Method for producing protein fine particles having antigen-binding activity

Abstract

To provide a method for producing protein fine particles having antigen-binding activity, from cocoons expressing a fusion protein of a single-chain antibody and fibroin protein.SOLUTION: The present invention provides a method for producing protein fine particles having antigen-binding activity. The method involves mixing: a protein solution prepared by dissolving cocoons comprising a fusion protein of a single-chain antibody and fibroin protein; and a salt solution comprising at least one salt selected from phosphates, hydrochlorides, sulfates, and citrates.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for producing protein microparticles having antigen-binding activity. [Background technology]

[0002] Using recombinant silkworm technology, silk proteins have been modified by introducing foreign genes to design and create silk with improved physical properties and added functionality. As a type of silk expressing such foreign proteins, "affinity silk" is known, which expresses a fusion protein of a single-chain antibody and a fibroin protein. Affinity silk is silk expressing a fusion protein of a fibroin protein and a single-chain antibody. Patent Document 1 discloses a technology that allows a composition containing a single-chain antibody that retains high antigen binding activity to be produced simply and quickly without fibrosis (gelation) by fusing a single-chain antibody (scFv) with a fibroin protein to express it in silk thread, dissolving the resulting silk thread without refining, and diluting the solution to reduce the salt concentration.

[0003] It is known that the fibroin protein that constitutes silk can be processed into various forms, such as not only fibers but also solutions, sponges, powders, films, and plastic-like molded bodies (Non-Patent Document 1). In addition, silk microparticles (e.g., nanoparticles) are forms that can be directly processed from the silk solution state. Silk microparticles are expected to be applied as drug delivery carriers (Non-Patent Document 2). In all preparations of silk microparticles, the cocoon thread is refined to remove the sericin protein, and then the fibroin protein is microparticulated, and there have been no reports of microparticulating the cocoon thread without refining it. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent No. 6870851 [Non-patent literature]

[0005] [Non-Patent Document 1] Katsura Kojima, Genetically Modified Silkworms Open the Frontier of Silk Utilization, Chemistry and Biology, The Agricultural Chemical Society of Japan, 2016, Vol. 54, No. 12, p. 915-919 [Non-Patent Document 2] DRUG DELIVERY, 2020, Vol.27, No.1, p.431-448 Summary of the Invention [Problem to be solved by the invention]

[0006] Silk filaments expressing a fusion protein of a single-chain antibody and a fibroin protein are refined before being microparticulated to inactivate the single-chain antibody.

[0007] Therefore, the present invention provides a method for producing protein microparticles having antigen binding activity from silk filaments in which a fusion protein of a single-chain antibody and a fibroin protein has been expressed. [Means for solving the problem]

[0008] The present inventors have conducted extensive research in view of the above problems, and have found that the above problems can be solved by a method for producing protein microparticles having antigen-binding activity, the method comprising mixing a protein solution in which a cocoon filament containing a fusion protein of a single-chain antibody and a fibroin protein is dissolved, with a salt solution containing at least one salt selected from the group consisting of phosphates, hydrochlorides, sulfates, and citrates, thereby completing the present invention. Effect of the Invention

[0009] The present invention provides a method for producing protein microparticles having antigen-binding activity from silk filaments expressing a fusion protein of a single-chain antibody and a fibroin protein. [Brief description of the drawings]

[0010] [Figure 1] 1 is a graph showing the results of activity measurement by ELISA of a protein solution in Test Example 1 (vertical axis: color absorbance (A492), horizontal axis: antigen human CRP concentration (μg / mL)). [Diagram 2] 1 is an electron microscope photograph (2500x magnification) showing the results of confirming the shape of protein microparticles in Test Example 1. [Diagram 3] 1 is a photograph showing the results of immune agglutination reaction of protein microparticles in Test Example 2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Hereinafter, the embodiment for carrying out the present invention will be described in detail. Note that the present invention is not limited to the following embodiment, and can be modified in various ways within the scope of the claims. In addition, in this specification, "X to Y" indicating a range includes X and Y, and means "X or more and Y or less". In this specification, "X and / or Y" means including at least one of X and Y, and includes "X alone", "Y alone", and "combination of X and Y". In addition, unless otherwise specified, the operation and measurement of physical properties are performed under the conditions of room temperature (20 to 25°C) / relative humidity 40 to 60% RH.

[0012] One aspect of the present invention relates to a method for producing protein microparticles having antigen-binding activity, the method comprising mixing a protein solution in which silk thread containing a fusion protein of a single-chain antibody and a fibroin protein is dissolved with a salt solution containing at least one salt selected from the group consisting of phosphates, hydrochlorides, sulfates, and citrates.

[0013] In the production method of this embodiment, protein microparticles having antigen binding activity can be produced from silk filaments in which a fusion protein of a single-chain antibody and a fibroin protein has been expressed.

[0014] In this specification, "silk filament containing a fusion protein of a single-chain antibody and a fibroin protein" is also simply referred to as "silk filament of the present invention."

[0015] A single-chain antibody (single-chain variable fragment: scFv) refers to a molecule in which variable regions (VH and VL) are taken from the heavy chain (H chain) and light chain (L chain) constituting the antigen-binding domain (Fab) of an antibody molecule and linked via a linker (peptide linker). Single-chain antibodies include antibodies having multiple VHs and VLs in a single polypeptide chain. An example of a single-chain antibody is a single-chain antibody (sc(FV)2) in which two VHs and two VLs are linked via a linker or the like. Such single-chain antibodies usually require multiple linkers. The linkers may be the same or different linkers. Single-chain antibodies also include dimers (diabodies) formed by setting the length of the linker short (for example, about 5 amino acids) to suppress the binding of VH and VL within the same polypeptide chain.

[0016] The single chain antibodies used may be the same or may be two or more different single chain antibodies.

[0017] The linker in the single chain antibody is not particularly limited as long as it does not inhibit the expression of VH and VL linked to both ends, the binding between VH and VL, etc. It is a peptide consisting of amino acids, preferably 1 to 50 amino acids, more preferably 1 to 30 amino acids, further preferably 12 to 18 amino acids, and particularly preferably 15 amino acids. As the linker according to the present invention, a polypeptide consisting of 15 amino acids, "(GGGGS) x 3" (G represents glycine and S represents serine), can be suitably used.

[0018] The method for preparing DNA encoding a single-chain antibody is not particularly limited, and any conventionally known method can be used. For example, first, mRNA is isolated from a hybridoma expressing a monoclonal antibody that specifically binds to a target antigen, and then cDNAs encoding the VH and VL of the monoclonal antibody are isolated from a cDNA library prepared using the mRNA. Next, DNA encoding a linker is inserted between the cDNA encoding VH and the cDNA encoding VL, and a recombinant DNA encoding a single-chain antibody is constructed by gene recombination technology.

[0019] Without preparing a cDNA library, genes encoding VH and VL can be directly amplified from mRNA by RT-PCR, and the amplified fragments can be used to construct DNA encoding a single-chain antibody by recombinant gene technology.

[0020] In this specification, the fibroin protein refers to at least one protein selected from the group consisting of fibroin L chain, fibroin H chain, and fibrohexamarin (P25). It is known that the fibroin protein is formed by disulfide bonding between the fibroin L chain and the fibroin H chain, and fibrohexamarin is bonded to every six of the bonded molecules, forming a complex (fibroin complex).

[0021] The fibroin L chain is a fibroin protein with a molecular weight of about 30,000 Da. A typical example of the fibroin L chain is a protein consisting of the 17th to 262nd amino acid sequence of the protein specified by GenBank Accession No. NP_001037488.1 (a gene consisting of the 90th to 827th base sequence of the DNA specified by GenBank Accession No. NM_001044023.1). This protein is a mature form obtained by cleaving and removing a signal peptide (a protein consisting of the 1st to 16th amino acid sequence of the protein specified by GenBank Accession No. NP_001037488.1) from a precursor, so that the fibroin L chain according to the present invention is, for example, a protein specified by GenBank Accession No. NP_001037488.1 (a gene specified by GenBank Accession No. NM_001044023.1).

[0022] The fibroin H chain is a fibroin protein with a molecular weight of about 350,000 Da. A typical example of the fibroin H chain is a protein consisting of the 22nd to 5263rd amino acid sequence of the protein specified by GenBank Accession No. NP_001106733.1 (a gene consisting of the 64th to 15789th base sequence of the DNA specified by GenBank Accession No. NM_001113262.1). This protein is a mature type obtained by cleaving and removing a signal peptide (a protein consisting of the 1st to 21st amino acid sequence of the protein specified by GenBank Accession No. NP_001106733.1) from a precursor, so that the fibroin L chain according to the present invention is, for example, a protein specified by GenBank Accession No. NP_001106733.1 (a gene specified by GenBank Accession No. NM_001113262.1).

[0023] Fibrohexamarin is a fibroin protein with a molecular weight of about 25,000 Da, and is also called "P25". Typical examples of fibrohexamarin include 17-proteins identified by GenBank accession number NP_001139413.1. Examples of the fibrohexamarin of the present invention include a protein consisting of the 220th amino acid sequence (a gene consisting of the 49th to 660th base sequence of the DNA specified by GenBank Accession No. NM_001145941.1). In addition, since this protein is a mature form obtained by cleaving and removing a signal peptide (a protein consisting of the 1st to 16th amino acid sequence of the protein specified by GenBank Accession No. NP_001139413.1) from a precursor, examples of the fibrohexamarin of the present invention include a protein specified by GenBank Accession No. NP_001139413.1 (a gene specified by GenBank Accession No. NM_001145941.1).

[0024] The fibroin protein of the present invention may be a mutant or a partial fragment, so long as it can form protein microparticles. The fibroin protein of the present invention may include mutants and partial fragments of fibroin L chain, fibroin H chain, or fibrohexamarin.

[0025] An example of a partial fragment of the fibroin L chain is a protein consisting of the amino acid sequence from 1 (methionine residue) to 242 (alanine residue) of the protein specified by GenBank Accession No. NP_001037488.1. The effectiveness of this partial fragment of the fibroin L chain has been demonstrated in a fibroin L chain-GFP fusion protein. An example of a partial fragment of the fibroin H chain is a protein consisting of the amino acid sequence from 1 (methionine residue) to 153 (alanine residue) of the protein specified by GenBank Accession No. NP_001106733.1 and a protein consisting of the amino acid sequence from 5205 (serine residue) to 5263 (cysteine ​​residue). This partial fragment of the fibroin H chain is the minimum unit of the fibroin H chain that has been confirmed to be capable of forming a heterodimer with the fibroin L chain (forming an S-S bond) and being secreted as a fibroin protein.

[0026] The fusion protein of the single-chain antibody and fibroin protein according to the present invention may be a protein in which the single-chain antibody is fused with at least one protein selected from the group consisting of fibroin L chain, fibroin H chain, and fibrohexamarin. The fibroin protein fused with the single-chain antibody is preferably at least one protein selected from the group consisting of fibroin L chain and fibroin H chain, and more preferably fibroin L chain, from the viewpoint that the single-chain antibody is easily arranged on the surface of the fibroin complex and a single-chain antibody with higher antigen specificity is easily obtained.

[0027] In the fusion protein according to the present invention, the fibroin protein may be fused to either the N-terminus or the C-terminus of the single-chain antibody, or may be fused to both the N-terminus and the C-terminus. Furthermore, the fibroin protein may be fused directly to the single-chain antibody, or may be fused indirectly via a linker (peptide linker). The number of amino acids in the linker peptide is, for example, 1 to 100, preferably 1 to 50, and more preferably 1 to 30.

[0028] The fusion protein of the single-chain antibody and the fibroin protein may further be fused with another functional protein. The other functional protein may be fused directly or indirectly to either or both of the N-terminus and C-terminus of the fusion protein, or between the single-chain antibody and the fibroin protein. There are no particular limitations on the other functional protein, and it may be appropriately selected depending on the function to be imparted to the fusion protein of the present invention. Examples of the other functional protein include proteins used for purification, detection, etc. of the fusion protein. Examples of the other functional protein include green fluorescent protein (GFP), luciferase protein, Myc-tag protein, His-tag protein, and the like. Examples of such proteins include hemagglutinin (HA)-tagged proteins, FLAG-tagged proteins (registered trademark, Sigma-Aldrich), and glutathione-S-transferase (GST) proteins.

[0029] The expression of the fusion protein in the cocoon filament can be carried out by a conventionally known method, such as a method of constructing a vector for expressing the fusion protein and introducing the vector into silkworms to express the fusion protein.

[0030] When expressing a fusion protein in the posterior silk gland of a silkworm, which synthesizes and secretes fibroin protein, the DNA encoding the fusion protein in the vector is linked downstream of a promoter that ensures expression in the posterior silk gland of a silkworm. As such a promoter, for example, the promoter of the fibroin L chain gene, the fibroin H chain gene, or the fibrohexamarin (P25) gene can be preferably used. The vector may have other expression control regions, enhancers, terminators, polyA signals, non-translated regions (5'-UTR and 3'-UTR), etc.

[0031] A vector for expressing a fusion protein may include DNA encoding another protein and its expression control region in addition to an expression cassette having DNA encoding the fusion protein and its control region. The DNA encoding another protein is not particularly limited, and may include, for example, genes encoding reporter proteins such as GFP and DsRed, drug resistance genes, etc. The expression control region of the DNA encoding the other protein may share a promoter and / or enhancer of the DNA encoding the fusion protein via an internal ribosome entry site (IRES), or may include a promoter different from the expression control region of the DNA encoding the fusion protein. Examples of the expression control region include the 3×P3 promoter, silkworm actin promoter, Drosophila-derived HSP70 promoter, enhancer, terminator, polyA signal, non-translated region (5'-UTR and 3'-UTR), etc.

[0032] As a vector for expressing a fusion protein, for example, a vector using a transposon can be suitably used. Examples of vectors using a transposon include vectors using DNA transposons such as piggyBac, Minos, and Mariner. In a vector using a transposon, the expression cassette is placed between a pair of terminal repeat sequences (inverted terminal repeat sequences) of a DNA transposon, whereby the expression cassette sandwiched between the terminal repeat sequences is excised from the vector by a transferase and is randomly inserted into a specific sequence (for example, the TTAA sequence in the case of using piggyBac) in the silkworm chromosome as a target.

[0033] When a vector using a transposon is used, a transferase corresponding to each transposon is also introduced into silkworms. The transferase may be directly introduced into silkworms, or an mRNA encoding the transferase or a plasmid vector encoding the transferase (helper plasmid) may be introduced. In the helper plasmid, examples of promoters for expressing the transferase include the promoter of an endogenous transferase gene, the silkworm actin promoter, and the HSP70 promoter derived from Drosophila melanogaster.

[0034] In the present invention, a vector for expressing the constructed fusion protein is introduced to prepare silkworms that produce cocoon filaments containing the fusion protein (in which the fusion protein is expressed). Methods for preparing such silkworms include, for example, injecting a vector for expressing the fusion protein into silkworm eggs, hatching and raising the eggs to obtain F0 silkworms, mating the obtained F0 silkworms with siblings or with wild-type silkworms to obtain F1 silkworms, and then crossing the obtained F0 silkworms with wild-type silkworms to obtain F1 silkworms. and selecting silkworms that express the fusion protein in their cocoon filaments from the F1 silkworms.

[0035] Injection of a vector for expressing a fusion protein into silkworm eggs can be carried out by appropriately selecting a method known to those skilled in the art. Known methods include, for example, a method of directly injecting (microinjecting) into silkworm eggs at the early stage of development using a DNA injection tube, and a method of physically or chemically drilling a hole in the eggshell using a needle, laser, etc., and then injecting a vector for expressing a fusion protein through the hole. In addition, when injecting a vector for expressing a fusion protein into silkworm eggs, a transferase, a helper plasmid, etc. may be injected together.

[0036] A method for selecting silkworms that express a fusion protein in their cocoon filaments from F1 silkworms includes, for example, irradiating F1 generation eggs or larvae with excitation light and using the fluorescence emitted from the eggs or larvae as an indicator when using an expression vector carrying a gene encoding a fluorescent protein such as GFP or DsRed. In particular, when using a fluorescent protein whose expression is controlled by the 3×P3 promoter, since the 3×P3 promoter has the effect of promoting gene expression in the nervous system (particularly the eyes), silkworms that produce cocoon filaments in which the fusion protein is expressed can be selected by selecting individuals that emit fluorescence in the nervous system and eyes of F1 generation eggs or larvae.

[0037] Methods for preparing cocoon threads containing (expressing) a fusion protein between a single-chain antibody and a fibroin protein from silkworms prepared in this manner include, for example, a method for recovering cocoon threads accumulated in the lumen of the silkworm's silk gland, a method for recovering silk threads spun by the silkworm, and a method for recovering cocoons formed by the silkworm.

[0038] The fibroin fibers synthesized in the posterior silk gland are sent to the middle silk gland, where they are concentrated, and are then wrapped in sericin synthesized in the middle silk gland. The cocoon threads (liquid silk) synthesized in this way are sent to the anterior silk gland, which is connected to the spinneret. The cocoon threads (liquid silk) accumulated in the lumen of the silk gland can be prepared by collecting the silk gland from the silkworm and removing the cells that constitute the silk gland (silk gland cells) from the collected silk gland. Methods for removing the silk gland cells include, for example, a method in which the silk gland is immersed in pure water to soften the silk gland cells and then the silk gland cells are removed, and a method in which the extracted silk gland is immersed in 30 to 100% ethanol to fix the fibroin and sericin, and then the silk gland cells are removed.

[0039] In this manner, the cocoon thread according to the present invention can be prepared. In this specification, "cocoon thread" refers to an animal fiber consisting of fibroin fibers composed of three types of fibroin proteins synthesized and secreted in the posterior silk gland of silkworms, and a sericin layer that envelops the fibroin fibers. In the present invention, the "cocoon thread" to be dissolved has a single-chain antibody that retains binding ability and antigen specificity. As described above, the single-chain antibody of the cocoon thread expressing a fusion protein of a single-chain antibody and a fibroin protein is inactivated by degumming. In a preferred embodiment of the present invention, the cocoon thread according to the present invention is not degummed (the sericin layer is not removed).

[0040] The method for preparing a protein solution according to the present invention includes the steps of mixing a cocoon thread containing a fusion protein of a single-chain antibody and a fibroin protein prepared by the above-mentioned method with a solution for dissolving the cocoon thread to prepare a cocoon thread dissolution solution, centrifuging the cocoon thread dissolution solution to remove precipitates, and diluting the cocoon thread dissolution solution from which the precipitates have been removed to prepare a protein solution.

[0041] In the step of preparing a solution for dissolving cocoon filaments, the cocoon filaments according to the present invention are mixed with a solution for dissolving cocoon filaments.

[0042] As the cocoon thread, the cocoons produced by the silkworms may be used as they are, or the cocoons may be cut with scissors or the like and moistened with ethanol (e.g., 70% ethanol) in order to increase the hydrophilicity and improve solubility.

[0043] Examples of dissolving components contained in the solution for dissolving cocoon filaments include lithium bromide, ethylenediamine chloride, sodium thiocyanate, lithium thiocyanate, magnesium nitrate, NMO (N-methylmorpholine N-oxide), lithium chloride / N,N-DMAc (N,N-dimethylacetamide), hexafluoroisopropanol, calcium nitrate / methanol, and mixtures thereof. The dissolving component is preferably lithium bromide, from the viewpoint of being able to more effectively exert the effects of the present invention.

[0044] The solution for dissolving the cocoon filaments may contain a buffer. Examples of the buffer include Tris-HCl buffer, phosphate buffer, Good's buffer, etc. The buffer is preferably Tris-HCl buffer.

[0045] The solution for dissolving the cocoon filaments is preferably an aqueous solution containing lithium bromide and a Tris-HCl buffer solution.

[0046] The pH, concentration of dissolved components and concentration of buffer agent of the solution for dissolving cocoon thread can be appropriately adjusted to the pH, concentration of dissolved components and concentration of buffer agent in the solution for dissolving cocoon thread described below.

[0047] In mixing the cocoon filaments of the present invention with the solution for dissolving the cocoon filaments, it is preferable to dissolve the cocoon filaments as much as possible (completely dissolve if possible) in order to facilitate the formation of protein microparticles. Mixing is preferably performed at about 20 to about 100°C (preferably about 37 to about 50°C) for 10 minutes to 12 hours while stirring (preferably shaking vigorously).

[0048] The pH of the solution for dissolving the cocoon filaments is, for example, 2-12, and preferably 7-10.

[0049] The concentration of the cocoon filaments in the solution in which the cocoon filaments are dissolved is, for example, 30 to 50 mg / mL.

[0050] The concentration of the dissolved components in the solution for dissolving the cocoon filaments is, for example, 7 mol / L or more, preferably 7 to 10 mol / L, more preferably 7 to 9 mol / L, and particularly preferably 7 mol / L.

[0051] The concentration of the buffer in the solution for dissolving the cocoon filaments is, for example, 10 to 200 mmol / L.

[0052] The concentrations of the dissolved components and the buffer in the solution for dissolving the cocoon filaments can be adjusted using alcohol (preferably ethanol), water (preferably pure water or RO water), and the like.

[0053] In the step of removing the precipitate, the solution of the cocoon filaments is centrifuged to remove the precipitate.

[0054] The conditions for centrifugation are not particularly limited, and may be, for example, 12,000 to 15,000 rpm for about 30 minutes.

[0055] The solution of the cocoon filaments from which the precipitate has been removed can be stored until it is used in the process of preparing a protein solution. In this case, in order to stably maintain the antibody activity, it is preferable to dilute the solution with water (preferably pure water or RO water) to a concentration of about 2 mol / L.

[0056] In the process of preparing the protein solution, the cocoon filament dissolution solution from which the precipitate has been removed is diluted. By diluting the cocoon filament dissolution solution, the concentration of the dissolved components is reduced. When diluting the cocoon filament dissolution solution, water (preferably pure water or RO water), the above-mentioned buffer agent, etc. can be used.

[0057] The temperature at which the dilution is carried out is preferably 4 to 60° C., from the viewpoint of being able to suppress denaturation, decomposition, aggregation, and the like of proteins.

[0058] The concentration of the cocoon filament in the protein solution is, for example, 1.0 to 20.0 mg / mL, preferably 1.0 to 10.0 mg / mL, and more preferably 2.5 to 5.0 mg / mL.

[0059] The concentrations of the dissolved components and the buffer in the protein solution do not affect the formation of protein microparticles so long as they are 1.0 mol / L or less and 750 mmol / L or less, respectively.

[0060] In one embodiment, the method comprises preparing a protein solution by mixing a solution of the cocoon filament of the present invention and a solution of the refined wild-type cocoon filament. By using a solution of the refined wild-type cocoon filament, the particle size of the protein microparticles can be made larger than when only the solution of the cocoon filament of the present invention is used (see Test Example 1).

[0061] In this specification, the protein solution obtained by mixing the dissolution solution of the cocoon filament according to the present invention with the dissolution solution of the refined wild-type cocoon filament is also referred to simply as the "protein solution (mixed solution)." In this specification, the contents described as the protein solution are also applied to the protein solution (mixed solution).

[0062] The solution of refined wild-type cocoon filaments can be prepared in the same manner as the solution of the cocoon filaments according to the present invention described above.

[0063] The concentration of the cocoon filaments in the protein solution (mixed solution) (total concentration of the cocoon filaments of the present invention and the wild-type cocoon filaments) is, for example, 1.0 to 20.0 mg / mL, preferably 1.0 to 10.0 mg / mL, and more preferably 2.5 to 5.0 mg / mL.

[0064] The concentration ratio (mass ratio) of the cocoon filament of the present invention to the refined wild-type cocoon filament in the protein solution (mixed solution) is not particularly limited and is, for example, 1:0.1 to 1:10, preferably 1:1 to 1:5. The concentration ratio may be 1:2 to 1:5.

[0065] The binding activity of the single-chain antibody contained in the protein solution to the antigen can be evaluated by carrying out an immunological method (eg, ELISA method) (see Test Example 1).

[0066] The production method of this embodiment includes mixing the above-mentioned protein solution with a salt solution containing at least one salt selected from the group consisting of phosphates, hydrochlorides, sulfates, and citrates, thereby producing protein microparticles.

[0067] The mixing ratio (volume ratio) of the protein solution to the salt solution is, for example, 1:2 to 1:7, preferably 1:3 to 1:6, and more preferably 1:4 to 1:5.

[0068] The salt according to the present invention is preferably a phosphate, more preferably potassium phosphate, and even more preferably potassium dihydrogen phosphate.

[0069] The salt concentration in the salt solution is, for example, 1.0 mol / L or more, preferably 1.0 to 2.0 mol / L, more preferably 1.2 to 1.3 mol / L, and even more preferably The concentration is usually 1.25 mol / L.

[0070] The method for preparing the salt solution according to the present invention is not particularly limited, and may be, for example, a method in which the above-mentioned salt is mixed with water (preferably pure water or RO water).

[0071] When mixing a protein solution and a salt solution, it is preferable to avoid physical stimulation as much as possible. Specifically, the protein solution and the salt solution are layered to form an interface, and then the protein solution and the salt solution are mixed. After the interface is formed, it is preferable to leave the solution standing for 5 minutes or more. When mixing, the solution is slowly inverted to mix uniformly. At this time, it is important to mix as slowly as possible so as not to generate bubbles. After mixing, the solution is left standing at room temperature overnight or at 2 to 8°C for 1 to 3 days to form protein microparticles. The formation of protein microparticles can be confirmed by an electron microscope.

[0072] In a preferred embodiment, from the viewpoint of being able to more effectively exert the effects of the present invention, a protein solution using lithium bromide as a soluble component contained in a solution for dissolving cocoon filaments, and a potassium phosphate (preferably potassium dihydrogen phosphate) solution (aqueous solution) are used as the salt solution.

[0073] The protein microparticles according to the present invention include nano-sized particles (less than 1000 nm). The protein microparticles can have a particle size in the range of, for example, 0.05 to 2.0 μm. The protein microparticles according to the present invention can contain at least 90% or more, preferably 95% or more, and more preferably 99% or more of protein microparticles having a particle size in the above range. The particle size of the protein microparticles according to the present invention means the maximum diameter (longest diameter) obtained by image analysis using an electron microscope. Protein microparticles having a particle size of more than 2.0 μm may be removed by a known method as necessary.

[0074] The protein microparticles can be collected as a precipitate by centrifuging at 3000 to 15000 rpm for 5 minutes or more. The collected protein microparticles are preferably washed with water (preferably pure water or RO water). The collected protein microparticles can be washed by adding an appropriate amount of water, suspending or dispersing by sonication, and then centrifuging at 3000 to 15000 rpm for 5 minutes or more to remove the supernatant. This makes it possible to remove proteins that are not involved in the formation of protein microparticles. During washing, it is preferable to avoid applying physical stimuli as much as possible. The number of washings is not particularly limited, and is, for example, 1 to 5 times, and preferably 3 times.

[0075] The protein microparticles recovered after washing can be made into a dispersion by adding water (preferably pure water or RO water) and treating with a sonicator.

[0076] The binding activity of the produced protein microparticles to an antigen can be evaluated by carrying out an immune agglutination reaction method (see Test Example 2).

[0077] The protein microparticles according to the present invention have antigen-binding activity and are therefore suitable as materials for drug delivery, immunochromatography, immunoassays, and the like.

[0078] One embodiment of the present invention is the use of the protein microparticles obtained by the production method of this embodiment as a reaction substrate in an immunoassay.

[0079] Immunoassay is a method for measuring an analyte in a sample by utilizing an antigen-antibody reaction, and is widely used because it can measure trace components specifically and accurately.

[0080] The latex agglutination immunoturbidimetry, which is one of the immunoassay methods, is a method in which the amount of the substance to be measured is measured by reacting the latex microparticles bound to antibodies, antigens, etc. with the object to be measured to agglutinate the latex microparticles, and measuring the difference in the degree of agglutination by absorbance, and is widely used in clinical tests, etc. When affinity silk is fixed to the surface of latex microparticles, self-agglutination occurs when an affinity silk solution is used, so it is difficult to fix affinity silk to the surface of latex microparticles in a solution state. On the other hand, since the protein microparticles according to the present invention have antibody activity, they can be used as a substitute for reaction substrates such as latex microparticles.

[0081] <Embodiment> The following describes an embodiment of the present invention. [1] A method for producing protein microparticles having antigen-binding activity, comprising the steps of: The production method includes mixing a protein solution in which silk thread containing a fusion protein of a single-chain antibody and a fibroin protein is dissolved with a salt solution containing at least one salt selected from the group consisting of phosphates, hydrochlorides, sulfates, and citrates. [2] The method according to [1], wherein the cocoon thread is unrefined. [3] A manufacturing method described in [1] or [2], which comprises mixing a lysate of cocoon filament containing the fusion protein with a lysate of refined wild-type cocoon filament to prepare the protein solution. [4] The method according to any one of [1] to [3], wherein the single chain antibodies are the same or two or more different single chain antibodies. [5] The method for producing a protein solution according to any one of [1] to [4], comprising layering the protein solution and the salt solution to form an interface, and then mixing the protein solution and the salt solution. [6] The method according to any one of [1] to [5], wherein the mixing ratio (volume ratio) of the protein solution to the salt solution is 1:4 to 1:5. [7] The method according to any one of [1] to [6], wherein the salt concentration in the salt solution is 1.0 mol / L or more. [8] The method according to any one of [1] to [7], wherein the concentration of the cocoon filament in the protein solution is 1.0 to 20.0 mg / mL. [9] The method according to any one of [1] to [8], wherein the protein microparticles include protein microparticles having a particle diameter of 0.05 to 2.0 μm.

[10] Use of protein microparticles obtained by the production method described in any one of [1] to [9] as a reaction substrate in an immunoassay. EXAMPLES

[0082] Hereinafter, the embodiments of the present invention will be described in more detail using examples, but the technical scope of the present invention is not limited to the following examples.

[0083] (Preparation of refined wild-type cocoon filaments) 1 g of cocoons produced by wild-type (W1) silkworms was finely chopped, 10 mL of 70% ethanol was added, and the mixture was stirred for 5 minutes, after which as much ethanol as possible was removed. 100 mL of scouring solution (0.02 M sodium carbonate water) was added, and the mixture was heated at 100°C for 30 minutes. After rinsing with 3 L of 60°C tap water and dehydrating with Wipeall, 100 mL of scouring solution was added again, and the mixture was heated at 100°C for 30 minutes. After rinsing with 3 L of 60°C tap water and then with purified water, the mixture was dried to prepare refined wild-type cocoon thread.

[0084] (Production of recombinant silkworm S27 strain expressing anti-human C-reactive protein (human CRP) single-chain antibody) Total RNA was purified from a hybridoma producing an anti-human CRP monoclonal antibody and analyzed by S Using the MART™ RACE cDNA amplification kit (Clontech), genes encoding the variable regions of the heavy and light chains (VH and VL) of the monoclonal antibody were isolated by the 5'-RACE method and the gene sequences were determined.

[0085] A single-chain antibody (scFv) was constructed by linking VH and VL DNA fragments via a flexible linker sequence (GGGGS×3).

[0086] Before using this single-chain antibody in silkworm genetic engineering, we expressed it in animal cells as a first screening and evaluated its binding activity. The single-chain antibody with a Myc tag added to the C-terminus was inserted into the animal cell expression vector pCAGGS-MCS vector to prepare an expression construct. For details on the pCAGGS-MCS vector, see Isolation and characterization of a novel HS1 SH3 domain binding protein, HS1BP3. Int. Immunol. 1999, 11, 1957-1964. The expression construct was transfected into cultured mouse T cells (DO-11.10) by electroporation. After 24 hours of culture, the cells were harvested and cell lysates were prepared using RIPA buffer (Nacalai Tesque). The cell lysate was added to a plate immobilized with human CRP, and the binding activity was evaluated by ELISA using an anti-Myc-tag-HRP-labeled antibody. As a result, anti-CRP-scFv-Myc with binding activity was obtained.

[0087] The DNA fragment of anti-CRP-scFv-Myc, whose binding activity was confirmed, was inserted between the fibroin L chain promoter and the sequence of fibroin L chain cDNA (FibLpro-FibL) and the fibroin L chain 3' untranslated region (FibL-3'-UTR) to construct a DNA construct (pLC / anti-CRP-scFv-Myc) expressing a fusion protein of fibroin L chain and anti-human CRP single chain antibody. Furthermore, this fusion protein expression unit was inserted into the recombinant silkworm vector pBac[3XP3-EGFPafm] to construct pBac[3XP3-EGFPafm]-LC-anti-CRP-scFv-Myc, which expresses the target protein in recombinant silkworms.

[0088] Silkworm genetic recombination was carried out according to the method described in Germline transformation of the silkworm Bombyx mori L-using a piggyBac transposon-derived vector, Nature Biotechnology 2000 Vol. 18 Issue 1 Pages 81-84. 952 fertilized eggs of the w1-pnd strain were microinjected with an injection buffer (0.5 mM phosphate buffer (pH 7.0), 5 mM KCl) containing DNA of FPafm-LC-anti-CRP-scFv-Myc and DNA of the helper plasmid pHA3PIG encoding the transferase of piggyBac, and incubated at 25°C until they hatched. 338 hatched larvae (G0) were reared and raised as parent moths, and then mated with sib or w1-pnd moths to obtain the next generation (G1) of 63 moth groups. G1 embryos were screened for expression of the introduced marker gene (EGFP), and EGFP-expressing individuals were found in 12 moth groups, so these were selected and reared in each moth group.

[0089] The resulting transgenic silkworms were reared in separate moth groups and broodstocks were bred by repeated sibling mating. The broodstocks were confirmed by a combination of Southern blot and quantitative PCR analysis of the number of introduced genes (see J. Silk Sci. Tech Jpn 29, 59-65(2021) for a method for verifying the number of inserted genes in transgenic silkworms using real-time PCR).

[0090] The obtained strains were differentiated by detecting differences in the size of the scFv gene using PCR (5'-CTACATCACCGCTCACTTACTTCC-3', 5'-CATTGTTGGTCAACTTCAAAGTCC-3'), which specifically amplifies the portion of the recombinant gene contained in the genomic DNA that contains the scFv.

[0091] The cocoons of these strains were lysed, and the binding activity was evaluated by ELISA to produce recombinant silkworm S27 strains expressing a fusion protein of fibroin L chain and anti-human CRP single-chain antibody.

[0092] The recombinant silkworm S27 strain was reared in an environment of 23 to 28°C using Silkmate original species 1-3 instar S (Nosan Corporation, Japan). Cocoons 7 to 10 days after hatching were cut open with a utility knife, prepared, and stored at room temperature after sealing in silica gel.

[0093] <Test Example 1> (1) Preparation of protein solution Cocoons produced by recombinant silkworms (S27) were cut with scissors, moistened with 70% ethanol to increase hydrophilicity, squeezed out the water, and weighed. 70% ethanol and 100 mM Tris-HCl (pH 9.0)-10 M lithium bromide (solvent for dissolving cocoon threads) were added to make the cocoon thread dissolving solution (90 mM Tris-HCl (pH 9.0)-9 M lithium bromide) at a concentration of 30-50 mg / mL. The cocoon threads were dissolved by vigorously shaking for 1 hour at 50°C. After dissolution, the solution was centrifuged at about 15,000 rpm for 30 minutes to remove the precipitate. The cocoon thread dissolving solution from which the precipitate had been removed was diluted with pure water to 20 mM Tris-HCl (pH 9.0)-2 M lithium bromide and stored.

[0094] (2) Activity measurement of protein solution by ELISA Human CRP antigen (4 μg / mL) diluted to 4 μg / mL with 20 mM Tris-HCl (pH 8.0)-5 mM CaCl2 was dispensed in 50 μl portions into a 96-well microplate and left to stand overnight at 4°C. After washing the plate with 0.05% Tween 20-150 mM NaCl aqueous solution, 100 μL of blocking solution (Blocking one (Nacalai Tesque, Inc.) diluted 4-fold with RO water) was dispensed into each well and left to stand at room temperature for 1 hour. After washing the plate with 0.05% Tween 20-150 mM NaCl aqueous solution, a dilution series of the protein solution was prepared using 10% Blocking one-0.05% Tween 20-150 mM NaCl aqueous solution (2-fold serial dilution, starting concentration 1.0 mg / mL). The diluted protein solution and anti-human CRP monoclonal antibody solution (control) were dispensed in 50 μL portions and left to stand at room temperature for 1.5 hours to allow the antibody to react. After washing the plate with 0.05% Tween 20-150 mM NaCl aqueous solution, 50 μL portions of 1000-fold diluted anti-Myc rabbit antibody (MBL 562-5) were dispensed as a secondary antibody and left to stand at room temperature for 1.5 hours. After washing the plate with 0.05% Tween 20-150 mM NaCl aqueous solution, 50 μL portions of 1000-fold diluted anti-rabbit IgG goat HRP (Abcam P0448) were dispensed as a tertiary antibody and left to stand at room temperature for 1 hour. After washing the plate with 0.05% Tween 20-150 mM NaCl aqueous solution, 50 μL portions of orthophenylenediamine substrate color development solution were dispensed and allowed to develop at room temperature for 20 minutes. The color development was stopped with sulfuric acid, and the absorbance at 492 nm was measured using a microplate reader (EPOCH: Biotek). The results are shown in Figure 1.

[0095] As shown in FIG. 1, a reaction curve depending on the protein concentration, that is, the concentration of the single-chain antibody, was confirmed.

[0096] (3) Preparation of protein microparticles A protein solution was prepared by adding pure water to the stored silk thread solution to dilute it to a protein concentration of 2.5-5.0 mg / mL. The protein solution was slowly layered on top of 1.25 M KH2PO4 (four times the amount of the protein solution) to form an interface. After leaving it to stand for 5 minutes, it was slowly mixed by inversion. It was left to stand for 3 days at 4°C to form protein microparticles. The formed protein microparticles were centrifuged at about 4000 rpm for 10 minutes, and the supernatant was removed. After gently adding pure water and leaving it to stand, the protein microparticles were washed by centrifuging at about 4000 rpm for 10 minutes and removing the supernatant, which was repeated three times. 0.1% Proclin water was added to the washed protein microparticles and thoroughly dispersed with a sonicator to prepare a dispersion of protein microparticles. The formation of protein microparticles was confirmed using an electron microscope (TM4000Plus, Hitachi High-Tech Corporation; magnification 2500x).

[0097] (4) Confirmation of the shape of protein microparticles Protein microparticles were prepared using a protein solution in which refined wild-type silk filaments were dissolved, a protein solution, and a protein solution (mixed solution).

[0098] 1 mL of 70% ethanol was added to 0.5 g of refined wild-type cocoon filaments, followed by 9 mL of 100 mM Tris-HCl (pH 9.0)-10 M lithium bromide solution. After stirring overnight at 37°C, the mixture was centrifuged at 15,000 rpm for 30 minutes to remove the precipitate, and a solution of refined wild-type cocoon filaments was prepared. The solution of refined wild-type cocoon filaments was diluted with pure water to 4 mM Tris-HCl (pH 9.0)-0.4 M lithium bromide, and a protein solution of refined wild-type cocoon filaments was prepared.

[0099] 1 mL of 70% ethanol was added to 0.5 g of cocoons produced by recombinant silkworms (S27), followed by 9 mL of 100 mM Tris-HCl (pH 9.0)-10 M lithium bromide solution. After stirring overnight at 37°C, the mixture was centrifuged at 15,000 rpm for 30 minutes to remove the precipitate, and a solution of the cocoon filaments was prepared. The solution of the cocoon filaments was diluted with pure water to 4 mM Tris-HCl (pH 9.0)-0.4 M lithium bromide to prepare a protein solution.

[0100] The purified wild-type cocoon filament lysate and the cocoon filament lysate were mixed in a 1:1 (volume ratio) and then diluted with pure water to 4 mM Tris-HCl (pH 9.0)-0.4 M lithium bromide to prepare a protein solution (mixed solution).

[0101] 5mL of each prepared protein solution was layered on 20mL of 1.25M KH2PO4 to form an interface. After mixing by slowly inverting, the mixture was left to stand overnight at room temperature and centrifuged at 4000 rpm for 10 minutes to remove the supernatant. The mixture was washed three times with RO water. After washing, 0.5mL of 0.1% Proclin water was added to the protein microparticles from which the supernatant had been removed, and the mixture was thoroughly pipetted and then thoroughly dispersed by sonication. The protein microparticles were observed under an electron microscope (TM4000Plus, Hitachi High-Tech Corporation; magnification 2500x). The results are shown in Figure 2.

[0102] As shown in Figure 2, when a protein solution in which refined wild-type cocoon filaments were dissolved was used, the particle size of the protein microparticles became large, and nano-sized particles could not be obtained. On the other hand, when a protein solution and a protein solution (mixed solution) were used, protein microparticles including nano-sized particles could be obtained. When a protein solution was used, protein microparticles with particle sizes of several tens of nm to 100 nm and with thin threads entangled around them were observed. In addition, by mixing the protein solution with a solution of refined wild-type cocoon filaments, protein microparticles with a larger particle size than the protein solution and dispersibility close to that of a single particle size were observed.

[0103] <Test Example 2> A blocking agent was added to the dispersion of protein microparticles obtained in the above Test Example 1 "Preparation of protein microparticles" and the mixture was shaken at room temperature for 1 hour to perform blocking to suppress non-specific reactions. As the blocking agent, a Block Ace aqueous solution prepared by dissolving KAC Cat. No. UKB80 in pure water was used. The turbidity of the protein microparticles was adjusted with 0.1% Proclin water so that the absorbance at 570 nm was about 20, and test liquid 1 (Example) was prepared. obtained.

[0104] As a control, a protein solution was prepared in the same manner as in "(1) Preparation of protein solution" in Test Example 1, except that cocoons produced by wild-type (W1) silkworms were used instead of cocoons produced by recombinant silkworms (S27), and a dispersion of protein microparticles was prepared in the same manner as in "(3) Preparation of protein microparticles". A blocking agent was added to the dispersion of protein microparticles, and the mixture was shaken at room temperature for 1 hour. The turbidity of the protein microparticles was adjusted with 0.1% Proclin water so that the absorbance at 570 nm was about 20, and test solution 2 (comparative example) was obtained.

[0105] The immune agglutination reaction of the protein microparticles was carried out by visually observing the agglutination image using an agglutination judgment plate.

[0106] Specifically, human CRP antigen was diluted to 50 mg / dL with diluent (20 mM Tris-HCl (pH 8.0), 10 mM EDTA, 0.1% BSA, 2 mM Ca). 200 μL of diluent or buffer (20 mM Tris-HCl (pH 8.0)) was dropped onto the agglutination test plate and spread inside the circle. 100 μL of test solution 1 or 2 was added on top and mixed with a shaker. The presence or absence of agglutination was observed after about 5 minutes. The results are shown in Figure 3.

[0107] As shown in FIG. 3, it is understood that the protein microparticles in Test Solution 1 cause a human CRP antigen-specific agglutination reaction.

[0108] Although the embodiments of the present invention have been described in detail, it is apparent that the same are illustrative and exemplary, not restrictive, and the scope of the present invention should be interpreted by the appended claims.

Claims

1. A method for producing protein microparticles having antigen-binding activity, comprising the steps of: The production method includes mixing a protein solution in which silk filaments containing a fusion protein of a single-chain antibody and a fibroin protein are dissolved with a salt solution containing at least one salt selected from the group consisting of phosphates, hydrochlorides, sulfates and citrates.

2. The method of claim 1 , wherein the silk thread is unrefined.

3. The method of claim 1, further comprising mixing a solution of the cocoon filament containing the fusion protein with a solution of the refined wild-type cocoon filament to prepare the protein solution.

4. The method of claim 1 , wherein the single chain antibodies are the same or two or more different single chain antibodies.

5. The method according to claim 1 , further comprising layering the protein solution and the salt solution to form an interface, and then mixing the protein solution and the salt solution.

6. The method according to claim 1, wherein a mixing ratio (volume ratio) of the protein solution to the salt solution is 1:4 to 1:

5.

7. The method according to claim 1 , wherein the salt concentration in the salt solution is 1.0 mol / L or more.

8. The method according to claim 1, wherein the concentration of the cocoon filament in the protein solution is 1.0 to 20.0 mg / mL.

9. The method according to claim 1, wherein the protein microparticles include protein microparticles having a particle size of 0.05 to 2.0 μm.

10. 10. Use of the protein microparticles obtained by the method according to any one of claims 1 to 9 as a reaction substrate in an immunoassay.

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