Protein molecule, method for producing protein molecule, and method

Abstract

A protein molecule which comprises a binding site for a binding target and in which the binding site includes the first to fourth immunoglobulin domains from the N-terminal side from among a plurality of immunoglobulin domains that have a shrimp / prawn-derived Dscam protein molecule, wherein the binding target is a substance derived from a virus that is pathogenic to mammals, a substance derived from a microbe that is pathogenic to mammals, or snake venom.

Description

Protein molecules, methods for producing protein molecules, and methods

[0001] The present invention relates to immunoglobulin family proteins derived from crustaceans. This application claims priority to Japanese Patent Application No. 2024-014783, filed February 2, 2024, the contents of which are incorporated herein by reference.

[0002] Due to economic constraints, the development of research reagents for diagnostics and treatments for bacterial toxin-caused infectious diseases, such as cholera, which are prevalent in developing countries, has been largely halted. Furthermore, inoculation of mice with these bacterial toxins to obtain antibodies capable of detecting them results in death, making antibody production and isolation difficult. Furthermore, simple genetic engineering techniques can only manipulate one gene at a time, making it difficult to isolate, improve, and mass-produce mouse antibodies synthesized from two separate genes, heavy chain and light chain, using genetic engineering techniques. For these reasons, research on bacterial toxins has lagged even further. Similar problems have been identified for research reagents for diagnostics, treatments, and treatments for snake venom in tropical regions. Therefore, antibodies derived from non-mammalian animals synthesized from a single gene are desirable as research reagents.

[0003] Recently, proteins with the same function as antibodies (referred to herein as crustacean antibodies for convenience) have been discovered in crustaceans. Non-Patent Document 1 reports crayfish Dscam, which can be called a crayfish antibody. Meanwhile, the present inventors confirmed that inoculation of kuruma shrimp with cholera toxin, which is lethal to mice, does not affect shrimp survival. Because crustacean antibodies are synthesized from a single gene, they can be isolated, mass-synthesized, and improved using simple genetic engineering techniques. Crustacean antibodies exhibit diversity through splicing alterations, and genome analysis to date has shown that shrimp possess the highest diversity among crustaceans. Furthermore, antibody gene sequences vary depending on the shrimp species, which may lead to differences in the likelihood of producing shrimp antibodies that specifically bind to antigens.

[0004] Fish & Shellfish Immunology Volume 92, September 2019, Pages 430-437

[0005] The present invention provides a protein molecule comprising at least a portion of the amino acid sequence of a shrimp-derived Dscam protein molecule, a method for producing the same, and a method using the protein molecule.

[0006] The present invention includes the following aspects. [1] A protein molecule having a binding site for a binding target, the binding site comprising at least a portion of the amino acid sequence of a shrimp-derived Dscam protein molecule. In this aspect, the binding site may comprise the first to fourth immunoglobulin domains from the N-terminus of the multiple immunoglobulin domains contained in the shrimp-derived Dscam protein molecule, and the binding target may be a substance derived from a virus pathogenic to mammals, a substance derived from a microorganism pathogenic to mammals, or snake venom. [2] The protein molecule according to [1], wherein the binding site comprises the first to seventh immunoglobulin domains from the N-terminus of the multiple immunoglobulin domains contained in the shrimp-derived Dscam protein molecule. [3] The protein molecule according to [1] or [2], wherein the binding site comprises the first to fourth immunoglobulin domains from the N-terminus of the multiple immunoglobulin domains contained in the shrimp-derived Dscam protein molecule. [4] The protein molecule according to any one of [1] to [3], which further comprises a reporter enzyme in addition to the binding site. [5] The protein molecule according to [4], wherein the reporter enzyme is luciferase or horseradish peroxidase (HRP). [6] The protein molecule according to any one of [1] to [5], which further comprises, in addition to the binding site, a labeling amino acid sequence that can be recognized by another antibody. [7] The protein molecule according to [6], wherein the labeling amino acid sequence is an HA tag. [8] The protein molecule according to any one of [1] to [7], which further comprises, in addition to the binding site, an amino acid sequence for purification that facilitates purification of the protein molecule. [9] The protein molecule according to [8], wherein the amino acid sequence for purification is a His tag or a GST tag.

[10] The protein molecule according to any one of [1] to [9], which further comprises, in addition to the binding site, a signal sequence for extracellular secretion.

[11] The protein molecule according to any one of [1] to

[10] , wherein the binding site has the ability to bind to a protein derived from a virus that infects humans.

[12] A polynucleotide comprising a base sequence encoding the protein molecule according to any one of [1] to

[11] and a promoter base sequence to which the base sequence is operably linked so as to promote expression of the protein molecule.

[13] The polynucleotide according to

[12] , wherein the polynucleotide is a plasmid DNA or an expression construct.

[14] A method for producing a protein molecule, comprising the step of expressing the protein molecule in a protein expression system into which the polynucleotide according to

[12] or

[13] has been introduced.

[15] A method for producing the protein molecule according to

[14] , wherein the protein expression system is a cultured cell.

[16] A method comprising the step of using the protein molecule according to any one of [1] to

[11] and detecting binding of the protein molecule to its binding partner.

[17] The method according to

[16] , wherein the protein molecule is used in place of a conventionally known antibody.

[18] The method according to

[16] or

[17] , comprising a treatment of contacting the binding partner with the protein molecule in the presence of a liquid component.

[19] The method according to

[18] , comprising a process of immobilizing the binding partner on a substrate surface and contacting the free protein molecule with the binding partner.

[20] The method according to

[18] , comprising a process of immobilizing the protein molecule on a substrate surface and contacting the free binding partner with the binding partner.

[21] The method according to any one of

[16] to

[20] , comprising performing ELISA.

[22] The method according to any one of

[16] to

[21] , wherein the binding partner is a substance that is lethal or toxic to mammals (e.g., a substance derived from a virus pathogenic to mammals, a substance derived from a microorganism pathogenic to mammals, or snake venom), or a substance that is conserved in mammals and therefore against which antibodies are difficult to produce in the mammalian body.

[23] The method according to

[22] , wherein the mammal is at least one species selected from the group consisting of human, mouse, rat, rabbit, goat, camel, alpaca, and llama.

[24] The method according to any one of

[16] to

[23] , wherein the binding partner is a substance known as an existing diagnostic marker.

[25] The method according to any one of

[16] to

[24] , wherein the binding target is a diagnostic marker for a disease specific to developing countries or a disease with a small number of patients.

[26] A testing method in which the binding target detects a substance related to a disease by the method according to any one of

[16] to

[25] , and determines the presence or absence of an abnormality based on any judgment criterion.

[27] The testing method according to

[26] , in which the substance related to a disease is a diagnostic marker.

[28] A method for producing a protein molecule, comprising the steps of injecting a substance into a crustacean and producing a Dscam protein molecule having a binding site capable of binding to the substance in the body of the crustacean. The crustacean in this embodiment may be a shrimp.

[29] The method for producing a protein molecule according to

[28] , in which the crustacean is at least one species selected from the group consisting of kuruma prawn, vannamei shrimp, black tiger shrimp, yama shrimp, striped shrimp, and swimming crab.

[30] The method for producing a protein molecule according to

[28] or

[29] , in which an anesthetic agent is administered to the crustacean before injection.

[31] The method for producing a protein molecule according to

[30] , wherein the anesthetic agent is a drug containing eugenol as an active ingredient.

[32] The method for producing a protein molecule according to

[30] or

[31] , wherein the treatment concentration of the anesthetic agent is obtained by diluting a stock solution containing 10.7 g of eugenol per 100 g of eugenol in a range of 1 / 500 to 1 / 2000.

[33] The protein molecule according to any one of [1] to

[11] , wherein the immunoglobulin domains possessed by the protein molecule are only four, i.e., the first to fourth immunoglobulin domains from the N-terminus of the protein molecule.

[34] The protein molecule according to any one of [1] to

[11] and

[33] , wherein the binding site comprises any one of the amino acid sequences of SEQ ID NOs: 5 to 18.

[35] The protein molecule according to any one of [1] to

[11] and

[33] , wherein the binding partner is a glycoprotein of Lassa virus, and the amino acid sequence comprises the amino acid sequences of SEQ ID NOs: 5 and 9, the amino acid sequences of SEQ ID NOs: 6 and 10, the amino acid sequences of SEQ ID NOs: 7 and 11, or the amino acid sequences of SEQ ID NOs: 8 and 12.

[36] The protein molecule according to any one of [1] to

[11] and

[33] , wherein the binding partner is p24 of HIV-1, and the amino acid sequence comprises the amino acid sequences of SEQ ID NOs: 13 and 14.

[37] The protein molecule according to any one of [1] to

[10] and

[33] , wherein the binding partner is cholera toxin, and the amino acid sequence comprises the amino acid sequences of SEQ ID NOs: 15 and 17, or the amino acid sequences of SEQ ID NOs: 16 and 18.

[38] A method comprising a step of detecting binding of the protein molecule to the binding partner using the protein molecule according to any one of [1] to

[11] and

[33] to

[37] .

[39] The method according to

[38] , wherein the detecting step comprises performing dot blotting.

[40] A method for producing the protein molecule according to any one of

[28] to

[32] , wherein the crustacean is kuruma prawn having a body length of 8 cm to 30 cm.

[41] A method for producing the protein molecule according to any one of

[28] to

[32] and

[40] , wherein the Dscam protein molecule is the protein molecule according to any one of [1] to

[11] and

[33] to

[37] .

[0007] According to the present invention, it is possible to provide a protein molecule containing at least a part of the amino acid sequence of a shrimp-derived Dscam protein molecule, a method for producing the same, and a method using the protein molecule.

[0008] Structure of Drosophila Dscam. Procedure for an exemplary embodiment of the present invention. Western blotting results using shrimp antibodies isolated from kuruma shrimp inoculated with Lassa virus glycoprotein (top). Virus particles with (left) and without (right) glycoproteins were analyzed. Equal amounts of virus particles are present in both lanes (bottom). Three cDNA clones of shrimp antibodies (clones 3, 4, and 9) were isolated from kuruma shrimp inoculated with HIV-1 p24 antibody as an antigen. A graph showing the results of ELISA performed on a 96-well plate using luciferase-shrimp antibody fusion proteins. A graph showing the results of measuring luciferase activity in the culture supernatant after transfection of 293T cells with expression plasmids for kuruma shrimp anti-Lassa glycoprotein (GP) antibodies bearing either shrimp signal peptide or human signal peptide. Contrary to our expectations, the human signal was 50-fold lower. However, the ELISA results (center and right) show that the human signal was 200-fold higher. Overall, the detection sensitivity was 10,000 times higher with the human signal. Fusion proteins were prepared between Ig domains 1-7 or Ig domains 1-4 and luciferase. ELISA analysis showed that the fusion protein containing Ig domains 1-4 had lower luciferase activity resulting from nonspecific binding to FBS. Diagram of the injection site into shrimp. Example of a procedure for producing shrimp antibodies in shrimp bodies. Plasmid structure for expressing a shrimp antibody-luciferase fusion protein. Example of a procedure for producing a shrimp antibody-luciferase fusion protein. Example of a procedure for identifying shrimp antibodies that bind to antigens by ELISA. Results of Experimental Example 1. Results of Experimental Example 2. Results of Experimental Example 3. Results of Experimental Example 6. Results of Experimental Example 7. Results of Experimental Example 8. Results of Experimental Example 9. Results of Experimental Example 10. Results of Experimental Example 10. Results of Experimental Example 11: dot blot of cholera toxin in PBS using shrimp antibodies.

[0033] Figure 11 shows the results of Experimental Example 11, where cholera toxin in PBS was quantified based on the dot blot results.

[0034] Figure 11 shows the results of Experimental Example 11, where cholera toxin in 10% FBS was quantified based on the dot blot results using shrimp antibody.

[0035] Figure 11 shows the results of Experimental Example 11, where cholera toxin in 10% FBS was quantified based on the dot blot results.11 shows the results of Experimental Example 11, in which a dot blot of cholera toxin in a microbial culture solution was performed using a shrimp antibody. 12 shows the results of Experimental Example 11, in which the amount of cholera toxin in a microbial culture solution was quantified from the dot blot results.

[0009] <Protein Molecule> A first aspect of the present invention is a protein molecule having a binding site for a binding target, wherein the binding site comprises at least a portion of the amino acid sequence of a shrimp-derived Dscam protein molecule (hereinafter, sometimes referred to as a shrimp antibody or shrimp Dscam). The binding site preferably comprises the first to fourth immunoglobulin domains from the N-terminus of the shrimp-derived Dscam protein molecule.

[0010] The binding target of shrimp Dscam may be any substance that can induce an immune response in shrimp, preferably a protein molecule foreign to shrimp, and more preferably a protein molecule lethal to mammals. Specific examples include proteins, lipids, sugar chains, and other substances derived from viruses that are pathogenic to mammals including humans (e.g., glycoprotein of Lassa virus, p24 of HIV-1), proteins, lipids, sugar chains, and other substances derived from pathogenic microorganisms that are pathogenic to mammals including humans (e.g., cholera toxin, diphtheria toxin), snake venom, and the like.

[0011] Shrimp Dscam is a homolog of human Down syndrome cell adhesion molecule (Dscam) and has a structure similar to that of Drosophila Dscam shown in Figure 1. Specifically, it has 10 immunoglobulin (Ig) domains at its N-terminus, with diversity in the second, third, and seventh Ig domains counting from the N-terminus. The specificity and binding strength of shrimp Dscam to its binding targets are primarily determined by the second and third Ig domains. As shown in the experimental examples described below, proteins containing the first to fourth Ig domains counting from the N-terminus of shrimp Dscam are capable of sufficiently binding to their binding targets. In other words, the four Ig domains, first to fourth from the N-terminus, are sufficient for the shrimp Dscam of this embodiment. The fifth to tenth Ig domains may or may not be included in shrimp Dscam of this embodiment. The amino acid sequences of the second and third Ig domains that have excellent binding properties to specific binding targets will be exemplified in the experimental examples described below.

[0012] The first and fourth Ig domains counting from the N-terminus of shrimp Dscam in this embodiment have little effect on the specificity of the binding target. As an example, a preferred amino acid sequence of the first Ig domain of kuruma prawn Dscam is shown in SEQ ID NO: 19, and a preferred amino acid sequence of the fourth Ig domain is shown in SEQ ID NO: 20. The amino acid sequences of the first and fourth Ig domains of shrimp Dscam in this embodiment may contain modifications as long as they do not significantly adversely affect the binding affinity or specificity to the binding target. Preferably, the sequences have at least 80%, preferably 90% or more, more preferably 95% or more, and even more preferably 99% or more sequence identity to the amino acid sequences exemplified here, and portions thereof may be deleted or truncated.

[0013] One example of this embodiment includes "crustacean antibody-like proteins using human virus-derived proteins as antigens, and methods for their production, isolation, and highly sensitive detection." By artificially inoculating shrimp with human virus-derived proteins as antigens, shrimp antibodies against the target antigen can be produced. The shrimp antibody genes can then be isolated and used as tools for medical and biological research, similar to mammalian antibodies (Figure 2). The inventors immunized kuruma shrimp twice with the antigen and amplified the Ig region (Ig) 1-7, which contains the diversity region, by RT-PCR. A plasmid expressing a fusion protein of this diversity region with luciferase was constructed using genetic engineering techniques. The plasmid was introduced into human 293T cells, and Western blotting or ELISA detection was performed using the culture supernatant as a shrimp antibody solution. As a result, shrimp antibodies detecting Lassa virus glycoprotein (Figure 3) and HIV-1 p24 protein (Figure 4) were successfully isolated. Shrimp antibodies have a signal peptide that allows them to be secreted extracellularly in shrimp cells. However, when the signal peptide was replaced with a human signal peptide for expression in human cells, the detection sensitivity increased by 10,000-fold (Fig. 5). The Ig domains 1-4 of shrimp Dscam are involved in antigen binding. Therefore, we constructed a fusion protein of the Ig domains 1-4 of the anti-HIV-1 p24 shrimp antibody with luciferase. This fusion protein showed higher specificity than the Ig domains 1-7 (Fig. 6). The detection sensitivity of shrimp antibodies that bind to HIV-1 p24 protein was comparable to that of a commercially available diagnostic kit using mouse antibodies.

[0014] <<Method>> A second aspect of the present invention is a method comprising the step of using the protein molecule of the first aspect to detect binding of the protein molecule to the binding target. Examples of methods for detecting the binding include conventional detection methods using antibodies, in which the protein molecule of the first aspect is used instead of the conventional antibody. Specific examples include dot blot, ELISA, immunochromatography, etc.

[0015] <Method for Producing Protein Molecules> A third aspect of the present invention is a method for producing protein molecules, comprising the steps of injecting a substance into a crustacean and producing a Dscam protein molecule having a binding site capable of binding to the substance within the body of the crustacean. Shrimp is preferred as a crustacean due to the high genetic diversity of Dscam. Examples of shrimp include kuruma prawn, vannamei shrimp, black tiger shrimp, amaranth shrimp, and striped shrimp. Among these, kuruma prawns with a body length of 8 cm or more are preferred because they have high resistance to injection and can achieve a survival rate of approximately 90% or more after injection. Here, the body length of kuruma prawns is defined as the length from the tip of the cephalothorax (the rear end of the eye) to the rear end of the telson.

[0016] Examples of substances to be injected into crustaceans include those exemplified as binding targets for the protein molecule of the first aspect. Preferably, an antigen solution containing the substance is injected into crustaceans as an antigen. For example, 1 to 100 μL of antigen solution is injected per injection per individual crustacean. The antigen solution can be obtained, for example, by dissolving the antigen in physiological saline at a desired concentration (e.g., 0.1 to 10 μg / μL). Bacterial lipopolysaccharide (LPS) may be added to the antigen solution as an adjuvant. The LSP concentration in the antigen solution can be, for example, 0.1 to 1 μg / 10 μL.

[0017] The number of injections of the antigen solution per individual crustacean is preferably one to three times, more preferably two times. The second injection is preferably given one to three weeks after the first injection. The efficiency of shrimp antibody production is higher when the number of injections is two rather than one. Since injections carry the risk of injuring the crustacean or burdening its physiological functions, it is preferable to limit the number of injections to three or less.

[0018] When injecting the antigen into shrimp, one method is to hold down the struggling shrimp with one hand and inject the antigen into the internal organs where lymphatic tissue is located (position in Figure 7(1)). However, if the shrimp struggles, the internal organs may be damaged by the needle tip, resulting in a high mortality rate. In this case, it is preferable to inject the antigen into the shrimp's muscle (position in Figure 7(2)), or into the area between the shrimp's shell and muscle or near the surface of the muscle (position in Figure 7(3)). Alternatively, the antigen can be injected after anesthetizing the shrimp with an anesthetic for crustaceans.

[0019] The body length of shrimp to be injected with the antigen is preferably 8 cm to 30 cm, more preferably 8 cm to 15 cm, for example, in the case of kuruma shrimp. In particular, when the body length of kuruma shrimp is 8 cm to 15 cm, it is easy to adjust the size of the rearing container and the individual density within the container, and it is easy to handle the shrimp during antigen inoculation.

[0020] After injecting shrimp with antigen a desired number of times, for example, three days after the second injection, shrimp samples can be taken from the whole body or internal organs containing lymphoid tissue. The shrimp Dscam mRNA contained in the sample can be cloned as cDNA by RT-PCR using standard methods (Figure 8). Shrimp antibody cDNA can be subjected to any genetic engineering technique. For example, a cDNA containing the first to fourth Ig domains from the N-terminus of a shrimp antibody can be cloned. A fusion protein of a human signal peptide and a shrimp antibody can be produced by incorporating the cDNA into a plasmid such as the one shown in Figure 9.

[0021] The cloned cDNA can be used to produce the desired shrimp antibody using any protein expression system. Figure 10 shows an example of a plasmid expressing a shrimp antibody-luciferase fusion protein transfected into human 293T cells. Because the shrimp antibody protein is bound to a human signal peptide, the desired fusion protein is secreted into the culture supernatant of human 293T cells.

[0022] ELISA is one method for identifying shrimp antibodies that bind to a target antigen (Figure 11). As a negative control, fetal bovine serum (FBS) alone is immobilized on the bottom of one well. As a positive control, a mixture of FBS and the target antigen protein is immobilized on the bottom of the other well. A shrimp antibody solution is added to these wells and incubated, for example, at 4°C for 2 hours to allow the shrimp antibody to bind to the antigen. The well is washed with PBS to remove unbound shrimp antibody. Since the shrimp antibody is bound to luciferase, the amount of bound shrimp antibody can be quantified by measuring luciferase activity. If higher luciferase activity is detected in wells immobilized with a mixture of FBS and the target antigen compared to wells immobilized with FBS alone, it can be concluded that the shrimp antibody binds to the target antigen.

[0023] Experimental Example 1: Materials and Methods: Lassa virus glycoprotein (GP) was synthesized at Sigma-Aldrich. Kuruma shrimp were purchased from Toko Sangyo Co., Ltd. Kuruma shrimp (≥8 cm body length) were immunized by injecting 50 μL of phosphate-buffered saline (PBS) containing Lassa virus GP (1 μg) and Escherichia coli-derived lipopolysaccharide (LPS) (Sigma-Aldrich) (10 μg). Ten days after the first immunization, shrimp were re-administered with the same solution. Three days after the second immunization, total RNA samples were extracted from shrimp. These RNA samples were used to amplify the Dscam sequence, including the N-terminal signal peptide and Ig1-Ig7 domains, by polymerase chain reaction (PCR) using the BamHI-Dscam-signal-S and Dscam-HA-XhoI-AS primers. The primer sequences were as follows: GGA TCC ATG GGC ACT ACC TAT ATG GTG (the BamHI restriction site is underlined) (SEQ ID NO: 1), CTC GAG TCA TGC GTA ATC CGG AAC ATC GTA CGG GTA GGT GGA GCG CTG GAC AGC (the XhoI restriction site is underlined; SEQ ID NO: 2). The PCR products were then cloned into the pTargeT mammalian expression plasmid (Promega) to construct eight independent expression clones. The expressed protein, designated Dscam-Ig1-7-HA here, contains an HA amino acid motif when expressed in mammalian cells and can be detected with an anti-HA antibody. Furthermore, the Dscam protein contains a shrimp signal peptide at its N-terminus, allowing it to be secreted into the culture supernatant.

[0024] Results: To identify clones encoding Dscam proteins capable of interacting with and binding to Lassa virus GP, 293T cells were transfected with the construction plasmid of a Lassa virus GP-pseudotyped murine leukemia virus (MLV) vector and individual Dscam-Ig1-7-HA-expressing clones. Cell lysates were prepared from transfected cells. Meanwhile, virion-containing fractions were collected by centrifugation of the culture supernatant through 10% sucrose. Western blot analysis was performed on the cell lysates (Fig. 12, top panel) and virion-containing fractions (Fig. 12, bottom panel) using anti-HA or anti-MLV p30 antibodies. If the Dscam Ig1-7-HA protein binds to Lassa virus GP, Dscam protein should be detected in the virion-containing fraction. Similar levels of expression of both Dscam-Ig1-7-HA and MLV p30 proteins were detected in cell lysates, regardless of the Dscam clone. Dscam protein did not affect MLV p30 expression, demonstrating consistent expression levels. Similarly, MLV p30 protein was expressed at similar levels in the virion-containing fractions, resulting in the production of similar amounts of Lassa virus-GP-pseudotyped MLV vectors, indicating that Dscam protein did not affect vector production. Although the pseudotyped vectors and expression levels of Dscam-Ig1-7-HA protein were uniform among the eight Dscam-Ig1-7-HA-expressing clones, only clones 3, 4, 6, and 8 contained detectable Dscam protein in the virion-containing fraction. This suggests that the Dscam protein expressed by clones 3, 4, 6, and 8 specifically binds to Lassa virus GP. Clone 6 had the highest abundance of Dscam protein in the virion-containing fraction, indicating its superior affinity for GP.

[0025] The results of Experimental Example 1 demonstrate that clone No. 3 has excellent binding ability to Lassa virus glycoprotein. The amino acid sequence of the Ig2 diversity region of clone No. 3 is shown in SEQ ID NO: 5, and the amino acid sequence of its Ig3 diversity region is shown in SEQ ID NO: 9. Protein molecules containing at least the Ig1-4 region of the Ig1-7 region of Kuruma prawn Dscam, which contains at least one of SEQ ID NO: 5 and SEQ ID NO: 9, have excellent binding ability to Lassa virus glycoprotein. The nucleotide sequences of the Ig2 diversity region and Ig3 diversity region of each clone were determined by the dideoxy method (NEB), and the amino acid sequences were determined based on the obtained nucleotide sequences.

[0026] The results of Experimental Example 1 demonstrate that clone No. 4 has excellent binding ability to Lassa virus glycoprotein. The amino acid sequence of the Ig2 diversity region of clone No. 4 is shown in SEQ ID NO: 6, and the amino acid sequence of its Ig3 diversity region is shown in SEQ ID NO: 10. Protein molecules having at least the Ig1-4 region of the Ig1-7 region of kuruma prawn Dscam, which includes at least one of SEQ ID NO: 6 and SEQ ID NO: 10, have excellent binding ability to Lassa virus glycoprotein.

[0027] The results of Experimental Example 1 demonstrate that clone No. 6 has particularly excellent binding ability to Lassa virus glycoprotein. The amino acid sequence of the Ig2 diversity region of clone No. 6 is shown in SEQ ID NO: 7, and the amino acid sequence of its Ig3 diversity region is shown in SEQ ID NO: 11. Protein molecules having at least the Ig1-4 region of the Ig1-7 region of kuruma prawn Dscam, which includes at least one of SEQ ID NO: 7 and SEQ ID NO: 11, have particularly excellent binding ability to Lassa virus glycoprotein.

[0028] The results of Experimental Example 1 demonstrate that clone No. 8 has excellent binding ability to Lassa virus glycoprotein. The amino acid sequence of the Ig2 diversity region of clone No. 8 is shown in SEQ ID NO: 8, and the amino acid sequence of its Ig3 diversity region is shown in SEQ ID NO: 12. Protein molecules having at least the Ig1-4 region of the Ig1-7 region of kuruma prawn Dscam, which includes at least one of SEQ ID NO: 8 and SEQ ID NO: 12, have excellent binding ability to Lassa virus glycoprotein.

[0029] Experimental Example 2: Materials and Methods. To examine the ability of Dscam protein to detect its target, Lassa virus GP, by Western blotting, 293T cells were transfected with an MLV vector construction plasmid and, as a control, a Lassa virus GP expression plasmid (Lassa-GP) or pcDNA3.1 (No-GP). MLV vector particles were collected by centrifugation through 10% sucrose and subjected to SDS-PAGE. The proteins were then transferred to a membrane. The membrane was then incubated overnight at 4°C with the culture supernatant of 293T cells transfected with each Dscam-Ig1-7-HA expression plasmid (clone 3, 4, 6, or 8) and washed with PBS. The membrane was then treated with a mouse anti-HA antibody followed by an HRP-conjugated anti-mouse IgG antibody. Dscam-binding proteins were visualized using ECL reagent.

[0030] Results: A protein band of the expected molecular size was detected on the membrane treated with Dscam proteins from clones 3, 6, and 8, but not with the protein from clone 4 (Fig. 13). These results demonstrate that Dscam-Ig1-7-HA clones 3, 6, and 8 can detect Lassa virus GP by Western blotting.

[0031] Experimental Example 3: Materials and Methods. To examine the ability of Dscam protein to detect Lassa virus GP in a direct ELISA, an expression plasmid was constructed for the fusion protein of clone 6 Dscam and nanoluciferase (Promega) (Dscam-Ig1-7-Luc). MLV vector particles pseudotyped with Lassa virus GP or lacking Lassa virus GP were prepared using the protocol described above. These particles were serially diluted (1 / 1, 1 / 2, 1 / 4) in PBS and plated onto a 96-well plate. Culture supernatant of 293T cells transfected with the Dscam-Ig1-7-Luc expression plasmid was added to the wells, and the plate was incubated at 4°C for 2 hours. The wells were washed with PBS, and nanoluciferase activity was measured using a nanoluciferase assay kit (Promega).

[0032] Results: Nanoluciferase activity in wells immobilized with Lassa virus GP-pseudotyped MLV vector particles was consistently higher than that in wells immobilized with GP-defective MLV vectors (Figure 14). Furthermore, nanoluciferase activity was detected in a dose-dependent manner in wells immobilized with Lassa virus GP-pseudotyped vectors. These results demonstrate that the Dscam-nanoluciferase fusion protein can detect Lassa virus GP by direct ELISA.

[0033] Experimental Example 4: Materials and Methods: The Dscam expression plasmid contains a shrimp signal peptide. To enhance the secretion efficiency of Dscam protein into the culture supernatant of human 293T cells, we replaced the shrimp signal peptide with the human interleukin-8 signal peptide. Human 293T cells were transfected with the modified Dscam expression plasmid containing the human signal peptide. Using the culture supernatant of transfected cells, direct ELISA was performed to detect Lassa virus GP using the same methodology.

[0034] Results: When shrimp signal peptide-containing Dscam was used, the luminescence value was approximately 1,000 (Figure 5), whereas when human signal peptide-containing Dscam was used, the luminescence value was approximately 220,000. The human signal peptide increased nanoluciferase activity 220-fold in wells immobilized with Lassa virus GP-pseudotyped MLV vectors.

[0035] Experimental Example 5: Materials and Methods To examine the ability of Kuruma shrimp Dscam to detect HIV-1 p24 protein, Kuruma shrimp (over 8 cm in length) were inoculated with 50 μL of PBS containing HIV-1 p24 protein (62.5 pg) and LPS (10 μg). Ten days after the first inoculation, shrimp were immunized with the same antigen solution. Three days after the second inoculation, total RNA samples were extracted from the inoculated shrimp. The Dscam sequence, containing the Ig1-7 domain but not the signal peptide, was amplified by PCR using the BamHI-Dscam-S and Dscam-XhoI AS primers. The nucleotide sequence of the BamHI-Dscam S primer is CAT ACA GGA TCC TGT GAT GAG AGC GGC CCC (SEQ ID NO: 3) (the BamHI cleavage site is underlined). Three clones were isolated, and expression plasmids for nanoluciferase fusion proteins containing Dscam Ig1-7 and the human signal peptide were constructed (Figure 15). Human 293T cells were transfected with each expression plasmid and the culture supernatant was collected. The wells of a 96-well plate were fixed with 100 μL of 2% FBS alone or 100 μL of 2% FBS with 625 pg / mL p24 solution. The culture supernatant was added to the wells and incubated at 4°C for 2 hours. The wells were then washed with PBS, and nanoluciferase activity was measured.

[0036] Results: When clone 3 Dscam protein was used, nanoluciferase activity in wells fixed with FBS and p24 was comparable to that in wells fixed with FBS alone (Fig. 4). On the other hand, when clone 4 or 9 Dscam protein was used, nanoluciferase activity in wells fixed with FBS and p24 exceeded that observed with FBS alone. These results indicate that clone 4 and 9 Dscam proteins can efficiently bind to HIV-1 p24 protein. In particular, clone 4 Dscam protein showed higher affinity for p24 than clone 9.

[0037] Experimental Example 6: Materials and Methods. In the previous experiment, we used the Kuruma shrimp Dscam protein containing the Ig1-7 region. In this experiment, we generated a Kuruma shrimp Dscam protein containing only the Ig1-4 region but lacking the Ig5-7 region. The Ig1-4 region of clone 4 was amplified by PCR using the BamHI-Dscam S and Dscam-Ig4-XhoI AS primers. The nucleotide sequence of the Dscam-Ig4-XhoI AS primer was CTC GAG CTC AAA TAG TCC TCC AAG (SEQ ID NO: 4). Expression plasmids for Dscam Ig1-4 and nanoluciferase fusion proteins containing the human signal peptide were then constructed (Figure 15). To determine which protein had higher affinity for HIV-1 p24, we performed direct ELISA using the same method as above.

[0038] Results: Kuruma shrimp Dscam Ig1-7 and Ig1-4, which contain the human signal peptide, showed comparable nanoluciferase activity in wells fixed with FBS and p24 (Figure 6). However, in wells fixed with FBS alone, nanoluciferase activity using the Dscam Ig1-4 protein was lower than that observed with Ig1-7. This result suggests that the Dscam Ig1-4 protein is more specific than Ig1-7.

[0039] The results of Experimental Examples 5 and 6 demonstrate that clone 4 has particularly excellent binding ability to HIV-1 p24. The amino acid sequence of the Ig2 diversity region of clone 4 is shown in SEQ ID NO: 13, and the amino acid sequence of its Ig3 diversity region is shown in SEQ ID NO: 14. Protein molecules having at least the Ig1-4 region of the Ig1-7 region of kuruma prawn Dscam, which contains at least one of SEQ ID NO: 13 and SEQ ID NO: 14, have particularly excellent binding ability to HIV-1 p24. Furthermore, protein molecules having the Ig1-4 region of kuruma prawn Dscam, which contains at least one of SEQ ID NO: 13 and SEQ ID NO: 14, also have excellent specificity for HIV-1 p24.

[0040] [Experimental Example 7] Materials and Methods To confirm the minimum concentration of HIV-1 p24 at which a protein having the Ig1-4 region of Kuruma shrimp Dscam could be detected, the p24 solution was serially diluted with 2% FBS and immobilized on a 96-well plate.

[0041] Results: Nanoluciferase activity at 1 pg / mL p24 was comparable to that of FBS alone (Figure 16). However, nanoluciferase activity at 10 pg / mL p24 was significantly higher than that of FBS alone, indicating that Dscam Ig1-4 protein can detect 10 pg / mL p24 protein. Commercially available sandwich ELISA kits using mouse anti-p24 antibodies can detect at least 7.5 pg / mL p24 protein according to the manufacturer's instructions. These results suggest that Dscam Ig1-4 has a detection sensitivity comparable to that of mouse antibodies.

[0042] Experimental Example 8: Materials and Methods. Isolation of mouse antibodies against activated cholera toxin is difficult because immunization of mice with activated cholera toxin results in lethality. Similarly, isolation of mouse antibodies against activated mammalian toxins, such as diphtheria toxin and snake venom, is also difficult. Indeed, inoculation of kuruma shrimp with 5 μg of cholera toxin, which results in 100% lethality in mice, produced no observable changes. Ten days after the initial inoculation of kuruma shrimp (8 cm or longer) with 5 μg of cholera toxin, a second immunization was performed with the same amount of cholera toxin. Three days after the second immunization, total RNA samples were extracted from two shrimp (CT1-2 and CT2-1). Nine clones containing the Dscam Ig1-4 region (18 clones in total) were isolated from each shrimp, and the nucleotide sequences of all clones were determined. Nine clones with different nucleotide sequences were selected to construct expression plasmids for Dscam Ig1-4 and nanoluciferase fusion proteins containing a human signal peptide. To identify clones that bind to cholera toxin, direct ELISA was performed as follows. 100 μL of 1% FBS solution (CAPRICORN) or 1% FBS and 10 μg / mL cholera toxin solution was added to each well of a 96-well plate (Microlite 2+, Thermo Scientific). 100 μL of coating buffer (Invitrogen) was then added and incubated overnight at 37°C to immobilize the protein. The wells were then washed four times with PBS, and a shrimp antibody Dscam solution was added and incubated at 4°C for 2 hours. The wells were then washed four times with PBS, and luciferase activity in each well was measured.

[0043] Results: Of the nine clones, CT1-2-3 and CT1-2-15 induced higher nanoluciferase activity in wells fixed with FBS and cholera toxin than with FBS alone, indicating the interaction of these Dscam Ig1-4 proteins with cholera toxin (Figure 17). Based on previously published exon classifications for black tiger shrimp, the exon numbers of the variable regions (Ig2 and Ig3) of the clones isolated in this study are listed. No clones had the same nucleotide sequence as CT1-2-15. However, of the nine CT1-2 shrimp clones, the nucleotide sequences of five clones (CT1-2-3, CT1-2-r1, CT1-2-r11, CT1-2-r16, and CT1-2-r22) were identical to CT1-2-3. Similarly, of the nine clones from CT2-1 shrimp, the nucleotide sequences of two clones (CT2-1-r11 and CT2-1-r23) were identical to those of CT1-2-3. The clones with the same nucleotide sequence as CT1-2-3, which interacts with cholera toxin, were the most abundant. These results indicate that Dscam isoforms that interact with target proteins share the same nucleotide sequence across different shrimp species and are present at a higher rate than other isoforms that do not interact with target proteins. CT1-2-r8, CT2-1-r6, and CT2-1-r13 were the second most abundant clones with the shared sequence. The CT1-2-r8 product showed higher nanoluciferase activity than the other clones in wells fixed with FBS and cholera toxin, but it also showed high activity in wells fixed with FBS alone (Figure 17). The CT1-2-r8 product may interact with an unknown protein present in both the FBS and cholera toxin solutions.

[0044] The results of Experimental Example 8 demonstrate that clone (CT1-2-3) has excellent binding ability to cholera toxin. The amino acid sequence of the Ig2 diversity region of this clone (CT1-2-3) is shown in SEQ ID NO: 15, and the amino acid sequence of its Ig3 diversity region is shown in SEQ ID NO: 17. Protein molecules having the Ig1-4 region of kuruma prawn Dscam, which includes at least one of SEQ ID NO: 15 and SEQ ID NO: 17, have excellent binding ability to cholera toxin.

[0045] The results of Experimental Example 8 demonstrate that clone (CT1-2-15) has excellent binding ability to cholera toxin. The amino acid sequence of the Ig2 diversity region of this clone (CT1-2-15) is shown in SEQ ID NO: 16, and the amino acid sequence of its Ig3 diversity region is shown in SEQ ID NO: 18. Protein molecules having the Ig1-4 region of kuruma prawn Dscam, which includes at least one of SEQ ID NO: 16 and SEQ ID NO: 18, have excellent binding ability to cholera toxin.

[0046] [Experimental Example 9] <Closed-circulation aquarium capable of rearing kuruma shrimp for a long period of time> An aquarium (length: 35 cm, width: 45 cm, height: 25 cm) was constructed by placing ceramic sand and approximately 30 L of seawater in a container (length: 43 cm, width: 60 cm, height: 37 cm) filled with tap water, and combining it with a circulating filter, UV sterilizer, and temperature controller. The temperature of the seawater in the rearing tank was regulated by tap water in the container. It was confirmed that kuruma shrimp could be reared in this closed-circulation aquarium for 2 to 3 months.

[0047] Establishing an Anesthesia Method Suitable for Kuruma Shrimp We investigated the optimal concentration, treatment time, and recovery time for Kuruma shrimp using FA100 (a solution containing 10.7 g of the active ingredient, eugenol, per 100 g; manufactured by DS Pharma Animal Health Co., Ltd.), an anesthetic for fish and crustaceans. For the experiment, commercially available FA100 (a solution containing 10.7 g of the active ingredient, eugenol, per 100 g) was diluted 1 / 500 to 1 / 4000. Significant differences in anesthesia duration were observed for each concentration, but the recovery time was less pronounced. Furthermore, a concentration of 1 / 4000, within the range typically used for crustaceans, had no anesthetic effect on Kuruma shrimp. These results demonstrated that a 1 / 2000 diluted anesthetic solution, the lowest concentration that exhibited an anesthetic effect, was optimal, taking into account the stress caused by anesthesia in Kuruma shrimp. The results are shown in Figure 18.

[0048] <Identifying the optimal site for antigen inoculation and blood collection in kuruma shrimp> In order to minimize stress on kuruma shrimp caused by handling, and to confirm the expression level of Dscam in each individual, we selected the optimal site for hemolymph collection. As a result, we found that the optimal site for antigen inoculation was the dorsal part of the first segment of kuruma shrimp, and the optimal site for blood collection was the first abdominal appendage.

[0049] [Experimental Example 10] 1. Preparation of E. coli E. coli JM109 strain (Promega) was cultured overnight in 50 mL of LB medium (Shioya MS). After culture, the E. coli was washed three times with PBS and fixed in 10% formalin solution for 24 hours. The E. coli was then washed three times with PBS, suspended in 25 mL of PBS, and stored at room temperature. 2. Preparation of Bacillus subtilis var. natto Commercially available natto was spread on an LB plate, and one resulting colony was cultured overnight in 50 mL of LB medium. After culture, the Bacillus subtilis var. natto was washed three times with PBS and fixed in 10% formalin solution for 24 hours. The B. subtilis was then washed three times with PBS, suspended in 25 mL of PBS, and stored at room temperature. 3. Preparation of human 293T cells. Human 293T cells (provided by ATCC) were cultured in D-MEM medium (Fujifilm Wako Pure Chemical Industries, Ltd.) in five 10-cm dishes (NEST). When the 293T cells reached confluence, they were scraped with a scraper (TPP), washed three times with PBS, and fixed in 10% formalin solution for 24 hours in a conical tube. The 293T cells were then washed three times with PBS, suspended in 5 mL of PBS, and stored. 4. Preparation of anti-E. coli shrimp antibody Dscam solution. Kuruma shrimp (approximately 15 cm in length) reared at the Faculty of Fisheries, Nagasaki University, were inoculated into the abdominal muscle tissue with 50 μL of the E. coli suspension obtained in step 1 above, twice at 10-day intervals. Plasmid clones (Ec-8-1, Ec-8-18, Ec-8-19, Ec-9-9, Ec-9-11, and Ec-9-13) expressing a fusion protein of the shrimp antibody Dscam and luciferase (secreted NanoLuc, Promega) were constructed from E. coli-inoculated kuruma shrimp using a commercially available plasmid clone construction kit (Thermo Fisher Scientific Inc.). The resulting plasmid clones were transfected into 293T cells using Fugene HD reagent (Promega). After 24 hours, the medium was replaced with fresh DMEM and cultured for another 24 hours. The culture supernatant was then removed and stored at -80°C. This culture supernatant was used as the shrimp antibody Dscam solution in the following experiments.5. Measurement of the Binding Activity of Shrimp Antibody Dscam: 1 mL of each suspension of E. coli, B. subtilis, and human 293T cells obtained in steps 1 to 3 above was transferred to a tube and centrifuged at 1500 rpm at room temperature for 5 minutes. The supernatant was removed, and the weight of the precipitate was measured. Based on the results of this measurement, each suspension of E. coli, B. subtilis, and 293T cells was transferred to a tube so that the weights were the same, and centrifuged again at 1500 rpm at room temperature for 5 minutes. The supernatant was removed, and the precipitate was suspended in 500 μL of the shrimp antibody Dscam solution obtained in step 4 above and incubated at 4°C for 2 hours. The precipitate was then washed three times with PBS, and the luciferase activity of the precipitate was measured using a commercially available luciferase activity assay kit (Promega) and a luminometer (ATTO). The results are shown in Figure 19. Preparation of a calibration curve: 50, 100, 150, and 200 mL of the E. coli suspension were transferred to tubes and centrifuged at 1500 rpm at room temperature for 5 minutes, and the supernatant was removed. The E. coli precipitate was suspended in 500 mL of shrimp antibody Dscam solution Ec-8-18 and incubated at 4°C for 2 hours. The suspension was then washed three times with PBS, and the luciferase activity of the E. coli precipitate was measured using the same method as in 5. The results are shown in Figure 20. These results demonstrate the feasibility of quantifying antigens using shrimp antibody Dscam solution Ec-8-18.

[0050] Experimental Example 11: 1. Preparation of anti-cholera toxin shrimp antibody Dscam solution. Kuruma shrimp (approximately 15 cm in length) reared at the Nagasaki University School of Fisheries were inoculated twice, 10 days apart, with 10 μg / 50 μL of cholera toxin (Fujifilm Wako Pure Chemical Industries) into the abdominal muscle tissue. Using the same procedure as for obtaining the anti-E. coli shrimp antibody Dscam, multiple plasmid clones expressing a fusion protein of the shrimp antibody Dscam and luciferase were constructed from the cholera toxin-inoculated kuruma shrimp. The resulting expression clones were transfected into 293T cells, and the culture supernatant was used as the shrimp antibody Dscam solution. 2. Identification of clones with binding affinity to cholera toxin by direct ELISA. Direct ELISA was used to identify clones that bound to cholera toxin among the clones obtained in step 1 above. CT1-2-15, the clone with the highest luciferase activity, was transfected into 293T cells. The culture supernatant of transfected 293T cells was then used as the anti-cholera toxin shrimp antibody Dscam solution in this experiment. 3. Dot blot method: A PVDF membrane (Milipore) was wetted with 100% ethanol, then treated with 20% ethanol and PBS, and dried until the surface was completely dry. Commercially available cholera toxin was diluted to concentrations of 6.25, 12.5, 25, 50, and 100 μg / mL, and 10 μL of each cholera toxin solution was applied dropwise to the PVDF membrane. After drying, the PVDF membrane was rewetted with 100% ethanol and then immersed in 5% skim milk solution and incubated at room temperature for 1 hour. The PVDF membrane was then washed with PBS and immersed in shrimp antibody Dscam solution at 4°C overnight. The PVDF membrane was then washed with PBS and incubated overnight in anti-luciferase mouse IgG antibody solution (Promega) at 4°C. The PVDF membrane was then washed with PBS and incubated at 4°C for 2 hours in a solution of anti-mouse IgG sheep antibody conjugated with HRP (Bio-Rad). The PVDF membrane was then washed with PBS, and ECL color reagent (Bio-Rad) was added, followed by imaging. The resulting photographs are shown in Figures 21, 23, and 25, respectively. The density of each spot was also measured using a densitometer. The results are shown in Figures 22, 24, and 26, respectively.These results clearly demonstrate that the dot blot method can be used to quantify the antigen cholera toxin using a shrimp antibody Dscam solution.

Claims

1. A protein molecule having a binding site for a binding target, the binding site including the first to fourth immunoglobulin domains from the N-terminus of the multiple immunoglobulin domains possessed by a shrimp-derived Dscam protein molecule, wherein the binding target is a substance derived from a virus pathogenic to mammals, a substance derived from a microorganism pathogenic to mammals, or snake venom.

2. The protein molecule according to claim 1, wherein the immunoglobulin domains contained in the protein molecule are only four immunoglobulin domains located at positions 1 to 4 from the N-terminus.

3. The protein molecule of claim 1, wherein the binding site comprises any one of the amino acid sequences of SEQ ID NOs: 5 to 18.

4. The protein molecule of claim 1, wherein the binding target is a glycoprotein of Lassa virus, and the amino acid sequence comprises the amino acid sequences of SEQ ID NOs:5 and 9, SEQ ID NOs:6 and 10, SEQ ID NOs:7 and 11, or SEQ ID NOs:8 and 12.

5. The protein molecule according to claim 1, wherein the binding target is HIV-1 p24 and the amino acid sequence comprises the amino acid sequences of SEQ ID NO:13 and SEQ ID NO:

14.

6. The protein molecule of claim 1, wherein the binding target is cholera toxin and the amino acid sequence comprises the amino acid sequences of SEQ ID NO: 15 and SEQ ID NO: 17, or the amino acid sequences of SEQ ID NO: 16 and SEQ ID NO:

18.

7. A method comprising the step of using a protein molecule according to any one of claims 1 to 6 and detecting that said protein molecule has bound to said binding target.

8. The method of claim 7, wherein the detecting step comprises performing a dot blot.

9. A method for producing a protein molecule, comprising the steps of injecting a substance into a shrimp and causing the shrimp to produce a Dscam protein molecule having a binding site capable of binding to the substance in the shrimp's body.

10. The method for producing a protein molecule according to claim 9, wherein the shrimp is a kuruma shrimp having a body length of 8 cm or more and 30 cm or less.

11. A method for producing a protein molecule according to claim 10, wherein the Dscam protein molecule is a protein molecule according to any one of claims 1 to 6.

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