WO2022107793A1

WO2022107793A1 - Immortalized pig-fetus small-intestinal macrophages

Info

Publication number: WO2022107793A1
Application number: PCT/JP2021/042171
Authority: WO
Inventors: 敬人竹之内; 俊一鈴木; 博英上西; 綾子宮▲崎▼; 賢太郎舛甚; 健浩國保; 道浩 ▲高▼木
Original assignee: 国立研究開発法人農業・食品産業技術総合研究機構
Priority date: 2020-11-20
Filing date: 2021-11-17
Publication date: 2022-05-27
Also published as: JP7477914B2; JPWO2022107793A1

Abstract

Attempts have been made to isolate macrophages from the small intestine of approximately one-month-old pigs and immortalize the macrophages, but microbial contamination was severe and isolation of macrophages from the porcine small intestine was not successful even when medium containing antibiotics and antifungals was used. However, it was discovered that the contamination is suppressed and primary culture of macrophages is possible if the small intestine of a pig fetus is used. Furthermore, it was clarified that the small-intestinal macrophages of the primary culture can be immortalized by causing SV40 large T antigen and telomerase reverse transcriptase to be expressed, and it became possible to provide a cell line of subculturable porcine small-intestinal macrophages.

Description

Immortalized pig fetal small intestine macrophages

The present invention relates to an immortalized pig small intestine macrophage and a method for producing the macrophage. The present invention also relates to a method for producing a pig infectious virus or a vaccine using the macrophages. Furthermore, the present invention relates to a method for detecting a pig infectious virus or a method for detecting a neutralizing antibody against a pig infectious virus using the macrophages.

In pig farming, diarrhea is one of the causes of livestock mortality and growth retardation, and is a serious problem in stable and efficient pork production. Diarrhea occurs alone or in association with various factors such as pathogen infection, nutrition, and host stress. Small intestinal macrophages, which are cells responsible for innate immunity, play a major role in the activation and termination of host immunity, and their response greatly affects the severity of diarrhea and the excretion of pathogens. It is also becoming clear that enteritis accompanied by diarrhea occurs due to changes in nutrition and the accompanying changes in the intestinal flora and its metabolites, and that the response of small intestinal macrophages plays a part in the pathological condition.

From these facts, it is considered that an in vitro immunological response analysis system for small intestinal macrophages is necessary as a basic tool for elucidating the pathophysiology of diarrhea in pigs and developing preventive methods. On the other hand, no cell line of porcine small intestine macrophages that can be passaged has been established so far, and its production has been required for constructing the analysis system.

The present invention has been made in view of the above-mentioned problems of the prior art, and an object of the present invention is to provide a cell line of porcine small intestine macrophages that can be passaged.

The present inventors have previously succeeded in isolating macrophages from the kidneys of pigs (6 days old) and immortalizing them (Non-Patent Document 1). Therefore, we also tried to isolate macrophages and immortalize them in the small intestine of pigs (about 1 month old). However, even when a medium containing an antibiotic and an antifungal drug was used, the contamination of microorganisms was severe, and it was not possible to isolate macrophages from the small intestine of pigs.

Therefore, first of all, in order to enable the primary culture of macrophages from the small intestine of pigs, we repeated diligent studies and research. As a result, it was found that the small intestine of the fetus does not cause the contamination as described above, and the small intestine macrophages can be isolated and cultured.

Then, when the SV40 large T antigen gene and the telomerase reverse transcriptase gene derived from pigs were introduced into the pig small intestine macrophages thus obtained and transformed, the obtained cells were divided at least 60 times or more. It became clear that it was possible. In addition, the immortalized cells may maintain the expression of marker molecules and the function as macrophages (activation of intracellular signal transduction molecules and induction of inflammatory cytokine production in response to bacterial-derived components). It was revealed.

Furthermore, the immortalized swine fever small intestine macrophages thus obtained were subjected to a susceptibility test against African swine fever virus (ASFV). As a result, it was revealed that the cells can detect ASFV in both the cytopathic effect (CPE) test and the blood cell adsorption (HAD) test. Furthermore, it was revealed that they are sensitive to all the ASFV strains tested and that these virus strains can also be propagated. The susceptibility was also surprisingly orders of magnitude higher than that of primary cultured pig alveolar macrophages (PAM) (100 to 10,000 times higher than PAM in the CPE test and 10 to 100 times higher than PAM in the HAD test). It was twice as expensive). Furthermore, susceptibility tests were also conducted for pig breeding / respiratory disorder syndrome virus and porcine circovirus type 2. As a result, it was confirmed that the immortalized pig fetal small intestinal macrophages show high susceptibility to these viruses as well as ASFV.

The present invention has thus found that the production of immortalized pig small intestine macrophages was successful for the first time, and that the produced immortalized pig fetal small intestine macrophages have high infection susceptibility to virus. It is based on the above, and specifically, it is as follows.
<1> Immortalized cells of small intestinal macrophages of porcine fetus.
<2> The cell according to <1>, wherein the small intestinal macrophage of a pig embryo expresses at least one protein selected from the group consisting of SV40 large T antigen and telomerase reverse transcriptase.
<3> The cell according to <2>, wherein the expression is an expression from a lentivirus encoding the protein.
<4> The cell according to <2> or <3>, wherein the telomerase reverse transcriptase is a pig-derived telomerase reverse transcriptase.
<5> The cell according to any one of <1> to <4>, wherein the pig fetus is a pig fetus having a fetal age of 30 to 114 days.
<6> A method for producing immortalized pig small intestine macrophages, which comprises a step of expressing at least one protein selected from the group consisting of SV40 large T antigen and telomerase reverse transcriptase in small intestine macrophages of pig embryo.
<7> The method according to <6>, wherein the step is a step of introducing a lentivirus encoding the protein into the small intestinal macrophages of the pig fetal body to express the protein.
<8> The method according to <6> or <7>, wherein the telomerase reverse transcriptase is a pig-derived telomerase reverse transcriptase.
<9> The method according to any one of <6> to <8>, wherein the pig fetus is a pig fetus with a fetal age of 30 to 114 days.
<10> A method for producing a pig-infecting virus, which comprises a step of contacting the cell according to any one of <1> to <5> with a pig-infecting virus and propagating the virus in the cell.
<11> A step of bringing the cell according to any one of <1> to <5> into contact with a pig-infecting virus and propagating the virus in the cell.
A method for producing a vaccine containing a pig infectious virus, which comprises a step of isolating the propagated pig infectious virus and a step of mixing the isolated pig infectious virus with a pharmacologically acceptable carrier or medium.
<12> The pig infectious virus is at least one virus selected from the group consisting of African pig fever virus, pig epidemic diarrhea virus, pig rotavirus, pig circovirus, and pig breeding / respiratory disorder syndrome virus. The manufacturing method according to <10> or <11>.
<13> The cell according to any one of <1> to <5>, which has a DNA to which a reporter gene is functionally bound downstream of the promoter region of a gene derived from a pig infectious virus.
<14> The pig infectious virus is at least one virus selected from the group consisting of African pig fever virus, pig epidemic diarrhea virus, pig rotavirus, pig circovirus, and pig breeding / respiratory disorder syndrome virus. The cell according to <13>.
<15> A method for detecting a pig infectious virus, which comprises the following steps, a step of culturing the cells according to <13> or <14> in the presence of a test sample.
A step of detecting the expression of the reporter gene in the cell, and a step of determining that the test sample contains a pig infectious virus when the expression of the reporter gene is detected.
<16> A method for detecting a neutralizing antibody against a pig infectious virus, which comprises the following steps. In the presence of a biological sample isolated from a test pig, any one of <1> to <5>. A step of contacting the cells described in the section with a pig infecting virus and propagating the virus in the cells.
The step of detecting the number of the propagated virus and the number of the virus detected in the step proliferate in the cell according to any one of <1> to <5> in the absence of the biological sample. A step of determining that the biological sample contains a neutralizing antibody against the virus when the number is small compared to the number of viruses.
<17> The pig infectious virus is at least one virus selected from the group consisting of African pig fever virus, pig epidemic diarrhea virus, pig rotavirus, pig circovirus, and pig breeding / respiratory disorder syndrome virus. The method according to <15> or <16>.

According to the present invention, it is possible to provide a cell line of a passageable pig fetal small intestine macrophage. In addition, since the immortalized pig fetal small intestine macrophages have high infectivity to viruses, various viruses can be propagated, and vaccines against the viruses can be produced and developed. In addition, it is possible to detect (test, diagnose) the virus because of its high infection susceptibility.

A photomicrograph showing the results of observing the adhesive primary cultured cells (porcine fetal small intestine primary cultured cells) that spread on the 22nd day after the small intestinal tissue pieces of the pig fetal were seeded by enzyme treatment and seeded. be. The scale bar in the figure represents 200 μm. 6 is a photomicrograph showing the results of observing spherical cells (macrophages, PIM) that are weakly adhered and proliferate on a cell sheet formed by peeling and reseeding primary cultured pig fetal small intestine cells. The scale bar in the figure represents 100 μm. It is a photograph showing the result of analysis of the primary cultured pig fetal small intestine cells after re-seeding and culturing by immunostaining. In the figure, the upper left side shows the result of staining the myofibroblast marker α-smooth muscle actin (αSMA), and the uppermost right side shows the result of staining the mesenchymal cell marker Vimentin. Is shown. The center shows the results of staining the epithelial marker cytokeratin (the left side shows the results of staining cytokeratin 18 (CK18), and the right side shows the results of staining cytokeratin 19 (CK19)). The lower left side shows the result of staining the macrophage marker CD172a, and the lowermost right side shows the result of staining the macrophage marker CD204. The scale bar represents 400 μm. In addition, under color display, brown represents antibody staining and bluish purple represents nuclear staining. It is a photograph which shows the result of the analysis of PIM by immunostaining. The upper left side shows the result of staining the macrophage marker Iba1, the upper right side shows the result of staining the macrophage marker CD172a, and the middle left side shows the result of staining the macrophage marker CD204. The result is shown. The right side in the middle shows the results of staining the mesenchymal cell marker Vimentin. The bottom shows the results of staining the epithelial marker cytokeratin (the left side shows the results of staining CK18, and the right side shows the results of staining CK19). The scale bar represents 400 μm. In addition, under color display, brown represents antibody staining and bluish purple represents nuclear staining. 6 is a photomicrograph showing the results of observing macrophages (IPIM) isolated from the small intestine of a pig fetal and immortalized. The scale bar in the figure represents 100 μm. It is a graph which shows the result of having tested the cell proliferation of IPIM. It is a photograph showing the result of analysis of IPIM by immunostaining. The upper part shows the results of staining the macrophage marker Iba1, the macrophage marker CD172a, the macrophage marker CD204, and the macrophage-containing antigen-presenting cell marker MHC-II, respectively, in order from the left side. The lower part shows the results of staining CD16, which is a macrophage marker, CD163, which is an M2 type macrophage marker, CD169, which is an M1 type macrophage marker, and CD203a, which is a pig macrophage marker, in order from the left side. The scale bar in the figure represents 200 μm. In addition, under color display, brown represents antibody staining and bluish purple represents nuclear staining. IPIM is a photograph showing activation of intracellular signaling molecules (phosphorylation of p38MAP kinase) in response to bacterial-derived components (muramyl dipeptide: MDP, lipopolysaccharide: LPS). IPIM is a photograph showing that it induces the production of inflammatory cytokines (IL-1β) in response to bacterially derived components (MDP, LPS). It is a micrograph showing the result of detecting the cytopathic effect (CPE) by inoculating IPIM with African swine fever virus (Armenia 07 strain) and observing it 2 days later. In the figure, the left panel "non-infected" shows the results of the negative control of the non-inoculated ASFV strain. In the presence of erythrocytes, IPIM was inoculated with the Armenia 07 strain and observed 2 days later, and it is a micrograph showing the result of detecting blood cell adsorption (HAD). In the figure, the left panel "non-infected" shows the results of the negative control of the non-inoculated ASFV strain. It is a micrograph showing the result of inoculating IPIM with the pig breeding / respiratory disorder syndrome virus (PRRSV), detecting the virus antigen by the indirect immunofluorescence method on the 3rd day, and detecting CPE on the 7th day. In the figure, the left panel "Non-inoculation" shows the results of the negative control of PRRSV strain non-inoculation. It is a graph which shows the result of having analyzed the time-dependent change of PRRSV 50% tissue culture infection amount (TCID ₅₀ ) in IPIM. It is a graph which shows the result of having analyzed the time course of the pig circovirus type 2 (PCV2) gene amount in IPIM.

The present invention relates to cells in which porcine fetal small intestine macrophages are immortalized (immortalized porcine fetal small intestine macrophages).

In the present invention, "immortalized pig fetal small intestine macrophages" means cells in which macrophages present in the small intestine of a pig (an animal of the family Cetartiodactyla, Mammalia) are immortalized. "Pig fetal" means a prenatal individual in a pig mother, preferably a pig with a fetal age of 30 to 114 days, and more preferably a pig with a fetal age of 90 to 114 days from the viewpoint of maturation in small intestinal tissue formation. be.

The "small intestine" from which macrophages are collected is not particularly limited, and examples thereof include the ileum, jejunum, and duodenum, but the ileum is preferable from the viewpoint of the development of lymphatic tissue in which immune cells are concentrated.

Immortalized "porcine small intestinal macrophages" are macrophages that are present in the pig small intestine and are differentiated from oval sac, fetal liver-derived macrophages or monospheres, and are active macrophages (inflammatory M1 type macrophages, anti-inflammatory). It may be a sex M2 type macrophage) or a resting type macrophage. Such macrophages remove the capsule from the small intestine collected from pig embryos, for example, as shown in Examples below. The isolated small intestinal tissue is then chopped, washed with buffer, subjected to enzymatic treatment and then cultured. Macrophage-like cells that are loosely adhered to a monolayer of cell sheets that occur during the culture process can be prepared by isolation.

The buffer solution used for washing the shredded small intestinal tissue is not particularly limited, and for example, dalbecco phosphate buffered saline (DPBS), phosphate buffered saline (PBS), and Tris hydrochloric acid buffer (TBS). , HEPES buffer.

The enzyme treatment is not particularly limited as long as cells can be separated and dispersed from the tissue, and examples thereof include collagenase, dispase, DNase (DNase I, etc.), trypsin, hyaluronidase, elastase, and pronase. , Preferably a combination of collagenase, dispase and DNase I. Further, the treatment temperature and time are not limited, and can be appropriately adjusted according to the type of enzyme used and the degree of cell separation / dispersion.

The medium used for culturing the cells dispersed by the enzyme treatment is not particularly limited as long as the small intestinal macrophages can be maintained, but it can be prepared by appropriately adding a well-known and conventional medium additive based on a known basal medium. can. Examples of the "basal medium" 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, and the like. Examples include ham medium, Fishers medium, BME medium, BGJb medium, CMRL 1066 medium, MEM Zinc option improvement medium, IMDM medium, medium 199 medium, and any mixed medium thereof. Examples of the "medium additive" include antibiotics (penicillin, streptomycin, gentamycin, bancomycin, etc.), antifungal agents (pimaricin, amphotericin B, etc.), functional proteins (insulin, transferase, lactoferrin, etc.), reducing agents (mono). Thioglycerol, 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 form) Glutathion, etc.), nucleotides, etc. (nucleoside, citidine, adenosine 5'-monophosphate, hypoxanthin, thymidin, etc.), metal salts (iron (III) nitrate, iron (II) 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 (paraaminobenzoic acid, ethanolamine, corti) Examples include, but are limited to, costerone, progesterone, lipoic acid, putresin, pyruvate, lactic acid, triiodotyronin, etc.), buffer compounds (HEPES, sodium bicarbonate, etc.), pH indicators (phenol red, etc.). It's not a thing.

The culture conditions using such a medium are not particularly limited, 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.

Then, by culturing the cells derived from the small intestine tissue dispersed by the enzyme treatment under such culture conditions, first, the primary cultured cells of the small intestine can be obtained. The culture time for obtaining the cells is not particularly limited, but is usually 1 week to 1 month, preferably 2 to 3 weeks.

Next, the obtained primary cultured cells of the small intestine are subjected to enzyme treatment, peeled from the incubator, dispersed, and re-sown. The enzyme treatment here can also be carried out using the above-mentioned enzyme, but preferably a combination of protease (enzyme having at least degrading activity for non-collagen protein), collagenase and DNase (for example, product name: Accumax (registered)). Trademark), manufactured by Sigma-Aldrich).

Then, in the culture after re-seed, a mixed culture system consisting of a cell sheet composed of myofibroblast-like cells and spherical cells (pork small intestine macrophages) that weakly adhere and proliferate on the cell sheet is constructed. To. The culture time for constructing the culture system is not particularly limited, but is usually between several days (2 to 4 days) and several weeks (2 to 3 weeks), preferably 1 to 2 weeks.

The method for immortalizing the small intestinal macrophages of the pig fetus thus obtained is not particularly limited, but it can be carried out by introducing at least one immortalizing gene. Examples of the immortalizing gene include SV40 large T antigen (SV40T antigen), telomerase reverse transcriptase (TERT), Myc, and Ras, and it is preferable to introduce SV40T antigen and TERT, and the immortalization efficiency of pig macrophages is preferable. From the viewpoint of enhancing the above, it is more preferable to introduce SV40T antigen and TERT derived from pig.

The immortalization gene can be introduced by using a vector encoding the gene. The vector may be linear or circular, and examples thereof include a viral vector, a plasmid vector, an episomal vector, an artificial chromosomal vector, and a transposon vector.

Examples of the virus vector include retrovirus vectors such as lentivirus, Sendai virus vector, adenovirus vector, adeno-associated virus vector, herpesvirus vector, vaccinia virus vector, poxvirus vector, poliovirus vector, sylvis virus vector, and labd. Examples include virus vectors, paramixovirus vectors, and orthomixovirus vectors. Examples of the plasmid vector include plasmid vectors for animal cell expression such as pcDNA3.1, pA1-11, pXT1, pRc / CMV, pRc / RSV, and pcDNAI / Neo. Among these vectors, retroviral vectors are preferable, and lentiviruses are more preferable, from the viewpoint of increasing the efficiency of gene transfer into porcine macrophages.

In addition to the immortalizing gene, the vector according to the present invention encodes an expression control sequence such as a promoter, enhancer, poly-A addition signal, terminator, etc., and a protein that binds to a replication initiation site or a replication initiation site to control replication. Nucleotide sequence, 5'cap structure, Shine-Dalgarno sequence, 5'untranslated region including Kozak sequence, 3'untranslated region including polyadenylation signal, AU rich element, GU rich element, etc., encoding other proteins It may contain nucleotides and the like.

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

Examples of the "nucleotide encoding other proteins" include marker genes such as reporter genes and drug resistance genes.

In addition, when introducing a plurality of immortalizing genes, these genes may be integrated into a single vector or may be integrated into separate vectors, but from the viewpoint of increasing expression efficiency, they may be integrated into separate vectors. It is desirable to incorporate it. Further, when incorporating into a single vector, for example, by inserting an IRES, 2A peptide sequence or the like into the vector, it becomes possible to express a plurality of immortalizing genes polycistronically.

Examples of the method for introducing the vector into cells include a lipofection method, a microinjection method, a calcium phosphate method, a DEAE-dextran method, an electroporation method, a particle gun method and the like. When the vector of the present invention is a retrovirus vector, appropriate packaging cells are selected based on the LTR sequence and packaging signal sequence possessed by the vector, and retrovirus particles are prepared using this. You may. Examples of the packaging cells include PG13, PA317, GP + E-86, GP + envelopeAm-12, and Psi-Crip. Furthermore, 293 cells and 293T cells having high transfection efficiency can also be used as packaging cells. Further, the virus particles thus prepared can be introduced into cells by the Polybrene method, Protamine method, RetroNectin method and the like.

The gene transfer and subsequent maintenance culture can be performed by the above-mentioned medium for primary culture of the fetal small intestine of pigs and the culture conditions using the same. In addition, the immortalized pig fetal small intestinal macrophages established by introducing the immortalizing gene in this way show proliferative properties of at least 1 month or longer, preferably 2 months or longer, and more preferably 3 months or longer. Shows the proliferative nature of. The doubling time for immortalized pig kidney macrophages is at least 4 days, preferably 2 days, more preferably 1 day. In addition, the immortalized pig fetal small intestine macrophages preferably maintain the characteristics of the macrophages, for example, at least among the macrophage-specific genes CD172a, CD16, Iba-1, CD204 (MSR-A) and CD203a. 1 gene is expressed, preferably 2 or more genes are expressed, more preferably 3 or more genes are expressed, still more preferably 4 or more genes are expressed, and particularly preferably. All these genes are expressed. Further, the immortalized pig fetal small intestinal macrophages may express at least one gene of CD163 and CD169, which are marker genes of a specific macrophage subpopulation, and MHC-II, which is an antigen-presenting cell marker gene. In addition, the immortalized pig fetal small intestinal macrophages according to the present invention have at least one of the functions of phagocytosis, LPS-stimulated inflammatory cytokine production, and IL-1β maturation associated with inflammasome activity. It retains, preferably two or more functions.

Unlike primary cultured pig macrophages, they develop densely (in sheet form) and proliferate. Therefore, since the macrophage can easily detect a change in morphology, it is also useful in an infection test (CPE test, HAD test, etc.) of African swine fever virus using degeneration of infected cells as an index, which will be described later.

(Pig infectious virus)
In the present invention, the "pig infectious virus (virus that infects pigs)" may be at least a virus that can infect pigs, and is a DNA virus (double-stranded (ds) DNA virus, single-stranded (ss) DNA virus. , A DNA virus containing both ss and dsDNA regions), RNA virus (single-stranded (ss) RNA virus (plus-stranded RNA virus or minus-stranded RNA virus), double-stranded (ds) RNA virus. ) May be.

Examples of the pig infectious virus include African pig fever virus (ASFV), pig epidemic diarrhea (PED) virus, pig rotavirus, pig circovirus (PCV2), and pig breeding / respiratory disorder syndrome (PRRS) virus. In Japan, on February 5, 2020, the name of "African swine fever" stipulated in the Domestic Animal Infectious Diseases Control Law was revised to "African swine fever".

(Manufacturing method of pig infectious virus)
The method for producing a pig infectious virus (proliferation method, amplification method) of the present invention comprises a step of contacting an immortalized pig fetal small intestine macrophage with a pig infectious virus and propagating the virus in the immortalized pig fetal small intestine macrophage. , The method.

The pig infectious virus to be brought into contact with immortalized pig fetal small intestine macrophages is as described above, but it may be a sample containing not only the isolated virus itself but also the virus. Examples of such "samples" include tissues and cells derived from pigs, their cultures, lavage fluids or extracts, or samples from pig breeding environments (breeding facilities, etc.), lavage fluids or their cultures.

"Contact" is usually performed by adding a pig-infecting virus to a medium for culturing immortalized pig fetal small intestinal macrophages. The "medium" is not particularly limited, and examples thereof include the above-mentioned medium for primary culture of the fetal small intestine of pigs.

"Proliferation" of a pig-infecting virus can be performed by culturing immortalized pig fetal small intestinal macrophages that the virus has come into contact with and infected. 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 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 pig-infecting virus is not particularly limited, but is usually 1 to 10 days, preferably 2 to 7 days, and more preferably 3 to 5 days.

Whether or not the pig infectious virus has propagated can be determined by a person skilled in the art by a known method. Examples of such a method include a CPE test using the cytopathic effect (CPE) as an index, as shown in Examples described later, and a method for detecting the degree of intracellular ATP depletion associated with CPE (for example, Promega). (Viral ToxGlo assay) provided by. In addition, a method for detecting a gene derived from a pig infectious virus or its expression can also be used. Here, the expression of the gene may be at the transcription level (RNA level) or the translation level (protein level). As a method for detecting a gene (genomic DNA, genomic RNA) or RNA, for example, PCR (RT-PCR, real-time PCR, quantitative PCR), sequencing, DNA microarray analysis method, Northern blotting or Southern blotting, in situ hybridization. , Dot blot, RNA protection assay, mass analysis. In addition, the gene or RNA level can be quantitatively detected by counting the number of reads in the so-called next-generation sequencing method. As a method for detecting a protein, for example, an antibody such as an ELISA method, an antibody array, immunoblotting, imaging cytometry, flow cytometry, radioimmunoassay, immunoprecipitation method, or immunohistochemical staining method is used for detection. Methods (immunological methods) and mass analysis methods can be mentioned.

(Vaccine manufacturing method)
The method for producing a vaccine containing a pig infectious virus of the present invention is a step of contacting an immortalized pig fetal small intestine macrophage with a pig infectious virus and propagating the virus in the immortalized pig fetal small intestine macrophage.
A method comprising the step of isolating the propagated porcine infectious virus and the step of mixing the isolated porcine infectious virus with a pharmacologically acceptable carrier or vehicle.

The step of multiplying the virus in the immortalized pig fetal small intestine macrophages is as described above. "Isolation" of the propagated porcine infectious virus means separation, purification and / or concentration of the immortalized porcine fetal small intestinal macrophages and / or the cells from the culture medium. Examples of the virus isolation method include filtration of culture medium, cell disruption (ultrasonic treatment, hypotonic solution treatment, freeze-thaw, etc.), centrifugation (ultracentrifugation method, density gradient centrifugation, etc.), and concentration (concentration (ultracentrifugation method, density gradient centrifugation, etc.)). Ammonium sulfate, resin column, polyethylene glycol salting out, etc.).

The pig infectious virus thus isolated may be used as it is as a vaccine (so-called live vaccine), may be used in an attenuated live form (so-called live attenuated virus), or may be used as a vaccine in an inactivated form. You may use it. Furthermore, as long as it has immunogenicity, some of these isolated pig infectious viruses (proteins, polypeptides, sugars, glycoproteins, lipids, nucleic acids, etc.) may be used as vaccines.

A live attenuated virus is a virus with a reduced virulence level compared to a virus isolated from the field. Attenuated virus can be propagated in known methods, such as in the presence of mutagens, acclimatization to cultured cells by continuous (long-term) passage in vitro, and conditions deviating from the natural growth environment (eg,). It can be obtained by feeding the pig infectious virus to the growth under high temperature conditions). In addition, a live attenuated virus can also be obtained by deleting or recombining a specific gene of a virus by using genome editing, gene modification technology, or the like.

A person skilled in the art can also inactivate the virus by using a known method. Examples of 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. ..

Examples of the "pharmacologically acceptable carrier" to be mixed with the isolated pig infectious virus include stabilizers, excipients, preservatives, surfactants, chelating agents, and binders. Examples of the "pharmacologically acceptable medium" include water, physiological saline, phosphate buffer, and Tris-HCl buffer. Those skilled in the art can select and use these carriers and vehicles as appropriate or in combination with known substances used in the art, depending on the dosage form and method of use of the vaccine. The form of the vaccine is not particularly limited, and may be, for example, a suspension form or a freeze-dried form.

From the viewpoint of enhancing the vaccine effect, an adjuvant may be further mixed. Examples of the adjuvant include inorganic substances such as aluminum gel adjuvants, microorganisms or substances derived from microorganisms (BCG, alum dipeptide, alum, pertussis toxin, cholera toxin, etc.), and surfactant substances (saponin, deoxycol). Acids, etc.), emulsions of oily substances (mineral oil, vegetable oil, animal oil, etc.), alum, etc. may be mentioned.

(How to detect pig infectious virus)
The present invention provides immortalized pig fetal small intestinal macrophages having DNA functionally bound to a reporter gene downstream of the promoter region of a pig infectious virus-derived gene.

Further, in the presence of the test sample, the step of culturing the immortalized pig fetal small intestine macrophages having the DNA, the step of detecting the expression of the reporter gene in the immortalized pig fetal small intestine macrophages, and the expression of the reporter gene Also provided is a method for detecting a pig infectious virus, which comprises a step of determining that the test sample contains a pig infectious virus when detected.

The "immortalized pig fetal small intestine macrophages" into which the DNA is introduced are as described above. Further, the DNA may be in the form of the vector described in the above-mentioned "immortalization gene". Further, the introduction of the DNA into the immortalized pig fetal small intestine macrophage can also be carried out by those skilled in the art using the methods listed in the above description regarding the "immortalization gene".

The "promoter region of a gene derived from a pig infectious virus" in the DNA is particularly limited as long as it is a region derived from a pig infectious virus and capable of activating the expression of a gene downstream thereof according to the infection / proliferation of the virus. It may be any of the earliest gene (immediate-early), the early stage gene (early), the late early stage gene (late), and the late stage gene (very-late).

The gene derived from the pig infectious virus to be used can be selected by a person skilled in the art with reference to known information as appropriate. For example, genes derived from ASFV are selected by referring to the list of "functions of proteins encoded by ASFV" in Table 1 of Virus Taxonomy International Committee on Taxonomy (ICTV) 9th Edition, 2012, pp. 155-157. However, the promoter region of p72, U104L, CD2v, DNA polymerase or p30 gene is preferably used (see Portal RS. et al., Virus, August 2017, Vol. 508, pp. 70-80).

The "reporter gene" that is functionally (operably) bound downstream of the promoter region is not particularly limited, and known ones are appropriately used. For example, a fluorescent protein gene, a luciferase gene, and a color-developing enzyme gene can be mentioned. Specific examples of the fluorescent protein gene include GFP (green fluorescent protein) gene, YFP (yellow fluorescent protein) gene, RFP (red fluorescent protein) gene and the like. Specific examples of the photoprotein / enzyme gene include an aequorin gene and a luciferase gene. Specific examples of the color-developing enzyme gene include a chloramphenicole acetyltransferase (CAT) gene, a β-glucuronidase (GUS) gene, a β-galactosidase gene, an alkaline phosphatase gene, and a SEAP gene.

Then, in the detection method of the present invention, the presence or absence of infection with the pig-infecting virus of the immortalized pig fetal small intestine macrophages, and eventually in the test sample, using the fluorescence, luminescence, color development, etc. generated in response to the expression of these reporter genes as indicators. The presence of swine infectious virus can be detected.

The test sample is not particularly limited as long as it is a sample in which a pig infectious virus can be present. For example, a pig-derived tissue, cells, their culture, a washing solution, or an extract, or a pig breeding environment ( A cleaning solution of a breeding facility, etc.) or a culture thereof can be mentioned.

The "medium" used for culturing in the detection method of the present invention is not particularly limited, and examples thereof include the above-mentioned medium for primary culturing of pig fetal small intestine. The temperature of the culture 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 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, and more preferably 2 to 5 days. be.

(Detection method of neutralizing antibody)
The method for detecting a neutralizing antibody against a virus that infects pigs of the present invention is
A step of contacting an immortalized pig fetal small intestine macrophage with a virus that infects a pig and propagating the virus on the immortalized pig fetal small intestine macrophage in the presence of a biological sample isolated from a test pig.
When the step of detecting the number of propagated viruses and the number of viruses detected in the steps are smaller than the number of viruses propagated in the immortalized pig fetal small intestine macrophages in the absence of the biological sample, the above-mentioned It is a method including a step of determining that the biological sample contains a neutralizing antibody against the virus.

The "neutralizing antibody" according to the present invention means an antibody that suppresses infection or proliferation of a pig infectious virus. Such antibodies also include all classes and subclasses of immunoglobulins. In addition, there are no particular restrictions on the "tested pig" as long as it is a pig, regardless of the experience of infection with the pig-infecting virus. The "biological sample" isolated from the test pig includes a sample derived from pig (for example, blood (serum, plasma, etc.), mucus (saliva, nasal juice, milk, gastrointestinal secretion, etc.), and purified from them. Antibodies).

"Contact" is as described above. Regarding the growth conditions, 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 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 the biological sample is not particularly limited, but is usually 1 to 10 days, preferably 2 to 7 days, and more preferably 3 to 5 days. In addition, the propagated virus can be detected by CPE test or the like, or by detecting a gene derived from a pig-infecting virus or its expression, as described above. As described above, in the detection method of the present invention, the presence or absence of a neutralizing antibody is determined using not only the number of viruses themselves but also the gene (genome DNA amount, etc.) reflecting the number of viruses or the expression level thereof as an index. May be.

Hereinafter, the present invention will be described in more detail based on Examples, but the present invention is not limited to the following Examples.

<Isolation of pig small intestine macrophages from pig fetus>
About 5 cm of the small intestine (ileum site) was collected from a pig fetus (about 108 days old), and after excision of the adhered film, it was cut in the vertical direction and opened in half. The small intestinal tissue was sufficiently shredded with scissors, transferred to a conical tube, washed with Dulbecco phosphate buffered saline (DPBS), and then immersed in DPBS containing collagenase / dispase / DNase I and enzymatically treated at 37 ° C. for 1 hour. .. Tissues are dispersed by pipetting, washed with DPBS, and then grown medium (DMEM High glucose: 10% bovine fetal serum, 10 μg / mL insulin, 25 μM monothioglycerol, 100 U / ml penicillin, 100 μg / mL streptomycin, 5 μg / mL. It was suspended in penicillin (product name: Fungin, manufactured by InvivoGen) and seeded in a T75 flask.

Initially, the small intestine collected from a pig (about 1 month old) was treated in the same manner as described above, and a primary culture was attempted. However, even if a medium containing the above-mentioned antibiotics (penicillin, streptomycin) and antifungal drug (pimaricin) is used, severe microbial contamination occurs 2-3 days after the start of culturing, and the first generation. The culture could not be performed.

On the other hand, for the small intestine collected from pig fetuses, such contamination could be suppressed and primary culture could be performed. Then, the primary cultured cells (FIG. 1A) that had adhered and spread after about 3 weeks were peeled off with a cell separation / dispersion solution (product name: Accumax (registered trademark), manufactured by Sigma-Aldrich) and collected, and a 100 mm tissue was collected. It was re-sown in the culture dish.

A cell sheet was formed in about one week, and spherical cells that weakly adhered and proliferated appeared on it (Fig. 1B). As a result of immunostaining, the cell sheet was composed of α-smooth muscle actin (αSMA) -positive myofibroblast-like cells, on which a mixed culture system in which macrophage marker (CD172a, CD204) -positive cells proliferated was constructed. It was confirmed that The epithelial cell markers (cytokeratin 18 and 19 (CK18, CK19)) were negative. (Fig. 2A).

In addition, as a result of collecting cells from the culture supernatant and isolating and immunostaining the cells adhering to the petri dish for suspension of Smilon culture, it was found that macrophage marker (Iba1, CD172a, CD204) -positive cells were isolated. .. The epithelial cell markers (CK18, CK19) were negative (FIG. 2B). Furthermore, in the recovered cells, the formation of multinucleated giant cells, which is a characteristic of primary cultured macrophages, was also confirmed (Fig. 2B). Therefore, pig small intestinal macrophages (Porcine integral macrophages: PIM) could be obtained by this method. The marker was detected by the following immunostaining.

(Immunostaining)
The cells were seeded on an 8-well chamber slide, cultured for a certain period of time, and then immersed in 4% paraformaldehyde phosphate buffer (manufactured by Nakarai) and fixed. After washing with PBST, it is treated with 1% Triton X-100 / PBS solution, blocked with a hydrogen peroxide solution (manufactured by DAKO) and a blocking agent (product name: Blocking Onehist, manufactured by Nacalai), and then the primary antibody [anti]. -ΑSMA (manufactured by Progen), antibody-Vimentin (manufactured by Progen), anti-CK18 (manufactured by Millipore), anti-CK19 (manufactured by Progen), anti-CD172a (manufactured by VMRD), anti-CD204 (TransGenic, Inc.), Iba1 (Wako), anti-MHC-II (Kingfiser Biotech), anti-CD163 (Bio-Rad), anti-CD169 (Bio-Rad), anti-CD203a ( Bio-Rad (manufactured by Bio-Rad)] was reacted for 1 hour. After washing with PBST, it was stained with diaminobenzidine using a detection system for immunohistochemical staining (product name: EnVision® system, manufactured by DAKO). In addition, the nuclei were stained with Meyer's hematoxylin solution (manufactured by Wako).

<Preparation of pig fetal small intestine macrophage immortalized cells>
Isolated PIMs do not grow. Therefore, we tried to immortalize PIM in order to impart proliferative ability. Specifically, first, PIM was inoculated into a petri dish for suspension culture of Smilon, and two types of immortalization genes (SV40 Large T antigen (SV40LT) gene and pig telomerase reverse transcriptase (pTERT) gene) were individually introduced the next day. It was exposed to a solution containing recombinant lentivirus particles for 2 hours. As described in Non-Patent Document 1, the recombinant lentivirus particles include a pLVSIN-EF1α neo vector encoding the SV40LT gene and the pTERT gene, respectively, together with the packaging vector, and a cell line for lentivirus packaging (product name:: It was prepared by introduction into Lenti-X 293T cells, Takara Bio, Inc.). Then, about one week later, PIM was exposed to the same lentivirus particles again, and the culture was continued.

As a result, about one month later, we succeeded in acquiring an immortalized PIM (Immortalized PIM: IPIM) that can be subcultured, and stored it frozen (Fig. 3A). Moreover, as a result of evaluating the proliferative ability of the IPIM cells by the test shown below, it was revealed that they can divide at least 60 times or more as shown in FIG. 3B.

(Cell proliferation test)
IPIM cells (1 × 10 ⁶ cells) were seeded in a 90 mm suspension culture dish (manufactured by SUMILON), peeled off with a TrypLE Express reagent (manufactured by Thermo Fisher) every 3 to 5 days, and subcultured. When the cells were collected, the number of cells was counted using a TC10 fully automatic cell counter (manufactured by Bio-Rad).

Furthermore, as with PIM, as a result of analysis by the above-mentioned immunostaining, various macrophage markers in IPIM cells were positive (Fig. 4). Specifically, Iba1, CD172a, CD204, CD16 and CD203a, which are macrophage markers common to M1 and M2 types, were positive in almost 100% of IPIM cells. In addition, regarding markers of specific macrophage subpopulations (M2-type macrophage marker: CD163, M1-type macrophage marker: CD169) and antigen-presenting cell markers (MHC-II), at a certain ratio depending on the state of individual cells. Positive IPIM cells were found.

In addition, as a result of evaluating the function as a macrophage by the method shown below, IPIM cells activate intracellular signal transduction molecules in response to bacterial-derived components (muramildipeptide: MDP, lipopolysaccharide: LPS) (Muramildipeptide: MDP, lipopolysaccharide: LPS). It was able to phosphorylate p38MAP kinase (FIG. 5A) and further induce the production of inflammatory cytokine (IL-1β) (FIG. 5B).

(Macrophage function evaluation test)
IPIM cells were seeded on a 24-well suspension culture plate (manufactured by SUMILON) at 3 × 10 ⁵ cells per hole. The next day, the medium was replaced with serum-free DMEM (400 μL) containing bacterial-derived components (MDP or LPS) at the respective concentrations shown in FIGS. 5A and 5B, and the cells were stimulated. After an additional 24 hours, the culture supernatant was collected and 50 mM Tris- containing lysis buffer (150 mM sodium chloride, 0.5% Triton X-100, 0.5% sodium deoxycholate, protease inhibitor, phosphatase inhibitor). Cell lysates were prepared using HCl buffer pH 7.5). The culture supernatant (300 μL) was enriched with protein components by the trichloroacetic acid / acetone precipitation method. The proteins contained in the culture supernatant and cell lysate were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrically transferred to a polyvinylidene fluoride (PVDF) membrane (manufactured by Merck). For the transcribed PVDF membrane, primary antibody [anti-phosphorylated p38MAP kinase (manufactured by Cell Signaling Technology), anti-p38MAP kinase (manufactured by Cell Signaling Technology), anti-IL-1β (manufactured by R & D Systems)) The target protein was detected by the Western blot method used.

Thus, the small intestinal macrophages (IPIM cells) of pig embryos that have been immortalized by introducing the SV40 large T antigen gene and the telomerase reverse transcriptase gene show high proliferative properties, while not only expressing the marker protein but also the expression thereof. It became clear that the function was also maintained.

<African swine fever virus susceptibility test>
Next, the virus susceptibility of IPIM cells was evaluated for African swine fever virus (ASFV) by the method shown below.

(About the ASFV strain used)
Three ASFV fields: European / Chinese epidemic strain Armenia07 (genotype: type II), strain Kenya05 / Tk-1 (genotype: type I) isolated from soft mites, and ASFV standard strain Espana75 (genotype: type I). Outbreak isolates were introduced from the World Organization for Animal Health of Madrid, Spin, ASF Reference Laboratory of the World Organization for Animal Health (OIE). Information such as the year of occurrence and the place of occurrence of each virus strain can be found in Fernandez-Pinero J, et al. (2013) (Transboundary and Emerging Diseases. 60 (2013) 48-58). The Vero cell-conditioned strain Lisbon60 / V was introduced from Plum Island Animal Disease Center (PIADC), USA. Armenia07, Kenya05 / Tk-1 and Espana75 strains were grown and cultured using PAM cells, and Lisbon60 / V strains were grown and cultured using Vero cells, which were used as inoculated virus samples. Inoculated virus samples were dispensed and stored at -80 ° C.

(About the cells used)
Pig alveolar macrophage (PAM) cells were recovered from pig lungs according to previously reported (Carrascosa AL. et al., Curr Protocol Cell Biol, December 2011, Chapter 26, UNIT 26.14, pp. 1-26). Vero (CCL-81) cells, COS-1 (CRL-1650) cells and pig alveolar macrophage-derived immortalized cell lines 3D4 / 21 (CRL-2843) cells, which are kidney-derived immortalized cell lines of African green monkeys, are American. Introduced from the Type Culture Collection (ATCC). Wild boar lung-derived cell line WSL cells used were those obtained from the Friedrich-Leffler Institute (FLI, Germany).

(Virus susceptibility test)
The susceptibility of cells to virus was determined by a CPE test using the cytopathic effect (CPE) as an index or a HAD test using a hemad adsorption (HAD) reaction specifically observed in ASFV-infected cells as an index. Specifically, first, WSL cells and 3D4 cells are arranged so that the number of PAM cells is 1 × 10 ⁵ and the number of Vero cells and COS-1 cells is 1.5 × 10 ⁴ per hole in a 96-well plate. / 21 cells were seeded to 2 × 10 ⁴ or IPIM cells to be 3 × 10 ⁴ . Then, after pre-culturing in each medium for 1 to 2 days, inoculate 8 holes of 25 μL of the virus solution diluted 10 ^- fold ( ^10-1 ) to 10 billion-fold (10-10) in the same medium. Then, a plate for detecting CPE was prepared. Further, 20 μL of 0.75% pig erythrocyte suspension suspended in PBS was added to the plate to prepare a plate for HAD detection. Then, both the CPE detection plate and the HAD detection plate were cultured at 37 ° C. in a 5% CO ₂ environment for 7 days and observed.

As a result, as shown in Table 1, CPE was detected in all the inoculated virus strains in PAM cells and IPIM cells. On the other hand, in Vero cells, COS-1 cells, WSL cells and 3D4 / 21 cells, CPE was not observed in the three field strains, but only in the cell-conditioned strain Lisbon60 / V (Table 1). As described above, it was clarified that the IPIM cells can be used not only for the CPE test (FIG. 6A) but also for the HAD test (FIG. 6B) like the PAM cells.

Next, the dilution limit (virus detection limit) at which the virus could be separated was evaluated for both PAM cells and IPIM cells. As a result, as shown in Table 2, in the CPE test, the detection limit concentration of IPIM cells was 100 to 10,000 times lower than that of PAM cells. In the HAD test, the detection limit concentration of IPIM cells was 10 to 100 times lower than that of PAM cells (Table 3). That is, it was revealed that IPIM cells are more sensitive to ASFV than PAM cells.

<Sensitivity test for pig breeding / respiratory disorder syndrome virus or porcine circovirus>
Next, the virus susceptibility of IPIM cells was evaluated for pig breeding / respiratory disorder syndrome virus (PRRSV) or porcine circovirus type 2 (PCV2) by the method shown below.

(About the virus strain used)
The EDRD-1 strain, which is a domestic isolate, was used for the pig breeding / respiratory disorder syndrome virus (PRRSV), and the Yamagata strain, which is a domestic isolate, was used for Porcine Circovirus Type 2 (PCV2).

(PRRSV susceptibility test)
IPIM cells were seeded on a 96-well suspension culture plate (manufactured by SUMILON) with 1 × 10 ⁵ cells / 0.1 mL medium per hole. Three days later, after removing the cell culture solution by suction, 0.1 mL of the virus solution diluted 10-fold to 10,000-fold from 10-fold to 10,000-fold was infused per hole for virus antigen detection by the indirect fluorescent antibody method. A plate was made. After removing the cell culture by suction on the 3rd day after virus inoculation, the cells were fixed with ice-cold 80% acetone, and the PRRS virus antigen was obtained by the indirect fluorescent antibody method using an anti-PRRS virus monoclonal antibody (clone: SR30-A). Was detected. The obtained results are shown in the upper part of FIG.

In addition, in order to observe virus growth, IPIM cells were inoculated on a 6-well suspension culture plate (manufactured by SUMILON) with 3 × 10 ⁶ cells / 3 mL medium per hole. After 3 days, the cell culture solution was aspirated and removed, and then 0.5 mL of a virus solution obtained by diluting the storage stock solution 100 times was inoculated per hole to adsorb the virus to the cells for 1 hour. After 1 hour, the medium containing the virus was removed, the cells were washed with 2 mL of medium per hole, 5 mL of new medium was added, and the cells were cultured at 37 ° C. in the presence of 5% CO ₂ . CPE was observed on the 1st, 2nd, 3rd, 5th, and 7th days after inoculation, and 0.5 mL of the culture supernatant was collected. The collected culture supernatant was diluted 10-fold stepwise from 10-fold to 10,000,000-fold, and then seeded in 1 × 10 ⁵ cells / 0.1 mL medium per hole and cultured for 3 days per hole in IPIM cells. CPE was observed by inoculating 0.1 mL (4 holes per dilution) and culturing at 37 ° C. in a 5% CO ₂ environment for 7 days. The observation results on the 7th day of inoculation are shown at the bottom of FIG. Furthermore, the 50% tissue culture infection amount (TCID ₅₀ ) was calculated based on the Behrens-Carber method. The obtained results are shown in FIG.

(PCV2 susceptibility test)
After suspending IPIM cells in 2 × 10 ⁵ cells / 180 μL medium per hole, mix with 20 μL of virus storage stock solution, and mix 200 μL of cell / virus mixture per hole with 48-hole suspension culture plate (manufactured by SUMILON). Was sown in. 2 hours after seeding, the medium containing the virus was removed, the cells were washed with 0.5 mL of medium per hole, 200 μL of new medium was added, and the cells were cultured at 37 ° C. in the presence of 5% carbon dioxide gas. The culture supernatant and cells immediately after the addition of the medium were collected together and used as a sample 2 hours after inoculation. Then, on the 2nd, 4th, and 6th days, the culture supernatant and the cells were collected together. From the collected sample, all DNA was extracted as a final 50 μL solution using DNeasy Blood & Tissue Kit (manufactured by QIAGEN), and 2 μL of the DNA was used for quantitative PCR targeting the PCV2 capsid gene. The obtained results are shown in FIG. 9 as relative values with the amount of virus gene 2 hours after virus inoculation as 1.

Regarding the PRRSV sensitivity of IPIM cells, as shown in FIG. 7, PRRSV is sensitive to IPIM cells, the viral antigen can be detected in the cytoplasm on the 3rd day after inoculation, and the clear CPE is on the 7th day after inoculation. Was observed. Furthermore, as shown in FIG. 8, the viral infectivity titer in the PRRSV-inoculated IPIM cell culture supernatant peaked 2-3 days after inoculation and increased nearly 100,000 times. The infection titer tended to be maintained without a significant decrease until the 7th day after inoculation.

Regarding the PCV2 sensitivity of IPIM cells, as shown in FIG. 9, it was observed that the amount of PCV2 gene tended to double with the doubling of IPIM cells after virus inoculation.

From these facts, it was confirmed that IPIM cells are also sensitive to the field strains of PRRSV and PCV2.

As described above, according to the present invention, it is possible to provide immortalized cells of porcine small intestine macrophages. In addition, since the immortalized cells have high infectivity to pig-infecting virus, various pig-infecting virus strains can be propagated, and vaccines against the virus can be produced and developed. In addition, it is possible to detect (test, diagnose) pig infectious virus because of its high susceptibility to infection.

Claims

Immortalized cells of small intestinal macrophages in pig fetuses.
The cell according to claim 1, wherein the small intestinal macrophages of pig embryos express at least one protein selected from the group consisting of SV40 large T antigen and telomerase reverse transcriptase.
The cell according to claim 2, wherein the expression is an expression from a lentivirus encoding the protein.
The cell according to claim 2 or 3, wherein the telomerase reverse transcriptase is a pig-derived telomerase reverse transcriptase.
The cell according to any one of claims 1 to 4, wherein the pig fetus is a pig fetus having a fetal age of 30 to 114 days.
A method for producing immortalized pig small intestine macrophages, which comprises a step of expressing at least one protein selected from the group consisting of SV40 large T antigen and telomerase reverse transcriptase in small intestine macrophages of pig fetal.
The method according to claim 6, wherein the step is a step of introducing a lentivirus encoding the protein into the small intestinal macrophages of the fetal pig and expressing the protein.
The method according to claim 6 or 7, wherein the telomerase reverse transcriptase is a pig-derived telomerase reverse transcriptase.
The method according to any one of claims 6 to 8, wherein the pig fetus is a pig fetus with a fetal age of 30 to 114 days.
A method for producing a pig-infecting virus, which comprises a step of contacting the cell according to any one of claims 1 to 5 with a pig-infecting virus and propagating the virus in the cell.
A step of bringing the cell according to any one of claims 1 to 5 into contact with a pig-infecting virus and propagating the virus in the cell.
A method for producing a vaccine containing a pig infectious virus, which comprises a step of isolating the propagated pig infectious virus and a step of mixing the isolated pig infectious virus with a pharmacologically acceptable carrier or medium.
10. The virus according to claim 10, wherein the pig infectious virus is at least one virus selected from the group consisting of African pig fever virus, porcine epidemic diarrhea virus, pig rotavirus, pig circovirus, and pig breeding / respiratory disorder syndrome virus. Or the manufacturing method according to 11.
The cell according to any one of claims 1 to 5, which has a DNA to which a reporter gene is functionally bound downstream of the promoter region of a gene derived from a pig infectious virus.
13. Claim 13 that the pig infectious virus is at least one virus selected from the group consisting of African pig fever virus, pig epidemic diarrhea virus, pig rotavirus, pig circovirus, and pig breeding / respiratory disorder syndrome virus. The cells described in.
A method for detecting a pig infectious virus, which comprises the following steps, a step of culturing the cells according to claim 13 or 14 in the presence of a test sample.
A step of detecting the expression of the reporter gene in the cell, and a step of determining that the test sample contains a pig infectious virus when the expression of the reporter gene is detected.
A method for detecting a neutralizing antibody against a pig infectious virus, which comprises the following steps. The cell according to any one of claims 1 to 5 in the presence of a biological sample isolated from a test pig. The step of bringing the virus into contact with the pig-infecting virus and propagating the virus in the cells,
The step of detecting the number of propagated viruses and the number of viruses detected in the steps are the viruses propagated in the cells according to any one of claims 1 to 5 in the absence of the biological sample. A step of determining that the biological sample contains a neutralizing antibody against the virus when the number is small compared to the number.
15. The pig infectious virus is at least one virus selected from the group consisting of African pig fever virus, pig epidemic diarrhea virus, pig rotavirus, pig circovirus, and pig breeding / respiratory disorder syndrome virus. Or the method according to 16.