JP2020076634A - Receptor for antigen recognition, antigen measuring kit, and antigen detecting method - Google Patents

Abstract

To provide a receptor for antigen recognition that contains a low molecular antibody, and that can be oriented and fixed on a substrate.SOLUTION: Provided is a receptor 10 for antigen recognition, that contains a low molecular antibody 12 and a peptide A14. In the receptor for antigen recognition, the peptide A has an α-helix structure. In the receptor for antigen recognition, the low molecular antibody is an scFv antibody, a VHH antibody derived from Camelidae animals, or an IgNAR antibody derived from sharks. In addition, the receptor for antigen recognition contains a peptide tag 16. In the receptor for antigen recognition, a low molecular antibody containing site, a peptide A containing site, and a peptide tag containing site are bonded in this order.SELECTED DRAWING: Figure 1

Description

  The present invention relates to an antigen recognition receptor, an antigen measurement kit, and an antigen detection method.

  Conventionally, it has been attempted to immobilize an antigen recognition receptor such as an antibody on a substrate and apply it to diagnostic applications, analytical applications, sensor applications and the like. In order for the antigen recognition receptor to exert its function, it is necessary to immobilize the functional site such as the antigen recognition site in an exposed state. If it is not immobilized in the exposed state, the antigen recognition receptor cannot bind to the target molecule such as the antigen, and the detection sensitivity and the detection accuracy are lowered. Therefore, it has been attempted to orient the antigen recognition receptor in a fixed direction and immobilize it on the substrate.

As an example of immobilizing by utilizing physical adsorption with a substrate, by creating a fusion protein into which a highly hydrophobic domain is introduced, the highly hydrophobic domain and the substrate are physically adsorbed, and the highly hydrophilic protein is oriented. A method of fixing has been reported (see Patent Document 1).
In addition, as an example of immobilizing by utilizing chemical bond with the substrate, amino acid containing carboxyl group is introduced into protein by genetic recombination, and the surface of substrate is treated with polylysine etc. to bind and fix the protein. A method of doing so has been reported (see Patent Document 2).

  The immobilization method utilizing physical adsorption or chemical bond with the substrate makes it possible to orient and immobilize the antigen recognition receptor on the substrate. However, most of the conventional immobilization methods as described above target full-length antibodies as antigen recognition receptors. Since the full-length antibody is produced by an experimental animal, it takes time and labor to produce the antibody, and the production cost is extremely high. For this reason, it has been required to orient and immobilize not a full-length antibody as an antigen recognition site but a low-molecular-weight antibody such as a single-chain antibody that can be produced inexpensively and on a large scale in Escherichia coli, yeast, etc.

JP, 2005-220028, A JP 2004-347317 A

  Low-molecular-weight antibodies can be inexpensively produced on a large scale, and can eliminate the influence of the Fc region and constant region, which are C-terminal fragments, and are therefore suitable as substances on the antigen-recognizing receptor side for investigating antigen-antibody reactions. It is a simple form. However, low-molecular-weight antibodies have a smaller molecular weight than full-length antibodies, and the proportion of functional sites such as antigen recognition sites is high. Therefore, the low-molecular-weight antibody is extremely disadvantageous for the orientation and immobilization on the substrate, and there is a problem that the structure is changed or inactivated depending on the binding site with the substrate.

  Therefore, an object of the present invention is to provide a receptor for antigen recognition, which contains a low molecular weight antibody and can be oriented and fixed on a substrate. Further, it is an object to provide an antigen measurement kit and an antigen detection method using this antigen recognition receptor.

  The antigen-recognizing receptor of the present invention is characterized by containing a low molecular weight antibody and peptide A, and the peptide A has an α-helix structure.

  According to the present invention, it is possible to provide a receptor for antigen recognition, which contains a low-molecular weight antibody and can be oriented and fixed on a substrate. Furthermore, an antigen measurement kit and an antigen detection method using this antigen recognition receptor can be provided.

It is a schematic diagram which shows the state at the time of fixing an example of the receptor for antigen recognition of this invention to a board | substrate. It is a figure which shows the relationship between the concentration and fluorescence intensity when an antigen is made to react with an example of the receptor for antigen recognition of this invention.

  The description of the constituents described below may be made based on a typical embodiment of the present invention, but the present invention is not limited to such an embodiment.

(Receptor for antigen recognition)
The antigen-recognizing receptor of the present invention may contain at least a low molecular weight antibody and peptide A, and other specific structures, materials, configurations such as morphology are not particularly limited.

  Here, the "antigen" in the receptor for recognizing an antigen may be a molecule capable of binding to the receptor for recognizing an antigen having a low molecular weight antibody. For example, enzymes, receptors, antibodies, antigens, viruses, environmental hormones, allergens, microorganisms, residual pesticides, cancer-related molecules, physiologically active substances, transcription factors, signal transduction factors, peptides that bind to specific sequences, specific sequences Examples thereof include proteins, nucleic acids, lipids, sugars and low molecular weight compounds that bind to.

<Low molecular weight antibody>
The "small molecule antibody" in the present invention is an antibody fragment in which a part of a whole antibody is deficient, and contains at least a variable region of a heavy chain or a light chain, and is a target molecule such as an antigen. It has only to have a binding ability to Therefore, as the low-molecular weight antibody to be used in the present invention, VHH antibody (VHH, VHH variable domain of heavy chain of heavy chain antibody), single chain antibody (scFv, single chain Fv), multivalent single chain antibody (sc (Fv) n), constant region fused single chain antibody (scFv-Fc) , Fab fragment, F (ab ′) ₂ fragment, immunoglobulin new antigen receptor (IgNAR), and antibody fragments obtained by linking these, and preferably VHH antibody, scFv antibody, IgNAR antibody. And an antibody fragment in which these are ligated.

  The VHH antibody is an antibody consisting only of the variable region of a heavy chain antibody, and the heavy chain may be an antibody in which a part of the constant region has been deleted or substituted, or a fragment thereof, as long as it has the variable region. The VHH antibody used in the present invention is not particularly limited, but an antibody derived from a camelid (Bactrian camel, dromedary, llama, alpaca, vicuna, guanaco, etc.) is preferable.

  The scFv antibody is an antibody in which each of the variable regions of the heavy chain and the light chain is linked by a linker, and as long as the heavy chain and the light chain have variable regions, an antibody or a fragment thereof in which a part of the constant region has been deleted or substituted May be The origin of the scFv antibody is not particularly limited, and antibodies of any origin can be preferably used.

  The IgNAR antibody is a heavy chain homodimer antibody, which is different from a mammal-derived antibody. It is an antibody derived from chondrocyte and binds to a target molecule such as an antigen only in the variable region of the heavy chain. Cartilage fish derived from cartilaginous fish include, for example, sharks and rays. As the low molecular weight antibody of the present invention, it is preferable to use shark-derived IgNAR antibody. The heavy chain of the IgNAR antibody may be an antibody having a part of the constant region deleted or substituted, or a fragment thereof, as long as it has a variable region.

<Peptide A>
The “peptide A” in the present invention is a peptide that supports a low-molecular weight antibody for maintaining the orientation of the low-molecular weight antibody when immobilized on a substrate and realizing improved detection sensitivity and stable reproducibility.

  Here, the peptide is a peptide in which two or more amino acids are bound by a peptide bond, and includes oligopeptides, polypeptides, proteins, homomeric peptides and heteromeric peptides. In addition, the peptide may take an electrically neutral form or a salt form, as a form consisting of only the amino acid sequence in the sequence listing, or as a fusion protein in which the peptide is fused to the terminal part or inside of a specific protein. Or a form such as a complex obtained by adding a sugar, polyethylene glycol, biotin, a fluorescent dye, or the like.

  The peptide A in the present invention is located on the C-terminal side of a low-molecular-weight antibody, and plays a role of supporting the target molecule even when the peptide A stably rises from the substrate in a solution and binds to the target molecule such as an antigen. Have. This enhances the presentation of the low-molecular weight antibody and can improve the detection sensitivity and the detection accuracy even with a small amount of the low-molecular weight antibody.

  Therefore, the peptide A of the present invention preferably has a structure containing a rigid and stable region, and the peptide A of the present invention is a peptide having an α-helix structure. The α-helix structure is a secondary structure frequently found in hemoglobin and various other proteins. The α-helix structure includes (i) a right-handed helical structure, and (ii) a rigid and stable region characterized by including 3.6 amino acid residues in one turn of the helix. It is a structure. Therefore, the orientation of the low molecular weight antibody can be made uniform, and the target molecule can be stably supported even when the target molecule binds to the target binding site.

  Furthermore, the peptide A of the present invention is preferably a hydrophilic peptide as a whole. Usually, the surface material of the substrate is a hydrophobic material. When the surface material of the substrate is hydrophobic, and if the hydrophilic peptide is contained as a whole, when the receptor for antigen recognition is immobilized on the substrate, the surface material of the substrate stably interacts with the surface material of the substrate to stabilize. Can stand up from. Therefore, the orientation of the low molecular weight antibody can be easily aligned. Furthermore, when a low-molecular antibody is produced, since it has a hydrophilic peptide, the low-molecular antibody is likely to be stably expressed in E. coli, and the expression amount of the low-molecular antibody is improved. In addition, inclusion body formation of the expressed low molecular weight antibody can be suppressed, and insolubilization of the antigen recognition receptor can be suppressed.

Examples of the peptide having an α-helix structure of the present invention include known peptides having an α-helix structure. Examples include peptides having structures such as a zinc finger structure, a leucine zipper structure, a helix turn helix structure, and an α stand structure. These structures may include only the α-helix site or may include other structures (eg β-sheet).
The peptide having an α-helix structure is preferably a peptide having an α-stand structure or a zinc finger structure, and particularly preferably a peptide having an α-stand structure.

  The α-stand structure in the present invention is based on the amino acid sequence of the A chain of 8 α helices (A to H chains) contained in myoglobin, and its secondary structure is more hydrophilic while maintaining the α helix. Is a sequence substituted with the amino acid of. The reason why myoglobin is used is that a peptide having a hydrophilic α-helix structure can be obtained by performing the above-mentioned substitution in the α-helix portion of myoglobin. Since it is hydrophilic and has a stable α-helix structure as a supporting portion, it is considered that the antigen-recognizing receptor having the peptide can raise the receptor without falling down on the substrate. It is considered that this makes it possible to align the orientation of the antigen recognition site and efficiently capture the target molecule. Further, since the height of the antigen recognition receptor is almost constant, it can be expected that the detection sensitivity is improved. Further, since the immobilization reaction occurs at a site separated from the low molecular weight antibody, the influence on the activity can be minimized, and the inactivation of the low molecular weight antibody can be suppressed.

As a specific α-stand structure, for example, a sequence in which the hydrophobic amino acid in the myoglobin A chain (A1 to A16) is replaced with glutamine (Gln) can be mentioned.
Here, an example of the α stand structure will be specifically shown. The following is a sequence (SEQ ID NO: 1 in the sequence listing) in which the hydrophobic amino acid in the myoglobin A chain (A1 to A16) is replaced with glutamine (Gln).

Sequence number 1 in the sequence listing
Array (N → C): SEGEWQQQQQHQWAHQE

The zinc finger structure in the present invention is one of the DNA binding motifs present in the DNA binding domains of many transcription factors, and is a structure having two antiparallel β sheet structures and one α helix structure. For example, the zinc finger structure of the C ₂ H ₂ class has a characteristic that approximately 30 amino acid residues are contained in each unit, and two Cys residues and His residues form a loop via a zinc ion. The structure has a rigid and stable region. Therefore, the orientation of the low-molecular weight antibody that is the target binding site can be made uniform, and the target molecule can be stably supported even when the target molecule such as an antigen binds to the target binding site.
When used as the peptide A of the present invention, it is preferably used only at the α-helix site of the zinc finger structure.

Examples of the α-helix part of the zinc finger structure include the following sequences.
Sequence number 2 in the sequence listing
Sequence (N → C): LAKHMASCRLRK

The peptide A of the present invention is preferably introduced at the C-terminal side of the low molecular weight antibody because it needs to be immobilized on the solid phase while maintaining the three-dimensional structure of the low molecular weight antibody.
Further, one or more of the peptide A of the present invention can be introduced as long as it maintains the orientation of the low molecular weight antibody when immobilized on a substrate and supports the low molecular weight antibody. By introducing the amino acid sequence at a plurality of positions, it may be possible to maintain the three-dimensional structure of the peptide in a more normal state. For example, if peptide A is introduced to the C-terminal side of each of the heavy chain and light chain of scFv, it will be easier to bind to the solid phase surface, and therefore the antigen-binding site will face outward and the target molecule can be efficiently captured. It is thought to be possible.
Furthermore, for example, SEQ ID NO: 1 and SEQ ID NO: 2 in the sequence listing can be introduced in combination.

  Peptide A of the present invention can be immobilized on the substrate at the C-terminal side, but the antigen recognition receptor of the present invention preferably further contains a "peptide tag" described below. If a peptide tag is added at the time of expressing a low-molecular-weight antibody, it can be used for immobilization on a substrate or a purification carrier to align the protein orientation on the substrate or purification carrier. Furthermore, since the immobilization reaction occurs at a site separate from the low molecular weight antibody, the influence on the activity can be minimized, and the inactivation of the low molecular weight antibody can be further suppressed.

<Peptide tag>
The “peptide tag” in the present invention may be any peptide as long as it has a function of binding to the material on the surface of the substrate, and other specific amino acid sequences and lengths are not particularly limited. Furthermore, the peptide tag of the present invention is more preferably a peptide which can easily purify an antigen recognition receptor and also has a function as an affinity tag. In the present invention, the term “bond” means that the peptide tag and the substrate interact with each other with a strength that can be utilized for the intended use of the present invention, and includes the case where the peptide tag has an affinity and is adsorbed.

  As the peptide tag, known tags can be used. For example, the affinity tag is His tag, PA tag, TARGET tag, FLAG tag, Myc tag, HA tag, GST tag, PS tag, PC tag, PMMA tag, SiN tag. , V5 tag, etc. can be used. Among these, it is preferable to use a His tag composed of a histidine residue from the viewpoint of purification efficiency and stability. Since the histidine residue that constitutes the His tag chelates several metal ions including nickel, cobalt and copper under specific buffer conditions, it can be expressed by using a hydrophobic substrate on which a metal is immobilized. The His-tag fusion antigen recognition receptor can be easily purified. In addition, when immobilized on a substrate, a commercially available nickel-containing hydrophobic substrate or the like can be used for stable and easy immobilization.

  Regarding the introduction position of the peptide tag of the present invention, it is preferable to introduce the peptide tag at the C-terminal side of peptide A, because it is necessary to immobilize it on the substrate while maintaining the three-dimensional structure of the low molecular weight antibody.

The substrate is not particularly limited in its material as long as it can immobilize the antigen recognition receptor. Examples of the material include resins, nylon, nitrocellulose, polysaccharides, glass and metals, metal oxides and the like. The substrate may be a substrate made of one kind of material or a composite substrate made by combining a plurality of materials.
When the antigen recognition receptor has an affinity tag as a peptide tag, it is preferable to use a substrate having a high affinity for the affinity tag. For example, in the case of a His tag, a substrate having nickel, cobalt, zinc, copper or the like on its surface can be used.

The form of the substrate is not particularly limited, and a substrate having an appropriate form may be used depending on the application. For example, it may have a flat plate shape, a film shape, a fibrous shape, a granular shape, a spherical shape, a lens shape, or the like, or may have a three-dimensional shape on a part of the surface.
Further, the substrate can be applied to various members, and may be petri dish, bag, tube, microtube, bottle, filter, porous body, or column.
Further, when it is assumed that the antigen recognition is performed electrically and optically, the substrate on which the antigen recognition site of the present invention is immobilized may be arranged on the electrode or the sensor. Alternatively, the antigen recognition site may be immobilized using the electrode or the sensor itself as a substrate.

FIG. 1 is a schematic diagram showing a state in which an example of the antigen recognition receptor of the present invention is immobilized on a substrate.
As shown in FIG. 1, the antigen recognizing receptor 10 of the present invention stands up without falling down on the substrate 20, because the peptide A14 containing a region such as an α-stand structure supports the low molecular weight antibody 12. As a result, the orientation of the low-molecular weight antibody 12 is uniform, and the low-molecular weight antibody 12 can be closely fixed to the substrate 20, so that target molecules such as antigens can be efficiently captured. Furthermore, since the height of the low molecular weight antibody 12 from the substrate 20 is substantially constant and the presentation of the functional site is further enhanced, the detection sensitivity and the detection accuracy can be further improved. Further, since the peptide tag 16 and the substrate 20, which are separate from the low molecular weight antibody 12, are immobilized, the influence on the activity of the low molecular weight antibody 12 can be minimized, and the detection accuracy can be improved. Can be improved.

<Method for producing antigen recognition receptor>
The method for producing the antigen recognition receptor 10 of the present invention will be described below.

  The antigen recognition receptor 10 of the present invention can be produced by a known cloning technique or chemical synthesis method. For example, by using cloning technology, DNAs encoding the respective fusion proteins are prepared, inserted into an expression vector capable of autonomous replication to give recombinant DNAs, which are then introduced into an appropriate host cell for transfection. By converting, the target antigen recognition receptor 10 can be prepared.

  The expression vector can be appropriately selected from those well known to those skilled in the art in consideration of the type of host cell, the method of introducing the recombinant expression vector into the host cell, the expression efficiency of the fusion protein, and the like. For example, it may be a plasmid vector or a viral vector derived from a retrovirus or an adenovirus. The expression vector preferably contains a promoter, terminator, etc. suitable for expressing the introduced DNA in advance.

  The host cell may be any cell that can express the desired low-molecular-weight antibody 12 from the recombinant expression vector. Examples thereof include Escherichia coli, Bacillus subtilis, actinomycetes, yeast, filamentous fungi, plant cells, insect cells, animal cells and the like.

  When the above-mentioned peptide tag 16 is introduced as a purification tag, a solution containing the product from the transformant is brought into contact with the surface of the carrier to be directly adsorbed on the surface of the carrier for separation and purification. You can The solution containing the product refers to all solutions containing the target low-molecular-weight antibody 12 and in which unnecessary contaminants due to the host are present. For example, cell lysate, soluble fraction obtained by centrifugation of cell lysate, solubilized insoluble fraction obtained by centrifugation of cell lysate, cell membrane fraction, cell wall fraction And secretory products secreted from cells, body fluids, or incompletely purified products thereof.

  The peptide A14 may be any peptide having an α-helix structure, and known peptides can be used as described above. When the peptide of SEQ ID NO: 1 in the sequence listing is used as the peptide A14, it can be prepared by replacing the hydrophobic amino acid of the myoglobin A chain (A1 to A16) with glutamine (Gln) as described above.

As the peptide tag 16, known tags can be used as described above.
Further, the low molecular weight antibody 12-containing site which is a site containing the low molecular weight antibody 12, the peptide A14 containing site containing the peptide A14 and the peptide tag 16 containing site containing the peptide tag 16 are sequentially provided on the substrate 20 according to a conventional method. be able to. The low molecular weight antibody 12, the peptide A14 and the peptide tag 16 may not be directly bound to each other, and may be bound via a linker, a spacer and another amino acid sequence, for example. If the low-molecular-weight antibody 12-containing site, the peptide A14-containing site, and the peptide tag 16-containing site are bound in this order and fixed to the substrate 20, the low-molecular-weight antibody 12-containing site rises without falling down on the substrate 20 as described above, This is preferable because the molecular antibodies 12 have the same orientation.

  Hereinafter, an example of a method for producing the antigen recognition receptor 10 of the present invention will be described more specifically.

<Preparation of low molecular weight antibody expression vector>
First, an expression vector for the low molecular weight antibody 12 is prepared. As a method for producing the expression vector of the low molecular weight antibody 12, for example, an inverse PCR method or a gene recombination method can be used.

  The inverse PCR method is one of PCR methods in which an unknown DNA sequence is amplified using a primer designed to extend from a known DNA sequence region toward an unknown DNA sequence region adjacent to the region. Is one. First, in a system containing at least a plasmid DNA encoding the genetic information of the constituent components of the antigen recognition receptor 10 and a DNA polymerase, an oligonucleotide and 4 kinds of dNTPs, primer extension using the above plasmid DNA as a template and the oligonucleotide as a primer A reaction is carried out to amplify the plasmid DNA encoding the genetic information of the components of the antigen recognition receptor 10. In this inverse PCR method, a commercially available highly efficient and highly accurate PCR enzyme can be used, and for example, KOD-Plus-Ver.2 (KOD-211, Toyobo Co., Ltd.) and the like can be preferably used.

  Then, T4 DNA Polymerase or the like is allowed to act on the DNA amplified by the inverse PCR method to carry out the blunting treatment of the DNA ends. As T4 DNA Polymerase, commercially available enzymes and buffers can be used. For example, MO203S (New England Biolabs) can be preferably used. After purification, the DNA ends are phosphorylated and the DNA ends are ligated to each other for circularization to prepare an expression vector of a low-molecular antibody fusion protein in which the low-molecular antibody 12, the peptide A14 and the peptide tag 16 are fused.

  Further, when the gene recombination technique is used, the DNA encoding the gene information of the low molecular weight antibody 12, the DNA encoding the gene information of the peptide A14, and the DNA encoding the gene information of the peptide tag 16 are A DNA that is continuously ligated to the 3'-end side of the DNA encoding the gene information is prepared. Expression of a low-molecular-weight antibody fusion protein in which a low-molecular-weight antibody, peptide A14 and peptide tag 16 are fused with a pET system plasmid widely used for high expression of a protein, for example, inserted into the cloning region of plasmid pET-11d DNA. Create a vector.

<Transformation of E. coli for recovery of expression vector plasmid DNA>
Next, Escherichia coli for recovery of the expression vector plasmid DNA is transformed. The strain of Escherichia coli for which the pET system can be used is not particularly limited, and for example, DH5, DH5α, BL21, BL21 (DE3) and the like can be used. As the host Escherichia coli, commercially available competent cells may be used, or they may be prepared by themselves. When using a commercially available competent cell, for example, but not limited to, Competent Cell DH5α (DS220, Biodynamics Research Institute, Inc.), Competent Cell BL21 (DE3) (DS250, Biodynamics Research, Inc.) Etc. can be preferably used. The procedure for introducing the plasmid DNA into E. coli may follow the procedure described in the kit. The transformed E. coli appears as colonies on the medium. Depending on the vector species used, it is preferable to add various antibiotics such as ampicillin to the LB agar medium.

<Confirmation of transformants by colony PCR>
Next, the transformant is confirmed by colony PCR. Escherichia coli appearing as a colony on the medium is collected, and the plasmid in the cells is amplified by a PCR gene amplification device. In the colony PCR method, a commercially available PCR enzyme having excellent amplification efficiency and extensibility can be used. For example, KOD Dash (LDP-101, Toyobo Co., Ltd.) and the like can be preferably used. With respect to the amplified DNA, the quality of the gene amplification process is confirmed from the molecular weight of the DNA fragment of the PCR-processed sample by agarose gel electrophoresis or the like. Furthermore, after colonies of transformant candidates carrying the target plasmid DNA are grown, the plasmid is extracted from the colonies. A commercially available plasmid extraction / purification kit can be used for extraction. For example, the plasmid mini kit (12125, Qiagen) and the like can be preferably used because it can be selectively used depending on the amount of plasmid that can be treated. The recovered plasmid is analyzed for its DNA sequence to confirm whether it is a transformant having the desired plasmid.

<Transformation of E. coli for protein expression>
Next, Escherichia coli for protein expression is transformed. Escherichia coli for protein expression can be transformed by the same method as the above-mentioned method for transforming E. coli for plasmid recovery.

<Protein expression in transformants>
Next, protein expression of the transformant is performed. For example, E. coli colonies transformed for protein expression are added to an antibiotic-containing medium and cultured. The protein is expressed in the transformant by further adding a protein inducer and culturing.

<Purification of expressed protein>
Next, the expressed protein is purified. The above culture solution is centrifuged to collect the cells, and the supernatant is removed to obtain a precipitate. The precipitate is subjected to cell lysis treatment using various surfactants. As the cell lysis treatment, a commercially available cell lysate containing a surfactant can be used. For example, B-PER Bacterial Protein Extraction Reagent (78248, Thermo Scientific) or B-PER II Bacterial Protein Extraction Reagent (78260, Thermo Scientific ) And the like can be preferably used. After the cell lysis treatment, the supernatant is collected by centrifugation. As a result, both soluble and insoluble (inclusion bodies, recovered as pellets) recombinant proteins can be recovered.

  In addition, it is preferable to use a His tag as the peptide tag 16 because the fused recombinant protein can be purified by utilizing the property that histidine binds to a divalent cation such as nickel at a pH around neutral. .. It is possible to use the principle that a protein once bound to a bound metal easily dissociates in a buffer under low pH conditions or in the presence of EDTA or imidazole. For purification, a commercially available His tag purification kit can be used, and for example, Ni-NTA Agarose (30210, QIAGEN) or Imidazole stock solution in QIAexpress (registered trademark) Kits (32149, QIAGEN) can be preferably used. It is also preferable because it can be directly purified / collected using a Ni-NTA affinity column or the like.

  The antigen recognition receptor 10 of the present invention obtained by the above steps can be easily immobilized by bringing it into contact with the substrate 20.

(Antigen detection method)
The receptor 10 for recognizing an antigen of the present invention can be suitably used for detecting or measuring an antigen in an immunoassay, for example. Specifically, an antigen is brought into contact with the antigen-recognizing receptor 10 of the present invention, and a labeled marker having a labeling substance such as an enzyme, a fluorescent dye, a luminescent dye, a radioactive substance, a magnetic substance, a conductive substance, and a quantum dot By incorporating it into a detection system, the target antigen can be detected based on the intensity of the signal obtained from the enzyme activity or various labeling substances.

  The labeling substance is not particularly limited, and usually, an enzyme, a fluorescent dye, a luminescent dye, a radioactive substance, a magnetic substance, a conductive substance, a quantum dot, etc. are used, and among them, a quantum dot (QD: Quantum Dot) Is preferably used. This is because quantum dots are particularly bright as probes for fluorescence imaging because they are much brighter and less likely to undergo photobleaching compared to other organic fluorescent dyes. Polymer Coat QD with various functional groups on the surface is commercially available from Life Technologies, and these are reacted with amino groups, carboxyl groups, and thiol groups in which disulfide bonds are reduced in protein molecules that compose antibodies. This is preferable because the antibody can be labeled with QD like other organic dyes and enzymes. Any method may be used as long as the label does not affect the antigen recognizability of the antibody, and the labeling can be easily performed by using a commercially available labeling kit. For example, for CdSe / ZnS quantum dots, Qdot 605 ITK Carboxyl Quantum Dots (Q21301MP, Life Technologies) and the like can be preferably used.

A known method may be used as a mechanism that acts on the detection of an antigen in a labeled marker having a labeling substance. For example, it may be a substance containing an antibody that recognizes an antigen of interest and marked by an antigen-antibody reaction, or a substance having a structure of adsorbing to a part of the antigen.
Alternatively, an affinity site may be added to the antigen itself, and a substance having an affinity for the affinity site may be used as the marker part. As the affinity site, a metal, a metal atom, a sulfur atom, a resin, various functional groups, or the like may be appropriately selected depending on the application.

  The antigen-recognizing receptor 10 of the present invention is not particularly limited, but can be suitably used for ELISA (Enzyme-Linked Immuno Sorbent Assay) which is an enzyme-linked immunosorbent method among immunoassays. ELISA is a method of detecting or measuring an antigen by utilizing a highly specific antigen-antibody reaction and using a color development or luminescence signal based on an enzyme reaction for detection. Although there are detection methods such as direct method, indirect method, sandwich method, and competition method in ELISA, any detection method can be used as the antigen detection method of the present invention. Among these, the same protein is sandwiched between two types of antibodies, a capture antibody and a detection antibody, for detection, and therefore the sandwich method is preferably used from the viewpoint of having extremely high specificity.

  As the substrate 20 on which the low molecular weight antibody 12 is immobilized, for example, a 96-well microtiter plate made of polystyrene can be used. As the microplate, a plate for ELISA is usually used. Among them, a plate that has been surface-treated in advance to allow high protein binding, or nickel that can selectively immobilize only the target fusion protein via a His tag is used. A chelate coated plate or the like can be preferably used. Specifically, maxisorp (high binding capacity type) Nunc immunoplate (437111, Thermo Fisher Scientific Co., Ltd.) and immobilizer (nickel chelate) Nunc immunoplate (436024, Thermo Fisher Scientific Co., Ltd.) And the like can be preferably used.

  As the blocking solution, a material that can suppress the influence of non-specific adsorption of proteins may be used. Among them, bovine serum albumin (BSA), skim milk, gelatin, synthetic polymer material and the like can be preferably used. Specifically, a skim milk solution diluted to 1 to 5% (W / V) with phosphate buffered saline can be preferably used.

  Moreover, the receptor 10 for recognizing an antigen of the present invention can be used as a sensor chip for the surface plasmon resonance method (SPR method) and the crystal resonator microbalance method (QCM method), and is suitable for the measurement of the SPR method and the QCM method. Can be used. For example, a gold substrate 20 may be coated with Ni-NTA (nickel-nitrilotriacetic acid) or the like to form a film, and the antigen-recognizing receptor 10 may be immobilized on the film to measure.

(Antigen measurement kit)
The configuration of the antigen measurement kit of the present invention is not particularly limited as long as it includes the antigen recognition receptor 10 of the present invention. The antigen measurement kit of the present invention can be manufactured using commonly used materials and methods. The antigen measurement kit of the present invention may include, for example, a sample tube, a plate, instructions for the user of the kit, a solution, a buffer, a reagent, a sample suitable for standardization or a control sample. More specifically, it may contain, for example, a reagent for immunoassay, a carrier for affinity purification, a filter or mask for capturing an antigen, a reagent for cell imaging and the like.

  As described above, the antigen recognition receptor 10 of the present invention contains the low molecular weight antibody 12 and can be oriented and fixed on the substrate 20. The antigen detection method and the antigen measurement kit using the antigen recognition receptor 10 of the present invention are suitable for diagnostic use, analytical use, sensor use, and the like.

  The present invention is not limited to the configuration described above, various modifications can be made within the range described in the specification, and an embodiment obtained by appropriately combining known technical means is also included in the present invention. It is included in the technical scope.

  Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

(Preparation of antigen recognition receptor)
According to the procedure described below, an antigen recognition receptor having the structure shown in Example 1 below was prepared.
Example 1 describes a VHH antibody that recognizes Troponin T derived from cardiac muscle as an antigen, and a peptide A consisting of the amino acid sequence represented by SEQ ID NO: 1 (described as PepA in the formula showing the sequence of Example 1). ) And a His tag having a histidine residue as a peptide tag are linked.
Example 1: VHH-PepA-His

<Preparation of peptide A>
Peptide A consisting of the amino acid sequence represented by SEQ ID NO: 1 was prepared by substituting glutamine (Gln) for the hydrophobic amino acid in the myoglobin A chain (A1 to A16).
Sequence number 1 in the sequence listing
Array (N → C): SEGEWQQQQQHQWAHQE

<Preparation of low molecular weight antibody expression vector>
DNA encoding VHH gene information that recognizes troponin T as an antigen and DNA encoding the gene information of His tag, which is a protein tag forming 6 consecutive histidine residues at the 3'end side thereof, are consecutively formed. For the plasmid DNA having the arranged DNA sequence, two types of short DNA chains (hereinafter referred to as primers) having sequences complementary respectively to the double strand of this plasmid DNA were prepared (forward primer and reverse primer). A DNA fragment obtained by dividing the gene information of peptide A having an α-stand structure into two at arbitrary positions was added to the 5'ends of these two kinds of primers in advance.

  Next, a mixture of dATP, dCTP, dGTP, and dTTP with plasmid DNA encoding gene information of VHH and His tag, two types of forward and reverse primers to which DNA fragments obtained by previously dividing gene information of peptide A were added respectively. Mixed dNTP (deoxyribonucleotide 5'-3 phosphate), DNA polymerase, and a dedicated buffer solution attached to an enzyme reagent necessary for the DNA polymerase to exhibit activity are mixed in a PCR gene amplification device to obtain a template. Was amplified by the inverse PCR method. As the PCR enzyme, KOD-Plus-Ver.2 (KOD-211, Toyobo Co., Ltd.) was used, and a designated PCR reaction solution was prepared using a dedicated reagent attached to this PCR enzyme reagent. PCR was performed.

  On the other hand, two kinds of primers were prepared in advance without adding DNA information encoding the gene information of peptide A (forward primer and reverse primer). The template plasmid DNA was amplified by the inverse PCR method using a PCR gene amplification device. The quality of the gene amplification treatment was confirmed by agarose gel electrophoresis of the sample after the PCR reaction from the molecular weight of the DNA fragment.

The DNA amplified by the inverse PCR method shows a double-stranded linear shape, and the T4 DNA Polymerase was allowed to act on this DNA chain to blunt the DNA ends. MO203S (New England Biolabs) was used as T4 DNA Polymerase. The DNA sample treated with Polymerase was purified by the QIAquick PCR Purification Kit (28104, QIAGEN). Next, T4 DNA Ligase (M0202S, New England Biolabs) and T4 Polynucleotide Kinase (M0201S, New England Biolabs) were allowed to act to ligate the DNA ends after the phosphorylation of the DNA ends to circularize. The circularized DNA sample was confirmed from the molecular weight of DNA by agarose gel electrophoresis.

<Transformation of E. coli for plasmid recovery>
Next, a plasmid DNA sample of the prepared expression vector was placed in a competent cell of Escherichia coli DH5α strain (DS220, Biodynamics Research Institute, Inc.) in a sample tube placed on ice and mixed. After being placed in ice for 30 minutes, they were thermally stimulated on a water bath at 42 ° C. for 30 seconds, and again placed in ice for 2 minutes. To the treated competent cells, SOC medium which had been kept at 37 ° C in advance was added, and the cells were cultured at 37 ° C for 1 hour. The liquid after culturing for 1 hour was dropped on an LB agar plate medium, spread with a conradi stick, and statically cultivated at 37 ° C. for 17 hours.

<Confirmation of transformants by colony PCR method>
Next, Escherichia coli that appeared as a colony on the medium was collected in a microtube, and the plasmid in the cells was amplified by a PCR gene amplification device. KOD Dash (LDP-101, Toyobo Co., Ltd.) was used as the PCR enzyme. Plasmid DNA of the transformant, T7 Promoter Primer (69348, Novagen) and T7 Terminator Primer (69337, Novagen) complementary to the plasmid used in the pET system, and dATP (dCTP, dGTP, dTTP) mixed dNTP (deoxyribonase). (Nucleotide 5'-3 phosphate), DNA polymerase, and a dedicated buffer solution attached to the enzyme reagent necessary for the DNA polymerase to exhibit activity are mixed, and the template plasmid DNA is amplified with a PCR gene amplification device. did. Regarding the amplified DNA, the quality of the gene amplification treatment was confirmed from the molecular weight of the DNA fragment by agarose gel electrophoresis of the sample after the PCR treatment. Further, a colony of a transformant candidate carrying the target plasmid DNA was cultured in a liquid LB medium containing ampicillin at 37 ° C. for one day to grow the transformant, and then the plasmid was prepared by the alkali-SDS method. DNA was extracted from the cells. Plasmid mini kit (12125, Qiagen) was used for extraction. The DNA sequence of the recovered plasmid was analyzed to confirm that it was a transformant having the desired plasmid.

<Transformation of E. coli for protein expression>
Next, put the prepared plasmid DNA sample of the expression vector whose gene sequence was confirmed in a competent cell of Escherichia coli BL21 (DE3) strain (DS220250, Biodynamics Research Institute, Inc.) in a sample tube placed on ice. , Mixed. After being placed in ice for 30 minutes, they were thermally stimulated on a water bath at 42 ° C. for 30 seconds, and again placed in ice for 2 minutes. The SOC medium preliminarily kept at 37 ° C. was added to the treated competent cells, and the cells were cultured at 37 ° C. for 1 hour. The liquid after culturing for 1 hour was dropped on an LB agar plate medium, spread with a conradi stick, and statically cultivated at 37 ° C. for 17 hours.

<Transformant protein expression (preculture)>
A colony of E. coli transformed for protein expression was placed in a 15-mL culture tube containing 1 mL of liquid LB medium containing an antibiotic such as ampicillin, and shake-cultured at 37 ° C. for 17 hours (preculture).

<Transformant protein expression (main culture)>
After pre-culturing, add the pre-culture liquid to a 50-mL culture tube containing 4 mL of liquid LB medium containing antibiotics such as ampicillin, and add about 1/13 of the pre-culture medium to the main culture medium at 29 ° C. The culture was carried out with shaking for about 3 hours. Then, isopropyl-β-D-galactopyranoside (IPTG) as a protein inducer was added, and the mixture was further shake-cultured at 29 ° C. for 17 hours (main culture).

<Extraction of expressed protein>
The main culture was centrifuged to collect the cells, and the supernatant was removed. The supernatant at this time was also collected as a solution containing extracellular fraction components. Phosphate buffered saline having a pH of 7.4 was added to the precipitate, and the precipitate was collected again by centrifugation and the supernatant was removed. This was repeated several times. A cell lysate containing a surfactant as a main component was added to the obtained precipitate for cell lysis treatment. A commercially available cell lysate can be used for the cell lysis treatment, and B-PER Bacterial Protein Extraction Reagent (78248, Thermo Scientific), which is a B-PER solution, was used. 2-4 mL of the B-PER solution was added per 1 g of the above-mentioned precipitate, and the cell lysis treatment was performed while the container was rotated and stirred for about 3 hours. The supernatant was collected by centrifugation. By this extraction treatment, both soluble and insoluble (inclusion bodies, recovered as pellets) and recombinant proteins were recovered.

<Purification with Ni-NTA resin and gel filtration column>
The Ni-NTA slurry solution was added to the supernatant collected by the cell lysis treatment, and the mixture was vortexed on a rotary shaker and then centrifuged at 1000 rpm for 1 minute. The supernatant was removed, phosphate buffered saline having a pH of 7.4 was added, and the mixture was gently inverted and stirred, and then centrifuged at 1000 rpm for 1 minute, and the supernatant was removed to obtain a precipitate. This was repeated 3 times. Next, a 20 mM imidazole solution was added to the obtained precipitate, gently inverted and stirred, and then gently centrifuged to remove the supernatant. This was repeated 3 times. Further, a 250 mM imidazole solution was added to the precipitate, the mixture was rotatively stirred on a rotatory shaker, and then centrifuged at 1000 rpm for 1 minute. The supernatant was removed, phosphate buffered saline having a pH of 7.4 was added, and the mixture was gently inverted and stirred, and then centrifuged at 1000 rpm for 1 minute to recover the supernatant. This was repeated 5 times, and the collected supernatant was collected each time. The supernatant was applied to a gel filtration column (PD-10 Desalting Columns, GE Healthcare), and the eluate from the column was collected.

<Confirmation of expressed protein>
To confirm the expressed protein, the protein molecules contained in the column eluate were confirmed from the molecular weight of the protein by SDS-polyacrylamide gel electrophoresis.

(Activity evaluation of antigen recognition receptor)
Next, the antigen-recognizing receptor of Example 1 was treated with a nickel-coated plastic substrate containing a nickel-coated 96-well microplate (for example, immobilizer (nickel chelate) Nunc immunoplate (436024, Thermo Fisher Scientific ( Strain))) and fixed on the substrate via the His tag. It was examined whether or not the immobilized antigen recognition receptor maintained its activity.
Specifically, the activity of the antigen recognition receptor having the structure shown in Example 1 was evaluated according to the procedure described below.

<Preparation of quantum dot labeled antibody>
Anti-troponin T, which is a CdSe / ZnS quantum dot surface-modified with a carboxyl group, has an amino group previously introduced into Qdot 605 ITK Carboxyl Quantum Dots (Q21301MP, Life Technologies) to partially reduce the disulfide bond constituting the antibody. Coupling with the thiol group of the antibody produced a quantum dot (QD) labeled anti-troponin T antibody.

<Antigen detection using low molecular weight antibody and QD-labeled antibody>
20 μg / mL of a purified low molecular weight antibody against troponin T was added dropwise to a 96-well microplate at 100 μL / well, and the wells were left at room temperature overnight after sealing the plate. After that, the solution in the well in which the low-molecular weight antibody was immobilized was removed, and a blocking solution (a skim milk solution diluted to 2% (W / V) with phosphate buffered saline having a pH of 7.4) was added dropwise at 300 μL / well. After the plate was sealed, the well was allowed to stand at 4 ° C. for 1 hour and then at room temperature for 1 hour.

  Next, the blocking solution in the wells was removed, and bovine serum albumin (BSA) solution diluted to 4% (W / V) with phosphate buffered saline of pH 7.4 was used to adjust the concentration of troponin T as an antigen to each concentration. 100 μL / well was added to each diluted solution, and the wells were subjected to plate sealing and then reacted at room temperature for 1.5 hours. After removing the diluted antigen solution, 300 μL / well of phosphate-buffered saline of pH 7.4 containing 0.05% (V / V) Tween 20 was added to each well to wash the wells three times in total. For detection, QD-labeled anti-troponin T antibody was added at 100 μL / well, and the wells were sealed with a plate and then reacted at room temperature for 1 hour. Subsequently, after removing the QD-labeled antibody solution, 300 μL / well of borate buffer solution of pH 7.5 containing 0.05% (V / V) Tween 20 was added to each well, and the wells were washed three times in total. did. The microplate on which a series of reactions was completed was measured at a excitation light wavelength of 450 nm, a detection wavelength of 605 nm, and a gain of 135 using a microplate reader capable of measuring fluorescence. A calibration curve was prepared from each antigen concentration and the obtained fluorescence intensity value.

The measurement results are shown in Table 1 and FIG.

When the antigen recognition receptor of the present invention was used, the fluorescence intensity increased depending on the troponin T (TnT) concentration. From this, it was confirmed that the immobilized antigen recognition receptor of the present invention maintained its activity.
From the above, it was shown that an antigen can be efficiently captured by using the receptor for antigen recognition of the present invention.

10 Receptor for antigen recognition 12 Low molecular weight antibody 14 Peptide A
16 peptide tag 20 substrate

Claims (6)

  An antigen-recognizing receptor containing a low-molecular antibody and peptide A, wherein the peptide A has an α-helix structure.   The antigen recognition receptor according to claim 1, wherein the low-molecular-weight antibody is an scFv antibody, a camelid-derived VHH antibody, or a shark-derived IgNAR antibody.   The antigen recognition receptor according to claim 1 or 2, further comprising a peptide tag.   The antigen recognition receptor according to claim 3, wherein the low-molecular-weight antibody-containing site, the peptide A-containing site, and the peptide tag-containing site are bound in this order.   An antigen measurement kit comprising the antigen recognition receptor according to any one of claims 1 to 4.   An antigen detection method comprising contacting an antigen with the receptor for recognizing an antigen according to any one of claims 1 to 4.

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