to BSA Observed (FIG. 1). In addition, A/H1N1/Michigan/45/2015/NA showed binding ability to the target protein in all 7 types of phages, and among them, it was observed that Michigan R2#22 phage showed relatively low binding ability to BSA ( Fig. 2).

A/H1N1/Beijing/22808/2009/HA and A/H1N1/Michigan/45/2015/NA proteins (concentration: 250 nM) were immobilized on plates with Beijing R4#1 and Michigan R2#22 by the same ELISA method as above. were treated by phage concentration (1×10 ⁸ to 1×10 ¹² PFU/mL) and their binding strength was compared. As a result, it was observed that the binding force increased in proportion to the increase in phage concentration (FIG. 3).

As a result of measuring and comparing the binding force of Beijing R4#1 according to the change in A/H1N1/Beijing/22808/2009/HA protein concentration (62 ~ 1000 nM) using the same ELISA method as above, the binding force increased in proportion to the increase in the concentration of the target protein. It was confirmed that it rose (FIG. 4).

A/H1N1/Beijing/22808/2009/HA, A/H1N1/Michigan/45/2015/HA, A/H1N1/Michigan/45/2015/ As a result of comparing binding strength with NA, A/H5N1/Anhui/1/2005/HA and A/H5N1/Hubei/1/2011/NA, both Beijing R4#1 and Michigan R2#22 were other influenza viruses in addition to their respective target proteins. It showed a relatively low binding force with related proteins (FIG. 5).

Since the peptide provided by the present invention binds to the influenza A H1N1 virus with very high specificity and affinity, it can be used very usefully in the development of a composition and detection kit for detecting influenza A H1N1 virus, and can be used to detect influenza A H1N1 virus infection. Influenza A H1N1 virus can be rapidly and accurately detected in a sample provided from a patient, so industrial applicability is high.

<110> CHUNG ANG University industry Academic Cooperation Foundation <120> Peptide specifically binds to H1N1 subtype of influenza A virus and uses it <130> NP20-0067 <160> 12 <170> KoPatentIn 3.0 <210> 1 <211> 12 <212> PRT <213> artificial sequence <220> <223> Beijing R3#7 <400> 1 Met Glu Thr Arg Pro Val Ala Pro His Glu Phe Arg 1 5 10 <210> 2 <211> 12 <212> PRT <213> artificial sequence <220> <223> Beijing R3#10 <400> 2 Tyr Pro Ala Ala Tyr His Thr Leu Thr Arg Glu Pro 1 5 10 <210> 3 <211> 12 <212> PRT <213> artificial sequence <220> <223> Beijing R3#15 <400> 3 Phe Glu Arg Gly Leu Arg Thr Asp Pro Tyr Ser Met 1 5 10 <210> 4 <211> 12 <212> PRT <213> artificial sequence <220> <223> Beijing R3#43 <400> 4 Ala Phe Ala Gln Ala Val Asn Tyr Gln Arg His Pro 1 5 10 <210> 5 <211> 12 <212> PRT <213> artificial sequence <220> <223> Beijing R4#1 <400> 5 Gln Met Gly Phe Met Thr Ser Pro Lys His Ser Val 1 5 10 <210> 6 <211> 9 <212> PRT <213> artificial sequence <220> <223> Michigan R2#1 <400> 6 Cys Thr Asn Gly Ser His Asn Leu Cys 1 5 <210> 7 <211> 9 <212> PRT <213> artificial sequence <220> <223> Michigan R2#6 <400> 7 Cys Ser Arg Ser Met Asp Ser Thr Cys 1 5 <210> 8 <211> 9 <212> PRT <213> artificial sequence <220> <223> Michigan R2#8 <400> 8 Cys Ser Ser Asn Thr Val Pro Ala Cys 1 5 <210> 9 <211> 9 <212> PRT <213> artificial sequence <220> <223> Michigan R2#17 <400> 9 Cys Asp Gly Arg Pro Asp Arg Ala Cys 1 5 <210> 10 <211> 9 <212> PRT <213> artificial sequence <220> <223> Michigan R2#19 <400> 10 Cys Thr Asp Lys Ala Ser Ser Ser Cys 1 5 <210> 11 <211> 9 <212> PRT <213> artificial sequence <220> <223> Michigan R2#22 <400> 11 Cys Gly Glu Gly Glu Ala Asp Val Cys 1 5 <210> 12 <211> 9 <212> PRT <213> artificial sequence <220> <223> Michigan R3#18 <400> 12 Cys Asp Gly Ala Asn Ala Arg Met Cys 1 5

Claims (11)

A peptide that specifically binds to the influenza A H1N1 virus, consisting of the amino acid sequence of SEQ ID NO: 5.
The peptide according to claim 1, wherein the peptide specifically binds to hemagglutinin or neuraminidase of influenza A H1N1 virus.
A polynucleotide encoding the peptide of claim 1.
A recombinant expression vector comprising the polynucleotide of claim 3.
A host cell transformed with the recombinant expression vector of claim 4.
A composition for detecting influenza A H1N1 virus comprising the peptide of claim 1 as an active ingredient.
The composition according to claim 6, wherein the peptide is labeled with one selected from the group consisting of a color enzyme, a radioactive isotope, a chromopore, a luminescent material, and a fluorescent material.
A kit for detecting influenza A H1N1 virus comprising the peptide of claim 1 as an active ingredient.
A method for detecting influenza A H1N1 virus comprising the following steps:
(a) contacting the peptide according to claim 1 with a sample to be detected; and
(b) confirming whether the peptide is bound to the influenza A H1N1 virus contained in the sample to be detected.
A composition for diagnosing influenza comprising the peptide of claim 1 as an active ingredient.
11. The composition according to claim 10, wherein the influenza is caused by influenza A H1N1 virus infection.

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