Immortalized porcine alveolar macrophage culture cell line, method for producing immortalized porcine alveolar macrophage culture cell line, reagent for preparing immortalized porcine alveolar macrophage culture cell line, and method for producing vaccine

Abstract

This immortalized porcine alveolar macrophage cultured cell line has a viral long terminal repeat, expresses a gene that induces cell immortalization, and is sensitive to porcine reproductive and respiratory syndrome virus and / or African swine fever virus.

Description

[Technical Field]

[0001] The present invention relates to an immortalized porcine alveolar macrophage culture cell line, a method for producing an immortalized porcine alveolar macrophage culture cell line, a reagent for preparing an immortalized porcine alveolar macrophage culture cell line, and a method for producing a vaccine. [Background technology]

[0002] Porcine reproductive and respiratory syndrome (PRRS) is the infectious disease that causes the most damage to the pig farming industry both in Japan and overseas. PRRS causes losses of approximately 28 billion yen per year in domestic pig farming. In addition to PRRS, African swine fever (ASF), which causes devastating damage in China and Southeast Asia, is also a problem. Although ASF has not occurred in Japan, the African swine fever virus (ASFV), which causes ASF, has been repeatedly detected during quarantine at airports and other locations.

[0003] The porcine reproductive and respiratory syndrome virus (PRRSV) and ASFV that cause PPRS can be detected and isolated by inoculating pig serum or organ emulsion into specific cells and using the cytopathic effect as an indicator.

[0004] The cells used for virus isolation using cultured cells are porcine alveolar macrophages (PAM), which are highly susceptible to PRRSV and other viruses. PAM are typically obtained by lung lavage of 6- to 12-week-old piglets, which requires a great deal of effort and the sacrifice of the piglets. Furthermore, because PAM are primary cultured cells, they have poor division and proliferation capabilities, making subculture extremely difficult.

[0005] MA104 cells are also used for virus isolation. MA104 is a cell line derived from monkey kidney. MA104 has the advantage of being easy to prepare due to its high proliferation capacity. However, because MA104 is derived from monkeys that are not hosts for PRRSV, it has low susceptibility to PRRSV and other viruses. Although susceptibility can be increased by adapting PRRSV to MA104, MA104 is not suitable for isolating new wild-type strains of PRRSV and other viruses.

[0006] Culturable cell lines derived from PAM have been proposed to enable subculture while maintaining the susceptibility of PAM to PRRSV. For example, Non-Patent Document 1 discloses PAM into which the SV40 large T antigen (SV40T) gene, a cell carcinogenesis-inducing factor, has been introduced by genetic recombination technology. Patent Document 1 also discloses a cell line that is susceptible to PRRSV and in which the SV40T gene has been introduced into PAM using a transposon. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Special Publication No. 2017-500029 [Non-patent literature]

[0008] [Non-Patent Document 1] HM Weingartl., et al., “Continuous porcine cell lines developed from alveolar macrophages Partial characterization and virus susceptibility”, Journal of Virological Methods, 2002, 104(2), p203-216 Summary of the Invention [Problem to be solved by the invention]

[0009] The cell line disclosed in Non-Patent Document 1 is not susceptible to PRRSV. Therefore, it cannot be used to isolate PRRSV. The cell line disclosed in Patent Document 1 requires macrophage colony-stimulating factor (M-CSF) for proliferation, so conditions such as the optimal concentration of M-CSF must be investigated.

[0010] The present invention has been made in view of the above circumstances, and aims to provide an immortalized porcine alveolar macrophage cultured cell line that is highly susceptible to PRRSV and ASFV and is easy to prepare, and a method for producing the immortalized porcine alveolar macrophage cultured cell line. It also aims to provide a reagent for preparing the immortalized porcine alveolar macrophage cultured cell line that is useful for preparing the immortalized porcine alveolar macrophage cultured cell line. It is yet another object of the present invention to provide a method for producing a vaccine using the immortalized porcine alveolar macrophage cultured cell line. [Means for solving the problem]

[0011] In the above-mentioned Patent Document 1, the use of a retroviral vector is avoided and a transposon is used because the viral long terminal repeat (hereinafter simply referred to as "LTR"), which is integrated into the cellular genome by the retroviral vector used to introduce the SV40T gene into cells, causes tumorigenesis. After extensive research, the inventors discovered that even when a retroviral vector is used, an immortalized porcine alveolar macrophage culture cell line highly susceptible to PRRSV and ASFV can be established by using porcine serum in the cultivation of primary cultured PAM, and thus completed the present invention.

[0012] The immortalized porcine alveolar macrophage culture cell line according to the first aspect of the present invention comprises: having a viral long terminal repeat sequence, expressing a cell immortalization-inducing gene, It is susceptible to at least one of the porcine reproductive and respiratory syndrome virus and African swine fever virus.

[0013] In this case, the cell immortalization-inducing gene is SV40 large T antigen gene, This may also be the case.

[0014] The viral long terminal repeat sequence is Moloney murine leukemia virus viral long terminal repeat sequence, This may also be the case.

[0015] A method for producing an immortalized porcine alveolar macrophage culture cell line according to a second aspect of the present invention comprises: A culturing step of culturing primary cultured alveolar macrophages collected from pigs in a medium containing pig serum; an introduction step of introducing a cell immortalization-inducing gene into the cells cultured in the culture step; Includes:

[0016] In this case, the cell immortalization-inducing gene is SV40 large T antigen gene, This may also be the case.

[0017] The cell immortalization-inducing gene is introduced into said cells together with a viral long terminal repeat sequence; This may also be the case.

[0018] The pig A 2-week-old pig, This may also be the case.

[0019] In addition, in the culturing step, The primary cultured alveolar macrophages are cultured for 10 to 24 hours. This may also be the case.

[0020] A reagent for preparing an immortalized porcine alveolar macrophage culture cell line according to a third aspect of the present invention comprises: Contains porcine serum.

[0021] A method for producing a vaccine according to a fourth aspect of the present invention comprises: an infection step of infecting the immortalized porcine alveolar macrophage cultured cell line according to the first aspect of the present invention with porcine reproductive and respiratory syndrome virus or African swine fever virus; a propagation step of propagating the porcine reproductive and respiratory syndrome virus or African swine fever virus by culturing the infected immortalized porcine alveolar macrophage culture cell line; obtaining the propagated porcine reproductive and respiratory syndrome virus or African swine fever virus; Includes: [Effects of the Invention]

[0022] According to the present invention, an immortalized porcine alveolar macrophage culture cell line that is highly susceptible to PRRSV and ASFV and can be easily prepared is obtained. The present invention also provides a reagent for preparing the immortalized porcine alveolar macrophage culture cell line, which is useful for preparing the immortalized porcine alveolar macrophage culture cell line. The present invention also provides a method for producing a vaccine using the immortalized porcine alveolar macrophage culture cell line. [Brief explanation of the drawings]

[0023] [Figure 1] FIG. 1 shows the structure of a plasmid according to an example. [Figure 2] FIG. 1 is a diagram showing the viral titer of a PAM cultured cell line according to an example. [Figure 3] FIG. 1 is a diagram showing the viral titer of each virus against a PAM cultured cell line according to an example. [Figure 4] FIG. 1 shows the viral titer after inoculation of each virus into a PAM cultured cell line according to an example. [Figure 5] FIG. 1 shows the expression of SV40T in a PAM cultured cell line according to an example. [Figure 6] FIG. 1 shows the results of esterase staining and sodium fluoride (NaF) inhibition tests of a PAM cultured cell line according to an example. [Figure 7]1A is a graph showing the phagocytic activity of a PAM-cultured cell line according to an example, and FIG. 1B is a graph showing the fluorescence intensity of a PAM-cultured cell line according to an example cultured at 4°C and 37°C. [Figure 8] FIG. 10 is a diagram showing the fluorescence intensity of a PAM cultured cell line according to an example, which is labeled via the CD163 molecule. [Figure 9] FIG. 1 shows the expression of SV40T in immortalized PAM bulk cells cultured in the presence or absence of pig serum. DETAILED DESCRIPTION OF THE INVENTION

[0024] Embodiments of the present invention will be described with reference to the drawings. Note that the present invention is not limited to the following embodiments and drawings. Note that in the following embodiments, the expressions "have," "include," or "contain" also include the meaning of "consisting of" or "consisting of."

[0025] (Embodiment 1) The immortalized porcine alveolar macrophage cultured cell line according to this embodiment (hereinafter simply referred to as the "cell line") is susceptible to at least one of PRRSV and ASFV. Hereinafter, "PRRSV, etc." means "PRRSV or ASFV." "Susceptible to PRRSV, etc." means that the cell can become a host cell infected by PRRSV, etc. Morphological changes (cytopathic effect) are observed in cultured cells infected with PRRSV, etc. The presence or absence of a cytopathic effect can be determined by observing cells exposed to PRRSV, etc. under a microscope, etc. The susceptibility of a cell line to PRRSV, etc. can be determined by measuring the 50% tissue culture infectious dose (TCID 50 ) can be evaluated by known methods such as.

[0026] In the genome of the cell line according to this embodiment, a cell immortalization-inducing gene is integrated together with the LTR by transformation. This allows the cell line to stably express the cell immortalization-inducing gene and thus become immortalized. Examples of cell immortalization-inducing genes include the SV40T gene, the human telomere reverse transcriptase (hTERT) gene, the human papillomavirus (HPV) E6 or E7 gene, the Cdk-4 gene, and the Bmi-1 gene. The cell immortalization-inducing gene may be a tumor suppressor gene, such as an siRNA or shRNA expression plasmid DNA that knocks down p53 and RB (retinoblastoma protein). Preferably, the cell immortalization-inducing gene is the SV40T gene.

[0027] A method for producing a cell line according to this embodiment will now be described. The method for producing a cell line includes a culturing step and an introducing step.

[0028] In the culturing step, primary cultured PAMs collected from pigs are cultured in a medium containing pig serum. Preferably, the pigs are piglets. The age of the piglets is not particularly limited, but may be, for example, 1 to 15 weeks, 1 to 10 weeks, 1 to 6 weeks, or 1 to 3 weeks. Preferably, the pigs are 2 weeks old. PAMs can be obtained from pigs by known methods such as lung lavage.

[0029] The medium is not particularly limited as long as it is capable of culturing PAM. Porcine serum may be collected from pigs or commercially available. The concentration of porcine serum in the medium is, for example, 3-20%, 5-15%, or 8-12%. The concentration of porcine serum in the medium is preferably 10%. In addition to porcine serum, the medium may contain fetal bovine serum, non-essential amino acids, a buffer, sodium pyruvate, antibiotics, and the like. Culture conditions are set appropriately, for example, at 37°C and 5% CO2. The culture time for primary culture of PAM in the culture step is not particularly limited and is, for example, 8-48 hours, 9-36 hours, or 10-24 hours.

[0030] In the introduction step, a cell immortalization-inducing gene is introduced into the PAM cultured in the culture step. Preferably, in the introduction step, the SV40T gene as the cell immortalization-inducing gene is introduced into the PAM together with an LTR. For example, the LTR is an LTR of a retrovirus, which is a single-stranded RNA virus. The LTR is a repeated sequence possessed by retroviruses and includes an enhancer sequence, promoter sequence, transcription initiation sequence, transcription terminator sequence, polyadenylation signal, etc., necessary for retroviral gene expression.

[0031] The major elements of a retroviral genome, from the 5' end to the 3' end, include the 5'LTR, SD sequence, packaging signal sequence, gag gene, pol gene, SA sequence, env gene, and 3'LTR sequence. LTR encompasses the 5'LTR and 3'LTR. LTR contains the U3, R, and U5 regions involved in transcription of viral genes, reverse transcription from the viral genome, and integration of double-stranded DNA synthesized through reverse transcription into host DNA. The U3 region has enhancer / promoter activity, and transcription from the R region of the 5'LTR to the R region of the 3'LTR is carried out by host cell RNA polymerase II.

[0032] The packaging signal sequence is known as the ψ sequence and is a non-coding, cis-acting sequence required for the encapsidation and packaging of retroviral RNA strands into viral particles during viral particle formation.

[0033] Preferably, a retroviral vector is used to transform the gene into the PAM in the introduction step. The retroviral vector may be a vector such as an oncoretrovirus or a lentivirus, which are included in the retrovirus. The retroviral vector used for gene transfer has at least a 5'LTR, a packaging signal sequence, an SV40T gene, and a 3'LTR in its genome. Gene products such as the gag gene, pol gene, and env gene may be supplied by packaging cells that harbor these genes.

[0034] The LTR and packaging signal sequences of the retroviral vectors are not particularly limited as long as they are derived from retroviruses and enable gene transfer into the PAM. These sequences may be derived from the same virus, or may be a combination of sequences derived from different viruses that enable viral particle formation and integration into the cellular genome when combined with appropriate packaging cells.

[0035] The term "retroviral-derived LTR" refers to a sequence that is a retroviral LTR, a fragment of a retroviral LTR, or a fragment into which a mutation has been introduced, as well as a sequence that is artificially designed and produced based on the sequence of a retroviral LTR.

[0036] The LTR and packaging signal sequence possessed by the retroviral vector of this embodiment may be sequences derived from, for example, oncoretroviruses such as Moloney murine leukemia virus (MMLV), mouse embryonic stem cell virus (MESV), murine stem cell virus (MSCV), myeloproliferative sarcoma virus (MPSV), spleen-limited focus-forming virus (SFFV), etc. The LTR of the retroviral vector is preferably an MMLV LTR or an MMLV-derived LTR.

[0037] Furthermore, the LTR and packaging signal sequences possessed by the retroviral vector of this embodiment may be sequences derived from, for example, human immunodeficiency viruses (HIV-1, HIV-2), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), equine infectious anemia virus (EIAV), and caprine arthritis-encephalitis virus (CAEV), which belong to the lentivirus family.

[0038] The LTRs of the retroviral vectors of this embodiment may be LTRs with mutated sequences, for example, the U3 region of the 3'LTR or 5'LTR may be replaced with enhancer and promoter sequences derived from a virus other than the virus from which the LTR sequence is derived.

[0039] To increase the efficiency of PAM transfection, the envelope gene of another virus may be used (pseudotyping). For pseudotyping, the G protein of vesicular stomatitis virus (VSV-G) is preferred, as it binds to phospholipids expressed on the surface of most mammalian cells and mediates intracellular entry of the viral vector.

[0040] To prepare retroviral vectors using pseudotyping, a retroviral vector packaging plasmid containing the SV40T gene and a vesicular stomatitis virus G protein expression plasmid are introduced into retroviral vector packaging cells. The cells containing the plasmids are cultured, and the SV40T expression retroviral vector produced in the culture supernatant is collected.

[0041] In the transfection step, a retroviral vector for SV40T expression is inoculated into the PAM. After inoculation, some cells survive the culture. These cells are then cloned using known methods to obtain cell lines.

[0042] The cell line according to this embodiment is a monocytic cell. Whether or not a cell is monocytic can be confirmed by evaluating a known marker specific to monocytic cells. Examples of monocytic cell markers include CD163, CD11b, and CD68. In particular, CD163 is a preferred marker for monocytic cells because it serves as a receptor for PRRSV. Furthermore, monocytic cells can also be identified by nonspecific esterase activity, which is inhibited by NaF.

[0043] The cell line according to this embodiment maintains the properties of macrophages, such as phagocytic activity, nitric oxide production, and cytokine production. The properties of macrophages can be evaluated using known methods and commercially available kits.

[0044] As shown in Test Example 1 below, the cell line according to this embodiment exhibits susceptibility to various PRRSV strains, including field isolates, equal to or greater than that of primary cultured PAM. Therefore, this cell line is useful for isolating and detecting PRRSV. Furthermore, this cell line can be used for monitoring the occurrence of PRRS, diagnosing PRRS, and developing vaccines for preventing PRRS.

[0045] Furthermore, the cell line according to this embodiment is also susceptible to ASFV, as shown in Test Example 5 below. Therefore, this cell line is useful for isolating and detecting ASFV. This cell line is also useful for monitoring the occurrence of ASFV, diagnosing ASFV, and developing vaccines for preventing ASFV.

[0046] (Embodiment 2) The reagent for preparing the PAM immortalized cultured cell line according to this embodiment contains porcine serum. Porcine serum can be obtained from blood collected from pigs by known methods. For example, porcine serum can be obtained by allowing porcine blood to stand without mixing with an anticoagulant to aggregate the clot, and then centrifuging the blood at a gravitational acceleration that does not cause hemolysis. The porcine serum may be purified using filter filtration or the like and sterilized by gamma irradiation or the like.

[0047] The reagent for preparing a PAM-immortalized culture cell line is preferably provided as a reagent to be added to a culture medium. The reagent for preparing a PAM-immortalized culture cell line may be provided as the above-mentioned culture medium. When the reagent for preparing a PAM-immortalized culture cell line is a culture medium, the preparation reagent may contain, in addition to porcine serum, a culture medium, amino acids, non-essential amino acids, a buffer, nutrients, antibiotics, and the like.

[0048] The reagent for preparing a PAM-immortalized cultured cell line according to this embodiment is suitable for preparing and culturing the PAM-immortalized cultured cell line according to the first embodiment above.

[0049] (Embodiment 3) The method for producing a vaccine according to this embodiment includes an infection step, a proliferation step, and an acquisition step. In the infection step, the PAM-immortalized cultured cell line according to the first embodiment is infected with PRRS or the like. Infection can be performed by a known method, for example, by inoculating PRRS or the like into a medium containing the PAM-immortalized cultured cell line.

[0050] In the propagation step, the infected PAM-immortalized cultured cell line is cultured to propagate PRRS and the like. The culture conditions for the PAM-immortalized cultured cell line are appropriately set, for example, at 37°C in 5% CO. The culture period is optional, but may be, for example, 1 to 10 days, 2 to 8 days, or 3 to 7 days.

[0051] In the obtaining step, the grown PRRS etc. are obtained. In the obtaining step, PRRS etc. may be separated from the PAM-immortalized cultured cell line or the medium in which the PAM-immortalized cultured cell line has been cultured, and the separated PRRS etc. may be further purified and concentrated. Methods for separating PRRS etc. include, for example, filtration of the medium, cell disruption and centrifugation, and concentration may also be performed using ammonium sulfate, a resin column, polyethylene glycol salting out, or the like.

[0052] The obtained PRRS etc. may be used as a live vaccine, an attenuated vaccine, or an inactivated vaccine. Furthermore, as long as it has immunogenicity, a part of the obtained PRRS etc., such as a protein, polypeptide, sugar, glycoprotein, lipid, or nucleic acid, may also be used as a vaccine.

[0053] An attenuated virus is a virus with reduced toxicity compared to the virus before attenuation. Virus attenuation can be performed by known methods. Attenuated viruses can be obtained, for example, by growing in the presence of a mutagen, acclimating to cultured cells by serial passage in vitro, or growing under conditions that deviate from the natural growth environment, such as high temperature conditions. The cultured cells used for acclimation by serial passage may be the above-mentioned PAM-immortalized cultured cell line, or other cells, such as MA104. Attenuated viruses can also be obtained by deleting or recombining specific viral genes using genome editing and gene modification techniques.

[0054] Viruses can be inactivated using known methods, including formaldehyde treatment, UV irradiation, X-ray irradiation, electron beam irradiation, gamma ray irradiation, alkylation treatment, ethyleneimine treatment, thimerosal treatment, β-propiolactone treatment, and glutaraldehyde treatment.

[0055] The vaccine according to this embodiment may contain a pharmacologically acceptable carrier. Examples of the carrier include stabilizers, excipients, preservatives, surfactants, chelating agents, and binders. The vaccine according to this embodiment may contain a pharmacologically acceptable medium. Examples of the medium include water, saline, phosphate buffer, and Tris-HCl buffer.

[0056] To enhance vaccine efficacy, the vaccine of this embodiment may be mixed with or used in combination with an immunostimulant (adjuvant). Examples of immunostimulants include inorganic substances such as aluminum gel adjuvant; microorganisms or microbial-derived substances such as BCG, muramyl dipeptide, Bordetella pertussis, pertussis toxin, and cholera toxin; surfactants such as saponin and deoxycholic acid; emulsions of oily substances such as mineral oil, vegetable oil, and animal oil; alum; and vitamin E acetate solution.

[0057] In addition, other substances such as emulsifiers and stabilizers may be added to the vaccine to improve its performance and stability. The form of the vaccine is not particularly limited, and may be, for example, in the form of a suspension or a freeze-dried form.

[0058] The vaccine according to this embodiment can be administered by intramuscular injection, subcutaneous injection, or intranasal, intratracheal, oral, or intradermal administration. The vaccine is administered to animals susceptible to PRRS or other infections, particularly to pigs aged 1 week, 3 weeks, 6 weeks, or 10 weeks. The dose is adjusted as appropriate. The number of times the vaccine is administered is optional, and a boosting effect may be achieved by one or more administrations.

[0059] The present invention will be explained in more detail with reference to the following examples, but the present invention is not limited to these examples. [Example]

[0060] Establishment of cell lines (Preparation of retroviral vector for SV40T expression) The SV40T gene was amplified by polymerase chain reaction (PCR) using cDNA synthesized from mRNA extracted from human kidney-derived 293T cells as a template according to standard methods. The nucleotide sequences of the forward and reverse primers are shown in SEQ ID NOs: 1 and 2, respectively. The SV40T gene was cloned into the retroviral vector packaging plasmid pMXs (Cell BioLabs) to construct pMXs-SV40T. The structure and nucleotide sequence of pMXs-SV40T are shown in Figure 1 and SEQ ID NO: 3, respectively.

[0061] pMXs-SV40T and the vesicular stomatitis virus G protein expression plasmid pCMV-VSV-G (Cell Bio Labs) were transfected into retroviral vector packaging cells Platinum-GP (Cell Bio Labs) using the plasmid transfection reagent TransIT-293 (Mirus Bio). The transfected cells were cultured in high-glucose D-MEM medium (Fujifilm Wako Pure Chemical Industries) containing 10% fetal bovine serum (Capricorn) and antibiotics, and the SV40T expression retroviral vector produced in the culture supernatant was collected.

[0062] (PAM immortalization and cloning) Primary cultured PAMs collected from 2-week-old PRRSV-negative piglets were resuspended in RPMI 1640 medium (Fujifilm Wako Pure Chemical Industries, Ltd.) containing 10% fetal bovine serum (Capricorn), 10% porcine serum (Cytiva), non-essential amino acids (Nacalai Tesque), sodium pyruvate (Nacalai Tesque), HEPES (Nacalai Tesque), and antibiotics. The cells were seeded onto cell culture plates and cultured at 37°C and 5% CO2. The following day, PAMs were inoculated with a retroviral vector for SV40T expression and further cultured. While many cells died, the surviving cell population was pooled into bulk cells. From these bulk cells, 12 cell lines were randomly cloned, and five clones with the highest proliferation rates were selected as immortalized PAM cell lines.

[0063] Test Example 1: PRRSV susceptibility evaluation A 10-fold serial dilution series of PRRSV (Zoetis vaccine strain) stock solution was prepared and inoculated onto the immortalized PAM cell lines of five selected clones (PAM-T1 to T5) and bulk cells, and cultured for 4 days. The viral titer (TCID ) in each cell line was determined based on the cytopathic effect. 50 ) was used to assess susceptibility to PRRSV.

[0064] The susceptibility of PAM-T4 and PAM-T5 to the Zoetis vaccine strain (hereafter referred to as "vaccine strain Z") as well as three PRRSV strains (the Boehringer Ingelheim vaccine strain (hereafter referred to as "vaccine strain B"), field isolate PGL9, and field isolate 156-K) was evaluated using the same method. For comparison, the susceptibility of untreated primary cultured PAM (primPAM), the monkey kidney-derived MA104 cells used in the development of vaccine strain Z, and the commercially available immortalized PAM cell lines 3D4 / 2, 3D4 / 21, and 3D4 / 31 (all manufactured by ATCC) was also evaluated using the same method.

[0065] (result) Figure 2 shows the viral titers of PAM-T1 to PAM-T5. High susceptibility was confirmed in particular for PAM-T4 and PAM-T5. Figure 3 shows the viral titers of each virus in each cell line. PAM-T4 and PAM-T5 exhibited sufficient susceptibility to all viruses. In particular, PAM-T4 and PAM-T5 exhibited susceptibility to the field isolates PGL9 and 156-K at the same level as primary cultured PAM. Compared to MA104, PAM-T4 and PAM-T5 exhibited high susceptibility to vaccine strains Z, PGL-9, and 156-K. Vaccine strain B, a virus attenuated with MA104 and adapted to monkey cells, exhibits high susceptibility to MA104. Viral infection was not confirmed for the immortalized PAM cell lines 3D4 / 2, 3D4 / 21, and 3D4 / 31 disclosed in Non-Patent Document 1.

[0066] Test Example 2: PRRSV proliferation evaluation A fixed number of primary cultures, PAM, MA104, PAM-T4, and PAM-T5, were inoculated with PRRSV vaccine strains Z, B, PGL9, and 156-K at a multiplicity of infection of 0.01, and the culture supernatants were collected over time. The virus titers in all the collected culture supernatants were measured using the TCID 2016 described above using MA104 cells. 50 The growth of the virus was assessed by assay determination.

[0067] (result) Figure 4 shows the time course of the viral titer for each virus on each cell line. PAM-T4 and PAM-T5 showed growth rates equal to or greater than that of primPAM.

[0068] Test Example 3: Analysis of basic properties of cell lines (Confirmation of SV40 T antigen expression) Primary cultured PAM seeded on cell culture plates, MA104 cells previously transfected with an SV40T expression plasmid, and PAM-T4 and PAM-T5 cells were fixed with Mildform 10N (Fujifilm Wako Pure Chemical Industries, Ltd.) and permeabilized with Triton X-100 diluted to 0.5% in sterile phosphate-buffered saline (PBS / BSA) containing 0.3% bovine serum albumin. These cells were reacted sequentially with an anti-SV40T mouse monoclonal antibody (clone PAb416, Merck Millipore) diluted 1:1000 in PBS / BSA solution and a DyLight 488-labeled anti-mouse IgG (H+L) secondary antibody (Vector Laboratories) diluted 2:2000 in PBS / BSA solution. DNA (nuclei) was visualized by treatment with Hoechst 33342 (Thermo Fisher Scientific) diluted 2:2000 in PBS solution, and the fluorescent signal was observed under a fluorescence microscope.

[0069] (esterase staining) The nonspecific esterase activity of PAM-T4 was evaluated using an esterase staining kit (Muto Chemical Co., Ltd.). Immortalized cell line PAM-T4 smears were prepared using a Cytospin 4 cell centrifuge (Thermo Fisher Scientific), fixed with the formalin-acetone buffer fixative provided with the kit, treated with α-NB reaction solution, and examined under a microscope for the presence of brown granules. The cells were then treated with NaF and examined under a microscope for the presence of brown granules.

[0070] (Evaluation of phagocytic activity) The phagocytic activity of PAM-T4 was evaluated using the pH indicator pHrodo Green E. coli BioParticles Conjugate for Phagocytosis Kit (Thermo Fisher Scientific). pHrodo BioParticles were added to the culture supernatant of PAM-T4 and porcine kidney-derived PK-15 cells seeded on cell culture plates, and the cells were cultured at 37°C and 5% CO2. The fluorescent signal was observed under a fluorescence microscope. The PK-15 cells were grown in E-MEM medium (Fujifilm Wako Pure Chemical Industries) containing 10% fetal bovine serum (Capricorn) and antibiotics. PAM-T4 cells reacted with pHrodo BioParticles at 4°C, and PAM-T4 cells reacted under the same conditions and then further cultured at 37°C and 5% CO2, were resuspended in a PBS solution (FACS buffer) containing 1% sodium azide and 0.5% BSA, and the fluorescence intensity of each cell was measured and analyzed using a BD FACS Calibur (BD Biosciences).

[0071] (Confirmation of CD163 molecule expression) PAM-T4 cells were resuspended in FACS buffer. These cells were reacted with an anti-porcine CD163 mouse monoclonal antibody (clone 2A10 / 11, Bio-Rad) or a mouse IgG1 isotype control antibody (clone MG1-45, BioLegend) diluted 1:1000 in FACS buffer, followed by an anti-mouse IgG1 phycoerythrin-conjugated secondary antibody (clone RMG1-1, BioLegend) diluted 1:1000 in FACS buffer to fluorescently label the CD163 molecules expressed on the cell surface. The fluorescence intensity of each labeled cell was measured and analyzed using a BD FACS calibur.

[0072] (result) As shown in FIG. 5, PAM-T4 and PAM-T5 were shown to stably express SV40T, which is a factor responsible for immortalization.

[0073] As shown in Figure 6, brown granules were confirmed by nonspecific esterase staining. These brown granules disappeared with NaF, indicating that PAM-T4 cells are monocytic cells.

[0074] Figure 7(A) shows images captured with a fluorescence microscope during the phagocytic activity assessment. No signal from the fluorescent probe, which emits light upon phagocytosis, was observed for PK-15, but was observed for PAM-T4. Figure 7(B) shows the fluorescence intensity measured for PAM-T4. Increased fluorescence intensity was observed for PAM-T4 cultured at 37°C compared to culture at 4°C, where many physiological functions of cells are attenuated. These results demonstrate that PAM-T4 possesses clear phagocytic activity, a characteristic of macrophages.

[0075] Figure 8 shows the fluorescence intensity of labeled cells. PAM-T4 was shown to stably express CD163 molecules on the cell surface. CD163 molecules are surface markers for monocytic cells and function as receptors for PRRSV. This also demonstrated that PAM-T4 cells are monocytic cells and are highly susceptible to PRRSV.

[0076] Test Example 4: Examination of the effect of the presence or absence of pig serum on the CD163 molecule expression of immortalized PAM bulk cells Immortalized PAM bulk cells were cultured in a similar manner to the cell line establishment described above, except that they were cultured in a medium without porcine serum. They were seeded onto cell culture plates together with immortalized PAM bulk cells in the presence of 10% porcine serum, fixed with Mildform 10N (Fujifilm Wako Pure Chemical Industries, Ltd.), and permeabilized with Triton X-100 diluted to 0.5% in PBS / BSA. These cells were then sequentially incubated with the above-mentioned anti-porcine CD163 molecule mouse monoclonal antibody diluted 1:1000 in PBS / BSA, followed by DyLight 488-labeled anti-mouse IgG (H+L) secondary antibody (Vector Laboratories) diluted 2:2000 in PBS / BSA. DNA (nuclei) was visualized by treatment with Hoechst 33342 diluted 2:2000 in PBS, and the fluorescent signal was observed under a fluorescence microscope.

[0077] (result) As shown in Figure 9, expression of the CD163 molecule was not observed in immortalized PAM bulk cells cultured in the absence of porcine serum. Because the CD163 molecule functions as a receptor for PRRSV, this suggests that immortalized PAM bulk cells cultured in the absence of porcine serum are not susceptible to PRRSV.

[0078] Test Example 5: ASFV susceptibility evaluation Ten-fold serial dilutions of wild-type ASFV stock were prepared and inoculated into immortalized PAM cell lines (PAM-T4 and its subclone, PAM-T43) and cultured for 4 days. Cytopathic effects were assessed by microscopic observation. Furthermore, ASFV was inoculated into both cell lines, and three days later, red blood cells isolated from pig blood were added and observed microscopically for the presence or absence of hemadsorption (HAD). When hemagglutinating ASFV replicates intracellularly, it is able to adsorb red blood cells to the cell surface, resulting in the formation of rosettes.

[0079] (result) Both PAM-T4 and PAM-T43 showed clear cytopathic effects on day 4 after ASFV inoculation. Furthermore, clear rosettes due to HAD were observed in PAM-T4 and PAM-T43 cells to which red blood cells were added on day 3 after ASFV inoculation. This test example demonstrated that the immortalized PAM cell line of this example is also susceptible to ASFV.

[0080] PAM-T43 was deposited on June 16, 2021, at the Patent Organism Deposit Center of the National Institute of Technology and Evaluation (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818, Japan) under the provisions of the Budapest Treaty under accession number NITE BP-03482.

[0081] The above-described embodiments are intended to explain the present invention and are not intended to limit the scope of the present invention. That is, the scope of the present invention is defined by the claims, not by the embodiments. Various modifications made within the scope of the claims and within the scope of the meaning of the invention equivalent thereto are considered to be within the scope of the present invention.

[0082] This application is based on Japanese Patent Application No. 2020-125310 filed on July 22, 2020. The entire specification, claims, and drawings of Japanese Patent Application No. 2020-125310 are incorporated herein by reference. [Industrial Applicability]

[0083] The present invention is useful in the pig farming industry, particularly in outbreak surveillance and vaccine development for porcine reproductive and respiratory syndrome and African swine fever.

Claims

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4. a first culturing step of culturing primary cultured alveolar macrophages collected from a pig in a medium containing pig serum; an introduction step of introducing a cell immortalization-inducing gene into the cells cultured in the first culture step; a second culture step of culturing the cells into which the cell immortalization-inducing gene has been introduced in a medium containing porcine serum; Including, The cells into which the cell immortalization-inducing gene has been introduced are Susceptible to porcine reproductive and respiratory syndrome virus, A method for producing immortalized porcine alveolar macrophage cell lines.

5. The cells into which the cell immortalization-inducing gene has been introduced are Susceptible to African swine fever virus, The method for producing the immortalized cultured porcine alveolar macrophage cell line according to claim 4.

6. The cell immortalization-inducing gene is SV40 large T antigen gene, The method for producing the immortalized cultured porcine alveolar macrophage cell line according to claim 4 or 5.

7. The cell immortalization-inducing gene is introduced into said cells together with a viral long terminal repeat sequence; A method for producing the immortalized cultured porcine alveolar macrophage cell line according to any one of claims 4 to 6.

8. The viral long terminal repeat sequence is Moloney murine leukemia virus viral long terminal repeat sequence, The method for producing the immortalized cultured porcine alveolar macrophage cell line according to claim 7.

9. The pig A two-week-old pig, A method for producing the immortalized cultured porcine alveolar macrophage cell line according to any one of claims 4 to 8.

10. In the first culture step, The primary cultured alveolar macrophages are cultured for 10 to 24 hours. A method for producing the immortalized cultured porcine alveolar macrophage cell line according to any one of claims 4 to 9.

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