KR20210038200A - High sensitive peptides for influenza virus, and the influenza virus detection chip using the peptides - Google Patents

Abstract

The present invention relates to: a peptide receptor for the detection of a novel influenza virus known as an acute respiratory disease-causing virus; a method of manufacturing a biosensor probe using the peptide receptor; and a method for detecting human-derived novel influenza virus antigen using the same. According to the present invention, a peptide having high-specific and high-selective binding ability to hemagglutinin, a novel influenza virus antigen, is any one of amino acid sequence listings 1 (GHPHYNNPSLQL), 2 (GHPHYNNPSLQLGGGGSC), and 7 (GHEHYNNESLQLGGGGSC).

Description

[High sensitive peptides for influenza virus, and the influenza virus detection chip using the peptides]

The present invention develops a molecular recognition element as a peptide receptor for highly sensitive detection of influenza A virus H5N1 subtype, and prevents and treats using its molecular conjugate, and further detects influenza virus present in trace amounts in a sample. It's about how to do it. The present invention performs performance verification of the peptide receptor discovered by peptidomimetics through spectroscopic detection, and then detects influenza virus present in a very small amount in a sample with high sensitivity and high specificity ( high selectivity).

The peptide receptor of the present invention can increase the binding affinity or binding constant to the target molecule by changing the amino acid composition in the molecular conjugate and replacing it using an In Silico-based simulation method. It relates to a method of discovering the best possible molecular conjugates, and using spectroscopic or electrochemical detection techniques from them to verify the functionality of the molecular conjugates to enable highly sensitive and highly specific detection of the actual influenza virus.

The method using the peptide-based molecular linker according to the present invention improves the manufacturing yield and simplifies the manufacturing process compared to the detection method using conventional antibodies, aptamers, PNAs, carbohydrates, enzymes, etc., so that spectroscopic or electrochemical detection When the technology is used, influenza viruses, which are known to cause acute respiratory diseases present in trace amounts in the sample, can be detected very simply, efficiently, and highly sensitively and highly specific. When the peptide receptor of the present invention is used, it is expected that it will be able to replace the existing influenza virus detection antibody.

Influenza virus is an RNA virus and belongs to the genus Orthomyxoviridae and influenza virus, according to the antigenic difference between the nucleocapsid protein (nucleocapsid, NP) and the matrix protein (matrix, M) of the virus. It is classified as type D.

Among them, A type is classified into H1 type to H18 type according to the antigenic characteristics of the HA protein, and N1 type to N11 type subtypes according to the antigenic characteristics of the NA protein. These virus particles have an envelope and are usually polymorphic spheres with a diameter of about 80 to 120 nm, but the virus immediately after separation from fertilized eggs and susceptible cells may have a filament shape of 400 nm in length. Specific glycoproteins exist in the outer coat of the virus, and typical outer coat glycoproteins present in type A and type B viruses are hemagglutinin (HA) and neuraminidase (NA). And plays an important role in antibody reaction. Genes are made up of segmented single-stranded RNA, wrapped in a helix capsid, and have 7-8 different RNA segments instead of one. Each RNA segment is composed of glycoproteins HA and NA, polymerase proteins PA, PB1, PB2, NP and M protecting genetic material, and finally non-structural proteins NS1 and NS2. Unlike other RNA viruses, the replication of these genetic materials occurs within the nucleus of the host cell. When the virus enters the host cell, HA, an envelope glycoprotein, binds to the cell receptor present in the cell membrane and allows the virus to enter the cell. And NA plays an important role in the virus being released from infected cells and spreading to new respiratory cells.

Since influenza virus type A has frequent antigenic variation and many subtypes exist, the labeling method proposed by the World Health Organization (WHO) is used for systematic management. The standard nomenclature is in the order of subtype/originate host/separated area/separation order/year of separation (type HA, type NA). If the virus is isolated from humans when indicating the origin host, do not indicate it.

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 as wild migratory birds, and it is presumed that the transition from birds to mammals occurs through interspecies infection. The epidemic cycle of type A virus due to antigenic mutation 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 emergence of the Spanish flu caused by the A/H1N1 type in 1918 killed about 50 million people worldwide, and in 1957 the Asian flu caused by the A/H2N2 type killed 2 million people worldwide, and in 1968, the A/H3N2 Hong Kong flu caused by his brother was the mildest of the pandemic, but killed about 1 million people worldwide.

The new influenza virus (A/H5N1) type to be detected in the present invention causes highly pathogenic bird flu and was originally known as a flu that is only infectious between birds, but it was found that it is transmitted from bird to human after 6 people died of Hong Kong bird flu in 1997. The virus is currently transmitted through direct contact with bird droppings and secretions, but the WHO warned that H5N1 mutations and human-to-human transmission are more dangerous than SARS. Influenza viruses like this are made up of only RNA, which is 100,000 times more likely to develop strains than DNA, and experts analyze that even strains that can be transmitted from person to person can occur.

In contrast, type B virus exhibits milder symptoms than type A, has less antigenic change, and is immunologically stable. Viruses of type C and D have not been known to be a problem in humans until now and are not related to influenza outbreaks.

Influenza viruses are common in late autumn to early spring, usually with the onset of winter. It is mainly spread directly or indirectly as a droplet infection, and is known to peak within 2 to 3 weeks and last for 5 to 6 weeks when the epidemic begins. The incubation period is 1 to 4 days, an average of 2 days, which differs depending on the infectivity of the virus and the immune status of the host. It is usually contagious from 1 day before the onset of symptoms to about 5 days after the onset, but in the case of children, the contagious period is longer, so it is contagious until about 7 days after the onset. When a new subtype of virus emerges, a pandemic can occur because most people are not immune to it.

The most obvious symptom when infected with the virus is a sudden high fever of 38-40℃ within 24 hours of infection, and systemic symptoms such as headache, muscle pain and fatigue, and respiratory symptoms such as sore throat, cough, sputum and rhinitis appear. Also, abdominal pain, vomiting, and cramps can occur infrequently. Healthy people recover after showing symptoms for several days, but people with chronic lung disease, heart disease and immunocompromised immunity may die due to complications such as pneumonia. Complications such as the back can also be accompanied. Children have similar symptoms as in adults, but have higher fever, febrile seizures, and otitis media, pseudomembranous laryngitis, and muscle pain are more common.

Differential diagnosis from respiratory infections caused by respiratory syncytial virus (RSV), parainfluenza virus, adenovirus, rhinovirus, pneumonia mycoplasma, and bacteria I need this. To this end, several diagnostic methods for influenza virus infection have been developed, the first of which is culture testing. This is a sample collected from animal cells and fertilized eggs (influenza virus), treated with antibiotics, and cultured to observe cytopathic effect (CPE), antigen detection by hemagglutination test, reverse transcription polymerase chain reaction method. (RT-PCR), Real-time RT-RCR, is a method of identifying 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 a gene (HA or NA site) specific for influenza virus. For type and subtype analysis, HA (hemagglutination assay) and HI (hemagglutination inhibition assay) are being conducted. In addition, RT-PCR (reverse transcription-polymerase chain reaction), Real-time RT-RCR, and EIA (enzyme linked immuno assay) methods were used to determine the A, B and subtypes. There is a diagnostic method that analyzes the nucleotide sequence of the NA gene and compares it with vaccine strains and foreign isolates. Third, serologic diagnosis of influenza virus can be diagnosed when the serum IgG influenza-specific antibody increases significantly more than 4 times in the acute phase (within 1 week of onset) and recovery (2-4 weeks of onset). However, these diagnostic methods are complex, take a long time, and require expert help. Finally, rapid influenza diagnostic tests (RIDTs) are a method to detect influenza virus antigens. Can be decided. For example, as an antibody-based electrochemical immune sensor recently developed, there is an electrode-based sensor in which a monoclonal antibody specific to H5 of influenza virus A type H5N1 developed by Lin et al. is immobilized (Lin et al., 2014), Eptamer beacon (short-range communication technology using Bluetooth) conjugated with quantum dots developed by Lee et al. as an optical immune sensor visualizes influenza virus H1N1 virus detection by smartphone It is a sensor with a sensitivity comparable to the detection limit of SPR-based technology and facilitates (Lee et al., 2018). However, it has a lower sensitivity than PCR, cannot characterize viral antigens, and is expensive. In addition, even if RIDTs are negative, influenza infection cannot be completely ruled out, so cell culture or genetic testing is necessary if necessary.

Accordingly, the present inventors conducted research to develop a diagnostic method capable of rapidly and accurately detecting influenza virus, and as a result, discovered a novel peptide receptor having high sensitivity and accuracy to HA, an influenza virus A/H5N1 envelope glycoprotein. Using this electrochemical detection method, a biosensor system capable of early diagnosis was developed.

The academic definition of a peptide is a form in which two to 50 amino acids are linked, which have the same constituents as proteins, but are much smaller in size, and have a lower molecular weight compared to antibodies widely used in disease diagnosis, making it easier to functionalize and manufacture. There are several advantages to the somewhat simpler process. However, there remains a problem to be overcome in the future, such as the possibility of degradation by various types of protease contained in a sample when used as a probe for early diagnosis of disease targeting bodily fluids. In addition, a sensor for molecular diagnosis using an antibody, which is one of the gold standards, is expensive to produce antibodies and has the possibility of cross-reaction, which increases the sensitivity and requires the development of a new type of detection that facilitates high-speed field access. Is being raised.

Overcoming the limitations of such existing detection methods is believed to be possible through the fusion of molecular evolution technology and peptidome technology. When a new concept of bioprobe is developed using this technology, it can be applied to the detection of disease-causing biomarkers, and at the same time, biomarker detection by enabling probe diversification, sensitivity improvement, multi-component measurement, real-time analysis, and non-label detection technology. In addition, it can be used for molecular diagnosis such as detection of harmful microorganisms, disease biomarkers, harmful substances, and environmental hormones.

However, despite the development of various antibody technologies and influenza virus detection technologies using the same as described above, the current immunization method technology using affinity antibodies has large disadvantages such as a complex chemical treatment method on the chip surface and non-specific protein binding. In addition, since its binding power is weak and can be affected by many chemicals, it is difficult to put it into practical use due to limitations in testing various protein-protein interactions, and high purity is required to immobilize antibodies on chips or microwells. It is required and has to include a complex purification process, so it has a disadvantage that the economy is inferior.

Therefore, there is an urgent need to develop a receptor capable of real-time detection of multiplexed disease-causing proteins or cells and having high and accurate recognition ability.

Although there are some challenges to overcome, it is believed that it is possible to develop a new source technology that can replace the existing detection method through the development of high-sensitivity probes using the ever-evolving convergence technology. In addition to the cancer diagnosis and early diagnosis of cardiovascular disease as exemplified above, many researchers are conducting applied research for diagnosing other diseases such as metabolic disease-related diseases and brain diseases. For this, it seems necessary to use peptide engineering technology and nano fusion technology, and it is considered that it is possible to develop a simple and highly sensitive integrated molecular diagnosis technology from body fluids in the future. Ultimately, it is expected to be able to develop a new concept integrated early diagnosis chip that can satisfy various demands such as reproducibility of diagnosis results, convenience and stability of tests, and economy.

In addition, using the peptide probe for early diagnosis, which is essential for the prevention and treatment of diseases including cancer, as a molecular diagnosis or molecular imaging tool using phage display technology, a molecular evolution technology, is used to diagnose early cancer, grasp the progress of cancer, target specific diseases or cancer cells, It can be applied to immunotherapy, gene delivery, etc. This possibility is considered to be of great significance in terms of preoccupying the peptide-based molecular conjugate development technology in the cancer-related diagnostic market and securing the related original technology, and through this, it is believed that it can contribute to the development of the medical and pharmaceutical-related industries. . At the same time, research on biomarkers that have an important influence on the pathogenesis of cancer or other diseases, cell aging, and growth of organs not only plays a pivotal role in elucidating the causal relationship of various life phenomena such as cancer occurrence and aging, but also occurs in the future. It is expected to become a core field of bio-research in Korea that can be applied to research such as differentiation and development of cell therapy products using stem cells.

In addition, based on this convergence-based technology, rapid and accurate diagnosis technology has become possible, but there are some challenges to overcome. For example, there are non-invasive, simple measurement, long life and stability, rapidity, improved sensor sensitivity, improved reliability, and the like. The technology in related fields is improving day by day and some limitations are suggested.However, as these problems are solved, molecular diagnostics or kits are also developed, enabling parallel verification according to new drug treatments, thereby increasing the possibility of success in new drug development. It can be expected to improve dramatically.

It is an object of the present invention to develop a simple, high sensitivity and high-specificity innovative biosensor that detects influenza viruses known as acute respiratory disease-causing viruses present in trace amounts in a sample, and is used to detect existing influenza viruses. It is to provide a technology that can replace expensive antibodies. In addition, it aims to provide a technology that enables the manufacture of a kit or biosensor for influenza virus detection that is spectroscopic or electrochemical so that it can be used in the field.

In order to achieve the above object, the present invention provides a peptide having a high specific and high selective binding ability to hemagglutinin, which is a new influenza virus antigen.

At this time, the peptide having a high specific, high selective binding ability to hemagglutinin, which is a new influenza virus antigen, is characterized in that it is any one of amino acid sequence list 1 (GHPHYNNPSLQL), 2 (GHPHYNNPSLQLGGGGSC), 7 (GHEHYNNESLQLGGGGSC).

In addition, the peptide is characterized in that it is a multiplex, triplex or multiplex.

In addition, the peptide is characterized in that it contains a cysteine at the N-terminus, the C-terminus, or the N-C middle.

In addition, the peptide is characterized in that it comprises a link (linker) at the C-terminal or N-terminal.

The present invention also provides a method of manufacturing a new influenza virus detection chip using a peptide receptor comprising the following steps.

(a) containing an amino acid sequence encoding a peptide receptor, all of which are prepared in a fusion protein or in a synthesized form;

(b) culturing and expressing a transformed microorganism in the form of a fusion protein of the water-soluble fraction containing the peptide receptor;

(c) site-specifically immobilizing a water-soluble synthesized peptide containing the amino acid sequence of the peptide receptor on a metal chip.

At this time, the peptide receptor is characterized in that it is any one of amino acid sequence list 1 (GHPHYNNPSLQL), 2 (GHPHYNNPSLQLGGGGSC), and 7 (GHEHYNNESLQLGGGGSC).

In addition, the metal is characterized in that the gold.

The present invention also provides a new influenza virus detection chip manufactured by the above manufacturing method.

According to the present invention, using peptidomimetics technology, peptide receptors are discovered and improved, specifically immobilized on a metal surface, and antibodies to hemagglutinin, an influenza virus antigen known as a virus causing acute respiratory diseases, present in a sample in a very small amount. It is possible to manufacture a biosensor that can detect highly efficient (sensitivity, specificity) with a simple process without the use of, and the present invention provides a technology that can detect influenza virus by replacing the existing influenza virus detection antibody. can do.

1 is an experimental result confirming the high specific binding of the peptide receptor used in the present invention to a target protein using an enzyme immunoassay method.
FIG. 2 is a result of an experiment for binding strength to a target protein according to the concentration of the peptide receptor used in the present invention by an enzyme immunoassay method.
3 is a result of an experiment of the binding force of the peptide receptor used in the present invention to a change in the concentration of a target protein by an enzyme immunoassay method.
FIG. 4 is a result of measuring binding force using electrochemical analysis (SWV) for a target protein of a peptide-based molecular conjugate used in the present invention.

The influenza virus that causes the pandemic flu, which causes numerous deaths, appears every season, and the risk factors such as the continuous occurrence of the mutation are increasing. The symptoms of this are similar to infections by other viruses and bacteria, so differential diagnosis is necessary, and development of a faster and more accurate diagnosis technology than the existing diagnosis method is urgently needed. Therefore, the present inventors have developed a new type of peptide-based biosensor that can efficiently detect influenza virus present in a very small amount in a sample at a very simple and low manufacturing cost, thereby replacing the existing antibody for molecular diagnosis of influenza virus. It is possible to provide a new type of high-sensitivity, high-specific detection technology.

Hereinafter, the present invention will be described in detail with examples.

Example 1 : Target protein (Influenza A/H5N1/Anhui/1/2005 Hemagglutinin (HA))

The target protein used in the present invention is A/H5N1/Anhui/1/2005 Hemagglutinin (HA), which was purchased and used from Sino biological, and was subjected to a biotinylation process using the Thermo scientific EZ-Link Sulfo-NHS-Biotinylation Kit. After the concentration was calculated by BCA assay, and stored in a -80°C freezer until use.

Example 2: Discovery of a new peptide receptor capable of highly specific and highly selective binding to A/H5N1/Anhui/1/2005 HA protein using M13 bacteriophage display

The bacteriophage random peptide library used in the present invention is Ph.D.-12 (New England BioLab), which is the end of the gene producing pIII, a kind of coat protein, in the genome of M13 bacteriophage. It consists of a recombinant bacteriophage expressing hundreds of millions of different peptides obtained by infecting E. coli after artificially inserting the gene sequence so that the peptides of 12 random amino acid sequences are expressed in E. coli.

Panning was performed three times in total, and streptavidin-coated 96-Well Plates (Thermo scientific, Cat. No. 15501) were used for panning.

First, biotinylated A/H5N1/Anhui/1/2005 HA protein and M13 bacteriophage are mixed in a 1.5 mL-eppendorf tube and then reacted at room temperature for 1 hour to form a pre-complex. Then, the plate coated with streptavidin was washed 3 times with PBS (phosphate buffered saline) for 5 minutes, and then the Pre-complex was put in a micro well and stirred at 150 rpm for 5 minutes at room temperature. . After 5 minutes, 1 μL of 0.1mM biotin was added and stirred at 150 rpm for 5 minutes at room temperature to block streptavidin, which is not bound to anything. After this process, the bacteriophage without binding force was washed 10 times with 0.1% Tween-20 in 200 μL of PBS (phosphate buffered saline), and finally, phage that specifically binds to A/H5N1/Anhui/1/2005 HA Was eluted in 100 μL of 0.2M Glycin-HCl (pH 2.2) solution, and 15 uL of Tris-HCl (pH 9.0) was added to neutralize the pH. One bacteriophage candidate group that binds well to A/H5N1/Anhui/1/2005 HA was firstly selected through a total of 3 replicates as described above, and the amino acid sequence was confirmed through DNA sequencing [Table 1].

Attached as amino acid sequence listing 1 Selected candidates Amino acid sequence (N→C) frequency Anhui R3#1 GHPHYNNPSLQL 29/46

Example 3: Measurement of avidity of candidate bacteriophages containing novel peptide probes capable of highly specific and highly selective binding to A/H5N1/Anhui/1/2005 HA using enzyme immunoassay (ELISA)

Amplification was performed to use one bacteriophage candidate group selected through three bacteriophage display in ELISA experiments. Phage stock (stock) was put in 20 mL LB medium with E. coli ER2738 cultured in advance, and incubated for 4-5 hours at 37°C and 190 rpm. The cultured bacteriophage candidate group was centrifuged at a rate of 12,000g for 10 minutes to recover the supernatant, and then 20% PEG/2.5M NaCl was added 1/6 (3.3 mL) of the supernatant volume and left in a refrigerator at 4℃ for 24 hours. I did. The precipitate obtained by centrifuging it again at a rate of 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 Eppendorf tube. One-quarter (about 166.7 μL) 20% PEG/2.5M NaCl was added and left on ice for 1 hour. After that, the precipitate obtained by centrifuging again at 14,000 rpm for 10 minutes was dissolved in 200 μL PBS (phosphate buffered saline) and then phage titration was performed to calculate the concentration of phage (PFU/mL: Plaque forming units/mL).

In order to compare the binding power of the selected phage and A/H5N1/Anhui/1/2005 HA, ELISA was performed as follows. First, biotinylated A/H5N1/Anhui/1/2005 HA 100 μL (HA concentration: 31.5 μg/mL) and BSA (Bovine serum albumin) were fixed on a plate coated with streptavidin for 1 hour at room temperature. Wash 6 times with 0.1% Tween-20 in PBS (Phosphate buffered saline). Then, 100 μL of Anhui R3#1 phage (concentration: 1×10 ¹¹ PFU/mL) was placed in a plate on which proteins were fixed, stirred at room temperature at 150 rpm, and reacted for 1 hour to induce binding. After washing 6 times with 0.1% Tween-20 in PBS (phosphate buffered saline), 200 μL M13-HRP antibody: PBS diluted 1: 500 was added and stirred at room temperature for 1 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 using ABTS. As a result, it was observed that A/H5N1/Anhui/1/2005 HA, which specifically binds, exhibits higher binding power than BSA that non-specifically binds. (Drawing 1)

A / H5N1 / Anhui, such as in the same ELISA method as described above / 1/2005 HA concentration (0.5 μM = 31.5 μg / mL ) to the plate fixed to Anhui R3 # 1 phage concentrations ^{(1 × 10 8 ~ 1 ×} 10 12 of protein PFU/mL) and compared the binding force, and as a result, it was observed that the binding force increased in proportion to the increase in the concentration of phage. (Drawing 2)

A/H5N1/Anhui/1/2005 HA concentration (0.062 ~ 1 μM) was measured and compared with the same ELISA method as above. It was confirmed that the bonding force increased in proportion. However, when the concentration of HA was 0.5 μM or more, it did not increase significantly. (Drawing 3)

These results show that the selected phage Anhui R3#1 specifically binds well to A/H5N1/Anhui/1/2005 HA, and the 12 peptide sequences'GHPHYNNPSLQL' possessed by Anhui R3#1 are peptide-derived influenza viruses. It shows that it can be used as an early infection diagnosis kit.

As shown above, the peptide props invented by the present inventors are highly specific and highly selectively capable of binding to A/H5N1/Anhui/1/2005 HA, which is an influenza virus envelope protein in the patient's blood, so that the amount of the target substance (increased or Gamma) can be accurately detected, and through this, early diagnosis of influenza virus infection becomes possible.

Example 4: Synthesis of peptide based on amino acid sequence discovered through M13 bacteriophage display

Based on the amino acid sequence Anhui R3#1 (GHPHYNNPSLQL) discovered through three bacteriophage display, a total of six peptides were synthesized by Peptron and produced [Table 2]. After synthesis, the purified and freeze-dried peptide was diluted to a high concentration in PBS (phosphate buffered saline), subdivided, and stored in a -20°C freezer until use.

The synthesized peptide sequence list is attached to each sequence list 2~7. Peptide name Amino acid sequence (N→C) IFA BP1 GHPHYNNPSLQLGGGGSC IFA BP2 LQLSPNNYHPHGGGGGSC IFA BP3 GHPHYNNPSLQLGHPHYNNPSLQLGGGGSC IFA BP4 GDPDYNNPSLQLGGGGSC IFA BP5 DHDHYNNDSDQDGGGGSC IFA BP6 GHEHYNNESLQLGGGGSC

Example 5: Measurement of binding force using electrochemical analysis (SWV) for a target protein of a peptide-based molecular conjugate

CHI 660E (Electrochemical Workstation, Ch Instruments Inc, USA) was used to measure the binding ability of the molecular conjugate to the target protein. Analysis was carried out at room temperature, and disposable gold chips were used.

SWV (Square Wave Voltammetry, Square Wave Voltammetry) is used in the presence of chemical crystals capable of oxidation/reduction reactions in the electrolyte. This is an analytical method to obtain thermodynamic and kinetic parameters of the electrochemical reaction of a substance that occurs in

_{First, a piranha solution made by mixing hydrogen peroxide (H 2} O ₂ ) and sulfuric acid (H ₂ SO ₄ ) in a 3:7 ratio after soaking in ethanol for 5 minutes to polish gold chips and then washing with 3rd distilled water. (Piranha solution) was applied at a time of 250 μL on the gold chip, allowed to stand at room temperature for 10 minutes, and then washed again with 3rd distilled water.

After that, the gold chip was immersed in 1mM HDT (1,6-Hexanedithiol) with thiol groups (-SH) on both sides and left at room temperature for 3 hours to fix the HDT on the gold chip and rinsed with 3rd distilled water. Immediately after HDT was fixed, to fill the surface of the remaining gold chip, it was immersed in 1mM MCH (Mercaptohexanol), left at room temperature for 1 hour, and washed with third distilled water. The gold chip on which HDT was immobilized was immersed in 50 μM BQ (1,4-Benzoquinone) and left at room temperature for 2 hours to allow the other thiol and BQ of HDT to disulfide bond. The gold chip with the functional group was washed with tertiary distilled water and fixed in the gold chip cell. Then, a PBS solution was added and a current of 0.3V was applied to activate the HDT/BQ functional group.

)을 넣고 상대전극(Counter Electrode), 기준전극(Reference Electrode), 작업전극(Working Electrode)를 이용하여 SWV를 측정하였다. 그다음 50μL의 타깃단백질인 A/H5N1/Anhui/1/2005 HA(농도: 0.25 μM = 15.75 μg/mL)와 비특이적으로 결합하는 BSA(농도: 0.25 μM = 16.25 μg/mL)를 IFA 펩타이드가 고정화된 골드칩과 1시간 동안 상온에서 반응시킨 후 전해용액을 넣고 펩타이드와의 결합능을 SWV로 측정하였다. In this way, 75 μL of IFA BP1-BP6 peptide (concentration: 100 μg/mL) was added to the activated gold chip, reacted at room temperature for 2 hours, washed 6 times with 3rd distilled water, and then w electrolytic solution (

) And measured SWV using a counter electrode, a reference electrode, and a working electrode. Then, 50 μL of the target protein A/H5N1/Anhui/1/2005 HA (concentration: 0.25 μM = 15.75 μg/mL) and BSA (concentration: 0.25 μM = 16.25 μg/mL) that non-specifically binds were added to the IFA peptide immobilized. After reacting with the gold chip at room temperature for 1 hour, an electrolytic solution was added and the binding ability with the peptide was measured by SWV.

A/H5N1/Anhui/1/2005 Each IFA BP1~BP6 peptide binding ability to HA was measured by electrochemical signal change, and the current value obtained from SWV was calculated (ΔI(%) = (I _b -I _a ) /I _b ×100) and finally, IFA BP6 showed the highest binding ability with the target protein. (Drawing 4)

Based on the experimental results of FIG. 4, amino acid sequence listing 1 (Anhui R3#1, GHPHYNNPSLQL) having high binding ability and high cognitive ability to hemagglutinin through the phage display method was discovered. phage surface). Amino acid sequence list 1 was basic chemical scaffolding, and sequence list 2 (IFA BP1, GHPHYNNPSLQLGGGGSC) and sequence list 7 (IFA BP6, GHEHYNNESLQLGGGGSC) synthesized in free peptide form using a chemical synthesis method were synthesized. 2 and SEQ ID NO: 7 aimed to increase the detection performance of the antigen, hemaglutinin, through the insertion of a specific amino acid motif (GGGGS) that can act as a flexibel linker. As a result, in the case of detection of the antigen, hemaglutinin, using SEQ ID NO. 7, the performance was the best, but any one of the performance sequence listings 1, 2, and 7 could be used to detect the antigen, hemaglutinin.

<110> Industry-academic cooperation foundation Daegu Hanny University <120> High sensitive peptides for influenza virus, and the influenza virus detection chip using the peptides <130> ULA-1712 <160> 7 <170> KopatentIn 2.0 <210> 1 <211> 12 <212> PRT <213> bacteriophage <400> 1 Gly His Pro His Tyr Asn Asn Pro Ser Leu Gln Leu 1 5 10 <210> 2 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 2 Gly His Pro His Tyr Asn Asn Pro Ser Leu Gln Leu Gly Gly Gly Gly 1 5 10 15 Ser Cys <210> 3 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 3 Leu Gln Leu Ser Pro Asn Asn Tyr His Pro His Gly Gly Gly Gly Gly 1 5 10 15 Ser Cys <210> 4 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 4 Gly His Pro His Tyr Asn Asn Pro Ser Leu Gln Leu Gly His Pro His 1 5 10 15 Tyr Asn Asn Pro Ser Leu Gln Leu Gly Gly Gly Gly Ser Cys 20 25 30 <210> 5 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 5 Gly Asp Pro Asp Tyr Asn Asn Pro Ser Leu Gln Leu Gly Gly Gly Gly 1 5 10 15 Ser Cys <210> 6 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 6 Asp His Asp His Tyr Asn Asn Asp Ser Asp Gln Asp Gly Gly Gly Gly 1 5 10 15 Ser Cys <210> 7 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 7 Gly His Glu His Tyr Asn Asn Glu Ser Leu Gln Leu Gly Gly Gly Gly 1 5 10 15 Ser Cys

Claims (9)

Peptides with high specific and high selective binding ability to hemagglutinin, a new influenza virus antigen. The method of claim 1,
A peptide having a high specific, high selective binding ability to hemagglutinin, a new influenza virus antigen, is a peptide characterized in that it is any one of amino acid sequence listing 1 (GHPHYNNPSLQL), 2 (GHPHYNNPSLQLGGGGSC), 7 (GHEHYNNESLQLGGGGSC). The method according to claim 1 or 2,
The peptide is a peptide, characterized in that the multiple, triple or multiple. The method of claim 3,
The peptide is a peptide, characterized in that it contains a cysteine in the N-terminal, C-terminal or NC intermediate. The method of claim 1,
The peptide is a peptide, characterized in that it comprises a link (linker) at the C-terminal or N-terminal. A method of manufacturing a new influenza virus detection chip using a peptide receptor comprising the following steps;
(a) containing an amino acid sequence encoding a peptide receptor, all of which are prepared in a fusion protein or in a synthesized form;
(b) culturing and expressing a transformed microorganism in the form of a fusion protein of the water-soluble fraction containing the peptide receptor;
(c) site-specifically immobilizing a water-soluble synthesized peptide containing the amino acid sequence of the peptide receptor on a metal chip. The method of claim 6,
The peptide receptor is any one of amino acid sequence listing 1 (GHPHYNNPSLQL), 2 (GHPHYNNPSLQLGGGGSC), and 7 (GHEHYNNESLQLGGGGSC). A method of manufacturing a novel influenza virus detection chip using a peptide receptor. The method of claim 7,
The method of manufacturing a new influenza virus detection chip, characterized in that the metal is gold. A method of manufacturing a new influenza virus detection chip manufactured by the method of claim 7 or 8.

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