KR102520151B1 - Monoclonal antibody specifically binding to ifitm3 protein of chicken and use thereof - Google Patents

Abstract

The present invention relates to a monoclonal antibody that specifically binds to chicken IFITM3 protein and uses thereof. Since the monoclonal antibody according to the present invention specifically binds to the epitope of the IFITM3 protein and can confirm the expression level of the IFITM3 protein at the protein level, it can be used for the development of biomarkers for early diagnosis of various viral infections associated with the IFITM3 protein. there is. In addition, since the IFITM3 protein shows antiviral activity against various viruses, it can be used to discover antiviral-related candidate materials by confirming the degree of IFITM3 protein expression enhancement.

Description

Monoclonal antibody specifically binding to chicken IFITM3 protein and its use

The present invention relates to a monoclonal antibody that specifically binds to chicken IFITM3 protein and uses thereof.

IFITM3 (Interferon-Induced Transmembrane Protein 3) protein is a key protein of the innate immune system that shows defense against a wide range of viruses. It has been reported to exhibit viral ability. In particular, it shows strong antiviral activity against RNA viruses and has been reported to have excellent protective effects against influenza viruses (Diamond MS et.al. , Nat. Rev. Immunol. , 13(1):46-57). , 2013). However, studies on the IFITM3 protein of chickens, which are one of the main hosts of avian influenza, whose mortality rate increases more than 300 times depending on the mutation of the virus, are insufficient, and antibodies capable of specifically detecting it have not been developed.

Meanwhile, in previous studies, the expression level of mRNA through primers specific to the chicken IFITM3 gene was confirmed using reverse transcription polymerase chain reaction (RT-PCR) technology. The ability of the virus has been confirmed. However, in experiments using RT-PCR, the expression level of the IFITM3 gene can be confirmed only at the mRNA transcription level, and the protein level expression level cannot be confirmed. In addition, it is difficult to analyze the location where the protein acts.

Diamond MS et. al., Nat. Rev. Immunol., 13(1):46-57, 2013

Accordingly, the present inventors studied to develop an IFITM3 protein-specific antibody to confirm the expression level of the IFITM3 protein at the protein level. As a result, a monoclonal antibody that specifically binds to an epitope present at the N-terminus of the chicken IFITM3 protein was found. Screening was performed using phage display technology, and the present invention was completed by confirming that the monoclonal antibody prepared based on the variable regions of the heavy and light chains of the screened antibody clone had excellent binding ability to IFITM3 protein extracted from various chicken tissues. .

In order to achieve the above object, one aspect of the present invention is an epitope of chicken IFITM3 (Interferon-Induced Transmembrane Protein 3) protein, a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 and a poly encoding the polypeptide provide nucleotides.

Another aspect of the present invention is a monoclonal antibody or antigen-binding fragment thereof that specifically binds to the epitope of IFITM3 represented by the amino acid sequence of SEQ ID NO: 2, a polynucleotide encoding the amino acid of the monoclonal antibody or antigen-binding fragment thereof, A recombinant expression vector containing the polynucleotide and a transformant transformed with the recombinant expression vector are provided.

Another aspect of the present invention, a) obtaining a sample isolated from a subject suspected of being infected with avian influenza virus; b) contacting the obtained sample with the monoclonal antibody; And c) detecting the reaction between the monoclonal antibody and the sample; it provides a method for providing information for diagnosing an avian influenza virus infection.

The novel antibody or antigen-binding fragment thereof specifically binding to the IFITM3 protein according to the present invention, particularly the chicken IFITM3 epitope present at the N-terminus, can be used for various purposes such as analysis of the mechanism of action of the IFITM3 protein and development of biomarkers for early diagnosis. can be used for research. In addition, it can be used in the field of developing a candidate material for enhancing the expression of IFITM3 protein, which shows strong antiviral activity against avian influenza virus, and thus can contribute to expanding the foundation for preemptive disaster prevention against avian influenza that can spread as a national disaster-type disease. It is expected that there will be

1 is a diagram showing alignment and comparison of IFITM3 amino acid sequences of chicken, duck, goose, ibis, human, chimpanzee, mouse, cow, pig and goat. Here, the color of the amino acid represents the chemical properties of the amino acid, blue represents acidity, red represents hydrophobicity, pink represents basicity, and green represents hydroxyl, sulfhydryl, amine, and glycine.
Figure 2 is a diagram showing the results of sequence alignment between chicken IFITM1 and IFITM3 proteins and the epitope region of the finally selected IFITM3 protein.
3 is a view showing the results of ELISA screening performed to select a target antigen peptide according to the present invention, that is, a monoclonal antibody scFv that specifically binds to an epitope of IFITM3 protein.
Figure 4 shows the result of confirming the binding ability with the protein isolated from chicken lung tissue by Western blot using a monoclonal antibody anti-IFITM3 scFv production clone that specifically binds to the epitope of the IFITM3 protein according to the present invention. it is a drawing Here, P is chicken IFITM3 protein from which the entire sequence was synthesized used as a positive control, L is chicken lung tissue, and B is bovine serum albumin (BSA) used as a negative control.
Figure 5a shows the result of confirming the binding ability with the protein isolated from chicken lung tissue by Western blot using the monoclonal antibody anti-IFITM3 scFv production clone 1A10 that specifically binds to the epitope of the IFITM3 protein according to the present invention. is the drawing shown.
Figure 5b is a result of confirming the binding ability with proteins isolated from various tissues of chicken by Western blot using the monoclonal antibody anti-IFITM3 scFv production clone 1E12 that specifically binds to the epitope of the IFITM3 protein according to the present invention. is the drawing shown. Here, 1 is a bronchus, 2 is a bursa of fabricius, 3 is a caecum, and 4 is a protein isolated from the rectum.

Hereinafter, the present invention will be described in detail.

Epitope of the IFITM3 protein

One aspect of the present invention provides a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 as an epitope of IFITM3 (Interferon-Induced Transmembrane Protein 3) protein.

As used herein, the term "Interferon-Induced Transmembrane Protein 3 (IFITM3) protein" is a key protein of the innate immune system that shows defense against a wide range of viruses, by limiting viral membrane hemifusion between the host and viral membranes. Prevent viral infections. Specifically, the IFITM3 protein is influenza A virus (IAV), Ebola virus (EBOV), Marburg virus (Marburg virus (MARV), severe acute respiratory syndrome coronavirus (SARS) -CoV), dengue virus (DEV), West Nile virus (WNV), and Zika virus (ZIKV), including enveloped viruses (enveloped viruses) exhibit a wide range of potent antiviral ability It is known.

As used herein, the term "epitope", also referred to as an epitope or antigenic determinant, is a specific part of an antigen that allows the immune system to identify an antigen, such as an antibody, B cell, or T cell. Specifically, an epitope is a set of amino acid residues of an antigen-binding site recognized by a specific antibody, or residues recognized by a T cell receptor protein and/or Major Histocompatibility Complex (MHC) receptor in T cells. An epitope is a molecule that forms a site recognized by an antibody, T cell receptor or HLA molecule, and refers to a primary, secondary and tertiary peptide structure, or charge. Also, isolated or purified protein or polypeptide molecules greater than and comprising the epitopes of the present invention are still included within the scope of the present invention.

The polypeptide may be the 12th to 29th amino acid sequence of the IFITM3 protein having the amino acid sequence of SEQ ID NO: 1 ( Gallus gallus , NCBI GenBank No. NP_001336990.1). Specifically, the epitope of the IFITM3 (Interferon-Induced Transmembrane Protein 3) protein may consist of the amino acid sequence of SEQ ID NO: 2.

In addition, the present invention provides a polynucleotide encoding the polypeptide. Specifically, the gene encoding the IFITM3 (Interferon-Induced Transmembrane Protein 3) epitope protein may be the nucleotide sequence represented by SEQ ID NO: 5.

Sequences used in the present invention are construed to include sequences exhibiting substantial identity with the sequences described in the Sequence Listing, considering mutations having biologically equivalent activity. The term 'substantial identity' means that when the sequence of the present invention and any other sequence are aligned so as to correspond to the maximum, and the aligned sequence is analyzed using an algorithm commonly used in the art, the minimum A sequence exhibiting 60% homology, more specifically 70% homology, even more specifically 80% homology, and most specifically 90% homology.

Therefore, an amino acid sequence having high homology with the sequence represented by SEQ ID NO: 2, for example, an amino acid sequence having a high homology of 70% or more, specifically 80% or more, more specifically 90% or more. It should be construed as included in the scope of the present invention.

Monoclonal antibody that specifically binds to an epitope of IFITM3

Another aspect of the present invention provides a monoclonal antibody or antigen-binding fragment thereof that specifically binds to the epitope of IFITM3 represented by the amino acid sequence of SEQ ID NO: 2.

As used herein, the term “monoclonal antibody that specifically binds to an epitope of IFITM3” refers to a specific binding to the IFITM3 protein, which is a key protein of the innate immune system that exhibits protective ability against a wide range of viruses, such as avian influenza virus. Means an antibody that can be detected. In the present invention, the monoclonal antibody is mixed with the anti-IFITM3 antibody.

The term "specifically binding" or "specifically recognizing" has the same meaning commonly known to those skilled in the art, and the antigen and antibody specifically interact to form an antigen-antibody complex. It can form, and it can also mean having an immunological reaction.

The monoclonal antibody or antigen-binding fragment thereof may specifically bind to the 12th to 29th amino acid sequence represented by SEQ ID NO: 1, that is, the amino acid sequence represented by SEQ ID NO: 2, but is not limited thereto.

The monoclonal antibody specifically binding to the epitope of chicken IFITM3 protein according to one embodiment of the present invention may be composed of the amino acid sequence of SEQ ID NO: 3 including a light chain variable region and a heavy chain variable region.

As used herein, the term "antigen-binding fragment" refers to a fragment having an antigen-binding function, and the antigen-binding fragment includes Fab, Fd, Fab', dAb, F(ab'), F(ab')2 It may be any one selected from the group consisting of, scFv, Fv, single-chain Fv dimer, and diabody, but is not limited thereto.

As used herein, the term "monoclonal antibody" is an art-recognized term and refers to a highly specific antibody directed against a single antigenic site. Monoclonal antibodies are also called monoclonal antibodies, monoclonal antibodies or monoclonal antibodies. Unlike polyclonal antibodies, which usually include different antibodies directed against different epitopes (antigenic determinants), monoclonal antibodies are directed against a single determinant on the antigen. Monoclonal antibodies have the advantage of improving the selectivity and specificity of diagnostic and analytical assays using antigen-antibody binding, and also have another advantage of not being contaminated by other immunoglobulins because they are synthesized by hybridoma culture.

The monoclonal antibody may be produced through various methods known in the art.

The antibodies include whole antibody forms as well as functional fragments of antibody molecules. A full antibody has a structure having two full-length light chains and two full-length heavy chains, and each light chain is connected to the heavy chain by a disulfide bond. A functional fragment of an antibody molecule refers to a fragment having an antigen-binding function, and examples of antibody fragments include (i) a light chain variable region (VL) and a heavy chain variable region (VH) and a light chain constant region (CL) and a Fab fragment consisting of the first constant region of the heavy chain (CH1); (ii) a Fd fragment consisting of the VH and CH1 domains; (iii) an Fv fragment consisting of the VL and VH domains of a single antibody; (iv) a dAb fragment consisting of the VH domain (Ward ES et al. , Nature , 341:544-546 (1989)); (v) an isolated CDR region; (vi) a bivalent fragment containing two linked Fab fragments. Phosphorus F(ab')2 fragment; (vii) a single-chain Fv molecule (scFv) joined by a peptide linker linking the VH domain and the VL domain to form an antigen-binding site; (viii) bispecific single-chain Fv dimer (PCT/US92/09965) and (ix) diabodies (WO94/13804), which are multivalent or multispecific fragments produced by gene fusion.

As used herein, the term "variable region" of an antibody refers to a region that exhibits many variations in sequence while performing a function of binding specifically to an antigen, and the variable region includes complementarity determining regions CDR1 and CDR2. and CDR3. There is a framework region (FR, framework region) between the complementarity determining regions, which serves to support the complementarity determining region ring.

As used herein, the term "complementarity determining region (CDR)" is a ring-shaped region involved in antigen recognition, and the specificity of an antibody for an antigen is determined as the sequence of this region changes.

As used herein, the term "scFv (single chain fragment variable)" refers to a single-chain antibody made by expressing only the variable region of an antibody through genetic recombination, and is a single-chain form in which the VH and VL regions of an antibody are connected with a short peptide chain. refers to the antibody of The term "scFv" is intended to include scFv fragments, including antigen-binding fragments, unless otherwise specified or otherwise understood from the context. This is obvious to a person skilled in the art.

According to one embodiment of the present invention, the present inventors bio-panned a naive chicken single chain Fv library using a phage display method to specifically bind to an epitope of IFITM3 protein, specifically chicken IFITM3 protein Antibodies were prepared.

In the present invention, the term "biopanning" refers to a peptide that has the property of binding to a target molecule (antibody, enzyme, cell surface receptor, etc.) It refers to the process of selecting only the phages expressed on the surface.

The monoclonal antibody or antigen-binding fragment thereof of the present invention is not particularly limited thereto, but may be subjected to glycosylation and/or PEGylation in order to increase residence time in vivo.

The term "glycosylation" of the present invention refers to a processing method of translocating a glycosyl group to a protein. The glycosylation is performed by linking a glycosyl group to a serine, threonine, asparagine or hydroxylysine residue of a target protein by a glycosyl transferase. It also plays an important role in cell recognition on the surface. Therefore, in the present invention, the effect of the antibody can be improved by changing the glycosylation or the pattern of the glycosylation of the monoclonal antibody or antigen-binding fragment thereof.

The term "PEGlation" of the present invention refers to a processing method for improving the residence time of an antibody in blood by introducing polyethylene glycol into the monoclonal antibody or antigen-binding fragment thereof. Specifically, by pegylating polymer nanoparticles with polyethylene glycol, the hydrophilicity of the surface of the nanoparticles is increased, and immune functions including macrophages in the human body that prey on and digest pathogens, waste materials, and foreign substances are activated. Rapid degradation within the body can be prevented through a so-called stealth effect that prevents recognition from the body. Thus, the blood retention time of the antibody can be improved by the pegylation. PEGylation used in the present invention may be formed by a method of forming an amide group by combining a carboxyl group of hyaluronic acid and an amine group of polyethylene glycol, but is not limited thereto, and pegylation can be performed in various ways. . At this time, the polyethylene glycol used is not particularly limited thereto, but preferably has a molecular weight between 100 and 1,000 and has a linear or branched structure.

As long as the function of the antibody of the present invention is maintained, various glycosylation and/or pegylation patterns may be modified by methods known in the art, and the antibody of the present invention may be modified by various glycosylation and/or pegylation methods. All of the mutant monoclonal antibodies or antigen-binding fragments thereof having a modified pegylation pattern are included.

A method for producing the monoclonal antibody is not particularly limited, and may be produced by a general method known in the art. As an example, it can be produced by a chemical peptide synthesis method, and the gene encoding the monoclonal antibody can be amplified by PCR (polymerase chain reaction) or synthesized by a known method, and then cloned into an expression vector and expressed. , It may be prepared using a hybridoma cell line derived from lymphocytes obtained by injecting the immunogen of the monoclonal antibody into an animal, and as another example, a protein derived from undifferentiated dental pulp stem cells to which the monoclonal antibody specifically binds. It may be prepared by inoculating an animal as an immunogen and using a hybridoma cell line prepared using lymphocytes obtained from the animal.

In addition, the present invention provides a polynucleotide encoding the amino acid of the monoclonal antibody or antigen-binding fragment thereof. Specifically, the gene encoding the monoclonal antibody specifically binding to the IFITM3 protein epitope may be the nucleotide sequence represented by SEQ ID NO: 4.

The polynucleotides encoding the light and heavy chains of the monoclonal antibody or antigen-binding fragment thereof of the present invention have codons preferred in organisms intended to express the light and heavy chains of the antibody or fragments thereof due to codon degeneracy. In consideration of this, various modifications can be made to the coding region within the range that does not change the amino acid sequences of the light and heavy chains of the antibody expressed from the coding region, and within the range that does not affect gene expression even in parts other than the coding region Various modifications or modifications may be made, and those skilled in the art will understand that such modified genes are also included in the scope of the present invention. That is, as long as the polynucleotide of the present invention encodes a protein having an activity equivalent thereto, one or more nucleic acid bases may be mutated by substitution, deletion, insertion, or a combination thereof, and these are also included in the scope of the present invention. The sequence of such a polynucleotide may be single- or double-stranded, and may be a DNA molecule or an RNA (mRNA) molecule.

Vector for the production of IFITM3 protein-specific monoclonal antibodies

Another aspect of the present invention provides a recombinant expression vector comprising a polynucleotide encoding the amino acid of the monoclonal antibody or antigen-binding fragment thereof.

As used herein, the term "vector" refers to a plasmid vector as a means for expressing a gene of interest in a host cell; phagemid vectors; cosmid vector; And viral vectors such as bacteriophage vectors, adenoviral vectors, retroviral vectors and adeno-associated viral vectors, etc. are included, but are not limited thereto, in order to achieve the purpose of expressing the anti-IFITM3 antibody or antigen-binding fragment thereof of the present invention. .

According to a preferred embodiment of the present invention, the polynucleotide encoding the anti-IFITM3 antibody or antigen-binding fragment thereof in the vector of the present invention is operatively linked to a promoter.

The term "operably linked" refers to a functional linkage between a nucleic acid expression control sequence (eg, a promoter, signal sequence, or array of transcriptional regulatory factor binding sites) and another nucleic acid sequence, whereby the control sequence It will regulate the transcription and/or translation of said other nucleic acid sequence.

The vector system of the present invention can be constructed through various methods known in the art, and specific methods for this are described in Sambrook et al. (2001), Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, which is incorporated herein by reference.

Vectors of the present invention may typically be constructed as vectors for cloning or vectors for expression. In addition, the vector of the present invention can be constructed using a prokaryotic cell or a eukaryotic cell as a host. When the vector of the present invention is an expression vector and a prokaryotic cell is used as a host, a strong promoter capable of promoting transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter and T7 promoter, etc.), a ribosome binding site for initiation of translation, and a transcription/translation termination sequence. When E. coli (eg, HB101, BL21, DH5α, etc.) is used as the host cell, the promoter and operator regions of the E. coli tryptophan biosynthetic pathway (Yanofsky, C. (1984), J. Bacteriol., 158: 1018- 1024) and the leftward promoter of phage λ (pLλ promoter, Herskowitz, I. and Hagen, D. (1980), Ann. Rev. Genet., 14:399-445) can be used as a regulatory region.

On the other hand, vectors that can be used in the present invention are plasmids often used in the art (e.g., pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14 , pGEX series, pET series, and pUC19, etc.), phagemids (eg, pComb3X), phage (eg, λgt4·λB, λ-Charon, and M13, etc.) or viruses (eg, SV40, etc.).

On the other hand, when the vector of the present invention is an expression vector and a eukaryotic cell is used as a host, a promoter derived from the genome of a mammalian cell (e.g., metallotionine promoter) or a promoter derived from a mammalian virus (e.g., adenovirus) viral late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter and four promoters of HSV) can be used, and usually have a polyadenylation sequence as a transcription termination sequence.

The vector of the present invention may be fused with other sequences as needed to facilitate protein purification, and the fused sequences are, for example, glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA) ), FLAG (IBI, USA) and 6x His (hexahistidine; Quiagen, USA) may be used, but are not limited thereto. In addition, the expression vector of the present invention may include an antibiotic resistance gene commonly used in the art as a selection marker, for example, ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin , and genes for resistance to neomycin and tetracycline.

Vector transfectant for preparation of IFITM3 protein-specific monoclonal antibody

In addition, the present invention provides a transformant transformed with the recombinant expression vector.

Any host cell known in the art may be used as a host cell capable of stably cloning and expressing the vector of the present invention continuously, for example, Escherichia coli, Bacillus subtilis and Bacillus thuringen. strains of the genus Bacillus, such as cis, Streptomyces, Pseudomonas (e.g. Pseudomonas putida), Proteus mirabilis or Staphylococcus (e.g. Pseudomonas putida) Examples include, but are not limited to, prokaryotic host cells such as Staphylocus carnosus). The host cell is preferably E. coli, more preferably E. coli ER2537, E. coli ER2738, E. coli XL-1 Blue, E. coli BL21(DE3), E. coli JM109, E. coli DH series, E. coli TOP10, E. coli TG1 and E. coli HB101. In the present invention, anti-IFITM3 antibodies or antigen-binding fragments thereof can be overexpressed using E. coli and mass-produced at low cost.

Methods for delivering the vector of the present invention into host cells include the CaCl ₂ method (Cohen, SN et al. (1973), Proc. Natl. Acac. Sci. USA, 9:2110-2114), the Hanhan method (Cohen, SN et al. (1973), Proc. Natl. Acac. Sci. USA, 9:2110-2114; and Hanahan, D. (1983), J. Mol. Biol., 166:557-580) and the electroporation method (Dower, WJ et al. (1988), Nucleic. Acids Res., 16: 6127-6145) and the like.

The vector injected into the host cell can be expressed in the host cell, and in this case, a large amount of the anti-IFITM3 antibody or antigen-binding fragment thereof of the present invention can be obtained.

Method for producing IFITM3 protein-specific monoclonal antibody

The present invention also, (a) preparing a culture medium by culturing the transformant; and (b) purifying the monoclonal antibody or antigen-binding fragment thereof of the present invention from the culture medium in step (a), wherein the monoclonal antibody or antigen thereof specifically binds to IFITM3 (Interferon-Induced Transmembrane Protein 3). Methods of making binding fragments are provided.

In the above production method, the transformant may be cultured according to an appropriate medium and culture conditions known in the art. This culture process can be easily adjusted and used by those skilled in the art according to the selected strain or animal cell.

The antibody obtained by culturing the transformant may be used in an unpurified state, and in addition, impurities are removed by various conventional methods, such as centrifugation or ultrafiltration, and the resultant is dialysis, salt precipitation, chromatography etc. can be used, and these can be used individually or in combination. Among them, affinity chromatography is most commonly used, and there are ion exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, and hydroxylapatite chromatography.

Use of IFITM3 protein-specific monoclonal antibodies

Another aspect of the present invention is a) obtaining a sample isolated from a subject suspected of being infected with avian influenza virus; b) contacting the obtained sample with a monoclonal antibody specifically binding to the epitope of the IFITM3 protein; And c) detecting the reaction between the monoclonal antibody and the sample; it provides a method for providing information for diagnosing an avian influenza virus infection.

As used herein, the term "avian influenza virus" refers to a causative virus that can cause avian influenza in chickens, ducks, wild birds, and the like. The virus has a variety of serotypes, such as 16 types of HA and 9 types of NA, depending on the genes of HA and NA in the outer membrane, and it is known that there is no cross-protection between different serotypes. Among them, highly pathogenic avian influenza (HPAI) that has occurred so far is known to be caused by H5 or H7 serotypes. The avian influenza virus is known to be able to infect humans through contact with infected birds, and it is known that the main infectious agent is feces of infected birds. When avian influenza virus infects humans, it is respiratory symptoms such as coughing and shortness of breath, and may be accompanied by symptoms throughout the body such as fever, chills, and muscle pain, and gastrointestinal symptoms such as diarrhea, headaches, and decreased consciousness. Symptoms related to the central nervous system may be accompanied, and there are cases in which only gastrointestinal symptoms or symptoms related to the central nervous system appeared without respiratory symptoms.

In the present invention, the avian influenza virus is not particularly limited thereto, but preferably may be various avian influenza viruses having genotypes including NP antigens such as H1N1, H5N3, H7N1, H7N7, and H9N2.

Step a) is a step of obtaining a sample isolated from a subject suspected of being infected with avian influenza virus.

The term "sample" may include tissues, cells, whole blood, serum, plasma, tissue autopsy samples (brain, skin, lymph nodes, spinal cord, etc.), cell culture supernatants, ruptured eukaryotic cells, bacterial expression systems, etc. It is not limited. By reacting these samples with the monoclonal antibody of the present invention with or without manipulation, the presence of the IFITM3 protein or the presence or absence of avian influenza virus infection can be confirmed.

Step b) is a step of contacting the sample obtained in step a) with a monoclonal antibody that specifically binds to the epitope of the IFITM3 protein, forming an antigen-antibody complex between the monoclonal antibody of the present invention and the IFITM3 protein antigen in the sample It is a step to

In the present invention, the term "antigen-antibody complex" refers to a combination of an IFITM3 protein antigen in a sample and a monoclonal antibody or antigen-binding fragment thereof according to the present invention recognizing the antigen.

Step c) is a step of detecting a reaction between the monoclonal antibody and the sample, and is a process of confirming whether an antigen-antibody complex is formed between the monoclonal antibody of the present invention and the IFITM3 protein antigen in the sample.

The formation of such an antigen-antibody complex can be measured according to a conventional immunoassay method, and ELISA (enzyme linked immunosorbent assay) using an antibody to the IFITM3 protein, immunoblotting, western blotting, immunoblot, It can be detected and measured through immunocytochemistry and FACS (Fluorescence-activated cell sorting), but is not limited thereto.

By analyzing the intensity of the final signal by the immunoassay process, the presence or absence of avian influenza virus infection can be diagnosed. That is, when the IFITM3 protein of the present invention is overexpressed in a sample isolated from a suspected individual and the signal is stronger than that of a sample isolated from a normal individual, it is diagnosed as being infected with avian influenza virus.

The method for diagnosing the avian influenza virus infection can be detected by reacting the IFITM3-specific monoclonal antibody of the present invention with an isolated sample of an individual suspected of avian influenza virus infection and analyzing the formation of an antigen-antibody complex, , it is possible to provide information for diagnosing the presence or absence of avian influenza virus infection.

In addition, the present invention provides a kit for diagnosing avian influenza virus infection, comprising a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to the epitope of the IFITM3 protein.

The kit according to the present invention includes an antibody against the IFITM3 protein of the present invention, and the presence or absence of avian influenza virus infection can be diagnosed by analyzing the signal generated by the reaction between the sample and the antibody. In this case, the signal may include, for example, alkaline phosphatase, β-galactosidase, horse radish peroxidase, luciferase, or cytochrome P450 coupled to the antibody, but is not limited thereto. .

In addition, the kit according to the present invention may include a label that generates a detectable signal, and the label may include a chemical (eg, biotin), an enzyme (eg, alkaline phosphatase, β-galactosidase, hose radish peroxidase and cytochrome P450), radioactive substances (eg 14C, 125I, 32P and 35S), fluorescent substances (eg fluorescein), luminescent substances, chemiluminescent substances and fluorescence resonance energy transfer (FRET) ), but is not limited thereto.

Measurement of the activity or signal of an enzyme used to diagnose the presence or absence of infection with avian influenza virus can be performed according to various methods known in the art. Through this, the expression of the IFITM3 protein can be analyzed qualitatively or quantitatively.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1. Sequence Comparison of IFITM3 Proteins

Protein sequence alignment was performed using ClustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalo). The protein sequence of IFITM3 protein is chicken ( Gallus gallus , NP_001336990.1), duck ( Anas platyrhynchos , AHL27882.1), goose ( Anser cygnoides , AQM74179.1), ibis ( Nipponia nippon , XP_009461927.1), human ( Homo sapiens , AFF60355.1), chimpanzee ( Pan troglodytes , NP_001185686.1), mouse ( Mus musculus , NP_079654.1), cow ( Bos taurus , NP_001071609.1), pig ( Sus scrofa , AFG25600.1) and goat ( Capra hircus , AIW39806.1) were obtained from GenBank of the National Center for Biotechnology Information (NCBI).

Multiple sequence alignment showed very low homology in the N-terminal domain (NTD) and C-terminal domain (CTD) between mammals and birds (Fig. 1). .

Example 2. Sequence Comparison of Chicken IFITM3 and IFITM1 Proteins

Protein sequence alignment was performed using ClustalW2. The protein sequences of the chicken IFITM3 ( Gallus gallus , NP_001336990.1) and IFITM1 ( Gallus gallus , NP_001336988.1) proteins were obtained from NCBI's GenBank.

Sequence alignment of the chicken IFITM3 and IFITM1 proteins showed low homology in the N-terminal domain and C-terminal domain between the IFITM3 and IFITM1 proteins (FIG. 2).

Example 3. Epitope selection of chicken IFITM3 protein

Through the Antibody Epitope Prediction Program (Immune Epitope Database and Analysis Resource, IEDB Analysis Resource), the top 20 for the chicken IFITM3 protein sequence (137a.a) [SEQ ID NO: 1] targeted in the present invention An epitope, that is, an antigenic determinant sequence was selected.

By applying the three prediction principles, three commonly predicted epitopes were first selected, and among them, the epitope 'PPYEPLMDGMDMEGKTRS [SEQ ID NO: 2]' with the highest score was selected as the final candidate epitope. (Fig. 2).

The epitope selected as the final candidate was determined by excluding variations due to genetic polymorphism and the possibility of being located inside the cell membrane in order to increase sensitivity with the reacting antibody.

Example 4. Synthesis of Target Antigen Peptide

The epitope sequence 'PPYEPLMDGMDMEGKTRS [SEQ ID NO: 2]' for the chicken IFITM3 protein finally selected in Example 3 was synthesized from the C-terminus using the Solid Phase Peptide Synthesis method. . Specifically, the peptide synthesis was performed by the solid phase Fmoc (9-Fluorenylmethoxycarbonyl) Chemistry method under continuous flow conditions using an organic chemical reaction. The amino acid sequence of the epitope 'PPYEPLMDGMDMEGKTRS' was synthesized one by one in a predetermined sequence through an automatic synthesizer. The synthesized crude peptide was purified using reversed phase-high performance liquid chromatography (HPLC) and dried using a lyophilizer. Thereafter, for QC (Quality Control) of the peptide, a final epitope was obtained using analytical reversed-phase HPLC and LC/Mass mass spectrometry.

Example 5. scFv construction and selection of anti-IFITM3 monoclonal antibodies against chicken IFITM3 protein

Phage displaying a chicken single-chain variable fragment (scFv) antibody library was constructed (cf. C.F. Barbas, Phage Display: a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001). Biopanning was performed on the epitope of the IFITM3 protein synthesized in Example 4 (PPYEPLMDGMDMEGKTRS [SEQ ID NO: 2]; 12th to 29th amino acid region of IFITM3 (GenBank Accession No. NP_001336990.1)) Afterwards, ELISA (enzyme-linked immunosorbent assay) was performed on the scFv-display phage (see CF Barbas, Phage Display: a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001).

As a result, an scFv binding to the synthetic peptide (PPYEPLMDGMDMEGKTRS [SEQ ID NO: 2]) was selected. Specifically, the degree of each binding reaction was confirmed on a plate coated with the chicken IFITM3 protein epitope as an antigen and a plate not coated with the antigen as a control (Background). Thereafter, clones were selected by deriving a final O.D value (Delta) from the difference between the obtained O.D. values.

As a result of the ELISA screening, 42 positive binders with no background were identified (FIG. 3). Among the identified 42 positive binders, the binding ability with the chicken tissue sample was confirmed using the periplasmic fraction of the top 20 clones (FIG. 4).

Example 6. Confirmation of binding ability of monoclonal antibody scFv to chicken IFITM3 protein

In Example 5, two clones named 1E12 and 1A10 showing binding affinity to protein samples extracted from chicken tissue, specifically lung tissue, were selected, and antibodies were prepared and sequenced.

In order to verify the detection ability of the antibody produced above, protein samples extracted from various tissues of chickens, such as lung, bronchus, bursa of fabricius, caecum, rectum, etc. Western blot was performed using . At this time, a protein sample synthesized from the entire chicken IFITM3 protein sequence was used as a positive control.

As a result of Western blotting, in the case of the antibody prepared from the 1A10 clone, the detection ability of the positive control was weak, but in the case of the antibody prepared from the 1E12 clone, the detection ability was excellent not only in the positive control but also in various tissue samples of actual chickens. (FIGS. 5A and 5B).

The amino acid sequence and base sequence of the monoclonal antibody anti-IFITM3 scFv prepared from the clone named 1E12 and having excellent specific binding ability to the chicken IFITM3 protein are summarized in Table 1 below.

anti-IFITM3 scFv sequence number scFV polypeptide LTQPSSVSANPGENVEITCSGGGSSSYYDHQQKAPGSAPVTLIYDNTNRPSNIPSRFSGSKLGSTGTLTITGVRADDEAVYFCDSEDCWPSLGSEPTTRLSISVTVKTAGSYAGIFGAGTTLTVLGQ SSRSSGGGGSSGGGGS AVTLDESGGGLQTPGGALSLVCKASGFTFSSYPMFWVRQAPGKGLEYVAAISKDGSFTLYGAVVKGRATISRDNGQSTVRLQLNNLRAEDTGTYYCARGGGGGSGCYSGFCHAGSIDAWGHGTEVIVSSTS 3 scFv gene CTGACTCAGCCGTCCTCGGTGTCAGCGAACCCGGGAGAAAACGTCGAGATCACCTGCTCCGGGGGTGGTAGCAGCAGCTACTATGACTGACACCAGCAGAAGGCACCTGGCAGTGCCCCTGTCACTCTGATCTATGACAACACCAACAGACCCTCGAACATCCCTTCACGATTCTCCGGTTCCAAACTTGGCTCCACGGGCACATTAACCATCACTGGGGTCCGAGCCGACGACGAGGCTGTCTATTTCTGTGACAGTGAAGACTGCTGGTAGCTGACTCAGCCGTCCTCGGTGTCAGCGAACCCGGGAGAAAACGTCGAGATCACCTGCTCCGGGGGTGGTAGCAGCAGCTACTATGACTGACACCAGCAGAAGGCACCTGGCAGTGCCCCTGTCACTCTGATCTATGACAACACCAACAGACCCTCGAACATCCCTTCACGATTCTCCGGTTCCAAACTTGGCTCCACGGGCACATTAACCATCACTGGGGTCCGAGCCGACGACGAGGCTGTCTATTTCTGTGACAGTGAAGACTGCTGGTAGCTATGCTGGTATATTTGGGGCCGGGACAACCCTGACCGTCCTAGGTCAGTCCTCTAGATCTTCCAGCGGTGGTGGCAGCTCCGGTGGTGGCGGTTCCGCCGTGACGTTGGACGAGTCCGGGGGCGGCCTCCAGACGCCCGGAGGAGCGCTCAGCCTCGTCTGCAAGGCCTCCGGGTTCACCTTCAGCAGTTATCCCATGTTCTGGGTGCGACAGGCACCCGGGAAGGGGCTGGAATACGTCGCAGCTATTAGCAAGGATGGTAGTTTCACACTCTACGGGGCGGTGGTGAAGGGCCGTGCTACCATCTCGAGGGACAACGGGCAGAGCACAGTGAGGCTGCAGCTGAACAACCTCAGGGCTGAGGACACCGGCACCTACTACTGCGCCAGAGGTGGTGGTGGTGGTAGTGGTTGTTATAGTGGTTTTTGTCATGCTGGTAGCATCGACGCATGGGGCCACGGGACCGAAGTCATCGTCTCCTCCACTAGT
4

Example 7. Mass production of monoclonal antibody scFv against chicken IFITM3 protein

A clone named 1E12 (scFV-IFITM3-expressing Ecoli TG1 clone) capable of producing a monoclonal antibody anti-IFITM3 scFv with excellent specific binding ability to chicken IFITM3 protein was seeded, and antibody was obtained through IPTG induction. was expressed. E.coil TG1 expressing the anti-IFITM3 scFv antibody was disrupted by sonication, and the obtained supernatant of E.coli bacterial cells was flowed through a Ni-NTA column. Anti-IFITM3 scFv antibody bound to Ni-NTA was eluted with elution buffer. The eluted anti-IFITM3 scFv antibody was purified by exchanging the buffer.

<110> INDUSTRIAL COOPERATION FOUNDATION JEONBUK NATIONAL UNIVERSITY <120> MONOCLONAL ANTIBODY SPECIFICALLY BINDING TO IFITM3 PROTEIN OF CHICKEN AND USE THEREOF <130> DHP20-546 <160> 5 <170> KoPatentIn 3.0 <210> 1 <211> 136 <212> PRT <213> artificial sequence <220> <223> chicken IFITM3 protein <400> 1 Met Glu Arg Val Arg Ala Ser Gly Pro Gly Val Pro Pro Tyr Glu Pro 1 5 10 15 Leu Met Asp Gly Met Asp Met Glu Gly Lys Thr Arg Ser Thr Val Val 20 25 30 Thr Val Glu Thr Pro Leu Val Pro Pro Pro Arg Asp His Leu Ala Trp 35 40 45 Ser Leu Cys Thr Thr Leu Tyr Ala Asn Val Cys Cys Leu Gly Phe Leu 50 55 60 Ala Leu Val Phe Ser Val Lys Ser Arg Asp Arg Lys Val Leu Gly Asp 65 70 75 80 Tyr Ser Gly Ala Leu Ser Tyr Gly Ser Thr Ala Lys Tyr Leu Asn Ile 85 90 95 Thr Ala His Leu Ile Asn Val Phe Leu Ile Ile Leu Ile Ile Ala Leu 100 105 110 Val Ala Ser Gly Thr Ile Met Val Ala Asn Ile Phe Asn His Gln Gln 115 120 125 Gln His Pro Glu Phe Ile Gly Pro 130 135 <210> 2 <211> 18 <212> PRT <213> artificial sequence <220> <223> IFITM3 epitope <400> 2 Pro Pro Tyr Glu Pro Leu Met Asp Gly Met Asp Met Glu Gly Lys Thr 1 5 10 15 Arg Ser <210> 3 <211> 274 <212> PRT <213> artificial sequence <220> <223> anti-IFITM3 scFV <400> 3 Leu Thr Gln Pro Ser Ser Val Ser Ala Asn Pro Gly Glu Asn Val Glu 1 5 10 15 Ile Thr Cys Ser Gly Gly Gly Ser Ser Ser Tyr Tyr Asp His Gln Gln 20 25 30 Lys Ala Pro Gly Ser Ala Pro Val Thr Leu Ile Tyr Asp Asn Thr Asn 35 40 45 Arg Pro Ser Asn Ile Pro Ser Arg Phe Ser Gly Ser Lys Leu Gly Ser 50 55 60 Thr Gly Thr Leu Thr Ile Thr Gly Val Arg Ala Asp Asp Glu Ala Val 65 70 75 80 Tyr Phe Cys Asp Ser Glu Asp Cys Trp Pro Ser Leu Gly Ser Glu Pro 85 90 95 Thr Thr Arg Leu Ser Ile Ser Val Thr Val Lys Thr Ala Gly Ser Tyr 100 105 110 Ala Gly Ile Phe Gly Ala Gly Thr Thr Leu Thr Val Leu Gly Gln Ser 115 120 125 Ser Arg Ser Ser Gly Gly Gly Gly Ser Ser Gly Gly Gly Gly Ser Ala 130 135 140 Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly Ala 145 150 155 160 Leu Ser Leu Val Cys Lys Ala Ser Gly Phe Thr Phe Ser Ser Tyr Pro 165 170 175 Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val Ala 180 185 190 Ala Ile Ser Lys Asp Gly Ser Phe Thr Leu Tyr Gly Ala Val Val Lys 195 200 205 Gly Arg Ala Thr Ile Ser Arg Asp Asn Gly Gln Ser Thr Val Arg Leu 210 215 220 Gln Leu Asn Asn Leu Arg Ala Glu Asp Thr Gly Thr Tyr Tyr Cys Ala 225 230 235 240 Arg Gly Gly Gly Gly Gly Ser Gly Cys Tyr Ser Gly Phe Cys His Ala 245 250 255 Gly Ser Ile Asp Ala Trp Gly His Gly Thr Glu Val Ile Val Ser Ser 260 265 270 Thr Ser <210> 4 <211> 1036 <212> DNA <213> artificial sequence <220> <223> anti-IFITM3 scFV_nucleotide <400> 4 ctgactcagc cgtcctcggt gtcagcgaac ccgggagaaa acgtcgagat cacctgctcc 60 gggggtggta gcagcagcta ctatgactga caccagcaga aggcacctgg cagtgcccct 120 gtcactctga tctatgacaa caccaacaga ccctcgaaca tcccttcacg attctccggt 180 tccaaacttg gctccacggg cacattaacc atcactgggg tccgagccga cgacgaggct 240 gtctatttct gtgacagtga agactgctgg tagctgactc agccgtcctc ggtgtcagcg 300 360 tgacaccagc agaaggcacc tggcagtgcc cctgtcactc tgatctatga caacaccaac 420 agaccctcga acatcccttc acgattctcc ggttccaaac ttggctccac gggcacatta 480 accatcactg gggtccgagc cgacgacgag gctgtctatt tctgtgacag tgaagactgc 540 tggtagctat gctggtatat ttggggccgg gacaaccctg accgtcctag gtcagtcctc 600 tagatcttcc agcggtggtg gcagctccgg tggtggcggt tccgccgtga cgttggacga 660 gtccgggggc ggcctccaga cgcccggagg agcgctcagc ctcgtctgca aggcctccgg 720 gttcaccttc agcagttatc ccatgttctg ggtgcgacag gcacccggga aggggctgga 780 atacgtcgca gctattagca aggatggtag tttcacactc tacggggcgg tggtgaaggg 840 ccgtgctacc atctcgaggg acaacgggca gagcacagtg aggctgcagc tgaacaacct 900 cagggctgag gacaccggca cctactactg cgccagaggt ggtggtggtg gtagtggttg 960 ttatagtggt ttttgtcatg ctggtagcat cgacgcatgg ggccacggga ccgaagtcat 1020 cgtctcctcc actagt 1036 <210> 5 <211> 56 <212> DNA <213> artificial sequence <220> <223> IFITM3 epitope_nucleotide <400> 5 ccaccgtatg aacccctgat ggacgggatg gacatggagg ggaagacccg cagcac 56

Claims (10)

delete delete delete A monoclonal antibody represented by the amino acid sequence of SEQ ID NO: 3 that specifically binds to the epitope of IFITM3 represented by the amino acid sequence of SEQ ID NO: 2. delete A polynucleotide encoding the amino acid of the monoclonal antibody of claim 4. A recombinant expression vector comprising the polynucleotide of claim 6. A transformant transformed with the recombinant expression vector of claim 7. a) obtaining a sample isolated from a subject suspected of being infected with avian influenza virus;
b) contacting the obtained sample with a monoclonal antibody represented by the amino acid sequence of SEQ ID NO: 3 that specifically binds to an epitope of the IFITM3 protein represented by the amino acid sequence of SEQ ID NO: 2; and
c) detecting a reaction between the monoclonal antibody and the sample; a method for providing information for diagnosing infection of avian influenza virus, comprising: According to claim 9,
The detection is detected by an assay selected from the group consisting of enzyme linked immunosorbent assay (ELISA), immunoblotting, western blotting, immunocytochemistry, and fluorescence-activated cell sorting (FACS). How to be.

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