KR102373670B1 - 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 detecting a novel influenza virus known as an acute respiratory disease-causing virus, a method for manufacturing a biosensor probe using the peptide receptor, and a method for detecting a human-derived novel influenza virus antigen using the same.
According to the present invention, the peptide having high specific and high selective binding ability to hemagglutinin, a novel influenza virus antigen, is any one of amino acid sequence list 1 (GHPHYNNPSLQL), 2 (GHPHYNNPSLQLGGGGSC), 7 (GHEHYNNESLQLGGGGSC) do.

Description

A peptide receptor capable of highly specific and highly selective binding to novel influenza virus and a method for manufacturing a biochip for virus detection using the same {High sensitive peptides for influenza virus, and the influenza virus detection chip using the peptides}

The present invention relates to the development of a molecular recognition element as a peptide receptor for high-sensitivity detection of influenza A virus H5N1 subtype, prevention and treatment using the molecular binder, and further detection of influenza virus present in a trace amount in a sample It's about how to do it. In the present invention, the performance of the peptide receptor discovered by peptidomimetics is verified through spectroscopic detection, and the influenza virus present in a trace amount in the sample has high sensitivity and high specificity ( It provides a method for detection with high selectivity).

The peptide receptor of the present invention is capable of increasing binding affinity or binding constant to a target molecule by changing and substituting the amino acid composition in the molecular coupler using an in silico-based simulation method. It relates to a method to discover the best possible molecular binding agent and to detect the actual influenza virus with high sensitivity and high specificity through the functional verification of the molecular binding agent using spectroscopic or electrochemical detection technology.

The method using the peptide-based molecular binder according to the present invention improves the production yield and simplifies the production process compared to the conventional detection method using antibodies, aptamers, PNA, carbohydrates, enzymes, etc., so that spectroscopic or electrochemical detection If the technology is used, influenza viruses known to cause acute respiratory diseases present in trace amounts in samples can be detected very simply, efficiently, with high sensitivity and with high specificity. When the peptide receptor of the present invention is used, it is expected to be able to replace the existing antibody for detecting influenza virus.

Influenza virus is an RNA virus and belongs to the Orthomyxoviridae family and influenza virus genus, and depending on the antigenic difference between the nucleocapsid protein (NP) and the matrix protein (matrix, M) of the virus, 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 80-120 nm. Viruses immediately after separation from fertilized eggs and susceptible cells sometimes have filamentous filaments with a length of 400 nm. Specific glycoproteins exist in the virus envelope. Representative envelope glycoproteins present in type A and B viruses are hemagglutinin (HA) and neuraminidase (NA), which are , and plays an important role in the antibody response. Genes consist of segmented single-stranded RNA, encased in a helical capsid, and contain not one, but seven or eight different RNA segments. Each RNA segment is composed of glycoproteins HA and NA, polymerase proteins PA, PB1, and PB2, NP and M protecting genetic material, and finally nonstructural proteins NS1 and NS2. Unlike other RNA viruses, the replication of these genetic materials occurs in the nucleus of the host cell. When the virus enters the host cell, the envelope glycoprotein HA binds to the cell receptor present in the cell membrane, allowing the virus to enter the cell. NA plays an important role in the release of the virus from infected cells and propagation to new respiratory cells.

Influenza virus type A has frequent antigenic mutations and many subtypes exist, so the labeling method proposed by the World Health Organization (WHO) is used for systematic management. The standard nomenclature is in the order of subtype/origin host/isolated region/isolation order/separation year (HA type, NA type). If the virus has been isolated from humans, it is not indicated when the origin host is indicated.

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 it has been the cause of influenza virus epidemics at least three times 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. Although the virus currently requires direct contact with bird droppings and secretions, the WHO warns that H5N1 is more dangerous than SARS if it mutates and spreads from person to person. Experts analyze that influenza viruses like this one are made up of RNA, which is 100,000 times more likely to mutate than DNA, so that there can be mutants that can be transmitted from person to person.

In contrast, type B virus exhibits milder symptoms than type A, has less antigenic change, and is immunologically stable. Hepatitis C and D viruses are not known to be a problem in humans so far and are not known to be associated with influenza epidemics.

Influenza viruses usually occur in late autumn to early spring, when winter begins. It is mainly transmitted directly or indirectly by droplet infection, and it is known that when an epidemic begins, it peaks within 2-3 weeks and lasts for 5-6 weeks. The incubation period is 1-4 days, on average, about 2 days, depending on the infectivity of the virus and the immune status of the host. Usually, it is 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. The emergence of a new subtype of the virus could cause a pandemic because most people do not have immunity to it.

When infected with the virus, the most obvious symptom is a sudden high fever of 38-40°C 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. Abdominal pain, vomiting, and convulsions may also occur rarely. Healthy people recover after showing symptoms for several days, but chronic lung disease, heart disease, and immunocompromised patients can die from complications such as pneumonia. Complications may also be present. In children, the symptoms are similar to those in adults, but the fever is higher, febrile convulsions may occur, and otitis media, pseudomembranous laryngitis, and myalgia are more common.

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, the first of which is culture testing. 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, hemagglutination assay (HA) and hemagglutination inhibition assay (HI) 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, serological diagnosis of influenza virus can be made when the serum IgG-influenza-specific antibody significantly increases by 4 times or more in the acute phase (within 1 week of onset) and during the recovery phase (within 2-4 weeks of onset). However, these diagnostic methods are complex, time-consuming, and require professional 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 As an example, a recently developed antibody-based electrochemical immune sensor includes an electrode-based sensor in which a monoclonal antibody specific for H5 of influenza virus type A H5N1 developed by Lin et al. is immobilized (Lin et al., 2014), an eptamer beacon with quantum dot spliced developed by Lee et al. as an optical immune sensor (short-distance communication technology using Bluetooth) is a visualization of influenza virus H1N1 virus detection by a smartphone. It is a sensor with sensitivity that facilitates the detection of SPR-based techniques and is comparable to the detection limit of SPR-based techniques (Lee et al., 2018). 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 ruled out, 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, and as a result, discovered a novel peptide receptor with high sensitivity and accuracy to HA, an influenza virus A/H5N1 envelope glycoprotein. A biosensor system capable of early diagnosis was developed using an electrochemical detection method using this.

The academic definition of a peptide is a form in which 2 to 50 amino acids are linked with the same composition as a protein but much smaller in size. There are several advantages that the process is rather simple. However, there are still challenges to be overcome, such as the possibility of degradation by various types of proteases contained in the sample when used as a probe for early diagnosis of diseases in bodily fluids. In addition, the molecular diagnostic sensor using an antibody, which is one of the classical methods (gold standard), is expensive to produce and has the possibility of cross-reaction, so the need for the development of a new type of detection that increases sensitivity and facilitates high-speed on-site access is being raised

It is judged that this limitation of the existing detection method can be overcome through the fusion of molecular evolution technology and peptidom technology. If a new concept bioprobe is developed using this technology, it can be applied to the detection of disease-causing biomarkers. 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 them, the current immunological method using affinity antibodies has major drawbacks such as complex chemical treatment of the chip surface and non-specific protein binding. In addition, since the binding force is weak and can be affected by many chemicals, there are many limitations in testing various protein-protein interactions, making practical use difficult. It also has a disadvantage in that economic feasibility is lowered because it is required and a complicated refining process must be included.

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 be overcome, it is thought that it will be possible to develop a new source technology that can replace the existing detection method through the development of high-sensitivity probes using the ever-developing fusion technology. Many researchers are conducting applied research not only for the cancer diagnosis and early diagnosis of cardiovascular disease, but also for the diagnosis of other diseases such as metabolic diseases and brain diseases. For this, it seems necessary to use peptide engineering technology and nano fusion technology, and it is judged that it is possible to develop a simple and highly sensitive integrated molecular diagnostic technology from body fluids in the future. Ultimately, it is expected that a new concept of integrated early diagnosis chip can be developed that can satisfy various needs such as reproducibility of diagnostic results, convenience and stability of tests, and economic feasibility.

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 evolutionary technology, early cancer diagnosis, understanding the progress of cancer, targeting specific diseases or cancer cells, It can be applied to immunotherapy, gene delivery, etc. This possibility is judged to be very meaningful in terms of preempting the peptide-based molecular binder development technology in the cancer-related diagnostic market and securing the related source technology, and it is thought that it can contribute to the development of the medical and pharmaceutical industries. . At the same time, research on biomarkers that have an important influence on the pathogenesis of cancer or other diseases, cellular aging, and growth of organs plays a pivotal role in identifying the causal relationship between various life phenomena, such as cancer occurrence and aging, as well as in the future. It is expected that it will become a core field of bio-research in Korea that can be applied to research such as differentiation and development of cell therapeutics using stem cells.

In addition, rapid and accurate diagnosis technology has become possible based on this convergence-based technology, but there are some challenges to overcome. For example, there are non-invasive, simple measurement, long life and stability, rapidity, improved sensor sensitivity, and improved reliability. Although the technology in the related field is improving day by day, and some limitations are suggested, as these problems are solved, molecular diagnostics or kit development continues, enabling parallel verification according to new drug treatment, raising the possibility of successful drug development. It can be expected to dramatically improve.

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

The present invention for achieving the above object provides a peptide having a high specific and highly selective binding ability to hemagglutinin, a novel influenza virus antigen.

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

In addition, the peptide is characterized in that it is a multimeric, triple or multimeric body.

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

In addition, the peptide is characterized in that it includes a linker at the C-terminus or the N-terminus.

The present invention also provides a method for manufacturing a novel 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 synthesized form;

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

(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 any one of amino acid sequence list 1 (GHPHYNNPSLQL), 2 (GHPHYNNPSLQLGGGGSC), 7 (GHEHYNNESLQLGGGGSC).

In addition, the metal is characterized in that gold.

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

According to the present invention, hemagglutinin, an influenza virus antigen known as an acute respiratory disease-inducing virus, present in a trace amount in a sample by excavating and improving a peptide receptor using peptidomimetics technology and specifically immobilizing it on a metal surface, is an antibody It is possible to manufacture a biosensor that can detect highly efficiently (sensitivity, specificity) with a simple process without the use of 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.
2 is a result of an enzyme immunoassay test for binding force to a target protein according to the concentration of the peptide receptor used in the present invention.
3 is a result of an enzyme immunoassay test for the binding force of the peptide receptor used in the present invention to the change in the concentration of the target protein.
4 is a measurement result of binding force using electrochemical analysis (SWV) for the target protein of the peptide-based molecular binder used in the present invention.

Influenza viruses that cause pandemic flu that cause numerous deaths appear every season, and risk factors such as the continuous occurrence of mutations are increasing. Its symptoms are similar to infections caused by other viruses and bacteria, so a differential diagnosis is required, and there is an urgent need to develop a faster and more accurate diagnostic technology than the existing diagnostic methods. Therefore, the present inventors developed a new type of peptide-based biosensor that can efficiently detect influenza virus present in trace amounts in a sample with very simple and low manufacturing cost, and can replace the existing antibody for molecular diagnosis of influenza virus. It has become possible to provide a new type of high-sensitivity and 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 was A/H5N1/Anhui/1/2005 Hemagglutinin (HA), which was purchased from Sino biological and used, and was biotinylated using Thermo scientific's EZ-Link Sulfo-NHS-Biotinylation Kit. After calculating the concentration by BCA assay, it was stored in a -80 ℃ freezer until use.

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

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

First, the biotinylated A/H5N1/Anhui/1/2005 HA protein and M13 bacteriophage are mixed in a 1.5 mL-eppendorf tube and 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 placed in a micro well and stirred at 150 rpm for 5 minutes at room temperature. . After 5 minutes, 1 μL of 0.1 mM biotin is added and stirred at 150 rpm for 5 minutes at room temperature to block unbound streptavidin. After this process, the bacteriophage without binding force was washed repeatedly 10 times with 0.1% Tween-20 in 200 μL of PBS (phosphate buffered saline), and finally, the phage specifically binding 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 for neutralization of pH. One bacteriophage candidate group that binds well to A/H5N1/Anhui/1/2005 HA was primarily selected through a total of three repeated experiments through the above process, and the amino acid sequence was confirmed through DNA sequencing [Table 1].

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

Example 3: A/H5N1/Anhui/1/2005 HA using enzyme immunoassay (ELISA) to measure the binding capacity of a bacteriophage candidate group containing a novel peptide probe capable of high specific, highly selective binding

Amplification was performed to use one bacteriophage candidate group selected through bacteriophage display three times in an ELISA experiment. The phage stock was placed in 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/2.5M NaCl was added to 1/6 (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 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. Add one-half (about 166.7 μL) 20% PEG/2.5M 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 ability of the selected phage and A/H5N1/Anhui/1/2005 HA. First, 100 μL of biotinylated A/H5N1/Anhui/1/2005 HA (HA concentration: 31.5 μg/mL) and bovine serum albumin (BSA) were immobilized on a plate coated with streptavidin at room temperature for 1 hour. 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 put on a plate on which the 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), add 200 μL M13-HRP antibody: a lysate diluted 1:500 in PBS and stir at room temperature for 1 hour. Even 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 that specifically binds has a higher binding affinity than BSA that binds non-specifically. (Drawing 1)

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

A/H5N1/Anhui/1/2005 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 binding force increased in proportion. However, when the concentration of HA was 0.5 μM or more, there was no significant increase. (Figure 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 sequence 'GHPHYNNPSLQL' of Anhui R3#1 is a peptide-derived influenza virus. It shows that it can be used as an early detection kit for infection.

As discussed above, the peptide probe invented by the present inventors can bind highly specific and highly selectively to A/H5N1/Anhui/1/2005 HA, which is an influenza virus envelope protein in the blood of a patient, so that the amount of the target substance (increase or ) can be accurately detected, making it possible to easily diagnose influenza virus early in the early stages.

Example 4: Synthesis of amino acid sequence-based peptides discovered through M13 bacteriophage display

Based on the amino acid sequence Anhui R3#1 (GHPHYNNPSLQL) discovered through bacteriophage display three times, a total of 6 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), then divided and stored in a -20°C freezer until use.

The synthesized peptide sequence list is attached as SEQ ID NOs 2 to 7, respectively. 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) 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 disposable gold chip 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.

First, to polish the gold chip, it is dipped in ethanol for 5 minutes, washed with tertiary distilled water, and then a piranha solution made by mixing hydrogen peroxide (H ₂ O ₂ ) and sulfuric acid (H ₂ SO ₄ ) in a 3:7 ratio. (Piranha solution) was applied on the gold chip by 250 μL, left at room temperature for 10 minutes, and washed again with tertiary 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 washed thoroughly with tertiary distilled water. Immediately after the HDT was fixed, it was immersed in 1 mM MCH (Mercaptohexanol) to fill the remaining surface of the gold chip, left at room temperature for 1 hour, and washed with tertiary distilled water. After immersing the HDT-immobilized gold chip in 50 μM BQ (1,4-Benzoquinone), it was left at room temperature for 2 hours, and other thiol groups of HDT and BQ were allowed to form disulfide bonds. After washing the gold chip with the functional group in tertiary distilled water and fixing it in a cell dedicated to the gold chip, PBS solution was added and a current of 0.3V was passed 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. Add 75 μL of IFA BP1 to BP6 peptide (concentration: 100 μg/mL) to the gold chip with activated functional groups, react at room temperature for 2 hours, wash 6 times with tertiary distilled water, and then

), and SWV was measured 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 non-specific BSA (concentration: 0.25 μM = 16.25 μg/mL) were mixed with IFA peptide immobilized thereon. After reacting with the gold chip at room temperature for 1 hour, an electrolyte solution was added, and the binding ability with the peptide was measured by SWV.

The binding capacity of each IFA BP1~BP6 peptide to A/H5N1/Anhui/1/2005 HA was measured as an electrochemical signal change, and the current value obtained from SWV was calculated (ΔI(%) = (I _b -I _a ) /I _b × 100) and finally IFA BP6 had the highest binding capacity with the target protein. (Figure 4)

Based on the experimental results of FIG. 4 , amino acid sequence list 1 (Anhui R3#1, GHPHYNNPSLQL) having high binding ability and high cognitive intelligence to hemaclutinin was discovered through the phage display method, which is that amino acid sequence list 1 is the phage surface ( It is a form expressed on the phage surface). SEQ ID NO: 2 (IFA BP1, GHPHYNNPSLQLGGGGSC) and SEQ ID NO: 7 (IFA BP6, GHEHYNNESLQLGGGGSC) synthesized in free peptide form were synthesized using a chemical synthesis method by using the basic chemical scaffold of amino acid sequence list 1 2 and SEQ ID NO: 7 were intended to enhance the detection performance of the antigen, hemaclutinin, through the insertion of a specific amino acid motif (GGGGS) that can act as a flexibel linker. As a result, when SEQ ID NO: 7 was used to detect the antigen hemaclutin, the performance was the best, but any one of performance SEQ ID NOs: 1, 2, and 7 could be used to detect the antigen hemaclutinin.

<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)

Any one amino acid sequence selected from the group consisting of SEQ ID NO: 1 (GHPHYNNPSLQL), SEQ ID NO: 2 (GHPHYNNPSLQLGGGGSC) and SEQ ID NO: 7 (GHEHYNNESLQLGGGGSC) having high specific and high selective binding ability to hemagglutinin, a novel influenza virus antigen composed of peptides. delete According to claim 1,
The peptide is a peptide, characterized in that a duplex, triplex or multiform. According to claim 1,
The peptide is a peptide, characterized in that it contains a cysteine in the N-terminal, C-terminal or NC-intermediate. According to claim 1,
The peptide is a peptide, characterized in that it comprises a linker at the C-terminus or the N-terminus. A method of manufacturing a novel influenza virus detection chip using a peptide receptor comprising the following steps;
(a) preparing a peptide receptor consisting of any one amino acid sequence selected from the group consisting of SEQ ID NO: 1 (GHPHYNNPSLQL), SEQ ID NO: 2 (GHPHYNNPSLQLGGGGSC) and SEQ ID NO: 7 (GHEHYNNESLQLGGGGSC);
(b) culturing and expressing the transformed microorganism in the form of a fusion protein containing the peptide receptor; and
(c) site-specifically fixing the expressed peptide receptor to a metal chip. delete 7. The method of claim 6,
The method of manufacturing a novel influenza virus detection chip, characterized in that the metal is gold. A novel influenza virus detection chip manufactured by the method of claim 6.

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