KR20180050261A - Novel fusion proteins comprising single chain fragment variable and viral coat protein, and uses thereof - Google Patents

Abstract

The present invention relates to fusion protein comprising a single chain variable fragment (scFv) and viral coat protein, and use thereof. Fusion protein with a single chain variable fragment and viral coat protein combined has a nanocluster formed through self assembly characteristic of the viral coat protein, and an antigen binding capacity of the single chain variable fragment is improved by a clustering effect of an antigen recognition site. As a result, when a fusion protein is manufactured by using a single chain variable fragment having various antigen recognition sites, securement for a polyclonal antibody system which is effective for detecting antigen and development of a diagnosis kit, a protein chip or the like using the same is possible. In addition, a treatment product or a diagnosing and detecting product are introduced to the inside or the surface of the nanocluster, and thus, the fusion protein are useful as a drug transporter for treating or diagnosing disease. Fusion protein having enhanced refinement purity and expression ratio, and noticeably improved solubility can be acquired by fusing maltose binding protein (MBP) and a histidine tag to an N-terminal and a C-terminal of the fusion protein respectively. Furthermore, since the fusion protein utilizes a colon bacillus protein expression system, production costs and time can be reduced in comparison with antibody production.

Description

{Novel fusion proteins comprising single chain fragment variable and viral coat protein, and uses thereof}

The present invention relates to a fusion protein comprising a single chain antibody fragment (scFv) and a viral envelope protein, and a use thereof.

The virus envelope protein is a protein that forms the structure of a virus, and has the ability to self-assemble, so it has a unique characteristic of forming a large spherical or rod-shaped three-dimensional structure by gathering tens to hundreds of monomers. For example, in the case of Brome Mosaic Virus (BMV), 180 monomers gather to form spherical particles, the size of which is about 30 nm in outer diameter and about 20 nm in inner diameter.

Viral envelope proteins may be dispersed into monomers depending on various conditions, or form nanoclusters in which monomers are bound. By using these properties, various substances can be captured in the viral envelope proteins. In general, nanocluster-shaped virus envelope proteins have nucleic acids, metals, and iron oxide inside, but in addition to manganese oxide, cobalt oxide, nickel oxide, indium oxide, iron sulfide, cadmium sulfide, selenocadmium, and selenoa Examples of including various inorganic metals such as lead have been reported. Therefore, various functional groups can be introduced inside and outside of the viral envelope protein through molecular biological methods, and the viral envelope protein structure into which various functional groups are introduced can be modified using an appropriate functional group as an optional method. It has the advantage of being able to impart a variety of necessary physicochemical properties.

On the other hand, the target-oriented drug delivery system refers to a technology designed to selectively deliver drugs to the treatment site so that healthy tissues are not exposed to drugs, and at the same time, excellent therapeutic effects can be exhibited with only a small amount of drugs. If a target-oriented drug delivery system is used, the drug treatment effect can be maximized by concentrating the drug on a specific part of the body with a disease, and side effects caused by highly toxic drugs such as anticancer drugs can be minimized. Recently, the technology development of Theranosis = Therapy+diagnosis, which can perform treatment and diagnosis at the same time using a target-oriented drug delivery system, is actively progressing. In other words, by attaching a molecular imaging probe to a target-oriented drug delivery system and imaging the tracer using non-invasive in vivo imaging, it is possible to monitor the treatment process in real time as well as drug delivery. It refers to the skills that can be done.

Signal peptides and antibodies are widely used as representative substances that impart target orientation. Antibodies are proteins produced by the immune response of vertebrates, and are immune proteins that specifically recognize and bind to a specific site of an antigen to inactivate or eliminate the action of the antigen. It is known that it is a very difficult technique to modify. Antibodies have two heavy chains and two light chains, and are basically Y-shaped. The variable regions of the light and heavy chains are combined to form an antigen binding site, which is an antibody using a single chain variable fragment (scFv), a small fragment that is artificially linked through a peptide linker. Research to perform the function of is in progress.

Because single-chain antibody fragments are smaller in size than normal antibodies, their penetration rate into tissues or cancer tissues is high, and they can be mass-produced in various expression systems including the E. coli system, and various molecular biological modifications are possible, and the time and cost of production. There is an advantage that can be reduced. However, since the size is small and there is no Fc region, it has the disadvantage of short half-life in the human body. For example, in the case of reported thyroid stimulating hormone (TSH)-ferritin, it is expressed in E. coli, but there is a problem in that a precipitate is formed and self-assembly is not performed, so a refolding process using an additional strong acid is required. This consumes a lot of time and cost for the production of functional protein nanoparticles, and may adversely affect the inherent functions (selectivity, binding force, stability) by denaturing the protein.

Nanostructures such as liposomes, which are mainly composed of phospholipids, which are components of biological cell membranes, are widely used as pharmaceutical delivery vehicles. However, liposomes are highly toxic in cells and are lost from the circulatory system due to the phagocytosis of macrophages when injected with drugs. The micelle, another nanostructure, is a carrier consisting of chain-type molecules having both hydrophilic and hydrophobic sites. In an aqueous solution, the hydrophobic parts gather together to form a spherical shape. It is used to increase the value. In the case of anticancer drugs, drug delivery systems using nanoparticles use a method that selectively accumulates in cancer tissues through EPR (enhanced permeability and retention) effect, a passive targeting method, but there is a disadvantage that it is difficult to always expect a certain effect.

Therefore, for more effective targeting, a method of using a drug delivery system to which an antibody capable of recognizing an antigen or receptor expressed at a target site is used has been developed. Currently, the method of forming covalent chemical bonds, which is used as a method of immobilizing an antibody for targeting, has poor reproducibility and may impair the unique function of the antibody. In the case of biologically-derived medicines, as well as general medicines, if structural changes are accompanied during the manufacturing process, accurate identification of structural changes, proof of drug efficacy, and toxicity tests should be performed in order to obtain approval as a medicine from the KFDA. Therefore, it is almost impossible to apply a target-oriented drug delivery system using an antibody to the human body without securing the reproducibility of antibody immobilization.

Korean Patent Publication No. 10-2013-0039672

In order to solve the conventional problems as described above, the present inventors have made a intensive study for the development of a method for improving the utility of single-chain antibody fragments, and produced a protein in which a single-chain antibody fragment and a viral envelope protein are fused, The fusion protein was experimentally confirmed to form a nanocluster by the self-assembly property of the viral envelope protein, thereby improving the antigen-binding ability, and based on this, the present invention was completed.

Accordingly, an object of the present invention is to provide a single-chain antibody fragment (scFv) and a viral envelope protein combined, a fusion protein, and a method for preparing the same.

Another object of the present invention is to provide a polynucleotide encoding the fusion protein, a recombinant expression vector comprising the polynucleotide, and a transformant transformed with the expression vector.

In addition, another object of the present invention is to provide a nanocluster comprising the fusion protein, a diagnostic kit comprising the nanocluster, and a protein chip.

In addition, it is another object of the present invention to provide a drug delivery system composition comprising a drug introduced into the nanocluster.

In addition, another object of the present invention is to provide a method for detecting an antigen using the nanocluster.

In order to achieve the object of the present invention as described above, the present invention provides a fusion protein in which a single chain antibody fragment (scFv) and a viral envelope protein are combined.

In one embodiment of the present invention, the single chain antibody fragment may be bound to the N-terminus or C-terminus of the viral envelope protein.

In another embodiment of the present invention, the single chain antibody fragment may include a heavy chain fragment and a light chain fragment of an antibody.

In another embodiment of the present invention, the viral envelope protein may be a protein derived from a brom mosaic virus or a cucumber mosaic virus.

In another embodiment of the present invention, the bromosaic virus and the oimosaic virus-derived envelope protein may be encoded by the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 8, respectively.

In another embodiment of the present invention, the fusion protein may be a histidine tag at the C-terminus or a maltose binding protein further fused at the N-terminus.

In addition, the present invention provides a polynucleotide encoding the fusion protein, a recombinant expression vector comprising the polynucleotide, and a transformant transformed with the recombinant expression vector.

In addition, the present invention provides a method for producing a fusion protein, comprising the following steps.

(a) preparing a recombinant expression vector comprising a polynucleotide encoding the fusion protein;

(b) introducing the prepared recombinant expression vector into a host cell to obtain a transformant; And

(c) obtaining the expressed fusion protein after expressing the fusion protein from the transformant.

In addition, the present invention provides a nanocluster comprising the fusion protein.

In one embodiment of the present invention, the nanocluster may be formed by self-assembly of viral envelope proteins constituting the fusion protein.

In addition, the present invention provides a drug delivery system composition comprising a drug introduced into the nanocluster.

In one embodiment of the present invention, the drug may be a therapeutic agent or a diagnostic/detection agent.

In another embodiment of the present invention, the therapeutic agent is a nucleic acid, a peptide, an antibody, an antibody fragment, a compound, a toxin, a nucleolytic enzyme, a hormone, an immunomodulator, an anticancer agent, a chelator, a boron compound, or a photoactive agent. I can.

In another embodiment of the present invention, the diagnostic/detection agent may be a radioisotope, a dye, a contrast agent, a fluorescent protein, a fluorescent compound, or a magnetic resonance imaging (MRI) contrast enhancer.

In another embodiment of the present invention, the drug is bound to the interior or surface of the nanocluster to enable targeted drug delivery.

In addition, the present invention provides a diagnostic kit or protein chip including the nanoclusters.

In addition, the present invention provides a method for detecting an antigen, comprising the step of detecting specific binding of an antigen-antibody by treating a clinical sample to the nanocluster.

In one embodiment of the present invention, the clinical sample may be patient-derived blood, serum, plasma, body fluid, saliva, urine, intestinal fluid, lymph fluid, or peritoneal fluid.

The fusion protein in which the single-chain antibody fragment and the viral envelope protein are combined according to the present invention form a nanocluster through the self-assembly property of the viral envelope protein, and the antigen-binding ability of the single-chain antibody fragment by the clustering effect of the antigen recognition site through this It was confirmed that this is improved. In the case of manufacturing a fusion protein using a single chain antibody fragment sequence having various antigen recognition sites, it is necessary to secure a polyclonal antibody system effective for antigen detection and to develop diagnostic kits and protein chips using the same. It is possible. In addition, by introducing a therapeutic agent or a diagnostic/detection agent into the interior or surface of the nanocluster, it can be usefully used as a drug delivery system for the treatment or diagnosis of a disease.

On the other hand, by fusing maltose binding protein (MBP) and histidine tag, respectively, to the N-terminus and C-terminus of the fusion protein, purification purity and expression level are improved, and a fusion protein with remarkably improved solubility can be obtained. Since the fusion protein uses an E. coli protein expression system, it has the advantage of relatively reducing production cost and time compared to antibody production.

1 is a conceptual diagram showing the configuration of the single-chain antibody fragment of the present invention-bromosaic virus envelope protein (scFv-BMV) fusion protein.
2 is a result of confirming the size and protein purity of the fusion protein through SDS-PAGE after purification of the scFv-BMV fusion protein.
FIG. 3 is an image of scFv-BMV fusion protein observed with a transmission electron microscope, and is a result showing that an aggregate structure of 60 monomers forms a spherical shape as a whole.
Figure 4 is a result showing the targeting of HER2-expressing cancer cells of the FITC fluorescence-labeled scFv-BMV fusion protein (NIH3T 6.7 (Her2/neu): mouse-derived muscle cells overexpressing HER2, MCF10A: breast tissue not expressing HER2) Cells, fibrocyst cells).
5 is a result of confirming the size and protein purity of the fusion protein through SDS-PAGE after purification of the single-chain antibody fragment-oimosaic virus envelope protein (scFv-CMV) fusion protein.
6 is a result showing that the fusion protein is separated with high purity through SDS-PAGE after purification of the scFv-BMV fusion protein in which the histidine tag is fused to the C-terminus.
7A and 7B show the expression level of the fusion protein through SDS-PAGE after purification of the scFv-ferritin fusion protein in which the N-terminal maltose binding protein (MBP) and the C-terminal histidine tag are fused. As a result showing an increase in the separation purity, FIG. 7A shows the results of separating the fusion protein using 10 mM maltose and FIG. 7B using 250 mM maltose.
8 is a result of confirming that the purified MBP-scFv-ferritin fusion protein can be separated from the fusion protein through MBP cleavage by treating caspase-3 at various ratios.

As a result of intensive research for the development of a method for improving the utility of single-chain antibody fragments, the present inventors produced a protein in which a single-chain antibody fragment and a viral envelope protein were fused, and the fusion protein is the self-assembly characteristic of a viral envelope protein. The present invention was completed by confirming that the nanoclusters were formed and thus the antigen-binding ability was improved.

Accordingly, the present invention provides a fusion protein in which a single chain antibody fragment (scFv) and a virus envelope protein are combined.

The term "antibody" used in the present invention includes immunoglobulin molecules having antigen-binding ability having reactivity with a specific antigen, and includes both polyclonal antibodies and monoclonal antibodies. In addition, the term includes forms produced by genetic engineering such as chimeric antibodies (eg, humanized murine antibodies) and heterologous antibodies (eg, bispecific antibodies). The antibody structurally consists of a heavy chain and a light chain, and each of the heavy and light chains includes a constant region and a variable region. The variable regions of the heavy and light chains include three multivariable regions called “complementarity-determining regions (CDRs)” and four “framework regions”.

The term "single chain variable fragment (scFv)" used in the present invention is not a general fragment formed from an antibody, but a fragment including the variable region of the heavy chain constituting the antibody and the variable region of the light chain. It is a fusion protein constructed by artificially fusing a fragment containing. The single chain antibody fragment may be a form in which heavy chain fragments and light chain fragments derived from various antibodies such as monoclonal antibodies or polyclonal antibodies are sequentially or reversely conjugated, A peptide linker composed of a sequence of 10 to 25 amino acids may be inserted between the heavy chain fragment and the light chain fragment, in this case, the linker used may be a peptide linker composed of serine (S) and glycine (G). The length of the linker is not particularly limited.

In the present invention, the type of the single-chain antibody fragment is not limited as long as the nucleic acid or amino acid sequence is known.

The antibody that is the source of the single chain antibody fragment is a therapeutic antibody, an antibody capable of binding to a therapeutic agent or a diagnostic/detection agent, an antibody for targeting to a specific cell or tissue, or simply an antibody for an antigen-antibody reaction. I can.

The term “viral coat protein” as used in the present invention is a protein constituting a virus particle, and is dispersed as a monomer depending on various conditions, and tens to hundreds of monomers are combined to form a large spherical or rod-shaped It shows the self-assembly property to form a nanocluster.

In the present invention, the viral envelope protein is a brom mosaic virus (BMV) derived envelope protein encoded by the nucleotide sequence of SEQ ID NO: 1 or a cucumber mosaic virus encoded by the nucleotide sequence of SEQ ID NO: 8 virus (CMV) derived envelope protein, but the type is not limited as long as it is a virus-derived envelope protein capable of forming nanoclusters with self-assembly characteristics.

In the present invention, the "fusion protein" is a combination of the single-chain antibody fragment and the viral envelope protein, and may be in a form in which the single-chain antibody fragment is bonded to the N-terminus or C-terminus of the viral envelope protein, and the single chain The antibody fragment may be directly bound to the viral envelope protein or through a peptide linker, and the spacing and orientation between single-chain antibody fragments may be controlled by adjusting the length and/or amino acid composition of the linker.

The present inventors actually prepared a single-chain antibody fragment (scFv)-viral envelope protein fusion protein and analyzed its characteristics.

In one embodiment of the present invention, a fusion protein in which a single-chain antibody fragment of Herceptin used as a target treatment for breast cancer and an envelope protein of bromosaic virus (hereinafter, referred to as BMV) or cucumber mosaic virus (hereinafter, referred to as CMV) are fused. Was prepared. The gene encoding the coat protein of BMV or CMV is amplified by PCR and cloned into a vector to prepare a platform vector for expression of the virus coat protein, and the gene for the Herceptin-derived scFv is cloned into the platform vector, and scFv-BMV or scFv- CMV fusion protein expression vectors were constructed (see Examples 1-1 to 1-2 and 3-1 to 3-2). Thereafter, the transformant produced by introducing the expression vector into E. coli was cultured to induce the expression of the fusion protein, and the fusion protein was isolated and purified (see Examples 1-3 and 3-3).

In another embodiment of the present invention, the characteristics were analyzed using the scFv-BMV fusion protein. Specifically, as a result of measuring the binding ability of Herceptin, scFv monomer, and scFv-BMV fusion protein to HER2 to determine the antigen-binding ability of the scFv-BMV fusion protein, scFv-BMV fusion protein in terms of affinity and avidity for HER2 It was confirmed that the antigen-binding ability was about 50 times higher than that of the Herceptin and scFv monomers (see Example 2-1).

In addition, as a result of observing the structure of the scFv-BMV fusion protein through an electron microscope, it was found that the fusion protein formed an aggregate structure in which 60 monomers were collected (see Example 2-2).

In addition, in order to investigate the cancer cell targeting function of scFv-BMV fusion protein, FITC fluorescence-labeled fusion protein on NIH3T-6.7 cells overexpressing HER2 and MCF10A fibrocyst cells, which are breast tissue cells that do not express HER2 on the cell surface. As a result of observing under a microscope after processing and culturing, it was confirmed that the fusion protein targets only cells overexpressing HER2 (see Example 2-3).

Through the above results, it was confirmed that the antigen-binding ability of the single-chain antibody fragment was improved by the clustering effect of the antigen recognition site due to the self-assembly characteristic of the viral envelope protein in the fusion protein prepared through the present invention.

In the present invention, a histidine tag is further fused to the C-terminus of the fusion protein to which the single-chain antibody fragment-viral envelope protein is bound to improve the purity of the protein obtained in the purification process, and the fusion protein It is possible to increase the expression level and water solubility of the prepared fusion protein by further fusion of a maltose binding protein (MBP) at the N-terminus of.

In one embodiment of the present invention, a vector expressing a scFv-BMV fusion protein in which a histidine tag is fused to the C-terminus is constructed using a pET 21 (a) vector in which a histidine tag is expressed at the C-terminus, and the vector After introducing E. coli, the protein was isolated and purified from the transformant. As a result, it was confirmed that a high-purity fusion protein was isolated (see Example 4).

In another embodiment of the present invention, a vector in which a maltose binding protein (MBP) is expressed at the N-terminus and a histidine tag is expressed at the C-terminus in order to prepare a fusion protein with improved expression level and water solubility. A fusion protein was prepared using (MBP-DEVD-28a vector). As a result, it was confirmed that a fusion protein having improved expression levels and water solubility can be obtained with high purity using maltose (see Examples 5-1 and 5-2). In addition, it was confirmed that the maltose-binding protein can be cleaved by treating the fusion protein with caspase-3 and isolated from the fusion protein (see Example 5-3).

As another aspect of the present invention, the present invention provides a polynucleotide encoding a fusion protein to which the single-chain antibody fragment-viral envelope protein is bound.

The nucleotide sequence constituting the polynucleotide is a nucleotide sequence that can encode the amino acid sequence of the fusion protein or a nucleotide sequence that encodes various amino acid sequences that can be added to the N-terminus or C-terminus of the amino acid sequence. It may be a form added to the 5'-end or 3'-end of the nucleotide sequence capable of encoding the sequence.

The polynucleotide may be modified by substitution, deletion, insertion, or a combination of one or more bases. When preparing by chemically synthesizing a nucleotide sequence, a synthesis method well known in the art can be used.

As another aspect of the present invention, the present invention provides a recombinant expression vector comprising the nucleotide.

The term "recombinant expression vector" used in the present invention includes a gene insert to express a target protein in an appropriate host cell, and a gene construct comprising essential regulatory elements operably linked to express the gene insert Can be The expression vector includes expression control elements such as a start codon, a stop codon, a promoter, and an operator, and the start and stop codons are generally considered to be part of the nucleotide sequence encoding the polypeptide, and when the gene construct is administered, the individual Must show an action in the coding sequence and can be provided in frame (in frame). The promoter of the vector can be constitutive or inducible. The type of promoter is not limited as long as the nucleic acid in the recombinant expression vector of the present invention is expressed in a host such as E. coli.

In the present invention, the expression vector is not particularly limited as long as it can express the polynucleotide to produce the fusion protein provided in the present invention, and mammalian cells (e.g., human, monkey, rabbit, rat, hamster, mouse Cells, etc.), plant cells, yeast cells, insect cells, or bacterial cells (e.g., E. coli, etc.). Preferably, the polynucleotide is operably linked to an appropriate promoter so that the polynucleotide can be expressed in a host cell, and may be a vector containing at least one selection marker, more preferably a commercially developed plasmid (pUC18, pBAD, pIDTSAMRT-AMP, etc.), E. coli-derived plasmids (pYG601BR322, pBR325, pUC118 and pUC119), Bacillus subtilis -derived plasmids (pUB110 and pTP5), yeast-derived plasmids (YEp13, YEp24 and YCp50), λ- Phage (Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11 and λZAP), retrovirus (retrovirus), adenovirus (adenovirus), vaccinia virus (vaccinia virus), baculovirus (baculovirus), and the like. Since the expression vector and the expression level of the protein differ depending on the host cell, it is preferable to select and use the most suitable host cell for the purpose.

As another aspect of the present invention, the present invention provides a transformant transformed with the recombinant expression vector.

The term “transformation” as used in the present invention means that a fragment of a DNA chain or plasmid containing a foreign gene of a different kind from that of the original cell penetrates between cells and binds to the DNA existing in the original cell. It refers to a molecular biological technique that changes the genotype. In the present invention, transformation refers to introducing the recombinant expression vector provided in the present invention into a host cell to express the fusion protein according to the present invention.

Transformation can be carried out by various methods, specifically, the Hanahan method, which improves efficiency by using a reducing material called dimethyl sulfoxide (DMSO) _{in the CaCl 2} precipitation method and the CaCl _{2 precipitation method, the electroporation method, and calcium phosphate.} Precipitation method, protoplasm fusion method, stirring method using silicon carbide fiber, Agrobacteria mediated transformation method, transformation method using PEG, dextran sulfate, lipofectamine, and drying/inhibition mediated transformation method can be used. have.

The host cell is not particularly limited in type as long as it is a cell capable of expressing the fusion protein by introducing the recombinant expression vector, and fungal cells such as E. coli , yeast cells, and Pichia pastoris; Insect cells such as Drosophila and Spodoptera Sf9 cells; Animal cells such as CHO, COS, NSO, 293, and Bow melanoma cells; Alternatively, plant cells or the like may be used, and more preferably, E. coli may be used.

In another aspect of the present invention, the present invention comprises the steps of: (a) preparing a recombinant expression vector comprising a polynucleotide encoding the fusion protein of the present invention; (b) introducing the prepared recombinant expression vector into a host cell to obtain a transformant; And (c) expressing the fusion protein from the transformant and then obtaining the expressed fusion protein.

In step (c), the step of obtaining the fusion protein is not limited to the method, and a person skilled in the art can perform it using a conventional method.

As another aspect of the present invention, the present invention provides a nanocluster comprising the fusion protein of the present invention.

The term "nanocluster" used in the present invention means that a fusion protein monomer is self-assembled and formed by the self-assembly property of the viral envelope protein constituting the fusion protein of the present invention, and the size and shape of the nanocluster May vary depending on the type of viral envelope protein used to prepare the fusion protein.

As another aspect of the present invention, the present invention provides a drug delivery system composition comprising a drug introduced into the nanocluster.

The nanocluster may bind a drug to the interior or surface to deliver a target-oriented drug, and the drug may be a therapeutic agent, a diagnostic agent, or a detection agent. The therapeutic agent may be a nucleic acid, a peptide, an antibody, an antibody fragment, a compound, a toxin, a nuclease, a hormone, an immunomodulator, an anticancer agent, a chelator, a boron compound, or a photoactive agent, and the diagnostic/detection agent May be a radioisotope, a dye, a contrast agent, a fluorescent protein, a fluorescent compound, or a magnetic resonance imaging (MRI) contrast enhancer, but is not limited thereto.

As another aspect of the present invention, the present invention provides a diagnostic kit or a protein chip comprising the nanocluster.

The diagnostic kit of the present invention may include a nanocluster comprising a single-chain antibody fragment capable of binding to a target protein capable of diagnosing a desired disease. At this time, the desired disease is not particularly limited as long as it includes a target protein that can be detected by the single-chain antibody fragment contained in the nanocluster. For example, a specific protein due to infectious diseases or genetic mutations caused by bacteria or viruses It may include hereditary diseases that may occur as they are expressed, metabolic diseases that may occur due to the introduction of substances that disturb metabolism in vivo, and the like.

The diagnostic kit of the present invention is composed of one or more other constituent compositions, solutions, or devices suitable for an analysis method, for example, a test tube or other suitable container, a reaction buffer, a secondary antibody, a preparation for detection, and sterile water. , Or an enzyme or the like may be further included.

The protein chip is a kind of diagnostic kit, and the fusion protein provided in the present invention or a nanocluster composed of the fusion protein may be fixed to a solid substrate. Although not limited, for example, gold, glass, modified silicone, tetrafluoroethylene, polystyrene, polypropylene, and the like may be commonly used polymers or gels. In addition, the surface of the substrate may be surface-treated with polymers, plastics, resins, carbohydrates, silica, silica derivatives, carbon, metals, inorganic glass and films. The substrate not only serves as a support, but also provides a place where the binding reaction between the immobilized antibody and the antigen takes place. The size and shape of the substrate and the position, size, and shape fixed on the substrate may be changed according to the purpose of analysis and devices such as a spotting machine and a scanner.

In addition to the protein chip, the diagnostic kit may be a kit using a polymerase chain reaction (PCR), an RT-PCR, a DNA chip, or a kit including essential elements necessary for performing ELISA, but is not limited thereto.

As another aspect of the present invention, the present invention provides a method for detecting an antigen comprising the step of detecting specific binding of an antigen-antibody by treating a clinical sample to the nanocluster of the present invention.

The clinical sample may be patient-derived blood, serum, plasma, body fluid, saliva, urine, intestinal fluid, lymph fluid, or peritoneal fluid, but is not limited thereto.

In the above method, after reacting the protein sample with the nanocluster, before confirming the occurrence of the antigen-antibody reaction, the target material that was not able to bind to the nanocluster and other substances in the sample that cannot be combined are washed and removed. It may further include the step of.

Hereinafter, a preferred embodiment is presented to aid the understanding of the present invention. However, the following examples are provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

[ Example ]

Example 1.Herceptin ( Herceptin ) Origin scFv - BMV Fusion protein Produce

1-1. Single chain antibody fragment - BMV Fusion protein For manufacturing BMV Platform vector creation

A platform vector for preparing a single chain variable fragment (scFv)-BMV virus fusion protein was constructed as follows, and FIG. 1 shows the composition of a viral fusion protein for expression of the scFv-BMV virus particle fusion protein. It shows a conceptual diagram showing.

Specifically, the gene (SEQ ID NO: 1) encoding the particles of the plant virus bromosaic virus (BMV) was amplified by performing a polymerase chain reaction (PCR) with the primers shown below. Thereafter, the restriction enzymes BamHI and NotI were used to clone into vector pET 28(a), and then the cloned viral particle (BMV) gene was confirmed through nucleotide sequence analysis.

P1: 5'-GTCGCGGATCCATGTCGACTTCAGGAACTGGTAA-3' (SEQ ID NO: 2)

P2: 5'-AAGGAAAAAAGCGGCCGCCTACCTATAAACCGGGG-3' (SEQ ID NO: 3)

1-2. Herceptin ( Herceptin ) Origin scFv - BMV Fusion protein Recombinant DNA production

The gene for the single-chain antibody fragment (hereinafter, scFv) of Herceptin, which is widely used as a platform vector for expression of a viral envelope protein derived from the bromosaic virus (hereinafter, BMV) produced in Example 1-1, and a breast cancer treatment (hereinafter, referred to as scFv). 4) was used to prepare a recombinant DNA to prepare a scFv-BMV fusion protein.

To this end, using Herceptin's scFv (HL) gene as a template, PCR was performed using the following primers (SEQ ID NOs: 5 and 6) to obtain an amplification product for the gene, and the restriction enzymes NdeI and BamHI were used. The amplified gene was cloned into a platform vector for expression of a virus envelope protein derived from bromosaic virus (BMV) produced in Example 1-1, and the cloned scFv(HL)-BMV gene (SEQ ID NO: 7) was subjected to nucleotide sequence analysis. Confirmed.

P3: 5'-gggaattccatatgaccgtggcccaggcggccgaagtg-3' (SEQ ID NO: 5)

P4: 5'-cgcggatccgctgccggccgcgtgctggccggcctg-3' (SEQ ID NO: 6)

On the other hand, using Herceptin's scFv (HL) gene as a template, PCR was performed using the primers (SEQ ID NOs: 5 and 6) to obtain an amplification product, and the amplification product was vector pET using restriction enzymes NdeI and BamHI. Cloned in 28(a), and the cloned scFv(HL) gene was confirmed through nucleotide sequence analysis.

1-3. scFv - BMV Fusion protein Expression and purification

The Herceptin-derived single-chain antibody fragment prepared according to the method of Example 1-2-bromosaic envelope protein (hereinafter, scFv-BMV) expression vector and pET 28 (a) vector cloned from the scFv (HL) gene were respectively used in Escherichia coli. By transforming the host and culturing the obtained transformant in a medium containing 0.1 to 1 mM of Isopropyl-β-D-thio-galactoside (IPTG) and 10 to 37° C. It induced overexpression of the protein. Thereafter, the proteins were separated and purified through the following process.

Specifically, the cultured E. coli cells were collected and suspended in a column buffer solution (50 mM Tris pH 7.5, 500 mM NaCl, 5% glycerol, 0.05% β-mercaptoethanol), crushed using an ultrasonic grinder, and then centrifuged. A supernatant was obtained. After that, the talon resin (1 ml, Talon resin, Clontech) was washed twice with 10 ml of the column buffer solution to be activated, and the supernatant was added and gently shaken at 4° C. for 1 hour to adsorb the protein to the Talon resin. Subsequently, the Talon resin was washed twice with a column buffer solution (10 ml), 10 ml of an elution buffer (100 mM imidazole) was added, and then slowly rocked again for 1 hour to obtain an eluate. . The protein contained in the obtained eluate was quantified through Bradford assay, and the purified protein was confirmed to be the size of the protein monomer and the purity of the purified protein through SDS-PAGE analysis.

As a result of SDS-PAGE analysis, it was confirmed that the purified scFv-BMV fusion protein as shown in FIG. 2 had a size of about 51 kDa and was separated with high purity.

Example 2. scFv - BMV Fusion protein Characterization

2-1. scFv - BMV Fusion protein Antigen avidity analysis

In order to analyze the antigen-binding ability of the scFv-BMV fusion protein prepared in Example 1, the fusion protein for HER2, an antigen, Herceptin-derived scFv monomer prepared through Examples 1-2 and 1-3, and Herceptin The binding force of was compared using SPR (Surface plasmon resonance) analysis.

Specifically, a Biacore T100 analyzer (GE Healthcare, Uppsala, Sweden) was used for the analysis, and in the biosensor analysis, the CM5 sensor activated with EDC / NHS (N'-(3-dimethylaminopropyl) carbodiimide hydrochloride / N-hydroxysuccinimide) A chip (GE Healthcare, Uppsala, Sweden) was immobilized by flowing HER2 protein (10 μg/ml in 10 mM Sodium acetate, pH 5.0) for 5 minutes at a flow rate of 10 μl/min. The activated portion remaining on the surface of the sensor chip was deactivated by adding 1.0 M ethanolamine (pH 8.5). In this state, Herceptin, Herceptin-derived scFv monomer, or scFv-BMV fusion protein was diluted in a buffer solution (1 X PBS, pH 7.4) for each concentration, and then flowed at a flow rate of 30 µl/min, while the binding sensorgram and dissociation sensor Gram was confirmed. At this time, to correct the sensorgram caused by the nonspecific binding with the sensor chip, a cell completely inactivated with only BSA (bovine serum albumin) was used, and the nonspecific binding was corrected by subtracting the sensorgram of BSA from the sensorgram of HER2. . The surface of the sensor chip was regenerated by flowing a regeneration buffer solution (20 mM NaOH).

As a result of the experiment, as shown in Table 1 below, the scFv-BMV fusion protein is about 50 times higher than that of Herceptin (Whole antibody) and Herceptin-derived scFv monomer in terms of affinity and binding power for HER2. It confirmed that it represents. The above results indicate that the scFv-BMV fusion protein has enhanced antigen-binding ability to a high level compared to the conventional Herceptin antibody and its monomer.

ka (1/Ms) kd (1/s) KD Whole antibody 9.64E+04 2.36E-04 2.45E-09 scFv monomer 2.15E+04 6.51-04 3.02E-08 scFv-HFL 2.60E+06 3.23E-04 1.24E-10 scFv - BMV 4.31+07 8.21E-04 1.90E -11

2-2. scFv - BMV Fusion protein Transmission electron microscopy analysis

The scFv-BMV fusion protein prepared in Example 1 was analyzed with a transmission electron microscope (TEM) to observe the shape of the protein.

As a result, as shown in FIG. 3, it was confirmed that the purified scFv-BMV fusion protein formed an aggregate structure in which 60 monomers were collected to form a spherical shape as a whole.

2-3. scFv - BMV Fusion protein Cancer cells Targeting Confirm

In order to confirm the cancer cell targeting function of the scFv-BMV fusion protein prepared and purified in Example 1 above, targeting was analyzed using cancer cells in which HER2 is overexpressed on the cell membrane surface and breast tissue cells in which HER2 is not expressed. .

To this end, the scFv-BMV fusion protein is treated with an excessive amount of FITC (Fluoresceine isothiocyanate; Thermo Scientific) solution, and FITC that has not reacted with the fusion protein is removed using a PD-10 Desalting column (GE Healthcare). Fluorescent labeling was performed by introducing FITC to Lysine exposed to. Next, NIH3T-6.7 cells over-expressing HER2 on the surface of mouse-derived muscle cells and MCF10A fibrocyst cells, which are breast tissue cells that do not express HER2 on the cell surface, were placed in an 8 well microslide 200 µl per well, 2x10 ⁴ /Well each. After seeding, culture, and the fluorescently labeled scFv-BMV fusion protein solution were treated with 200 µl at a concentration of 500 uM to each cell, and then reacted for 30 minutes in a light-shielded state at room temperature. Upon completion of the reaction, the reaction was repeated three times with PBST, and the cell nuclei were stained through DAPI staining. Thereafter, the cells were rinsed again with PBST three times, and finally, the cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature, and then fluorescence images on the cell surface were observed under a microscope.

As a result, as shown in FIG. 4, it was confirmed that the scFv-BMV fusion protein was bound to HER2 on the cell surface, as FITC fluorescence was well detected in NIH3T-6.7 cells overexpressing HER2. On the other hand, FITC fluorescence was hardly observed in MCF10A cells not expressing HER2. The results indicate that the scFv-BMV fusion protein targets only cells overexpressing HER2.

Example 3. Herceptin ( Herceptin ) Origin scFv - CMV Fusion protein Produce

3-1. Single chain antibody fragment - CMV Fusion protein For manufacturing CMV Platform vector creation

A platform vector for preparing a single chain variable fragment (scFv)-CMV virus fusion protein was constructed in the following manner.

Specifically, a gene (SEQ ID NO: 8) encoding particles of a plant virus, cucumber mosaic virus (CMV), is amplified by performing a polymerase chain reaction (PCR) with a primer shown below. Made it. Thereafter, the restriction enzymes BamHI and NotI were used to clone into vector pET 28(a), and then the cloned viral particle (CMV) gene was confirmed through nucleotide sequence analysis.

P5: 5'-ctcgcggatccatggacaaatctgaatcaacc-3' (SEQ ID NO: 9)

P6: 5'-aaggaaaaaagcggccgctcagactgggagcactccaga-3' (SEQ ID NO: 10)

3-2. Herceptin ( Herceptin ) Origin scFv - CMV Fusion protein Recombinant DNA production

Using the gene for the CMV-derived viral envelope protein expression platform vector prepared in Example 3-1 and the same Herceptin single-chain antibody fragment (hereinafter, scFv) as used in Example 1-2, scFv- In order to produce the CMV fusion protein, it was intended to produce recombinant DNA.

For this, an amplification product for the gene was obtained by using Herceptin's scFv (HL) gene as a template and PCR using primers (SEQ ID NOs: 5 and 6), and the amplification using restriction enzymes NdeI and BamHI. The resulting gene was cloned into a platform vector for expression of CMV-derived virus envelope protein prepared in Example 3-1, and the cloned scFv (Herceptin)-CMV gene (SEQ ID NO: 11) was confirmed through nucleotide sequence analysis.

3-3. scFv - CMV Fusion protein Expression and purification

The Herceptin-derived single-chain antibody fragment prepared according to the method of Example 3-2-Oymosaic envelope protein (hereinafter, scFv-BMV) expression vector was transformed into an E. coli host, respectively, and the same method as in Example 1-3. According to the following, scFv-CMV fusion protein expression was induced in E. coli transformants, followed by isolation and purification.

As a result of confirming the size of the protein monomer and the purity of the purified protein through SDS-PAGE analysis, it was confirmed that the scFv-CMV fusion protein of about 51 kDa was purified with high purity, as shown in FIG. 5.

Example 4. For improving the purity of tablets scFv -Protein nanoparticles Fusion protein Produce

4-1. C-terminal histidine tag fused scFv - BMV Fusion protein Produce

In order to improve the purification purity of the expressed protein, in the preparation of the scFv-BMV fusion protein of Example 1-1, pET 21 (Histidine tag) is expressed at the C-terminus instead of pET 28(a) vector ( a) The scFv-BMV fusion protein was prepared using a vector (Novagen). This is to exclude proteins that are early terminated during the protein expression process from the purification process.

More specifically, the gene encoding the particles of BMV (SEQ ID NO: 1) was amplified by performing a polymerase chain reaction (PCR) with the primers shown below. Thereafter, the restriction enzymes BamHI and NotI were used to clone into the vector pET 21(a), and then the cloned viral particle (BMV) gene was confirmed through nucleotide sequence analysis.

P7: 5'-gtcgcggatccatgtcgacttcaggaactggtaa-3' (SEQ ID NO: 12)

P8: 5'-aaggaaaaaagcggccgcgtccacttcgtccctataaaccgggg-3' (SEQ ID NO: 13)

Next, using Herceptin's scFv (HL) gene as a template, PCR was performed using primers (SEQ ID NOs: 5 and 6) to obtain an amplification product for the gene, and the amplification using restriction enzymes NdeI and BamHI The resulting gene was cloned into the platform vector pET 21 (a) for expression of the BMV-derived virus envelope protein produced by the above method, and the cloned scFv(HL)-BMV gene (SEQ ID NO: 7) was confirmed through nucleotide sequence analysis.

4-2. C-terminal histidine tag fused scFv - CMV Fusion protein Produce

The scFv-CMV fusion protein was prepared similarly to the method of preparing the scFv-BMV fusion protein in which the C-terminal histidine tag of Example 4-1 was fused.

First, the gene encoding CMV particles (SEQ ID NO: 8) was amplified by performing a polymerase chain reaction (PCR) with the primers shown below. Thereafter, the restriction enzymes BamHI and NotI were used to clone into the vector pET 21(a), and then the cloned viral particle (CMV) gene was confirmed through sequencing.

P9: 5'-ctcgcggatccatggacaaatctgaatcaacc-3' (SEQ ID NO: 14)

P10: 5'-aaggaaaaaagcggccgcgtccacttcgtcgactgggagcactccaga-3' (SEQ ID NO: 15)

Next, using Herceptin's scFv (HL) gene as a template, PCR was performed using primers (SEQ ID NOs: 5 and 6) to obtain an amplification product for the gene, and the amplification using restriction enzymes NdeI and BamHI The resulting gene was cloned into a platform vector for expression of CMV-derived viral envelope protein prepared in Example 3-1, and the cloned scFv(HL)-CMV gene (SEQ ID NO: 11) was confirmed through nucleotide sequence analysis.

4-3. C-terminal histidine tag fused scFv - BMV And scFv - CMV Fusion protein Purification efficiency analysis

Each of the scFv-BMV and scFv-CMV fusion protein expression vectors prepared according to the methods of Examples 4-1 and 4-2 was transformed into an E. coli host, and each fusion protein was expressed in the same manner as in Example 1-3. After separation and purification. For the purified protein, the size of the protein monomer and the purity of the purified protein were confirmed through SDS-PAGE analysis.

As a result, as shown in Figure 6, compared to the scFv-BMV (Figure 2) and scFv-CMV (Figure 5) fusion proteins purified in Examples 1-3 and 2-3, a protein using a C-terminal histidine tag It was confirmed that more high-purity proteins were isolated through expression and purification methods.

Example 5: for enhancing the expression level of soluble protein MBP Tagging scFv -Protein nanoparticles Fusion protein Produce

5-1. N-terminal MBP And C-terminal histidine tag fused scFv -Ferritin Fusion protein Produce

In order to increase the expression level and improve the water solubility of the expressed scFv-protein nanoparticle fusion protein, a maltose binding protein (MBP) is expressed at the N-terminus, and a histidine tag is expressed at the C-terminus (MBP). -DEVD-28a vector (available in laboratory) was used to prepare a fusion protein.

Specifically, an amplification product for the gene was obtained by using Herceptin's scFv (HL) gene as a template and PCR using primers (SEQ ID NOs: 5 and 6), and the amplification using restriction enzymes NdeI and BamHI The resulting gene was cloned into the platform vector MBP-DEVD-28a for ferritin expression, and the cloned scFv(HL)-ferritin gene was confirmed through sequencing.

5-2. N-terminal MBP And C-terminal histidine tag fused scFv - ferritin Fusion protein Purification efficiency analysis

The scFv-ferritin fusion protein expression vector prepared according to the method of Example 5-1 was transformed into an E. coli host, and expression of the MBP-scFv-ferritin fusion protein in the transformant was performed in the same manner as in Example 1-3. After induction, the fusion protein was separated and purified through the following process.

Specifically, the cultured E. coli cells were collected and suspended in a column buffer solution (50 mM Tris pH 7.5, 500 mM NaCl, 5% glycerol, 0.05% β-mercaptoethanol), crushed using an ultrasonic grinder, and then centrifuged. A supernatant was obtained. Then, the amylose resin (10 ml, Amylose resin, NEB) filled in the column was washed and activated with 50 ml of the column buffer solution, and the supernatant was added to adsorb the protein to the amylose resin. Subsequently, the amylose resin was washed with a column buffer solution (100 ml), 20 ml of a washing buffer solution (column buffer solution containing 1 mM maltose) was added, and then a column elution solution containing 10 mM maltose was added. The protein was separated and purified from resin using. The protein contained in the obtained eluate was quantified through Bradford assay, and the purified protein was confirmed for the size of protein monomers and the purity of the purified protein through SDS-PAGE analysis.

As a result of SDS-PAGE analysis, as shown in Figs. 7A and 7B, the total protein (lane 1) before introduction into the amylose resin, the residual protein that does not bind to the resin (lane 2), and washing due to non-specific binding Unlike the proteins (lane 3), when using maltose at a concentration of 10 mM or 250 mM, it was confirmed that the MBP-scFv-ferritin fusion protein was sequentially eluted from the column (lanes 4 to 13, respectively). Through the above results, it was confirmed that the MBP-scFv-ferritin fusion protein was purified with high efficiency and purity.

5-3. N-terminal MBP Removal through tag cutting

The protein purified in Example 　5--2 additionally has an MBP tag at the N-terminus. After protein purification, it was determined that removing the tag would be beneficial for the function as an antibody or for self-assembly of the protein particles, and thus the protein was degraded. The MBP tag was removed using an enzyme. The vector for expression of the fusion protein prepared in Example 5-1 has a DEVD sequence between the MBP tag and the target protein, which is cleaved by the proteolytic enzyme Caspase-3. Therefore, when Caspase-3 is treated, the MBP tag can be removed from the target protein by cleaving the DEVD sequence.

As a result of the experiment, as shown in FIG. 8, when the MBP-scFv-ferritin fusion protein purified through Example 5-2 was treated with Caspase-3, the ratio of the fusion protein and caspase-3 was from 250:1 to 2000: In the case of 1, it was confirmed that the MBP protein was completely cleaved after 13 hours and the scFv-ferritin fusion protein was completely separated from MBP.

The above-described description of the present invention is for illustrative purposes only, and those of ordinary skill in the art to which the present invention pertains can understand that it is possible to easily transform it into other specific forms without changing the technical spirit or essential features of the present invention. There will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and are not limiting.

<110> Dongguk University Industry-Academic Cooperation Foundation <120> Novel fusion proteins comprising single chain fragment variable and viral coat protein, and uses thereof <130> MP16-244_division <160> 15 <170> KoPatentIn 3.0 <210> 1 <211> 570 <212> DNA <213> Brome mosaic virus <400> 1 atgtcgactt caggaactgg taagatgact cgcgcgcagc gtcgtgctgc cgctcgcaga 60 aatcgttgga ccgctagggt ccaaccagta attgtcgaac cactcgctgc tggccaaggc 120 aaggccatta aagcgattgc aggatacagc atatcaaagt gggaggcgtc ttcggacgcg 180 attacagcga aagccaccaa tgccatgagt atcactctgc cccatgagct ctcttctgaa 240 aagaataagg agcttaaggt cggcagggtg ctgctttggt tgggacttct tcctagcgtt 300 gctgggagga ttaaggcttg tgttgctgag aaacaggcac aggccgaggc tgcttttcaa 360 gtagccttgg cggttgcaga ctcctcgaaa gaggtggtcg cggccatgta tacggacgcc 420 tttcgagggg cgactctggg ggatttgctt aatctccaga tttatctgta tgcatctgaa 480 gcagtgcctg ctaaggcggt cgttgtacat ctagaagttg agcacgtaag gcctacgttc 540 gatgacttct tcaccccggt ttataggtag 570 <210> 2 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> BMV_F (P1) <400> 2 gtcgcggatc catgtcgact tcaggaactg gtaa 34 <210> 3 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> BMV_R (P2) <400> 3 aaggaaaaaa gcggccgcct acctataaac cgggg 35 <210> 4 <211> 843 <212> DNA <213> Artificial Sequence <220> <223> Herceptin_scFv <400> 4 atgaccgtgg cccaggcggc cgaagtgcag ttagtcgaat ccggtggagg cttagttcag 60 cctggtggca gccttcgtct gtcatgcgct gcttctggtt ttaacatcaa agatacttat 120 atccactggg tacggcaggc cccaggaaaa ggtctggaat gggtggcccg tatttatccc 180 actaatggat atacccgtta tgccgactca gtaaaaggtc gtttcactat ttcggcagat 240 actagtaaaa atacagctta tctgcaaatg aactctctca gagcagagga taccgcggtt 300 tattattgtt ctcgctgggg tggtgatggt ttctatgcta tggattattg gggtcaaggt 360 actttggtta cggtgtctgg tggtggtggt tcaggcggag gtggtagtgg tggtggaggt 420 agcgacattc agatgacgca atctcctagc agtctttcag cgagtgtcgg tgatcgtgtc 480 accattacat gtcgtgcatc acaagatgtt aataccgcag tggcgtggta tcagcagaag 540 ccgggtaaag caccgaaatt gctgatatat tccgcctcat tcttatatag cggtgttcct 600 tctcgcttta gcggatcgcg aagcggaaca gactttaccc tgacaatatc gtctctgcag 660 ccggaggatt ttgcaacgta ttattgccaa cagcattata caaccccacc gacctttggt 720 cagggtacga aagtagaaat taagggtgga ggtggttccg gtggtggtgg tagtggtgga 780 ggtggtagtg gccaggccgg ccagcacgcg gccggcagcg gatccggcag cagcggcagc 840 agc 843 <210> 5 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Herceptin_F (P3) <400> 5 gggaattcca tatgaccgtg gcccaggcgg ccgaagtg 38 <210> 6 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Herceptin_R (P4) <400> 6 cgcggatccg ctgccggccg cgtgctggcc ggcctg 36 <210> 7 <211> 1395 <212> DNA <213> Artificial Sequence <220> <223> scFv(Herceptin)-BMV <400> 7 atgaccgtgg cccaggcggc cgaagtgcag ttagtcgaat ccggtggagg cttagttcag 60 cctggtggca gccttcgtct gtcatgcgct gcttctggtt ttaacatcaa agatacttat 120 atccactggg tacggcaggc cccaggaaaa ggtctggaat gggtggcccg tatttatccc 180 actaatggat atacccgtta tgccgactca gtaaaaggtc gtttcactat ttcggcagat 240 actagtaaaa atacagctta tctgcaaatg aactctctca gagcagagga taccgcggtt 300 tattattgtt ctcgctgggg tggtgatggt ttctatgcta tggattattg gggtcaaggt 360 actttggtta cggtgtctgg tggtggtggt tcaggcggag gtggtagtgg tggtggaggt 420 agcgacattc agatgacgca atctcctagc agtctttcag cgagtgtcgg tgatcgtgtc 480 accattacat gtcgtgcatc acaagatgtt aataccgcag tggcgtggta tcagcagaag 540 ccgggtaaag caccgaaatt gctgatatat tccgcctcat tcttatatag cggtgttcct 600 tctcgcttta gcggatcgcg aagcggaaca gactttaccc tgacaatatc gtctctgcag 660 ccggaggatt ttgcaacgta ttattgccaa cagcattata caaccccacc gacctttggt 720 cagggtacga aagtagaaat taagggtgga ggtggttccg gtggtggtgg tagtggtgga 780 ggtggtagtg gccaggccgg ccagcacgcg gccggcagcg gatccatgtc gacttcagga 840 actggtaaga tgactcgcgc gcagcgtcgt gctgccgctc gcagaaatcg ttggaccgct 900 agggtccaac cagtaattgt cgaaccactc gctgctggcc aaggcaaggc cattaaagcg 960 attgcaggat acagcatatc aaagtgggag gcgtcttcgg acgcgattac agcgaaagcc 1020 accaatgcca tgagtatcac tctgccccat gagctctctt ctgaaaagaa taaggagctt 1080 aaggtcggca gggtgctgct ttggttggga cttcttccta gcgttgctgg gaggattaag 1140 gcttgtgttg ctgagaaaca ggcacaggcc gaggctgctt ttcaagtagc cttggcggtt 1200 gcagactcct cgaaagaggt ggtcgcggcc atgtatacgg acgcctttcg aggggcgact 1260 ctgggggatt tgcttaatct ccagatttat ctgtatgcat ctgaagcagt gcctgctaag 1320 gcggtcgttg tacatttaga agttgagcac gtaaggccta cgttcgatga cttcttcacc 1380 ccggtttata ggtag 1395 <210> 8 <211> 657 <212> DNA <213> Cauliflower mosaic virus <400> 8 atggacaaat ctgaatcaac cagtgctggt cgtaaccatc gacgtcgtcc gcgtcgtggt 60 tcccgctccg ccccctcctc cgcggatgct aactttagag tcttgtcgca gcagctttcg 120 cgacttaata agacgttagc agctggtcgt ccaactatta accacccaac ctttgtaggg 180 agtgaacgct gtagacctgg gtacacgttc acatctatta ccctaaagcc accaaaaata 240 gaccgtgagt cttattacgg taaaaggttg ttactacctg attcagtcac ggaatatgat 300 aagaagcttg tttcgcgcat tcaaattcga gttaatcctt tgccgaaatt tgattctacc 360 gtgtgggtga cagtccgtaa agttcctgcc tcctcggact tatccgttgc cgccatctct 420 gctatgttcg cggacggagc ctcaccggta ctggtttatc agtatgccgc atctggagtc 480 caagccaaca acaaactgtt gtttgatctt tcggcgatgc gcgctgatat aggtgacatg 540 agaaagtacg ccgtcctcgt gtattcaaaa gacgatgcgc tcgagacgga cgagctagta 600 cttcatgttg acatcgagca ccaacgcatt cccacatctg gagtgctccc agtctga 657 <210> 9 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> CMV_F (P5) <400> 9 ctcgcggatc catggacaaa tctgaatcaa cc 32 <210> 10 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> CMV_R (P6) <400> 10 aaggaaaaaa gcggccgctc agactgggag cactccaga 39 <210> 11 <211> 1485 <212> DNA <213> Artificial Sequence <220> <223> scFv(Herceptin)-CMV <400> 11 catatgaccg tggcccaggc ggccgaagtg cagttagtcg aatccggtgg aggcttagtt 60 cagcctggtg gcagccttcg tctgtcatgc gctgcttctg gttttaacat caaagatact 120 tatatccact gggtacggca ggccccagga aaaggtctgg aatgggtggc ccgtatttat 180 cccactaatg gatatacccg ttatgccgac tcagtaaaag gtcgtttcac tatttcggca 240 gatactagta aaaatacagc ttatctgcaa atgaactctc tcagagcaga ggataccgcg 300 gtttattatt gttctcgctg gggtggtgat ggtttctatg ctatggatta ttggggtcaa 360 ggtactttgg ttacggtgtc tggtggtggt ggttcaggcg gaggtggtag tggtggtgga 420 ggtagcgaca ttcagatgac gcaatctcct agcagtcttt cagcgagtgt cggtgatcgt 480 gtcaccatta catgtcgtgc atcacaagat gttaataccg cagtggcgtg gtatcagcag 540 aagccgggta aagcaccgaa attgctgata tattccgcct cattcttata tagcggtgtt 600 ccttctcgct ttagcggatc gcgaagcgga acagacttta ccctgacaat atcgtctctg 660 cagccggagg attttgcaac gtattattgc caacagcatt atacaacccc accgaccttt 720 ggtcagggta cgaaagtaga aattaagggt ggaggtggtt ccggtggtgg tggtagtggt 780 ggaggtggta gtggccaggc cggccagcac gcggccggca gcggatccat ggacaaatct 840 gaatcaacca gtgctggtcg taaccatcga cgtcgtccgc gtcgtggttc ccgctccgcc 900 ccctcctccg cggatgctaa ctttagagtc ttgtcgcagc agctttcgcg acttaataag 960 acgttagcag ctggtcgtcc aactattaac cacccaacct ttgtagggag tgaacgctgt 1020 agacctgggt acacgttcac atctattacc ctaaagccac caaaaataga ccgtgagtct 1080 tattacggta aaaggttgtt actacctgat tcagtcacgg aatatgataa gaagcttgtt 1140 tcgcgcattc aaattcgagt taatcctttg ccgaaatttg attctaccgt gtgggtgaca 1200 gtccgtaaag ttcctgcctc ctcggactta tccgttgccg ccatctctgc tatgttcgcg 1260 gacggagcct caccggtact ggtttatcag tatgccgcat ctggagtcca agccaacaac 1320 aaactgttgt ttgatctttc ggcgatgcgc gctgatatag gtgacatgag aaagtacgcc 1380 gtcctcgtgt attcaaaaga cgatgcgctc gagacggacg agctagtact tcatgttgac 1440 atcgagcacc aacgcattcc cacatctgga gtgctcccag tctga 1485 <210> 12 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> BMV_F (P7) <400> 12 gtcgcggatc catgtcgact tcaggaactg gtaa 34 <210> 13 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> BMV_R (P8) <400> 13 aaggaaaaaa gcggccgcgt ccacttcgtc cctataaacc gggg 44 <210> 14 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> CMV_F (P9) <400> 14 ctcgcggatc catggacaaa tctgaatcaa cc 32 <210> 15 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> CMV_R (P10) <400> 15 aaggaaaaaa gcggccgcgt ccacttcgtc gactgggagc actccaga 48

Claims (20)

A fusion protein comprising a single chain antibody fragment (scFv) and a viral envelope protein,
The fused protein is further fused with a histidine tag at the C-terminal of the protein, a maltose binding protein is further fused to the N-terminus of the protein,
The viral envelope protein is a protein derived from brome mosaic virus or cucumber mosaic virus,
Wherein the single chain antibody fragment (scFv) is a Herceptin-derived single chain antibody fragment. The method according to claim 1,
Wherein said single chain antibody fragment is attached to the N-terminus or C-terminus of the viral envelope protein. The method according to claim 1,
Wherein the single chain antibody fragment comprises a heavy chain fragment and a light chain fragment of the antibody. The method according to claim 1,
Characterized in that the brome mosaic virus coat protein is encoded by the nucleotide sequence of SEQ ID NO: 1. The method according to claim 1,
Characterized in that the cucumber mosaic virus coat protein is encoded by the nucleotide sequence of SEQ ID NO: 8. A polynucleotide encoding the fusion protein of any one of claims 1 to 5. A recombinant expression vector comprising the polynucleotide of claim 6. A transformant transformed with the recombinant expression vector of claim 7. A method for producing a fusion protein, comprising the steps of:
(a) preparing a recombinant expression vector comprising the polynucleotide of claim 6;
(b) introducing the prepared recombinant expression vector into a host cell to obtain a transformant; And
(c) expressing the fusion protein from the transformant and obtaining the expressed fusion protein. 6. A nanocluster comprising the fusion protein of any one of claims 1 to 5. 11. The method of claim 10,
Wherein the nanoclusters are formed by self-assembly of a viral coat protein constituting the fusion protein. A drug delivery composition comprising a drug introduced into a nanocluster of claim 10. 13. The method of claim 12,
Wherein said medicament is a therapeutic agent or a diagnostic / detection agent. 14. The method of claim 13,
Wherein the therapeutic agent is a nucleic acid, a peptide, an antibody, an antibody fragment, a compound, a toxin, a nucleic acid hydrolase, a hormone, an immunomodulator, an anticancer agent, a chelator, a boron compound or a photoactive agent. 14. The method of claim 13,
Wherein the diagnostic / detection agent is a radioactive isotope, a dye, a contrast agent, a fluorescent protein, a fluorescent compound, or a magnetic resonance imaging (MRI) enhancement agent. 13. The method of claim 12,
Wherein the drug is conjugated to the interior or surface of the nanoclostomer and is capable of target-directed drug delivery. 10. A diagnostic kit comprising the nanoclusters of claim 10. A protein chip comprising the nanocluster of claim 10. 11. A method for detecting an antigen, comprising treating a clinical sample with the nanoclusters of claim 10 to detect specific binding of the antigen-antibody. 20. The method of claim 19,
Wherein the clinical sample is a patient-derived blood, serum, plasma, body fluid, saliva, urine, intestinal fluid, lymph, or peritoneal fluid.

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