KR20220039422A - Peptide specifically binding to H5N1 subtype of influenza A virus and uses thereof - Google Patents

Abstract

The present invention relates to a peptide specifically binding to H5N1 subtype of influenza A virus and uses thereof. More particularly, the present invention relates to a peptide specifically binding to neuraminidase of H5N1 subtype of influenza A virus and a method for detecting H5N1 subtype of influenza A virus using the same. The peptide provided in the present invention binds to H5N1 subtype of influenza A virus with very high specificity and affinity and thus can be very useful in developing compositions and kits for detecting H5N1 subtype of influenza A virus. In addition, with the present invention, H5N1 subtype of influenza A virus can be detected with speed and accuracy from a sample provided from a patient suspected of infection with H5N1 subtype of influenza A virus.

Description

Peptide specifically binding to H5N1 virus and uses thereof {Peptide specifically binding to H5N1 subtype of influenza A virus and uses thereof}

The present invention relates to a peptide that specifically binds to influenza A H5N1 virus and uses thereof, and more particularly, to a peptide that specifically binds to neuraminidase of influenza A H5N1 virus and influenza A H5N1 using the same It relates to a virus detection method.

Influenza virus belongs to the genus Orthomyxoviridae influenza virus as an RNA virus, and A, B, C, classified as type D. Among them, type A is classified into subtypes from H1 to H18 according to the antigenic characteristics of the HA protein, and from N1 to N11 according to the antigenic characteristics of the NA protein. These virus particles have an envelope and are usually polymorphic spherical shapes with a diameter of about 80 to 120 nm. Viruses immediately after separation from fertilized eggs and susceptible cells may have a filamentous filament with a length of 400 nm. Certain glycoproteins exist in the virus envelope. Representative envelope glycoproteins present in type A virus 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, the most representative influenza virus in terms of infectivity and pathogenicity is type A virus. The virus can infect a variety of species, including birds and humans. Its natural host is known as wild migratory birds, and it is speculated that transmission from birds to mammals occurs through cross-species infection. The epidemic period of type A virus due to antigenic mutation is estimated to be about 10 to 40 years, and has been the cause of at least three influenza virus epidemics in the past. In 1918, about 50 million people died worldwide due to the outbreak of Spanish flu caused by A/H1N1 type. In 1957, 2 million people died from Asian influenza caused by A/H2N2 type worldwide. The Hong Kong flu, caused by his brother, was the mildest of the pandemic, but killed about 1 million people worldwide. The novel influenza virus (A/H5N1) type to be detected in the present invention was known to cause highly pathogenic avian influenza and was originally known to infect only birds. The virus is currently transmitted only through direct contact with bird droppings and secretions, but the WHO warns that human-to-human transmission through mutations in H5N1 is more dangerous than SARS.

Respiratory diseases caused by influenza virus infection are not specific, so differential diagnosis from respiratory syncytial virus (RSV), parainfluenza virus, adenovirus, rhinovirus, pneumococcal mycoplasma, and bacteria I 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. This is a sample (influenza virus) collected from animal cells and fertilized eggs, treated with antibiotics and then cultured, followed by cytopathic effect (CPE) observation, antigen detection by hemagglutination test, and reverse transcription polymerase chain reaction method. (RT-PCR) and real-time RT-RCR are methods for identifying specific genes of influenza virus. The second is a gene detection test, which is a method to analyze the type and subtype of influenza virus by amplifying a gene (HA or NA region) specific to influenza virus. For type and subtype analysis, HA (hemagglutination assay) and HI (hemagglutination inhibition assay) are being used. In addition, types A and B and subtypes are determined using RT-PCR (reverse transcription-polymerase chain reaction), real-time RT-RCR, and EIA (enzyme linked immunoassay) methods, and HA or 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 significantly increases more than 4 times between the acute (within 1 week onset) and convalescent (2-4 weeks onset) sera. However, these diagnostic methods are complex, time-consuming, and require expert help. Lastly, rapid influenza diagnostic tests (RIDTs) are a method to detect influenza virus antigens. Recently, new diagnostic reagents have been developed. By collecting a sample from a patient in the clinic and diagnosing it within 30 minutes, it is possible to quickly check whether antiviral drugs are administered or not. can decide However, the disadvantage is that it is less sensitive than PCR, cannot characterize the virus antigen, and is expensive. In addition, even if RIDTs are negative, influenza infection cannot be completely excluded, so cell culture or genetic testing is required if necessary.

Accordingly, the present inventors conducted research to develop a diagnostic method capable of rapidly and accurately detecting influenza virus A H5N1, and as a result, high sensitivity to influenza virus A/H5N1 envelope glycoprotein neuraminidase (NA) and novel peptides with accuracy were discovered, and the present invention was completed by developing a system capable of early diagnosis of influenza virus A H5N1 using this.

Accordingly, an object of the present invention is to provide a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 6, which specifically binds to influenza A H5N1 virus.

Another object of the present invention is to provide a polynucleotide encoding the 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 H5N1 virus comprising the peptide as an active ingredient.

Another object of the present invention is to provide a kit for detecting influenza A H5N1 virus comprising the peptide as an active ingredient.

Another object of the present invention is to provide a method for detecting influenza A H5N1 virus comprising the steps of: (a) contacting the peptide according to claim 1 with a sample to be detected; and (b) determining whether the peptide and the influenza A H5N1 virus contained in the detection target sample are bound.

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 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 6, which specifically binds to influenza A H5N1 virus.

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 comprising 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 H5N1 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 H5N1 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 H5N1 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 and the influenza A H5N1 virus contained in the detection target sample are bound.

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 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 6, which specifically binds to influenza A H5N1 virus.

The scope of the peptide that specifically binds to influenza virus according to the present invention includes a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 6 and functional equivalents of the peptide. "Functional equivalent" means at least 60% or more, preferably 70% or more, more preferably 80% or more, even more preferably the amino acid sequence represented by SEQ ID NO: 3 as a result of the addition, substitution or deletion of amino acids. is 90% or more of sequence homology, refers to a peptide that exhibits substantially the same physiological activity as the peptide represented by the amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 6.

In a preferred embodiment of the present invention, the peptide may be a peptide having the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, and most preferably, a peptide having the 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 neuraminidase of influenza A H5N1 virus, and exhibits high specificity not binding to influenza A viruses of other subtypes.

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 chemistry. In addition, the peptide of the present invention can be prepared by a genetic engineering method. First, a DNA sequence encoding the peptide is prepared according to a conventional method. A DNA sequence can be prepared by PCR amplification using appropriate primers. Alternatively, the DNA sequence may be synthesized by standard methods known in the art, for example, using an automatic DNA synthesizer (eg, those sold by Biosearch or Applied Biosystems). The constructed DNA sequence is a vector comprising one or more expression control sequences (eg, promoter, enhancer, etc.) that are operatively linked to the DNA sequence and control the expression of the DNA sequence , and transform the host cell with the recombinant expression vector formed therefrom. The resulting transformant is cultured under an appropriate medium and conditions to express the DNA sequence, thereby recovering a substantially pure peptide encoded by the DNA sequence from the culture. The recovery may be performed using a method known in the art (eg, chromatography). As used herein, the term 'substantially pure peptide' means that the peptide according to the present invention does not substantially contain any other proteins derived from the host. The genetic engineering method for synthesizing the peptide of the present invention may be referred to the following literature: Maniatis et al., Molecular Cloning;

A laboratory Manual, Cold Spring Harbor laboratory, 1982.

The production yield of the peptide according to the present invention is improved compared to the detection method using an existing antibody, aptamer, PNA, carbohydrate, enzyme, etc., and the production process is simple. Existing influenza viruses can be detected very simply, efficiently, with high sensitivity and with high specificity.

The present invention also provides a polynucleotide encoding the peptide.

As used herein, the terms “DNA”, “polynucleotide” and “nucleic acid” refer to a DNA molecule isolated from the total genomic DNA of a particular species. Thus, a DNA fragment (segment) encoding a polypeptide is one or more coding sequences that have been substantially isolated or purified from the total genomic DNA of a species from which the DNA fragment can be obtained. refers to a piece of DNA 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 understood by those skilled in the art, the polynucleotide sequence of the present invention expresses, or is modified to express, a protein, a polypeptide, a peptide, etc., and is encoded in a genomic sequence, an extra-genomic sequence, a plasmid sequences and smaller engineered gene fragments. Such fragments may be isolated naturally, or they may be synthetically modified by human hands.

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

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

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

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

To express a desired polypeptide, a nucleotide sequence encoding a polypeptide or functional equivalent can be inserted into an appropriate expression vector (i.e., a vector containing elements (elements) necessary for transcription and translation of the inserted coding sequence). An expression vector comprising a sequence encoding a polypeptide of interest, and appropriate transcriptional control elements (elements) and translational control elements (elements) can be constructed by methods 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 vector; 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.

The “control elements (elements)” or “regulatory sequences” present in an expression vector 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 strength and specificity. Depending on the vector system and host used, any number of appropriate transcription elements and translation elements (including constitutive promoters and inducible promoters) may be used.

For mammalian host cells, a number of virus-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 linked within an adenovirus transcription/translation complex consisting of a late promoter and a tripartite leader. there is. Insertion into a nonessential E1 or E3 region of the viral genome can be used to obtain a viable virus capable of expressing the polypeptide in an infected host cell. (Logan and Shenk, Proc. Natl. Acad. Sci. U.S.A.81: 3655–3659 (1984)). Also, transcription enhancers (eg, Rous Sarcoma Virus (RSV) enhancers) may be used to enhance expression in mammalian host cells.

A host cell may be selected for its ability to modulate expression of the inserted sequence or to process (process) the expressed protein in a desired manner. Such alterations of the polypeptide 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 may be selected to ensure processing.

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

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

The present invention also provides a composition for detecting influenza A H5N1 virus comprising a peptide as an active ingredient.

In order to easily confirm, detect and quantify whether the peptide of the present invention forms a bond with influenza A H5N1 virus, the peptide of the present invention may be provided in a labeled state. That is, it may be provided by being linked (eg, covalently bonded or cross-linked) 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 the link. As the linker, any known in the art may be used, and the sequence of a suitable peptide linker may be selected in consideration of the following factors: (a) ability to be applied to a flexible extended conformation; (b) the ability not to create secondary structures that interact with the epitope; and (c) the absence of a hydrophobic moiety or a charged moiety capable of reacting with the epitope.

In one embodiment 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 (peroxidase), alkaline phosphatase (alkaline phosphatase) corresponding to the chromogenic enzyme); 124I, 125I, 111In, 99mTc, 32P,35S corresponding to radioactive isotopes; chromophore; It may be FITC, RITC, rhodamine, Texas Red, fluorescein, phycoerythrin, quantum dots corresponding to a light emitting material or a fluorescent material, and limited thereto. doesn't happen

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

The peptide containing the detectable label can be detected using a conventionally known detection method for each label. Specifically, for example, when the fluorescent signal FITC is used as a label, the fluorescence intensity for each peptide concentration at the maximum wavelength (Excitation: 495 nm, Emission: 520 nm) of the FITC linked to the peptide is measured using a fluorometer. measurement method can be used.

The present invention also provides a kit for detecting influenza A H5N1 virus comprising the peptide as an active ingredient.

The influenza A H5N1 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 are those obtained by chemical or genetic engineering methods as described above. can The peptide is the same as described above for the 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 powder state, dissolved in an appropriate buffer, or maintained at 4°C.

The influenza A H5N1 virus detection kit of the present invention can be quickly and conveniently diagnosed in the field compared to the existing PCR method, and may be provided in the form of an influenza fluorescence detection kit containing the influenza A H5N1 virus. In the form of a system that can increase the sensitivity to influenza by linking the peptide of the present invention to other conventionally known probes, and can specifically detect only influenza, and can detect influenza by imaging it through a fluorescence microscope. can be provided.

In order to facilitate the detection of influenza A H5N1, the peptide 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 H5N1 detection kit of the present invention detects whether the peptide of the present invention binds to the influenza A H5N1 virus in vitro or contained in the detection target or its location. It may further include a component for The component is a known compound for labeling the peptide of the present invention, or an antibody to a specific receptor that binds to the peptide of the present invention or the peptide of the present invention for screening through antigen-antibody reaction, or a secondary antibody therefor, 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 the process is the same. For example, ELISA (enzyme-linked immunosorbant assay) is performed with a slight modification to ELONA (enzyme-linked oligonucleotide assay).

In addition, the peptide of the present invention may be provided in the form of coating on the surface of the plate. In this case, the influenza A H5N1 virus can be detected by inoculating the target sample directly on the plate and reacting it under suitable conditions, and then observing the binding of the influenza A H5N1 virus and the peptide contained in the target sample on the surface of the plate.

The influenza A H5N1 virus detection kit of the present invention may take the form of a bottle, a tub, a small sachet, an envelope, a tube, an ampoule, etc., which are partially or entirely plastic, glass , paper, foil, wax, and the like. The container may be equipped with a fully or partially removable closure which is part of it or may 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 a needle.

The present invention also provides a method for detecting influenza A H5N1 virus comprising the steps of:

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

(b) determining whether the peptide and the influenza A H5N1 virus contained in the detection target sample are bound.

The "detection target sample" of the present invention means a target sample in which influenza A H5N1 virus is likely to be present, and it is necessary to confirm the presence or absence of the influenza A H5N1 virus. Specifically, it may be, for example, blood, urine, tears, sweat, saliva, lymph, and cerebrospinal fluid, but is not limited thereto.

For the determination of binding in step (b) of the present invention, reference may be made to the above with respect to the composition for detecting influenza A H5N1 virus, which is another aspect of the present invention, and will be omitted to avoid excessive complexity of the description of the present specification.

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

In one embodiment of the present invention, the flu may be characterized by infection with influenza A H5N1 virus.

In addition to the peptide of the present invention, the diagnostic composition of the present invention may contain other subcomponents that can be used to detect influenza A H5N1 virus, and the foregoing may be applied in the same manner.

Since the peptide provided by the present invention binds with influenza A H5N1 virus with very high specificity and affinity, it can be very usefully utilized in the development of a composition and detection kit for influenza A H5N1 virus detection, and influenza A H5N1 virus infection is suspected. Influenza A H5N1 virus can be detected quickly and accurately in a sample provided from a patient.

1 is an experimental result confirming the high specific binding of the peptide used in the present invention to A/H5N1/Hubei/1/2011/NA using an enzyme immunoassay method.
Figure 2 shows the results of experiments on the binding force to A/H5N1/Hubei/1/2011/NA according to the concentration of the peptide used in the present invention by an enzyme immunoassay method.
Figure 3 shows the results of experiments on the binding force of the peptides used in the present invention to changes in the concentration of A/H5N1/Hubei/1/2011/NA by enzyme immunoassay.
4 is A/H1N1/Beijing/22808/2009/HA, A/H5N1/Anhui/1/2005/HA and target protein A/H5N1/Hubei/1 to confirm the specificity of the peptide used in the present invention. This is the result of comparing the binding force to /2011/NA by enzyme immunoassay.
5 is an experimental result confirming the high specific binding of the synthesized peptide used in the present invention to A/H5N1/Hubei/1/2011/NA using an electrochemical measurement method.

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

Example 1: Discovering new peptides capable of highly specific and highly selective binding to A/H5N1/Hubei/1/2011/NA protein using M13 bacteriophage display

The target protein used in the present invention was A/H5N1/Hubei/1/2011/NA, purchased from Sino biological, and BCA assay after biotinylation using Thermo scientific's EZ-Link Sulfo-NHS-Biotinylation Kit. The concentration was calculated by , and stored in a -80°C freezer until use.

Bacteriophage random peptide library (Phage display peptide library) used in the present invention is Ph.D.-12 (New England BioLab), which is a gene terminus that produces pIII, a kind of coat protein, in the genome of M13 bacteriophage. It consists of recombinant bacteriophage expressing more than hundreds of millions of different peptides obtained by artificially inserting a gene sequence so that peptides of 12 random amino acid sequences are expressed in E. coli and then infected with E. coli.

A total of 4 pannings were performed, and streptavidin High Binding Capacity Coated 96-Well Plates (Thermo scientific, Cat. No. 15501) were used for panning.

First, the plate coated with streptavidin was washed 3 times with PBS (phosphate buffered saline) for 5 minutes, and then 0.5 μM of A/H5N1/Hubei/1/2011/NA protein with biotin was added in 100 μL volume. It was fixed at room temperature for 1 hour. After that, block the remaining streptavidin portion without binding of the target protein with 0.1 mM biotin, and add 100 μL of 1×10 ¹¹ PFU/mL of M13 bacteriophage with a 12-mer peptide to the target protein at room temperature for 1 hour. and reacted After this process, bacteriophages without binding force were washed repeatedly 10 times with 0.1% Tween-20 in 200 μL of PBS (phosphate buffered saline), and finally, A/H5N1/Hubei/1/2011/NA specifically binding to Phage was 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 neutralization of pH. Bacteriophages binding to A/H5N1/Hubei/1/2011/HA through a total of 4 repeated experiments with the above process confirmed the amino acid sequence through DNA sequencing, among which three bacteriophage candidates that bind repeatedly were primary was selected (Table 1)

Example 2: Measurement of binding affinity of a bacteriophage candidate group containing a novel peptide probe capable of high specific, highly selective binding to A/H5N1/Hubei/1/2011/NA using enzyme immunoassay (ELISA)

The amplification of each phage was performed in order to use the three bacteriophage candidate groups selected through bacteriophage display 4 times for ELISA experiments. The phage stock was put into 20 mL LB medium together with the previously cultured E. coli ER2738, and then incubated at 37° C. and 190 rpm for 4-5 hours. The cultured bacteriophage candidate group was centrifuged at 12,000 g speed for 10 minutes to recover the supernatant, then 20% PEG/2.5 M NaCl was added to one-sixth (3.3 mL) of the supernatant, and left in a refrigerator at 4 ° C for 24 hours. did. The precipitate obtained by centrifugation again at 12,000 g for 15 minutes is dissolved in 1 mL PBS (phosphate buffered saline), centrifuged again at 14,000 rpm for 5 minutes, and the supernatant is transferred to a new 1.5 mL-micro tube and the supernatant Add one-sixth of the volume (approximately 166.7 μL) 20% PEG/2.5 M NaCl and place on ice for 1 hour. After that, the precipitate obtained by centrifugation again at 14,000 rpm for 10 minutes was dissolved in 200 μL PBS (phosphate buffered saline) and phage titration was performed to calculate the phage concentration (PFU/mL: Plaque forming units/mL).

ELISA was performed as follows to compare the binding strength of the selected phage and A/H5N1/Hubei/1/2011/NA. First, biotinylated A/H5N1/Hubei/1/2011/NA and 100 μL of biotinylated BSA (Bovine serum albumin) (concentration: 0.25 μM) were immobilized on a streptavidin-coated plate at room temperature for 1 hour, followed by 5% BSA. Blocking was done After that, wash 6 times with 0.1% Tween-20 in PBS (Phosphate buffered saline), add 100 μL of phage candidates (concentration: 1×10 ¹¹ PFU/mL) to a plate on which proteins are fixed, 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 200 μL of anti-M13-HRP antibody: 5% BSA in NaHCO ₃ diluted 1:5000 to 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 the color development process using ABTS.

As a result, it was observed that Hubei R3#13 and Hubei R3#43 phages specifically bind to A/H5N1/Hubei/1/2011/NA and exhibit relatively low binding affinity to BSA ( FIG. 1 ).

By the same ELISA method as above, Hubei R3#13 and Hubei R3#43 were mixed with the same A/H5N1/Hubei/1/2011/NA protein (concentration: 0.5 μM) on a plate by phage concentration (1×10 ¹¹ to 1× 10 ¹² PFU/mL) to compare the binding force. As a result, it was observed that both types of phage increased in proportion to the concentration of the phage, and Hubei R3#43 phage showed a higher binding force to the target than Hubei R3#13 phage ( FIG. 2 ).

As a result of measuring and comparing the binding force of Hubei R3#43 according to the change in A/H5N1/Hubei/1/2011/NA concentration (62 ~ 1000 nM) by the same ELISA method as above, the binding force of Hubei R3#43 was determined by the concentration of the target protein. It was confirmed that the binding force increased in proportion to the increase (FIG. 3).

In order to confirm the specificity of Hubei R3#43 for the target protein by the same ELISA method as above, A/H1N1/Beijing/22808/2009/HA, A/H5N1/Anhui/1/2005/HA and the target protein A/ The binding force to H5N1/Hubei/1/2011/NA was compared. As a result, Hubei R3#43 had a relatively high binding affinity to the target protein compared to other proteins (Fig. 4).

These results show that the selected phage Hubei R3#43 specifically binds well to A/H5N1/Hubei/1/2011/NA, and the 12 peptide sequences 'MPLRYPTQVTLD' possessed by Hubei R3#43 are peptide-derived influenza It shows that it can be used as an early diagnosis kit for viral infection.

Example 3: Synthesis of amino acid sequence-based peptides discovered through M13 bacteriophage display

Based on the amino acid sequence Hubei R3#43 (MPLRYPTQVTLD) discovered through bacteriophage display three times, a total of six peptides were synthesized by requesting from Peptron (Table 2). After synthesis, the purified and freeze-dried peptides were diluted to a high concentration in PBS (phosphate buffered saline) and then subdivided and stored in a -20°C freezer until use.

Example 5: Measurement of binding force using electrochemical analysis (SWV) on target protein of peptide-based molecular binders

CHI 660E (Electrochemical Workstation, Ch Instruments Inc, USA) was used to measure the binding ability of the molecular binder to the target protein. The analysis was carried out at room temperature, and a carbon chip coated with disposable streptavidin (Streptavidin modified screen-printed carbon electrode, Metrohm, Switzerland) was used.

In SWV (Square Wave Voltammetry), when a high-speed voltage is scanned to the working electrode in the presence of chemical crystals capable of oxidation/reduction reactions in the electrolyte, the resulting current value closes the electrode surface It is an analysis method to obtain thermodynamic and kinetic parameters of the electrochemical reaction of a substance.

+

in

)에 넣어 SPCE칩의 상대전극(Counter Electrode), 기준전극(Reference Electrode), 작업전극(Working Electrode)를 이용하여 SWV를 측정하였다. 그 다음 15 μL의 목적단백질인 A/H5N1/Hubei/1/2011/NA(농도: 0.25 μM = 15.75 μg/mL)와 비특이적으로 결합하는 BSA(농도: 0.25 μM = 16.25 μg/mL)를 INA 펩타이드가 고정화된 탄소칩과 1시간 동안 상온에서 반응시킨 후 전해용액을 넣고 펩타이드와의 결합능을 SWV로 측정하였다. First, after washing with tertiary distilled water and flowing nitrogen gas, the peptide candidates with 15 μL biotin (INA BP1 ~ BP5, concentration: 100 μg/mL) were allowed to react with the carbon chip at room temperature for 1 hour. After the reaction, rinse with tertiary distilled water 6 times, then flow nitrogen gas again, and

+

in

) to measure SWV using a counter electrode, a reference electrode, and a working electrode of the SPCE chip. Then, 15 µL of the target protein A/H5N1/Hubei/1/2011/NA (concentration: 0.25 µM = 15.75 µg/mL) and non-specific BSA (concentration: 0.25 µM = 16.25 µg/mL) were mixed with INA peptide. After reacting with the immobilized carbon chip at room temperature for 1 hour, an electrolyte solution was added and the binding ability with the peptide was measured by SWV.

The results for this are shown in FIG. 5 .

Figure 5 is each INA BP1 ~ BP5 peptide binding capacity for A / H5N1/Hubei/1/2011/NA was measured by electrochemical signal change, and the current value obtained from SWV was calculated (ΔI (%) = (I _a - I _b )/I _a × 100).

Since the peptide provided by the present invention binds with influenza A H5N1 virus with very high specificity and affinity, it can be very usefully utilized in the development of a composition and detection kit for influenza A H5N1 virus detection, and influenza A H5N1 virus infection is suspected. Influenza A H5N1 virus can be rapidly and accurately detected in a sample provided from a patient, which has high industrial applicability.

<110> CHUNG ANG University industry Academic Cooperation Foundation <120> Peptide specifically binding to H5N1 subtype of influenza A virus and uses it <130> NP20-0065 <160> 6 <170> KoPatentIn 3.0 <210> 1 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Hubei R3#7 <400> 1 Asp Gly Ser Met Leu Asn Arg Met Arg Gly Phe Ser 1 5 10 <210> 2 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Hubei R3#13 <400> 2 Lys Val Val Gly Ser Gly Tyr Val Gly His Arg Thr 1 5 10 <210> 3 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Hubei R3#43 (INA BP1) <400> 3 Met Pro Leu Arg Tyr Pro Thr Gln Val Thr Leu Asp 1 5 10 <210> 4 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> INA BP3 <400> 4 Glu Pro Glu Arg Tyr Pro Thr Gln Val Thr Glu Asp 1 5 10 <210> 5 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> INA BP4 <400> 5 Cys Gly Met Pro Leu Arg Tyr Pro Thr Gln Val Thr Leu Asp Gly Cys 1 5 10 15 <210> 6 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> INA BP5 <400> 6 Cys Gly Met Pro Leu Arg Tyr Pro Thr Gln Val Thr Leu Asp Gly Cys 1 5 10 15 Gly Gly Gly Gly Ser 20

Claims (11)

A peptide that specifically binds to influenza A H5N1 virus, having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 6.
The peptide according to claim 1, wherein the peptide specifically binds to neuraminidase of influenza A H5N1 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 H5N1 virus comprising the peptide of claim 1 as an active ingredient.
The composition of claim 6, wherein the peptide is labeled with one selected from the group consisting of a chromogenic enzyme, a radioactive isotope, a chromopore, a luminescent material, and a fluorescent material.
A kit for detecting influenza A H5N1 virus comprising the peptide of claim 1 as an active ingredient.
A method for detecting influenza A H5N1 virus comprising the steps of:
(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 H5N1 virus contained in the sample to be detected.
A composition for diagnosing influenza comprising the peptide of claim 1 as an active ingredient.
The composition according to claim 10, wherein the flu is caused by influenza A H5N1 virus infection.

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