Immortalized macrophage of caprinae subfamily animal

Abstract

Provided is an immortalized macrophage of a Caprinae subfamily animal obtained by causing a macrophage of a Caprinae subfamily animal to express at least one protein selected from the group consisting of SV40 large T antigen and telomerase reverse transcriptase.

Description

Immortalized macrophages of Caprinae animals

[0001] The present invention relates to cells obtained by immortalizing macrophages of Caprinae animals (immortalized macrophages of Caprinae animals) and a method for producing the macrophages. The present invention also relates to a method for producing a Caprinae animal pathogen or a vaccine using the macrophages. Furthermore, the present invention relates to a method for detecting a Caprinae animal pathogen or a method for detecting a neutralizing antibody against a Caprinae animal infectious virus using the macrophages.

[0002] Goats, sheep, etc. belonging to the Caprinae family are animals subject to monitoring for 30 out of 99 diseases of monitored infectious diseases defined by the Law on Prevention of Livestock Infectious Diseases (Japan). The number of animals kept in Japan is showing an increasing trend from a plateau, with about 31,000 goats and about 24,000 sheep in 2022. In addition, there are many cases where these animals are raised by livestock farmers or individuals as companion animals, and from the perspective of preventing the occurrence and spread of infectious diseases, the importance of monitoring these diseases is increasing.

[0003] Here, macrophages are one of the immune cells that act on the front line of the body's defense in animals and are also known as target cells for infection by various pathogens. Therefore, isolating such macrophages and establishing a cultured cell line with continuous cell proliferation ability (immortalized macrophages) is important for analyzing the properties of infectious pathogens.

[0004] However, currently, for Caprinae animals such as goats and sheep, cell lines of immortalized macrophages have not been established, and there is a need for their production.

[0005] Japanese Patent Application Laid-Open No. 2021-61772 International Publication No. 2022 / 107793

[0006] Takato Takenouchi et al. , Front Vet Sci. 2017 Aug 21:4:132. Takato Takenouchi et al. , Front Vet Sci. 2022 Jul 18:9:919077. Takato Takenouchi et al. , Front Vet Sci. 2022 Nov 18:9:1058124. Juan F. Munoz-Gutierrez et al. , Virus Res. 2015 Feb 16:198:35-43.

[0007] This invention has been made in view of the problems of the prior art described above, and aims to provide immortalized macrophages of goat subfamily animals.

[0008] To achieve the above objective, the inventors diligently studied macrophages from animals belonging to the Bovidae family (goats, sheep, and cattle). As a result, they succeeded in isolating and culturing primary cultured macrophages from the blood of these animals. Furthermore, they attempted to induce immortalization by introducing two genes (SV40 large T antigen gene and porcine-derived telomerase reverse transcriptase gene) into these primary cultured macrophages. As a result, they succeeded in obtaining macrophages that exhibited sustained cell proliferation from goats and sheep, i.e., animals belonging to the Caprinae subfamily. On the other hand, from cattle, which belong to a different subfamily (Borinae) within the same family (Bovidae), they were unable to obtain macrophages that exhibited sustained cell proliferation (cell proliferation maintained for at least two months) through the above induction.

[0009] Furthermore, analysis of the characteristics of macrophages derived from goat subfamily animals that were immortalized in this way revealed the expression of macrophage marker molecules Iba1, CD172a, and CD204. In addition, the induction of inflammatory cytokine production and promotion of phosphorylation in signal transduction, which are characteristic of macrophages, were detected in response to bacterial cell wall components (lipopolysaccharide (LPS) and muramyl dipeptide (MDP)). Thus, we succeeded in producing immortalized cells derived from goat subfamily animals in which the characteristics of macrophages were maintained.

[0010] Furthermore, it was revealed that one strain of immortalized macrophages derived from goats, produced in this manner, was already infected with bovine viral diarrhea virus (BVDV). In other words, it was confirmed that immortalized macrophages derived from goat subfamily animals are susceptible to BVDV and can even propagate the virus.

[0011] Furthermore, as described above, we confirmed that both immortalized macrophages derived from goats and immortalized macrophages derived from sheep could propagate multiple types of arboviruses. In addition, we confirmed that these immortalized macrophages derived from goat subfamily animals are also susceptible to canine arthritis and encephalitis viruses.

[0012] This invention is based on the first successful production of immortalized macrophages derived from goat subfamily animals, and the discovery that these immortalized macrophages are susceptible to infection by pathogens such as viruses. Specifically, the invention is as follows:

[0013] In other words, the present invention provides the following embodiments.

[0014] [1] Immortalized cells of macrophages from goat subfamily animals.

[0015] [2] The cell according to [1], wherein macrophages of goat subfamily animals express at least one protein selected from the group consisting of SV40 large T antigen and telomerase reverse transcriptase.

[0016] [3] The cell according to [2], wherein the expression is from a lentivirus encoding the protein.

[0017] [4] The cell according to [2] or [3], wherein the telomerase reverse transcriptase is a porcine-derived telomerase reverse transcriptase.

[0018] [5] A method for producing immortalized macrophages, comprising the step of expressing at least one protein selected from the group consisting of SV40 large T antigen and telomerase reverse transcriptase in macrophages of goat subfamily animals.

[0019] [6] The method according to [5], wherein the step is to introduce a lentivirus encoding the protein into a macrophage of a goat subfamily animal and express the protein.

[0020] [7] The method according to [5] or [6], wherein the telomerase reverse transcriptase is porcine-derived telomerase reverse transcriptase.

[0021] [8] A method for producing a pathogen of a goat subfamily, comprising the step of bringing cells described in any one of [1] to [4] into contact with a pathogen of a goat subfamily, and growing the pathogen in the cells.

[0022] [9] A method for producing a vaccine containing a goat subfamily pathogen, comprising the steps of: contacting cells described in any one of [1] to [4] with a goat subfamily pathogen and growing the pathogen in the cells; isolating the grown pathogen; and mixing the isolated pathogen with a pharmacologically acceptable carrier or medium.

[0023]

[10] A cell according to any one of [1] to [4], having DNA in which a reporter gene is functionally bound downstream of the promoter region of a gene derived from a goat subfamily pathogen.

[0024]

[11] A method for detecting a pathogen of a goat subfamily, comprising the steps of: culturing the cells described in

[10] in the presence of a test sample; detecting the expression of the reporter gene in the cells; and determining that the test sample contains a pathogen of a goat subfamily if the expression of the reporter gene is detected.

[0025]

[12] A method for detecting neutralizing antibodies against viruses infecting goat subfamily animals, comprising the steps of: contacting cells described in any one of [1] to [4] with a virus infecting goat subfamily animals in the presence of a biological sample isolated from a goat subfamily animal under test, and growing the virus in the cells; detecting the number of viruses grown; and determining that the biological sample contains neutralizing antibodies against the virus if the number of viruses detected in the first step is less than the number of viruses grown in cells described in any one of [1] to [4] in the absence of the biological sample.

[0026] The present inventors have previously developed a method for producing immortalized cells by introducing two genes (SV40 large T antigen gene and porcine-derived telomerase reverse transcriptase gene) into primary cultured porcine macrophages (Patent Documents 1 and 2, Non-Patent Documents 1-3). They have also revealed that immortalized porcine macrophage cell lines exhibit high susceptibility to porcine-infecting viruses (Patent Documents 1 and 2, Non-Patent Documents 2 and 3).

[0027] However, the goats and sheep (Bovidae, Caprinae) from which we successfully created macrophages in this study are completely different animals at the family level from wild boars (Suidae). It is not easy to imagine that this method can be applied to animals of the Caprinae subfamily, or that the created immortalized cells can maintain their macrophage characteristics. Furthermore, as mentioned above, considering that we were unable to establish immortalized macrophages in cattle (Bovidae, Borinae subfamily), even though they belong to the same family, there is no reasonable expectation that we will succeed in creating immortalized macrophages in animals of the Caprinae subfamily, and from this perspective as well, it is not something that can be easily imagined.

[0028] Furthermore, there is a report that sheep brain macrophages can be immortalized by transfecting them with a human-derived telomerase reverse transcriptase gene, and that natural prion isolates (infectious proteins) can be amplified (Non-Patent Literature 4). However, the cell characteristics were not sufficiently analyzed in that report, and it is unclear whether the characteristics of macrophages were maintained.

[0029] According to the present invention, it is possible to provide a cell line of macrophages from goat subfamily animals that possesses continuous cell proliferation. Furthermore, since such immortalized macrophages from goat subfamily animals are susceptible to pathogens of goat subfamily animals, it becomes possible to proliferate such pathogens, and thus enable the manufacture and development of vaccines against said pathogens. In addition, because they are susceptible to infection, it is also possible to detect (test, diagnose) the pathogens.

[0030] These are photographs showing the results of observing mixed cultures of feeder cells (porcine kidney cells or B46 cells) with goat or sheep blood. In the figures, A shows the results of observing mixed cultures of porcine kidney cells and goat blood, and B shows the results of observing mixed cultures of porcine kidney cells and sheep blood. In these figures, the arrows indicate spherical cells (macrophages) that weakly adhere to and proliferate on porcine kidney cells. C shows the results of observing mixed cultures of B46 cells and goat blood, and D shows the results of observing macrophages attached to suspension culture dishes isolated and recovered from the supernatant of the said mixed culture. These are photographs showing the results of observing two types of goat-derived macrophages (iGEM-1, iGBM-7) and one type of sheep-derived macrophage (iOBM) that were immortalized by the introduction of two types of genes (SV40 large T antigen (SV40LT) gene and porcine-derived telomerase reverse transcriptase (pTERT) gene). This photograph shows the results of immunostaining of iGBM-1, iGBM-7, and iOBM to detect the expression of macrophage marker molecules (Iba1, CD172a, CD204). In the figure, "NC" indicates the result of immunostaining performed without the primary antibody (negative control). This graph shows the results of analyzing the cell proliferation of iGBM-1, iGBM-7, iOBM, and iOBM-2. iOBM-2 cells were established by exposing iOBM cells to a lentiviral solution containing the SV40LT gene and pTERT gene two additional times (a total of three times). This photograph shows the results of detecting the insertion of the SV40LT gene and pTERT gene in iGBM-1, iGBM-7, and iOBM by RT-PCR. This graph shows the results of quantitative RT-PCR detection of inflammatory cytokine (IL1A, IL1B, IL6) gene expression in iGBM-1, iGBM-7, and iOBM treated with LPS. This photograph shows the results of Western blotting detection of phosphorylation of p38 MAP kinase, NFκBp65, and p44 / 42 MAP kinase in iGBM-7 treated with LPS or MDP. This photograph shows the results of immunohistochemistry and RT-PCR detection of BVDV infection in iGBM-1, iGBM-7, and iOBM.In the figures, A to C show the results of immunohistochemical staining, and in these figures, "NC" indicates the results of immunohistochemical staining performed without using a primary antibody (BVDV-specific antibody) (negative control). Also, in A, the cells indicated by the arrows are BVDV-positive cells. D shows the results of RT-PCR analysis, "PC" indicates the results using culture supernatant (upper panel) or goat fetal lung (FGL) cells (lower panel) from the bovine kidney-derived cell line (MDBK) of the BVDV type 1 standard strain NOSE strain as the sample (positive control), and "NC" indicates the results using water as the sample (negative control). This is a graph showing the results of testing the arbovirus proliferation activity in iGBM-7. In the figure, "AKAV" indicates the replication of Akabane virus, "AINOV" indicates the replication of Aino virus, "CHUV" indicates the replication of Chuzan virus, "DAGV" indicates the replication of Diagula virus, "BTV-1" indicates the replication of Bluetongue virus serotype 1, and "EHDV-2" indicates the replication of epidemic hemorrhagic disease virus serotype 2. The notation in the figure is the same in Figure 9B. This is a graph showing the results of testing the arbovirus replication in iOBM-2. These are photographs showing the results of detecting canine arthritis encephalitis virus (CAEV) infection in iGBM-7 and iOBM-2 by Giemsa staining. In the figure, "CAEV 2dpi" shows photographs of cells on day 2 of CAEV infection, and "Mock" shows photographs of cells on day 2 of non-virus infection. These are photographs showing the results of detecting the cytopathic effect of CAEV in iGBM-7 and iOBM-2. In the figure, "CAEV 3dpi" shows photographs of individual cells 3 days after CAEV infection, and "Mock" shows photographs of individual cells 3 days after non-infection with the virus. These are photographs showing the results of detecting CAEV infection in iGBM-7 and iOBM-2 using the nested PCR method.In the figure, "M" indicates the lane where the 100 bp DNA ladder marker was run; "1" shows the results of analyzing genomic DNA extracted from CAEV-infected iGBM-7 (iGBM-7 + CAEV); "2" shows the results of analyzing genomic DNA extracted from CAEV-infected iOBM-2 (iOBM-2 + CAEV); "3" shows the results of analyzing genomic DNA extracted from persistently infected sheep fetal kidney cells (positive control); and "4" shows the results of analyzing genomic DNA extracted from sheep fetal lung cells (negative control).

[0031] This invention relates to immortalized macrophages from caprinae animals (immortalized macrophages from caprinae animals). In this invention, "caprinae animals" refers to animals belonging to the Caprinae subfamily of the Bovidae family in the Artiodactyla order of the Mammalia class. Furthermore, when the constituent species of the Bovidae family excluding the Bovidae subfamily are grouped together as the Antilopinae subfamily, this can be rephrased as animals belonging to the Caprini tribe. Examples of caprinae animals according to this invention include animals belonging to the genus Capra (goats, etc.), animals belonging to the genus Sheep (sheep, etc.), and animals belonging to the genus Antelope (Japanese serow, etc.).

[0032] In the present invention, the "macrophages" to be immortalized may be active macrophages (inflammatory M1-type macrophages, anti-inflammatory M2-type macrophages) or resting macrophages. Furthermore, there are no particular restrictions on the tissues in which the macrophages exist. Examples of macrophages according to the present invention (so-called tissue macrophages) include blood macrophages, kidney macrophages, alveolar macrophages, intestinal macrophages, liver macrophages, brain macrophages, and osteoclasts.

[0033] "Blood macrophages" can be prepared, for example, by mixing blood collected from goat subfamily animals with feeder cells in a culture, as shown in the examples described below, and then isolating the spherical cells that adhere to and proliferate on the feeder cells. There are no particular restrictions on the form of the "blood" used in the mixed culture; for example, anticoagulant-treated blood (e.g., heparin-treated peripheral blood), coagulated blood, or hemolyzed blood can be used.

[0034] The "feeder cells" used in the mixed culture described above are not particularly limited as long as they can maintain blood macrophages, but examples include kidney-derived cells, liver-derived cells (e.g., hepatic sinusoidal endothelial cells), lung-derived cells, intestinal-derived cells, brain-derived cells, and bone-derived cells. More preferably, however, are monolayer cell sheets obtained by culturing cells from each tissue (a primary culture system (mixed culture system) obtained by culturing cells from each tissue, from which macrophage-like cells have been removed), as described below. Furthermore, there are no particular restrictions on the origin of the feeder cells, but animals belonging to the Suidae family are preferred, and pigs are more preferred. More specifically, a primary culture system derived from pig kidneys, as shown in the examples described later, is an example. Animals belonging to the Bovidae family are also preferred as the origin of the feeder cells, animals belonging to the Bovidae or Caprinae subfamilies are more preferred, and cattle, sheep, or goats are even more preferred, with cattle being more preferred. More specifically, cattle hepatic sinusoidal endothelial cells (B46 cells, etc.), as shown in the examples described later, are an example.

[0035] "Other tissue macrophages" can be prepared, for example, as follows: First, various tissues collected from goat subfamily animals (e.g., kidney (renal cortex, etc.), lung (lung parenchyma, etc.), intestine (small intestine, etc.), liver (hepatic parenchyma, etc.), brain (cerebrum, etc.), bone (bone marrow, etc.)) are finely chopped, washed with buffer, subjected to enzymatic treatment, and then cultured. Macrophage-like cells can be isolated from the mixed culture system (a monolayer of cell sheets and macrophage-like cells adhering to it) that is produced during the culture process. There are no particular restrictions on the "buffer solution" used to wash the finely chopped tissue; examples include Dulbecco's phosphate-buffered saline (DPBS), phosphate-buffered saline (PBS), Tris-HCl buffer (TBS), and HEPES buffer. The aforementioned "enzyme treatment" is not particularly limited in terms of the enzymes used, as long as they can separate and disperse cells from the tissue. Examples include collagenase, dispase, DNase (DNase I, etc.), trypsin, hyaluronidase, elastase, or pronase, but preferably a combination of collagenase, dispase, and DNase I. There are also no restrictions on the temperature and time of treatment, and these can be adjusted as appropriate depending on the type of enzyme used and the degree of cell separation and dispersion. Furthermore, in order to produce the aforementioned mixed culture system, the cells cultured after the enzyme treatment may be subjected to further enzyme treatment to detach them from the culture vessel, disperse them, and re-seed them. This enzyme treatment can also be carried out using the enzymes mentioned above, but preferably a combination of protease (an enzyme having at least degrading activity against non-collagenous proteins), collagenase, and DNase (for example, product name: Accumax®, manufactured by Sigma-Aldrich).

[0036] The culture medium used for the isolation and proliferation of the above-mentioned "blood macrophages" and "other tissue macrophages" (hereinafter also referred to as "primary culture medium for tissue macrophages") is not particularly limited as long as it can maintain each tissue macrophage, but it can be prepared by appropriately adding well-known and conventional culture medium additives to a known basal medium. Examples of "basal media" include DMEM medium, DMEM medium (high glucose), DMEM medium (low glucose), RPMI 160 medium, RPMI 1640 medium, Ham F12 medium, KSOM medium, Eagle MEM medium, Glasgow MEM medium, αMEM medium, Ham medium, Fishers medium, BME medium, BGJb medium, CMRL 1066 medium, MEM Zinc option improved medium, IMDM medium, Medium 199 medium, and any mixed medium of these. Examples of "culture medium additives" include antibiotics (penicillin, streptomycin, gentamicin, vancomycin, etc.), antifungal agents (pimaricin, amphotericin B, etc.), functional proteins (insulin, transferrin, lactoferrin, etc.), reducing agents (monothioglycerol, 2-mercaptoethanol, catalase, superoxide dismutase, N-acetylcysteine, etc.), lipids other than fatty acids (cholesterol, etc.), amino acids (alanine, L-glutamine, non-essential amino acids, etc.), peptides (glutathione, reduced glutathione, etc.), nucleotides, etc. (nucleosides, cytidine, adenosine 5'-1) Examples of pH indicators include, but are not limited to, phosphoric acid, hypoxanthine, thymidine, etc., metal salts (ferrous nitrate, ferrous sulfate, copper sulfate, zinc sulfate, etc.), inorganic salts (sodium, potassium, calcium, magnesium, phosphorus, chlorine, etc.), carbon sources (glucose, galactose, fructose, sucrose, etc.), vitamins, inorganic compounds (selenic acid), organic compounds (para-aminobenzoic acid, ethanolamine, corticosterone, progesterone, lipoic acid, putrescine, pyruvate, lactic acid, triiodothyronine, etc.), buffer compounds (HEPES, sodium bicarbonate, etc.), and pH indicators (phenol red, etc.).

[0037] Furthermore, there are no particular restrictions on the culture conditions using such a medium, but the culture temperature is usually 30 to 40°C, preferably 37°C. The concentration of carbon dioxide in the gas in contact with the medium is usually 1 to 10% by volume, preferably 2 to 5% by volume.

[0038] Furthermore, by culturing cells derived from each tissue under these culture conditions, primary cultured macrophages derived from each tissue can be obtained. There are no particular restrictions on the culture time for obtaining these cells, but it is usually 1 week to 2 months, preferably 2 to 3 weeks. In addition, since the adhesion between these primary cultured macrophages and the feeder cells or cell sheets is weak, the macrophages can be recovered by centrifugation of the culture supernatant. Moreover, since macrophages readily adhere to culture vessels for suspension cell culture or non-tissue culture, more selective isolation from other cells becomes possible by reseeding the primary cultured macrophages in such culture vessels.

[0039] There are no particular restrictions on the method for immortalizing the macrophages of goat-minae obtained in this way, but it can be done by introducing at least one immortalization gene. Examples of immortalization genes include SV40 large T antigen (SV40T antigen), telomerase reverse transcriptase (TERT), Myc, and Ras. There are no particular restrictions on the origin of TERT, and examples include mammals such as Suidae (e.g., pigs, wild boars), Bovidae (e.g., goats, sheep, and other goat-minae, and cattle, and other bovine-minae), and Hominidae (e.g., humans). Furthermore, in the present invention, it is preferable to introduce SV40T antigen and TERT, and from the viewpoint of increasing the immortalization efficiency of macrophages of goat-minae, it is more preferable to introduce SV40T antigen and porcine-derived TERT.

[0040] The introduction of an immortalization gene can be carried out by using a vector that encodes the gene. The vector can be linear or circular, and examples include viral vectors, plasmid vectors, episomal vectors, artificial chromosome vectors, and transposon vectors.

[0041] Examples of viral vectors include retroviral vectors such as lentivirus, Sendai virus vectors, adenovirus vectors, adeno-associated virus vectors, herpesvirus vectors, vaccinia virus vectors, poxvirus vectors, poliovirus vectors, silbis virus vectors, rhabdovirus vectors, paramyxovirus vectors, and orthomyxovirus vectors. Examples of plasmid vectors include plasmid vectors for animal cell expression such as pcDNA3.1, pA1-11, pXT1, pRc / CMV, pRc / RSV, and pcDNAI / Neo. Among these vectors, from the viewpoint of enhancing the gene transfer efficiency into macrophages of Caprinae animals, retroviral vectors are preferred, and lentivirus is more preferred.

[0042] In addition to the immortalizing gene, the vector according to the present invention may contain expression control sequences such as a promoter, an enhancer, a polyA addition signal, a terminator, a nucleotide sequence encoding a protein that binds to a replication origin and controls replication, a 5' non-translated region containing a 5' cap structure, a Shine-Dalgarno sequence, a Kozak sequence, etc., a 3' non-translated region containing a polyadenylation signal, an AU-rich element, a GU-rich element, etc., and nucleotides encoding other proteins.

[0043] By operably arranging the immortalizing gene downstream of the promoter, each polynucleotide can be efficiently transcribed. Examples of such "promoters" include the EF1α promoter, CMV promoter, SRα promoter, SV40 early promoter, LTR promoter, RSV promoter, HSV-TK promoter, MSCV promoter, hTERT promoter, β-actin promoter, CAG promoter, metallothionein promoter, heat shock promoter, etc.

[0044] Examples of "nucleotides encoding other proteins" include marker genes such as reporter genes and drug resistance genes.

[0045] Furthermore, when introducing multiple types of immortalization genes, these genes may be incorporated into a single vector or into separate vectors, but from the viewpoint of increasing expression efficiency, it is preferable to incorporate them into separate vectors. Also, when incorporating into a single vector, for example, by inserting IRES, 2A peptide sequences, etc. into the vector, it becomes possible to express multiple types of immortalization genes polycistronically.

[0046] Methods for introducing the vector into cells include lipofection, microinjection, calcium phosphate, DEAE-dextran, electroporation, and particle gun. Furthermore, if the vector of the present invention is a retroviral vector, appropriate packaging cells may be selected based on the LTR sequence and packaging signal sequence of the vector, and retroviral particles may be prepared using these cells. Examples of packaging cells include PG13, PA317, GP+E-86, GP+envAm-12, and Psi-Crip. Additionally, 293 cells or 293T cells, which have high transfection efficiency, can be used as packaging cells. The viral particles thus prepared can then be introduced into cells by methods such as Polybrene, Protamine, and RetroNectin.

[0047] Furthermore, such gene introduction and subsequent maintenance culture can be carried out using the primary culture medium for tissue macrophages and the culture conditions using it described above. In addition, the immortalized macrophages of goat subfamily animals established by introducing the immortalization gene in this way exhibit proliferative activity for at least one month, preferably two months or more, more preferably three months or more, even more preferably four months or more, even more preferably five months or more, even more preferably six months or more, and even more preferably seven months or more. The doubling time of the immortalized macrophages of goat subfamily animals is, for example, within eight days, preferably within seven days, more preferably within six days, even more preferably within five days, and even more preferably within four days.

[0048] Furthermore, it is preferable that immortalized macrophages from goat subfamilies retain the characteristics of macrophages. For example, at least one of the macrophage-specific genes Iba1, CD172a, CD204 (MSR-A), CD203a, and CD16 is expressed, preferably two or more genes are expressed, more preferably three or more genes are expressed, and even more preferably at least Iba1, CD172a, and CD204 are expressed. In addition, immortalized macrophages from goat subfamilies may express at least one of the marker genes CD163 and CD169, which are specific macrophage subpopulations, and the antigen-presenting cell marker gene MHC-II. Furthermore, immortalized macrophages from goat subfamilies retain, as a macrophage characteristic, at least one of the following functions: production of inflammatory cytokines in response to stimulation by bacterial cell wall components (such as LPS), and / or promotion of phosphorylation and phagocytosis in signal transduction, and IL-1β maturation associated with inflammasome activity. Preferably, they retain the production of inflammatory cytokines in response to stimulation by bacterial cell wall components, and / or promotion of phosphorylation in signal transduction.

[0049] (Pathogens of Caprinae Animals) In the present invention, "pathogens of Caprinae Animals" means microorganisms that can infect animals belonging to the Caprinae subfamily and can proliferate within their bodies (for example, inside cells such as macrophages). Examples of such microorganisms include viruses, bacteria (eubacteria), fungi, protozoa, and the like.

[0050] Viruses that can infect animals of the subfamily Caprinae (hereinafter also referred to as "viruses that infect animals of the subfamily Caprinae") may be DNA viruses (double-stranded (ds)DNA viruses, single-stranded (ss)DNA viruses, DNA viruses containing both ss and dsDNA regions) or RNA viruses (single-stranded (ss)RNA viruses (positive-strand RNA viruses or negative-strand RNA viruses), double-stranded (ds)RNA viruses). More specifically, examples include arboviruses (for example, orthobunyaviruses such as Akabane virus, Aino virus, and Schmarenberg virus, orbiviruses such as Chuzan virus, Diagula virus, Bluetongue virus, and Epidemic Hemorrhagic Disease virus), bovine viral diarrhea virus (BVDV), canine arthritis-encephalitis virus (CAEV), foot-and-mouth disease virus, ruminant disease virus, and Aarf virus (parapoxvirus). Examples of bacteria that can infect goat-minae include Mycobacterium species (such as Mycobacterium bovis, Mycobacterium avium subsp. Paratuberculosis, etc.), Brucella species (such as B. abortus, B. melitensis, B. suis, B. ovis, etc.), and Ehrlichia ovine. Examples of protozoa that can infect goat-minae include Toxoplasma gondii and Neospora caninum.

[0051] (Method for producing pathogens of goat subfamily animals) The present invention provides a method for producing pathogens of goat subfamily animals (growth method, amplification method), which includes the step of bringing immortalized macrophages of goat subfamily animals into contact with a pathogen of goat subfamily animals and growing the pathogen in the immortalized macrophages.

[0052] The goat subfamily pathogens to be brought into contact with immortalized macrophages are as described above, but may include not only the isolated pathogen itself, but also samples that may contain the pathogen. Examples of such "samples" include tissues, cells, cultures, washing solutions, or extracts or processed products (food products, etc.) derived from animals (goat subfamily animals, etc.), or samples, washing solutions, or cultures thereof from the habitat or rearing environment of animals (goat subfamily animals, etc.).

[0053] "Contact" is typically performed by adding a goat-in-the-subfamily pathogen to a culture medium used to cultivate immortalized macrophages of goat-in-the-subfamily animals. There are no particular restrictions on such "culture medium," but the aforementioned primary culture medium for tissue macrophages is an example.

[0054] The "proliferation" of goat subfamily pathogens can be carried out by culturing immortalized macrophages that have come into contact with and infected by the pathogen. The culture temperature is not particularly limited, but is usually 30 to 40°C, preferably 37°C. The concentration of carbon dioxide in the gas in contact with the culture medium is not particularly limited, but is usually 1 to 10% by volume, preferably 2 to 5% by volume. The culture period after contact with the goat subfamily pathogen is usually 1 to 10 days, preferably 2 to 7 days, more preferably 3 to 5 days, but is not particularly limited, and the culture period may be longer (for example, 6 months or more) depending on the pathogen.

[0055] Whether or not a pathogen of the Caprinae subfamily has proliferated can be determined by methods known to those skilled in the art. Such methods include, for viruses, a CPE test using cytopathic effects (CPE) as an indicator, and a method for detecting the degree of intracellular ATP depletion associated with CPE (e.g., the Viral ToxGlo assay provided by Promega). For bacteria, for example, a method of counting the colonies formed in a culture suitable for the proliferation of each pathogen (e.g., agar culture). For protozoa, for example, a method of measuring the number of parasites by observation under a microscope. Methods for detecting genes derived from the Caprinae subfamily pathogen or their expression can also be used. Here, gene expression may be at the transcriptional level (RNA level) or the translational level (protein level). Methods for detecting genes (genomic DNA, genomic RNA) or RNA include, for example, PCR (RT-PCR, real-time PCR, quantitative PCR), sequencing, DNA microarray analysis, Northern blotting or Southern blotting, in situ hybridization, dot blotting, RNase protection assay, and mass spectrometry. Furthermore, gene or RNA levels can be quantitatively detected by counting the number of reads in so-called next-generation sequencing. Methods for detecting proteins include, for example, antibody-based detection methods (immunological techniques) such as ELISA, antibody arrays, immunoblotting, imaging cytometry, flow cytometry, radioimmunoassay, immunoprecipitation, and immunohistochemical staining, as well as mass spectrometry.

[0056] (Method for producing a vaccine) The method for producing a vaccine containing a goat subfamily pathogen of the present invention is a method comprising the steps of: contacting an immortalized macrophage of a goat subfamily with a goat subfamily pathogen and growing the pathogen in the immortalized macrophage of the goat subfamily; isolating the grown goat subfamily pathogen; and mixing the isolated goat subfamily pathogen with a pharmacologically acceptable carrier or medium.

[0057] The process of growing the pathogen in the immortalized macrophages is as described above. "Isolation" of the grown goat subfamily pathogen means separation, purification, and / or concentration from the culture medium of the immortalized macrophages and / or the cells. Examples of methods for isolating the pathogen include filtration of the culture medium, cell disruption (sonication, hypotonic treatment, freeze-thaw cycle, etc.), centrifugation (ultracentrifugation, density gradient centrifugation, etc.), and concentration (ammonium sulfate, resin column, polyethylene glycol salting-out, etc.).

[0058] The goat subfamily pathogens isolated in this manner may be used as vaccines (so-called live vaccines), in a weakened live form (so-called weakened live pathogens), or in an inactivated form as vaccines. Furthermore, as long as they possess immunogenicity, some of these isolated goat subfamily pathogens or their products (proteins, polypeptides, sugars, glycoproteins, lipids, nucleic acids, toxins, etc.) may be used as vaccines.

[0059] Attenuated pathogens are pathogens that have a reduced toxicity level compared to pathogens isolated from the field. Attenuated pathogens can be obtained by known methods, such as growth in the presence of mutagenic agents, acclimation to cultured cells through continuous (long-term) passage in vitro, or growth under conditions deviating from natural growth environments (e.g., high-temperature conditions). Attenuated pathogens can also be obtained by deleting or recombining specific genes in pathogens using genome editing, gene modification technologies, etc.

[0060] Pathogens can also be inactivated by known methods if you are skilled in the art. Such inactivation methods include formaldehyde treatment, UV irradiation, X-ray irradiation, electron beam irradiation, gamma ray irradiation, alkylation treatment, ethylene-imine treatment, thimerosal treatment, β-propiolactone treatment, and glutaraldehyde treatment.

[0061] Examples of "pharmacologically acceptable carriers" to be mixed with isolated goat subfamily animal pathogens include stabilizers, excipients, preservatives, surfactants, chelating agents, and binders. Examples of "pharmacologically acceptable media" include water, physiological saline, phosphate buffer, and Tris-HCl buffer. Those skilled in the art can appropriately select or combine known carriers and media used in the art, depending on the vaccine dosage form and method of use. Furthermore, there are no particular restrictions on the form of the vaccine; for example, it may be in the form of a suspension or in a lyophilized form.

[0062] From the perspective of enhancing the vaccine's effectiveness, adjuvants may be added. Examples of adjuvants include inorganic substances such as aluminum gel adjuvants, microorganisms or substances derived from microorganisms (such as BCG, muramyl dipeptide, Bordetella pertussis, pertussis toxin, and cholera toxin), surfactants (such as saponins and deoxycholic acid), emulsions of oily substances (such as mineral oil, vegetable oil, and animal oil), and alum.

[0063] (Method for detecting pathogens in goat subfamily animals) The present invention provides immortalized macrophages of goat subfamily animals having DNA in which a reporter gene is functionally bound downstream of the promoter region of a gene derived from a goat subfamily pathogen.

[0064] The present invention also provides a method for detecting pathogens of goat subfamily animals, comprising the steps of: culturing immortalized macrophages of goat subfamily animals having the DNA in the presence of a test sample; detecting the expression of the reporter gene in the immortalized macrophages; and determining that the test sample contains a pathogen of goat subfamily animals if the expression of the reporter gene is detected.

[0065] The "immortalized macrophages of the goat subfamily" into which the DNA is introduced are as described above. Furthermore, the DNA may be in the form of a vector as described above in the section on "immortalization genes." Moreover, the introduction of the DNA into the immortalized macrophages of the goat subfamily can also be carried out by a person skilled in the art using the methods listed above in the description of "immortalization genes."

[0066] The "promoter region of a gene derived from a goat subfamily pathogen" in the aforementioned DNA is not particularly limited as long as it is a region derived from a goat subfamily pathogen that can activate the expression of downstream genes in response to the infection and proliferation of the pathogen. If it is a virus, it may be any of the genes: immediate-early, early, late, or very-late. A person skilled in the art can select the goat subfamily pathogen gene to be used by referring to publicly known information as appropriate.

[0067] There are no particular restrictions on the "reporter gene" functionally (operably) bound downstream of the promoter region, and known genes can be used as appropriate. Examples include fluorescent protein genes, luminescent enzyme genes, and chromogenic enzyme genes. Specific examples of fluorescent protein genes include GFP (green fluorescent protein) genes, YFP (yellow fluorescent protein) genes, RFP (red fluorescent protein) genes, etc. Specific examples of luminescent protein / enzyme genes include aequorin genes, luciferase genes, etc. Specific examples of chromogenic enzyme genes include chloramphenicol acetyltransferase (CAT) genes, β-glucuronidase (GUS) genes, β-galactosidase genes, alkaline phosphatase genes, SEAP genes, etc.

[0068] Furthermore, in the detection method of the present invention, fluorescence, luminescence, color development, etc., which occur in response to the expression of these reporter genes, can be used as indicators to detect whether or not immortalized macrophages are infected with a pathogen of the goat subfamily, and consequently, the presence of the pathogen in the test sample.

[0069] There are no particular restrictions on the test samples, as long as they may contain pathogens from goat subfamily animals. Examples include tissues, cells, cultures, washing solutions, extracts, or processed products (food products, etc.) derived from animals (goat subfamily animals, etc.), or samples, washing solutions, or cultures of animals (goat subfamily animals, etc.) collected from their habitats or breeding environments.

[0070] Furthermore, there are no particular limitations on the "culture medium" used for culturing in the detection method of the present invention, but examples include the primary culture medium for tissue macrophages described above. The culture temperature is not particularly limited, but is usually 30 to 42°C, preferably 37°C. The concentration of carbon dioxide in the gas in contact with the culture medium is not particularly limited, but is usually 1 to 10% by volume, preferably 2 to 5% by volume. The culture period until the expression of the reporter gene is detected in the presence of the test sample is not particularly limited, but is usually 1 to 10 days, preferably 2 to 7 days, more preferably 2 to 5 days.

[0071] (Method for detecting neutralizing antibodies) The present invention provides a method for detecting neutralizing antibodies against viruses that infect goat subfamily animals, comprising the steps of: contacting immortalized macrophages of a goat subfamily animal with a virus that infects goat subfamily animals in the presence of a biological sample isolated from a goat subfamily animal, and allowing the virus to grow in the immortalized macrophages of the goat subfamily animal; detecting the number of viruses that have grown; and determining that the biological sample contains neutralizing antibodies against the virus if the number of viruses detected in the first step is less than the number of viruses that grew in the immortalized macrophages of the goat subfamily animal in the absence of the biological sample.

[0072] The term "neutralizing antibody" in this invention refers to an antibody that suppresses infection or proliferation of viruses that infect goat subfamily animals. Such antibodies include all classes and subclasses of immunoglobulins. Furthermore, there are no particular restrictions on the "tested goat subfamily animal," as long as it is a goat subfamily animal, regardless of whether it has previously been infected with a goat subfamily virus. Examples of "biological samples" isolated from the test goat subfamily animal include samples derived from goat subfamily animals (e.g., blood (serum, plasma, etc.), mucus (saliva, nasal secretions, milk, gastrointestinal secretions, etc.), and antibodies purified from these).

[0073] The "contact" is as described above. Regarding the conditions for proliferation, the culture temperature is not particularly limited, but is usually 30 to 40°C, preferably 37°C. The concentration of carbon dioxide in the gas in contact with the culture medium is not particularly limited, but is usually 1 to 10% by volume, preferably 2 to 5% by volume. The culture period in the presence or absence of a biological sample is not particularly limited, but is usually 1 to 10 days, preferably 2 to 7 days, more preferably 3 to 5 days. Furthermore, the proliferated virus can be detected as described above by a CPE test or by detecting a gene derived from a virus that infects goat subfamily animals or its expression. Thus, in the detection method of the present invention, the presence or absence of neutralizing antibodies may be determined not only by the number of viruses themselves, but also by using the gene (genomic DNA amount, etc.) or its expression level that reflects the number of viruses as an indicator.

[0074] The present invention will be described more specifically below based on examples and comparative examples, but the present invention is not limited to the following examples.

[0075] (Example 1) Construction of a mixed culture system of goat or sheep blood and feeder cells We attempted to prepare blood macrophages derived from these animals by mixing and culturing goat or sheep blood and feeder cells.

[0076] The goats used were hybrid, male, and aged 6-8 years. The sheep used were hybrid, female, and aged 6-8 years. The blood from these animals was purchased from veterinarian Risako Maruyama of Tamura Hoof Trimming Shop (Obihiro City, Hokkaido, Japan) (approximately 5 ml of blood was obtained from each animal).

[0077] The feeder cells used were porcine kidney cells as described in Non-Patent Literature 1. Specifically, a primary culture system derived from porcine kidney cells, mainly containing epithelial cells, fibroblasts, and myofibroblasts, with macrophages removed, was used. The porcine kidney cells were then cultured at 150 cm². ２ Using tissue culture flasks (manufactured by Falcon), the tissue was pre-cultured in the growth medium described below, and then mixed culture was started by adding 2 ml each of goat or sheep blood.

[0078] Additionally, bovine liver sinusoidal endothelial cell line B46 was used as feeder cells (the same as that used by Hiramatsu et al., Cell. Biol. Int. 48:76-83, 2024). The B46 cells were then placed in a 75 cm chamber. ２ Using a tissue culture flask (manufactured by Falcon), the tissue was pre-cultured in the growth medium described below, and then mixed culture was started by adding 1 ml of goat blood.

[0079] As a result, approximately two weeks after starting mixed culture with porcine kidney cells, spherical cells that weakly adhered to and proliferated on the sheet consisting of these feeder cells were observed (Figure 1A: mixed culture with goat blood, Figure 1B: mixed culture with sheep blood, cells indicated by arrows in each photograph). Then, the cells recovered from the culture supernatant were seeded onto a suspension culture dish (Sumitomo Bakelite), and the attached macrophages were isolated and collected.

[0080] Furthermore, macrophages adhering to suspension culture dishes could also be isolated and recovered from the supernatant of a mixed culture system of B46 cells and goat blood (Figure 1C) (Figure 1D).

[0081] (Example 2) Preparation of immortalized blood macrophage cell lines derived from goats or sheep Blood macrophages isolated and recovered in the above procedure were seeded in 60 mm suspension culture dishes (manufactured by Sumitomo Bakelite Co., Ltd.). The attached cells were then exposed for 2 to 24 hours to a solution containing recombinant lentiviruses into which two types of immortalization genes (SV40 large T antigen (SV40LT) gene and porcine-derived telomerase reverse transcriptase (pTERT) gene) were individually introduced (hereinafter also simply referred to as "recombinant lentivirus solution"). Exposure to the recombinant lentivirus solution was performed 1 to 3 times at intervals. After observing under a microscope for more than one month, cells were detached from dishes in which proliferative cell colonies were confirmed, collected, and subcultured.

[0082] As a result, in the case of goats, primary cultured macrophages seeded in five 60 mm dishes were immortalized, and proliferative cells appeared from all five dishes. From these, immortalized goat blood-derived macrophages (hereinafter also referred to as "iGBM")-1 was established from a dish in which cells recovered from a mixed culture system with B46 cells were exposed three times to a recombinant lentivirus solution (Figure 2A). In addition, iGBM-7 was established from a dish in which cells recovered from a mixed culture system with porcine kidney cells were exposed once to a recombinant lentivirus solution (Figure 2B).

[0083] For sheep, primary cultured macrophages seeded in six 60 mm suspension culture dishes were immortalized, and proliferative cells appeared in one of them. This was from a dish in which cells recovered from a mixed culture with porcine kidney cells were exposed once to a recombinant lentivirus solution, and these cells were subcultured as an immortalized ovine blood-derived macrophage cell line (also referred to as "iOBM") (Figure 2C).

[0084] (Comparative Example 1) Preparation of bovine-derived blood macrophage immortalized cell line Similar to the goat and sheep described above, bovine blood and feeder cells were mixed and cultured to prepare blood macrophages derived from this animal. Then, the above immortalization gene was introduced into these primary cultured macrophages to attempt to create a macrophage immortalized cell line.

[0085] The cattle used were either Japanese Black breed, female, 4 years old, or Japanese Black breed, male, 1 year old. The blood samples were obtained from the National Agriculture and Food Research Organization, Animal Health Research Division, Kagoshima Research Station (approximately 5 ml of blood was obtained per animal).

[0086] According to the method described by Hiramatsu et al. in Cell. Biol. Int. 48:76-83, 2024, bovine blood and porcine kidney cells or B46 cells were mixed and cultured, and primary cultured macrophages were isolated and recovered. The obtained bovine blood macrophages were seeded in a 60 mm suspension culture dish. The attached cells were then periodically exposed to the above-mentioned recombinant lentivirus solution 3 to 4 times.

[0087] As a result, three dishes seeded with cells recovered from a mixed culture with porcine kidney cells and six dishes seeded with cells recovered from a mixed culture with B46 cells were observed for approximately two months after immortalization induction, but no proliferative cells were observed, and it was not possible to create cell lines that could be subcultured. The results of Examples 1 and 2, as well as Comparative Example 1, are summarized in the table below.

[0088]

[0089] As described above, we succeeded in creating immortalized cells from macrophages of animals belonging to the Caprinae subfamily (goats and sheep). On the other hand, we were unable to establish immortalized cells from cattle, which belong to the same Bovidae family.

[0090] Next, iGBM-1, iGBM-7, and iOBM obtained above were subjected to the following analytical experiments.

[0091] (Cell Culture) Cells were cultured in growth medium (DMEM High Glucose: containing 10% fetal bovine serum, 100 U / mL penicillin, 100 units / mL streptomycin, 100 μg / mL insulin, 25 μM monothioglycerol, and 5 μg / mL fungin). Cells (1 × 10⁶) ６ The cells were seeded in 90 mm suspension culture dishes (Sumitomo Bakelite Co., Ltd.) and periodically subcultured. At each subculture, cells were detached using TrypLE Express solution (Thermo Fisher Scientific), and the cell count was measured using a Bio-Rad TC20 automated cell counter.

[0092] (Immunostaining) Place cells on an 8-well chamber slide (Asahi Glass Co., Ltd.) at approximately 1 x 10⁶ ５ Cells were seeded in wells and cultured. After washing once with DPBS, cells were fixed with 4% paraformaldehyde phosphate buffer (Nacalai Tesque), permeabilized with 1% Triton X-100 / PBS solution, and blocked with Blocking One Hist (Nacalai Tesque). Next, the cells were incubated with primary antibody at room temperature for 1 hour and then stained using the EnVision system (DAKO) (stained brown). Cell nuclei were counterstained with Meyer hematoxylin solution (Fujifilm Wako) (stained blue). Stained slides were observed under a microscope. The following primary antibodies were used. - Anti-ionized calcium-binding adapter molecule 1 (Iba1) rabbit polyclonal antibody (manufactured by Fujifilm Wako Co., Ltd.) - Anti-CD172a mouse monoclonal antibody (clone DH59B) (manufactured by VMRD Co., Ltd.) - Anti-CD204 mouse monoclonal antibody (clone SRA-E5) (manufactured by Transgenic Co., Ltd.) - Anti-BVDV-1 & 2 mouse monoclonal antibody (manufactured by VMRD Co., Ltd.).

[0093] (Detection of insertions of SV40LT and pTERT genes) Genomic DNA was extracted from cells using the NucleoSpin kit (Takara Bio). Forward primers were designed within the lentiviral vector sequence, and reverse primer sequences were designed within the SV40LT or pTERT gene. Gene insertions into the genome were detected by PCR. The lengths of the PCR products derived from the SV40LT and pTERT genes were 128 bp and 143 bp, respectively. PCR amplification was performed using KOD FX DNA polymerase (Toyobo Co., Ltd.), and the obtained PCR products were separated by agarose gel electrophoresis and detected by GelGreen™ staining (Biotium, Inc.).

[0094] (RT-PCR and Quantitative RT-PCR) Total RNA was extracted from cells using the NucleoSpin RNA Kit (Takara Bio Inc.). Then, using this as a template, a reverse transcription reaction was performed using the PrimeScript RT reagent with gDNA Eraser Kit (Takara Bio Inc.) to prepare complementary DNA (cDNA).

[0095] Using the prepared cDNA as a template, PCR reactions were performed using primers that specifically recognize the goat or sheep GAPDH gene and the genome of bovine viral diarrhea virus (BVDV). The resulting PCR products were analyzed by agarose gel electrophoresis.

[0096] Furthermore, quantitative RT-PCR was performed on the aforementioned cDNA using Thermo Scientific™ SYBR™ Green qPCR Master Mix (manufactured by Thermo) in accordance with its product protocol, using primers that specifically recognize goat or sheep GAPDH, IL1A, IL1B, and IL6, and measured in QuantStudio3.

[0097] (Induction of inflammatory cytokine expression) Total RNA was extracted from cells treated with 1 μg / mL lipopolysaccharide (LPS) for 3 hours and from untreated cells using the NucleoSpin RNA Kit (Takara Bio Inc.). Using these as templates, the gene expression changes of IL1A, IL1B, and IL6 were analyzed by quantitative RT-PCR as described above.

[0098] (Western blotting) Lysates prepared from cells were subjected to SDS polyacrylamide gel electrophoresis and fractionated. Next, proteins were transferred from the gel to a PVDF membrane, treated with Blocking One Hist (Nacalai), and then treated with a primary antibody followed by a horseradish peroxidase (HRP) labeled secondary antibody. The HRP-labeled PVDF membrane was immersed in a solution of Chemiluminescent One Ultra Kit (Nacalai) and analyzed with a C-DiGit chemiluminescence scanner.

[0099] (Activation of molecules in intracellular signal transduction) After treating iGBM-7 cells with LPS and muramyl dipeptide (MDP), which are components of the bacterial cell wall, for 30 minutes, a cell lysate was prepared, and phosphorylation of p38 MAP kinase, NFκBp65, and p44 / 42 MAP kinase was detected by the Western blotting method described above.

[0100] Based on the analysis experiments conducted on iGBM-1, iGBM-7, and iOBM, first, immunohistochemical staining revealed the expression of macrophage marker molecules (Iba1, CD172a, CD204) in all three cells: iGBM-1 (Figure 3A), iGBM-7 (Figure 3B), and iOBM (Figure 3C).

[0101] Analysis of cell proliferation revealed that, as shown in Figure 4, proliferation was maintained for at least two months in all three cells: iGBM-1, iGBM-7, and iOBM. Furthermore, the doubling time for iGBM-1 cells was approximately 5.2 days (Figure 4A), for iGBM-7 cells approximately 3.8 days (Figure 4B), and for iOBM cells approximately 7.2 days (Figure 4C). In addition, iOBM-2 cells, which were subjected to two additional exposures (a total of three exposures) to a recombinant lentivirus solution, showed improved proliferative capacity with a doubling time of approximately 4.9 days (Figure 4D).

[0102] Furthermore, the insertion of the SV40LT and pTERT genes into the genome was confirmed in all cell lines by PCR (Figure 5).

[0103] Furthermore, treatment with LPS resulted in increased expression of inflammatory cytokines (IL1A, IL1B, IL6) in all cells (Figure 6). In addition, treatment of iGBM-7 cells with LPS and MDP resulted in enhanced phosphorylation of p38 MAP kinase, NFκBp65, and p44 / 42 MAP kinase in a concentration-dependent manner with these bacterial cell wall components (Figure 7).

[0104] Furthermore, analysis of BVDV infection in iGBM-1 cells, iGBM-7 cells, and iOBM cells revealed that, as is evident from a comparison of immunostaining results using antibodies against BVDV ("BVDV" in Figures 8A, B, and C) and those without primary antibodies ("NC" in Figures 8A, B, and C), BVDV-positive cells were detected only in iGBM-1 cells (Figure 8A, arrow). Similarly, analysis by RT-PCR also revealed a specific band derived from BVDV only in iGBM-1 cells.

[0105] (Arbovirus proliferation test) Place iGBM-7 cells or iOBM-2 cells in a 24-well cell culture plate (Sumitomo Bakelite Co., Ltd.) at a rate of 1 x 10⁶ ５ Seeds were seeded in / well. The following day, the cells were washed three times with Earl's solution, and then each arbovirus was inoculated at a multiple of infection (MOI) of 0.1. The arboviruses used were Akabane virus (AKAV) strain OBE-1, Aino virus (AINOV) strain JaNAr28, Chuzan virus (CHUV) strain 31, Diagula virus (DAGV) strain KSB-29 / E / 01, Bluetongue virus (BTV) serotype 1 ON-24 / E / 17, and Epidemic hemorrhagic disease virus (EHDV) serotype 2 (Ibaraki virus) strain No. 2. After culturing at 37°C for 1 hour, the inoculated viruses were removed by washing three times with Earl's solution, and growth medium with 2% fetal bovine serum was added. The culture supernatant was collected at 24, 48, 72, 96, and 120 hours (hpi) after inoculation, and the viral titer (TCID) was determined by CPE activity against hamster lung cell line (HmLu-1 cells). ５０ The amount ( / ml) was measured.

[0106] As a result, an increase in viral titers in the culture supernatant was observed in iGBM-7 cells and iOBM-2 cells 24 or 48 hours after inoculation with Akabane virus, Aino virus, Chuzan virus, Diagula virus, Bluetongue virus, and epidemic hemorrhagic disease virus (Figures 9A and 9B).

[0107] (Goat Arthritis / Encephalitis Virus Infection Test) iGBM-7 cells or iOBM-2 cells are placed in 6-well cell culture plates (Sumitomo Bakelite Co., Ltd.) in 4 x 10⁶ wells. ５ Seeds were seeded using / well. The following day, the cells were washed three times with Earl's solution, and then inoculated with 1 ml each of canine arthritis / encephalitis virus (CAEV). Two days after infection, the cells were washed twice with PBS, fixed with methanol for 5 minutes, and stained with Giemsa stain (Sigma-Aldrich).

[0108] (Detection of CAEV provirus insertion) Genomic DNA was extracted from the CAEV-infected cells using the DNeasy Blood & Tiss kit (QIAGEN). Provirus insertion into the genome was detected by nested PCR using known primers specific to the gag region of CAEV. The length of the PCR product was 184 bp after second PCR. PCR amplification was performed using KOD FX DNA polymerase (Toyobo Co., Ltd.), and the obtained PCR products were separated by agarose gel electrophoresis and detected by GelGreen™ staining (Biotium, Inc.).

[0109] As a result, both iGBM-7 and iOBM-2 cells showed the formation of multinucleated giant cells (synthiam), characteristic of retroviruses, upon CAEV infection (Figure 10). Cell detachment was also observed (Figure 11), indicating the induction of cytopathic effects (CPE) by CAEV infection. Furthermore, insertion of the CAEV provirus into the genomic DNA of both cells was confirmed (Figure 12).

[0110] As described above, the present invention makes it possible to provide immortalized macrophages from goat subfamily animals. Furthermore, since these immortalized macrophages are susceptible to goat subfamily animal pathogens such as BVDV, arbovirus, and CAEV, it becomes possible to proliferate such pathogens, enabling the manufacture and development of vaccines against these pathogens. In addition, because they are susceptible to infection, it becomes possible to detect (test, diagnose) the aforementioned pathogens.

Claims

1. Immortalized cells of macrophages from goat subfamily animals.

2. The cell according to claim 1, comprising a macrophage of a goat subfamily expressing at least one protein selected from the group consisting of SV40 large T antigen and telomerase reverse transcriptase.

3. The cell according to claim 2, wherein the expression is from a lentivirus encoding the protein.

4. The cell according to claim 1 or 2, wherein the telomerase reverse transcriptase is porcine-derived telomerase reverse transcriptase.

5. A method for producing immortalized macrophages, comprising the step of expressing at least one protein selected from the group consisting of SV40 large T antigen and telomerase reverse transcriptase in macrophages of goat subfamily animals.

6. The method according to claim 5, wherein the step is to introduce a lentivirus encoding the protein into a macrophage of a goat subfamily animal and express the protein.

7. The method according to claim 5 or 6, wherein the telomerase reverse transcriptase is porcine-derived telomerase reverse transcriptase.

8. A method for producing a pathogen of a goat subfamily, comprising the step of bringing the cells described in claim 1 into contact with the pathogen of a goat subfamily, and growing the pathogen in the cells.

9. A method for producing a vaccine containing a goat subfamily pathogen, comprising the steps of: contacting cells described in claim 1 with a goat subfamily pathogen and growing the pathogen in the cells; isolating the grown pathogen; and mixing the isolated pathogen with a pharmacologically acceptable carrier or medium.

10. The cell according to claim 1, having DNA in which a reporter gene is functionally bound downstream of the promoter region of a gene derived from a pathogen of a goat subfamily animal.

11. A method for detecting pathogens of goat subfamily animals, comprising the steps of: culturing the cells described in claim 10 in the presence of a test sample; detecting the expression of the reporter gene in the cells; and determining that the test sample contains pathogens of goat subfamily animals if the expression of the reporter gene is detected.

12. A method for detecting neutralizing antibodies against viruses infecting goat subfamily animals, comprising the steps of: contacting cells described in claim 1 with a virus infecting goat subfamily animals in the presence of a biological sample isolated from a goat subfamily animal under test, and allowing the virus to grow in the cells; detecting the number of viruses that have grown; and determining that the biological sample contains neutralizing antibodies against the virus if the number of viruses detected in the first step is less than the number of viruses that grew in the cells described in claim 1 in the absence of the biological sample.

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