WO2012015270A2 - Lignée cellulaire sensible au virus du syndrome reproducteur et respiratoire porcin - Google Patents

Lignée cellulaire sensible au virus du syndrome reproducteur et respiratoire porcin Download PDF

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WO2012015270A2
WO2012015270A2 PCT/KR2011/005605 KR2011005605W WO2012015270A2 WO 2012015270 A2 WO2012015270 A2 WO 2012015270A2 KR 2011005605 W KR2011005605 W KR 2011005605W WO 2012015270 A2 WO2012015270 A2 WO 2012015270A2
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virus
prrs virus
respiratory syndrome
pcd163
tailless
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WO2012015270A3 (fr
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이창희
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경북대학교 산학협력단
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C12N2510/02—Cells for production
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/10011—Arteriviridae
    • C12N2770/10034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/10011—Arteriviridae
    • C12N2770/10051—Methods of production or purification of viral material

Definitions

  • the present invention is a pig respiratory syndrome virus (PRRS virus) susceptible cell line, pig respiratory syndrome virus (PRRS virus) production method, pig respiratory syndrome syndrome (PRRS virus) antigen production method, pig respiratory syndrome syndrome (PRRS virus) production
  • PRRS virus pig respiratory syndrome virus
  • Porcine reproductive and respiratory syndrome is a swine infectious disease that has been prevalent in the swine industry of the world as well as in Korea since it was first reported in North America in the late 1980s. In terms of the economic damage caused by the disease, it is reported to be about $ 600 million (about 540 billion won) annually in the United States, and it is estimated that about 100 billion won is lost annually in Korea.
  • PRRS virus Porcine respiratory syndrome
  • PRRS virus the causative agent of PRRS, is divided into European type 1 (European genotype) and North American type 2 (North American genotype) according to antigenic and genetic characteristics.
  • Swine Respiratory Syndrome Virus causes infection mainly in porcine alveolar macrophage (PAM), a natural target host, alters normal immune function and decreases host defense response to infection. May cause persistent infections up to 5-6 months.
  • PAM porcine alveolar macrophage
  • RNA viruses a variety of viral variants have been reported.
  • the disease is characterized by genital insufficiency (late lactate, stillbirth and mummies) in sows and gilts, high mortality in piglets and respiratory disease in pigs of all ages.
  • porcine respiratory syndrome virus PRRS virus
  • PAM cell lines have been recently constructed by inducing immortalization using supercultured pig alveolar macrophages.
  • PAM cell lines (ATCC CRL-2843, -2844, -2845), which have been constructed with the results of the PRRS virus susceptibility test, have not been able to infect and proliferate.
  • porcine sialoadhesin pSn
  • porcine CD163 pCD163
  • PRRS virus porcine respiratory syndrome virus
  • the present inventors overcome the shortcomings of swine lung-derived primary cultured macrophages or MARC-145 (monkey kidney embryonic cells) used for isolation of porcine respiratory syndrome virus (PRRS virus), and are highly infectious against swine respiratory syndrome virus.
  • MARC-145 monkey kidney embryonic cells
  • PRRS virus porcine respiratory syndrome virus
  • a cell line with improved proliferative capacity of the European and North American PRRS viruses was developed, and the present invention was completed by revealing that the cell line can be usefully used for the production of a vaccine capable of preventing swine respiratory syndrome.
  • the present invention provides a porcine respiratory syndrome virus (PRRS virus) sensitive cell line, wherein the tailless pCD163 polypeptide is expressed.
  • PRRS virus porcine respiratory syndrome virus
  • PRRS virus infecting swine respiratory syndrome virus
  • PRRS virus susceptible cell line expressing tailless pCD163 polypeptide
  • PRRS virus swine respiratory syndrome virus
  • PRRS virus porcine respiratory syndrome virus
  • It provides a method for producing swine respiratory syndrome virus (PRRS virus) antigen comprising the step of separating the pig respiratory syndrome syndrome (PRRS virus) antigen from the cell culture.
  • PRRS virus swine respiratory syndrome virus
  • an immunogenic composition comprising a PRRS antigen produced in a porcine respiratory syndrome virus (PRRS virus) sensitive cell line, wherein a tailless pCD163 polypeptide is expressed.
  • PRRS virus porcine respiratory syndrome virus
  • Preventing or treating Swine Respiratory Syndrome Virus (PRRS virus) infection by administering to the animal an immunogenic composition comprising a PRRS virus antigen produced in a pig respiratory syndrome virus (PRRS virus) sensitive cell line expressing a tailless pCD163 polypeptide.
  • PRRS virus Swine Respiratory Syndrome Virus
  • the cell line of the present invention can improve the proliferation rate of the virus with high infectivity against porcine respiratory syndrome virus (PRRS virus) through the expression of tailless pCD163 lacking the cytoplasmic domain of porcine CD163. Swine respiratory syndrome virus (PRRS virus) can be obtained with high efficiency, and thus obtained swine respiratory syndrome virus can be useful in the production of vaccines that can prevent swine respiratory syndrome.
  • PRRS virus porcine respiratory syndrome virus
  • FIG. 1 is a schematic diagram of the structural domains of wild type pCD163 and tailless pCD163 (WT CD163: wild type pCD163, tailless CD163: tailless pCD163).
  • 2 is an electrophoretic image of a tailless pCD163.
  • FIG. 3 is a schematic diagram of the pFB-Neo-tailless pCD163 vector gene map.
  • Figure 4 is a photograph comparing each expression protein in BHK-pCD163 (control) and BHK-tailless pCD163 transformed cell line of the present invention using a fluorescent antibody method (WT CD163: wild type pCD163, tailless CD163: tailless pCD163).
  • FIG. 5 is a photograph comparing each expressed protein in BHK-pCD163 (control) and BHK-tailless pCD163 transformed cell line of the present invention using Western blot (WT CD163: wild type pCD163, tailless CD163: tailless pCD163).
  • FIG. 6 is a photograph comparing the infection rate after PRRS virus inoculation to BHK-pCD163 (control) and BHK-tailless pCD163 transformed cell line of the present invention using fluorescent antibody method (WT CD163: wild type pCD163, tailless CD163: tailless pCD163).
  • FIG. 7 is a graph showing the percentage of virus infected cells after PRRS virus inoculation into BHK-pCD163 (control) and BHK-tailless pCD163 transformed cell lines of the present invention (WT CD163: wild type pCD163, tailless CD163: tailless pCD163).
  • FIG. 8 is a comparison of infection rate using FACS analysis after PRRS virus inoculation to BHK-pCD163 (control) and BHK-tailless pCD163 transformed cell line of the present invention (WT CD163: wild type pCD163, tailless CD163: tailless pCD163).
  • FIG. 9 is a comparison of viral and CD163 protein expression using Western blot after PRRS virus inoculation to BHK-pCD163 (control) and BHK-tailless pCD163 transgenic cell lines of the present invention (WT CD163: wild type pCD163, tailless CD163: tailless). pCD163).
  • FIG. 10 is a graph comparing virus proliferation rates of BHK-pCD163 (control) and BHK-tailless pCD163 transformed cell lines of the present invention (WT CD163: wild type pCD163, tailless CD163: tailless pCD163).
  • WT CD163 wild type pCD163, tailless CD163: tailless pCD163
  • the present invention provides a porcine respiratory syndrome virus (PRRS virus) sensitive cell line, wherein the tailless pCD163 polypeptide is expressed.
  • PRRS virus porcine respiratory syndrome virus
  • CD163 is a glycoprotein of 130 kDa (kilodaltons), which is characterized by expression only at the cell surface of monocytes and macrophage.
  • the glycoprotein consists of an extracellular region consisting of nine cysteine-rich scavenger receptor cysteine-rich (SRCR) domains, a single transmembrane domain and a short cytoplasmic tail.
  • SRCR cysteine-rich scavenger receptor cysteine-rich
  • the main role of CD163 is to remove hemoglobin from plasma. Because the extracellular region of CD163 contains an important component for the ligand-binding process, it was logically assumed that a site that could interact with the swine respiratory syndrome virus would be located in a similar region.
  • the term 'tailless pCD163' means that the cytoplasmic tail of porcine CD163, which is a cell receptor of porcine respiratory syndrome, is defective.
  • 'swine respiratory syndrome virus (PRRS virus) susceptible cell line' refers to a cell line with a high virus infection rate and high proliferation rate due to easy infection with swine respiratory syndrome virus.
  • the PRRS virus sensitive cell line of the present invention may be made by introducing a tailless pCD163 gene into an animal cell and then inducing the expression of the tailless pCD163 to increase susceptibility to PRRS virus infection.
  • a method of introducing a tailless pCD163 gene into an animal cell includes transformation, transfection, electroporation, nuclear injection, liposomes, micelles, and ghost cells, which are well known in the art and commonly performed in the art.
  • a method of fusion to a carrier such as a protoplast may be used, and preferably, transformation is used.
  • the animal cell into which the tailless pCD163 gene is introduced may be, but is not limited to, porcine respiratory syndrome virus (PRRS virus) insoluble animal cell line in a preferred embodiment.
  • PRRS virus porcine respiratory syndrome virus
  • 'pig respiratory syndrome virus (PRRS virus) proliferative insoluble animal cell line' refers to an animal cell line that is not infected by swine respiratory syndrome virus.
  • Said porcine respiratory syndrome virus (PRRS virus) insoluble animal cell line in a preferred embodiment BHK-21 (cub hamster kidney), PK15 (pig kidney), PAM (ATCC CRL-2843, -2844, -2845), ST One cell selected from the group consisting of (pork), Vero (monkey kidney cells), Marc-145 (from monkey kidney cells), Madin-Darby bovine kidney (MDBK) and Madin-Darby canine kidney (MDCK) cells
  • PRRS virus porcine respiratory syndrome virus
  • BHK-21 cub hamster kidney
  • PK15 pig kidney
  • PAM ATCC CRL-2843, -2844, -2845
  • PCK Madin-Darby canine kidney
  • Porcine Respiratory Syndrome Virus (PRRS virus) sensitive cell lines expressing the tailless pCD163 polypeptide of the present invention can be used for the production of PRRS virus, wherein the PRRS virus produced using the cell line of the present invention is a killed vaccine or attenuated live vaccine. It can be used as.
  • PRRS virus Porcine Respiratory Syndrome Virus
  • porcine respiratory syndrome virus (PRRS virus) susceptible cell lines expressing the tailless pCD163 polypeptide of the present invention can be used to grow viruses for the purpose of producing viral antigens for diagnostic kits.
  • lysates from uninfected cells may be coated onto an ELISA plate to detect and quantify antibodies to viruses in pig serum.
  • live or inactivated viruses grown on porcine respiratory syndrome virus (PRRS virus) sensitive cell lines which express tailless pCD163 polypeptide, can be used to immunize animals to produce polyclonal, monospecific or monoclonal antibodies. Can be.
  • the antibodies thus produced can be used as the basis for diagnostic assays for the detection and quantification of viruses in porcine serum and other biological samples.
  • the present invention also provides
  • PRRS virus porcine respiratory syndrome virus
  • PRRS virus sensitive cell line expressing tailless pCD163 polypeptide
  • PRRS virus swine respiratory syndrome virus
  • the present invention also provides
  • 'pig respiratory syndrome virus (PRRS virus) antigen' refers to an amino acid sequence that induces an immune response to PRRS in the host.
  • an antigen is a concept comprising the full length of a PRRS protein, an analog thereof or an immunogenic fragment thereof.
  • immunogenic fragment refers to a fragment of a protein that induces an immune response in the host, including one or more epitopes.
  • Such fragments can be identified using any number of epitope mapping techniques known in the art. See, eg, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66 (Glenn E. Morris, Ed., 1996) Humana Press, Totowa, New Jersey.
  • linear epitopes can be determined by the simultaneous synthesis of multiple peptides corresponding to a portion of a protein molecule on a solid support, reaction of the peptides with the antibody while the peptides are attached to the support, and the like.
  • the tailless pCD163 gene of step (a) has a nucleotide sequence of SEQ ID NO: 1.
  • PRRS virus proliferative insoluble animal cell line of step (a) is BHK-21 (small hamster kidney), PK15 (pig kidney), PAM (ATCC CRL-2843, -2844, -2845), ST (pork ring), Vero ( Monkey kidney cells), Marc-145 (from monkey kidney cells), Madin-Darby bovine kidney (MDBK) and Madin-Darby canine kidney (MDCK) cells, but may be one selected from the group consisting of, but not limited to.
  • the vector used to prepare the recombinant vector containing the tailless pCD163 gene of step (a) includes a pFB-Neo retroviral vector, a pFB-Zeo retroviral vector and a mammalian expression vector (including an antibiotic resistant open reading frame).
  • mammalian expression vector may be selected from, but is not limited to.
  • the expression can be induced by a method generally used in the art.
  • a mammalian expression vector containing a common antibiotic (neomycin, zeocin, puromycin, hygromycin, etc.) resistant open reading frame may be used to safely transmit to a target animal cell line using a transfection reagent.
  • a transfection reagent By transfection, genes can be continuously expressed.
  • a method of expressing a tailless pCD163 gene by using a stable cell line using a retroviral vector system was used.
  • the present invention also provides
  • an immunogenic composition comprising a PRRS antigen produced in a porcine respiratory syndrome virus (PRRS virus) sensitive cell line, wherein a tailless pCD163 polypeptide is expressed.
  • PRRS virus porcine respiratory syndrome virus
  • the PRRS viral antigen may be a full length sequence of a PRRS protein, an analog thereof or an immunogenic fragment thereof.
  • immunogenic composition refers to any pharmaceutical composition comprising a PRRS viral antigen and is used synonymously with a vaccine.
  • immunogenic compositions can be used to prevent or treat diseases or conditions associated with PRRS virus infection in a subject.
  • Preferred immunogenic compositions can induce, stimulate or enhance an immune response against the PRRS virus.
  • immunogenic composition as used herein includes compositions containing completely killed or attenuated PRRS virus as well as the immunogenic fragment compositions described below.
  • an 'immunogenic fragment composition does not contain all of the antigens derived from or homologous to an antigen derived from a PRRS virus but comprises at least one immunogenic polypeptide or antigen, ie, an immunogenic fragment antigen. It means the composition containing. Such compositions are substantially free of complete PRRS virus.
  • immunogenic fragment compositions are prepared from immunogenic polypeptides or recombinant analogs thereof that have been at least partially purified or fractionated from a PRRS virus.
  • the immunogenic composition of the present invention may comprise one or more pharmaceutically acceptable carriers for the PRRS viral antigen.
  • Pharmaceutically acceptable carriers used herein include any and all solvents, dispersion media, coatings, adjuvant, stabilizer, diluent, preservative, antibacterial, antifungal, isotonic, absorbent, and the like.
  • compositions used in the present invention may comprise known injectable physiologically acceptable sterile solutions.
  • isotonic aqueous solutions such as saline or corresponding plasma protein solutions
  • the immunogenic compositions (vaccines) of the present invention may comprise diluents, tablets, stabilizers or adjuvant.
  • Diluents can include water, saline, dextrose, ethanol, glycerol, and the like.
  • Isotonic agents, in particular sodium chloride, dex may include mannitol, sorbitol and lactose.
  • Stabilizers include, in particular, alkali salts of albumin and ethylenediaminetetraacetic acid.
  • Adjuvants may include aluminum hydroxide and aluminum phosphate, saponins, water emulsions, water emulsions.
  • the immunogenic composition of the present invention may further contain a pharmaceutically acceptable salt, preferably phosphate, in physiologically acceptable concentrations, in addition to the diluents, climbers, stabilizers and adjuvant.
  • a pharmaceutically acceptable salt preferably phosphate
  • immunogenic compositions of the present invention may further comprise one or more other immunomodulators such as interleukin, interferon or other cytokines.
  • the killing vaccine can be prepared by conventional vaccine preparation methods in the art. For example, if the virus propagates to high titers, virus antigenic masses are obtained and treated with formalin, betapropriolactone (BPL), bicomponent ethyleneimine (BEI) or gamma rays, or other known to those skilled in the art. Deactivate by the method. The inactivated virus is then mixed with a pharmaceutically acceptable carrier (such as saline solution) and any adjuvant.
  • a pharmaceutically acceptable carrier such as saline solution
  • adjuvants include, but are not limited to, aluminum hydroxide, oil-in-water and water-in-oil emulsions, AMPHIGEN, saponins, interleukins, interferons and cytokines.
  • Inactivation by formalin may be performed by mixing the virus suspension with 37% formaldehyde and a final formaldehyde concentration of 0.05%.
  • the virus-formaldehyde mixture is tested for residual live virus by mixing by constant stirring at room temperature for about 24 hours and then inactivating the virus mixture for growth on the appropriate cell line.
  • Inactivation by BEI can be performed by mixing the virus suspension with 0.1M BEI (2-bromo-ethylamine in 0.175 N NaOH) to a final BEI concentration of 1 mM. After the virus-BEI mixture is mixed by constant stirring at room temperature for about 48 hours, 1.0 M sodium thiosulfate is added at a final concentration of 0.1 mM. Continue mixing for an additional 2 hours. Inactivated virus mixtures are tested for residual live virus by assaying for growth on appropriate cell lines.
  • the immunogenic composition of the present invention is a composition containing an attenuated PRRS virus (hereinafter referred to as 'attenuated live vaccine')
  • a method for preparing an attenuated live vaccine may be performed using a conventional vaccine preparation method in the art.
  • a conventional vaccine preparation method in the art.
  • the virus produced through the PRRS virus production method described above can be concentrated, frozen and stored before being vaccinated to mix the virus with a pharmaceutically acceptable carrier (such as saline solution) and any adjuvant. .
  • the immunogenic composition (vaccine) of the present invention may be in any form known in the art, such as, but not limited to, liquid and injection.
  • propylene glycol and, if necessary, may contain an amount of sodium chloride sufficient to prevent hemolysis (eg about 1%).
  • the immunogenic compositions (vaccines) of the invention can be administered by any means of administration known in the art.
  • the administration can be administered directly intravenously, intramuscularly, transdermal, mucosal, intratracheal or subcutaneous.
  • the vaccine may be administered systemically or locally.
  • the immunogenic composition (vaccine) of the present invention may be administered to a mammal, and examples of the mammal may be humans, cows, pigs, goats, dogs, sheep, and the like, but are not limited thereto.
  • the mammal is a pig.
  • the immunogenic composition (vaccine) of the present invention can be administered in an effective amount.
  • An 'effective amount' means an amount required to alleviate or eliminate a symptom to be treated or prevented and may be appropriately selected by those skilled in the art.
  • the present invention also provides
  • Preventing or reinforcing swine respiratory syndrome virus (PRRS virus) infection in animals by administering to the animal an immunogenic composition comprising a PRRS virus antigen produced in a pig respiratory syndrome virus (PRRS virus) sensitive cell line expressing a tailless pCD163 polypeptide.
  • PRRS virus pig respiratory syndrome virus
  • the animal may be a mammal, and the mammal may be human, cow, pig, goat, dog, sheep, or the like, but is not limited thereto.
  • the mammal is a pig.
  • the invention also provides the use of an immunogenic composition for preventing or treating an infection caused by a PRRS virus in a mammal.
  • CD163 gene manipulation was performed using pGEM-T-pCD163 plasmid DNA into which a previously cloned wild-type CD163 open reading frame (ORF) was inserted.
  • ORF open reading frame
  • the PCR-based site-directed mutagenesis was used to replace the 1067th glutamine codon CAG, the amino acid sequence immediately after the transmembrane domain (the amino acid sequence where the cytoplasmic tail begins), with the stop codon TAG.
  • protein translation no longer proceeds at this site, resulting in a loss of the cytoplasmic tail region, resulting in the synthesis of a tailless pCD163 protein that is shorter than the wild-type CD163.
  • FIG. 1 A schematic diagram of the structure and domain composition of the full-length wild-type pCD163 and tailless pCD163 proteins is shown in FIG. 1.
  • a mutagenic sense primer (5'-CTCATTTGGACT T AG AAGCGAAGAC-3 ') and an antisense primer (5'-GTCTTCGCTT CT A AGTCCAAATGAG-3') were designed.
  • DNA template (5ng /) 3, 10 pmole sense and anti-sense primer and high-fidelity pfx DNA polymerase using 95 30 seconds after reaction of 95 30 seconds, 55 1 minute, 68 12 minutes 16 cycles using the primers PCR was carried out.
  • the entire PCR product was treated with DpnI restriction enzyme for 2 hours and then transformed into E. coli XL1 Blue using the final product of 2.
  • the site was extracted using a sense primer (5'-CGGGATCCATGGACAAACTCAGAATGG-3 ') and an antisense primer (5'-GTCTTCGCTTGGATCCCTAAGTCCAAATGA-3').
  • the amplified gene was detected by electrophoresis on 0.8% agarose gel (see FIG. 2). Also, the DNA sequence analysis confirmed the deletion of the corresponding region.
  • the entire nucleotide sequence of the constructed tailless pCD163 is shown in SEQ ID NO: 1.
  • pFB-Neo retroviral vector (Stratagene) including a neomycin resistant open reading frame was used.
  • pGEM-pCD163, pGEM-tailless pCD163 and pFB-Neo plasmid were cut with BamHI and electrophoresed on 0.8% agarose gel to identify the bands, and then sections were separated using a gel extraction kit.
  • Wild type pCD163 (about 3.3 kb) and tailless pCD163 (about 3.2 kb) truncated with BamHI were ligation with pFB-Neo vector (about 6.6 kb) to finally prepare pFB-Neo-pCD163 and pFB-Neo-tailless pCD163 plasmids, respectively.
  • the gene map of the pFB-Neo-tailless pCD163 vector is shown in FIG. 3.
  • Example 3 Selection of transformed cell lines expressing tailless pCD163 and wild-type pCD163 using Retroviral gene transfer system
  • PFB-Neo-pCD163 (plasmid encoded with wild-type pCD163) or pFB-Neo-tailless pCD163 (plasmid encoded with tailless pCD163), pVPack-VSV-G Stratagene, a plasmid encoded with vesicular stomatitis virus G glycoprotein) and pVPack-GP (plasmid encoded with Stratagene, Molony Murine Leukemia virus (MMLV) gag / pol gene) were transfected using Lipofectamine 2000 (Invitrogen).
  • pVPack-VSV-G and pVPack-GP serve to encode viral proteins and enzymes that retroviruses play a necessary role in replication mechanisms including cell adsorption.
  • vesicular stomatitis virus G glycoprotein forms the surface of the virus during assembly of retroviruses and plays a role in binding and adsorption of target cells.
  • McMolony Murine Leukemia virus (MMLV) gag / pol gene is a retrovirus capsid and viral enzymes (reverse).
  • transcriptase responsible for reverse transcription and insertion of target genes, allowing them to assemble retroviral particles containing viral proteins and enzymes that play a role in replication mechanisms, including cell adsorption, by inserting the encoded plasmids. do.
  • a fluorescent antibody method was performed to verify cell surface expression of CD163 protein.
  • the cells were fixed with 4% paraformaldehyde, and the anti-CD163 antibody was reacted with the fixed cells.
  • the secondary antibody expressed as goat anti-mouse IgG-Alexa Fluor as a fluorescent antibody.
  • strong cell surface staining was confirmed in the BHK-pCD163 and BHK-tailless pCD163 cell clones as shown in FIG. 5, which demonstrated that the respective cells expressed high levels of the corresponding CD163 protein (see FIG. 4). ).
  • Western blot techniques were also used to detect wild type and tailless CD163 proteins in BHK-pCD163 and BHK-tailless pCD163 cells. Obtained by cell protein fraction in SDS-PAGE, transfer of cell fraction to nitrocellulose membrane and expression of protein using anti-CD163 antibody as primary antibody and goat anti-mouse IgG-HRP as secondary antibody And the molecular weight was confirmed. As shown in FIG. 6, wild-type CD163 and slightly smaller tailless CD163 protein bands of 130 kDa were identified in each cell clone (see FIG. 5).
  • PRRS virus (VR-2332) was performed using established BHK-pCD163 and BHK-tailless pCD163 cell lines. After the virus inoculation, each cell was infected with the PRRS virus nucleocapsid protein-specific monoclonal antibody (SDOW17), and the virus-infected cell clusters were identified. It could be confirmed (see FIG. 6).
  • the percentage of cells stained with PRRS virus-specific monoclonal antibody was determined by FACS analysis, which was about 28% in BHK-pCD163 (control) and 75% in BHK-tailless pCD163 after virus inoculation. It became. This result shows about 3 times the elevated viral infectivity in BHK-tailless pCD163 cells as in the fluorescent antibody method (see Fig. 8).
  • virus proliferation rate in each cell line showed an increased viral titer of about 2 log or more in the BHK-tailless pCD163 cell line, and the increased growth kinetics was also confirmed in the one-step growth curve analysis (see FIG. 10).

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Abstract

La présente invention concerne une lignée cellulaire sensible au virus du syndrome reproducteur et respiratoire porcin (PRRS) qui exprime le polypeptide pCD163 dépourvu de queue. La lignée cellulaire sensible au virus du syndrome reproducteur et respiratoire porcin de la présente invention est fortement infectieuse vis-à-vis du virus du syndrome reproducteur et respiratoire porcin et améliore ainsi le taux de prolifération du virus du syndrome reproducteur et respiratoire porcin par l'expression de pCD163 dépourvu de queue à des défauts dans le domaine cytoplasmique porcin. Par conséquent, la lignée cellulaire de la présente invention peut générer des virus du syndrome reproducteur et respiratoire porcin ayant une efficacité élevée. Les virus du syndrome reproducteur et respiratoire porcin obtenus ainsi peuvent être efficacement utilisés dans des vaccins pour la prévention du syndrome reproducteur et respiratoire porcin.
PCT/KR2011/005605 2010-07-30 2011-07-29 Lignée cellulaire sensible au virus du syndrome reproducteur et respiratoire porcin WO2012015270A2 (fr)

Applications Claiming Priority (2)

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KR10-2010-0073887 2010-07-30
KR1020100073887A KR101253692B1 (ko) 2010-07-30 2010-07-30 돼지생식기호흡기증후군 바이러스 감수성 세포주

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WO2012015270A3 WO2012015270A3 (fr) 2012-04-19

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KR101703783B1 (ko) * 2014-12-18 2017-02-08 제주대학교 산학협력단 참돔 지느러미 유래 세포주의 개발

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KR20070017361A (ko) * 2004-04-23 2007-02-09 파마시아 앤드 업존 캄파니 엘엘씨 바이러스에 대한 세포성 증식 허용성 인자, 및 이의 용도

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KR20070017361A (ko) * 2004-04-23 2007-02-09 파마시아 앤드 업존 캄파니 엘엘씨 바이러스에 대한 세포성 증식 허용성 인자, 및 이의 용도

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021070785A1 (fr) * 2019-10-11 2021-04-15 国立研究開発法人農業・食品産業技術総合研究機構 Procédé de production de virus infectieux porcin

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KR20120012030A (ko) 2012-02-09
WO2012015270A3 (fr) 2012-04-19

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