WO2012015270A2

WO2012015270A2 - Permissive cell line to the porcine reproductive and respiratory syndrome virus

Info

Publication number: WO2012015270A2
Application number: PCT/KR2011/005605
Authority: WO
Inventors: 이창희
Original assignee: 경북대학교 산학협력단
Priority date: 2010-07-30
Filing date: 2011-07-29
Publication date: 2012-02-02
Also published as: KR101253692B1; WO2012015270A3; KR20120012030A

Abstract

The present invention relates to a permissive cell line to the porcine reproductive and respiratory syndrome (PRRS) virus, which expresses the tailless pCD163 polypeptide. The permissive cell line to the porcine reproductive and respiratory syndrome virus of the present invention is highly infectious to the PRRS virus, and thus improves the proliferation rate of the PRRS virus through tailless pCD163 expression with porcine cytoplasmic domain defects. Therefore, the cell line of the present invention may yield porcine reproductive and respiratory syndrome viruses with high efficiency. The thus-obtained PRRS viruses can be effectively used in vaccines for preventing porcine reproductive and respiratory syndrome.

Description

Swine Respiratory Syndrome Virus Susceptible Cell Line

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 A method for preventing or treating swine respiratory syndrome virus (PRRS virus) infection in an animal by administering to the animal an immunogenic composition and an immunogenic composition comprising a viral (PRRS virus) antigen produced according to the method.

Porcine reproductive and respiratory syndrome (PRRS) 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.

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 (PRRS 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. Furthermore, due to the high mutation rate of genes that are characteristic of 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.

To date, the isolation and cultivation of porcine respiratory syndrome virus (PRRS virus) has been carried out using primary cell culture methods using PAM cells collected directly from pig lungs and Marc-145 cells derived from existing monkey kidney cells. .

Most North American viruses are cultured using the Marc-145 cell line, while European viruses are difficult to isolate. Occasionally, isolated European viruses can be adapted to Marc-145 cells via blind-passage for further cultivation, but isolation and cultivation of European and some North American Reproductive Syndrome Syndrome Virus (PRRS virus) is possible in Marc-145 cells. It is known to be quite limited.

Therefore, it is common to use swine lung-derived primary cultured macrophages to isolate European viruses. However, since the effective number of primary cultured PAM cells capable of virus propagation is 1-2 times short and the sensitivity is low during thawing after cryogenic freezing, the use of the primary cultured cells once produced is limited. There is a problem of the hassle and the economic cost of extracting from the negative pig lung.

In order to overcome this drawback, PAM cell lines have been recently constructed by inducing immortalization using supercultured pig alveolar macrophages. However, 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. As a result of the establishment of a pig-derived cell line susceptible to PRRS virus, it is still limited to virus isolation and in vitro studies.

Recently porcine sialoadhesin (pSn) and porcine CD163 (pCD163) have been identified as porcine respiratory syndrome virus (PRRS virus) cell receptors and co-expressing these pSn and pCD163 in swine respiratory syndrome virus (PRRS virus) non-sensitive cells. It has been reported that pCD163 alone expression can produce susceptible cell lines that allow virus propagation. In addition, susceptibility to both European and North American viruses has been confirmed by inducing pCD163 expression in existing virus-insensitive immortalized PAM cell lines. These cell lines are expected to be important materials for the development of the next generation of vaccines.

In addition, in order to develop high-efficiency vaccines, it is necessary to build a virus proliferation and culture system using receptor-expressing cell lines, as well as to establish a cell line capable of producing a high titer virus by enhancing the proliferative and infectious ability of the PRRS virus (PRRS virus). Situation.

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. 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.

In addition, the present invention

infecting swine respiratory syndrome virus (PRRS virus) susceptible cell line expressing tailless pCD163 polypeptide (PRRS virus); And

It provides a method for producing swine respiratory syndrome virus (PRRS virus) comprising culturing the cells infected with the swine respiratory syndrome virus (PRRS virus).

In addition, the present invention

Transforming porcine respiratory syndrome virus (PRRS virus) insoluble animal cell line with a recombinant vector comprising a tailless pCD163 gene;

Inducing expression of tailless pCD163 in said transformant to increase susceptibility to PRRS virus infection of porcine respiratory syndrome virus (PRRS virus) proliferative insoluble animal cell lines;

Infecting the cell line with increased porcine respiratory syndrome virus (PRRS virus) susceptibility with PRRS virus ex vivo or in vitro;

Culturing the cells infected with the porcine respiratory syndrome virus (PRRS virus); And

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.

In addition, the present invention

Provided is 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.

In addition, the present invention

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. Provide a method.

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.

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.

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).

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).

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).

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).

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).

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).

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).

[Description of Symbols in Drawing]

WT CD163: wild type pCD163, tailless CD163: tailless pCD163

First, the CD163 will be described in detail.

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. 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. Recent molecular functional studies have shown that SRCR domain 5 of CD163 is required for infection of porcine respiratory syndrome. (Van Gorp H, Van Breedam W, Van Doorsselaere J, Delputte PL, Nauwynck HJ (2010) Identification of the CD163 protein domains involved in infection of the porcine reproductive and respiratory syndrome virus, J Virol 84: 3101-3105).

In the present invention, the term 'tailless pCD163' means that the cytoplasmic tail of porcine CD163, which is a cell receptor of porcine respiratory syndrome, is defective.

In the present invention, '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.

In the present invention, 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. Alternatively, 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.

In the present invention, '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 However, it is not limited thereto.

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.

In addition, 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. For example, lysates from uninfected cells (optionally purifying viral particles or extracting selected viral proteins) may be coated onto an ELISA plate to detect and quantify antibodies to viruses in pig serum.

In addition, 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

infecting porcine respiratory syndrome virus (PRRS virus) sensitive cell line expressing tailless pCD163 polypeptide (PRRS virus); And

The present invention also provides

(a) transforming porcine respiratory syndrome virus (PRRS virus) insoluble animal cell line with a recombinant vector comprising a tailless pCD163 gene;

(b) inducing expression of tailless pCD163 in said transformant to increase susceptibility to PRRS virus infection of porcine respiratory syndrome virus (PRRS virus) proliferative insoluble animal cell lines;

(c) infecting the cell line with increased porcine respiratory syndrome virus (PRRS virus) susceptibility with PRRS virus ex vivo or in vitro;

(d) culturing the cells infected with the porcine respiratory syndrome virus (PRRS virus); And

(e) providing a method for producing swine respiratory syndrome virus (PRRS virus) antigens, comprising the step of separating the swine respiratory syndrome syndrome (PRRS virus) antigen from the cell culture.

In the present invention, 'pig respiratory syndrome virus (PRRS virus) antigen' refers to an amino acid sequence that induces an immune response to PRRS in the host. As used herein, an antigen is a concept comprising the full length of a PRRS protein, an analog thereof or an immunogenic fragment thereof.

In the present invention, "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. For example, 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. Such techniques are known in the art and are described, for example, in US Pat. No. 4,708,871; Geysen et al. (1984) Proc. Natl. Acad. Sci. USA 81: 3998-4002; Geysen et al. (1986) Molec. Immunol. 23: 709-715. Also included in this definition are synthetic antigens, examples include polyepitopes, flanking epitopes, and other recombinant or synthetic antigens. See, eg, Bergmann et al. (1993) Eur. J. Immunol. 23: 2777-2781; Bergmann et al. (1996), J. Immunol. 157: 3242-3249; Suhbier, A. (1997), Immunol, and Cell Biol. 75: 402-408; Gardner et al., (1998) 12th World AIDS Conference, Geneva, Switzerland, June 28-July 3, 1998.

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.

In order to induce the expression of step (b), the expression can be induced by a method generally used in the art. For example, 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. By transfection, genes can be continuously expressed. In an embodiment of the present invention, 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

The PRRS viral antigen may be a full length sequence of a PRRS protein, an analog thereof or an immunogenic fragment thereof.

As used herein, the term “immunogenic composition” refers to any pharmaceutical composition comprising a PRRS viral antigen and is used synonymously with a vaccine.

Such 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. Thus, the term immunogenic composition as used herein includes compositions containing completely killed or attenuated PRRS virus as well as the immunogenic fragment compositions described below.

In the present invention, 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. Thus, 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.

Those skilled in the art will appreciate that the compositions used in the present invention may comprise known injectable physiologically acceptable sterile solutions. In the preparation of ready-to-use solutions for parenteral injection or infusion, isotonic aqueous solutions, such as saline or corresponding plasma protein solutions, are readily available. In addition, 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.

In addition, the immunogenic compositions of the present invention may further comprise one or more other immunomodulators such as interleukin, interferon or other cytokines.

If you look at this in more detail:

When the immunogenic composition of the present invention is a composition containing a fully killed PRRS virus (hereinafter referred to as 'killing vaccine'), 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. Examples of 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.

When 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. Can be. For example, 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. In the case of solutions or injections, 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. For example, 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. Preferably 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. For example, a therapeutically or prophylactically effective amount when the vaccine of the attenuated day the virus strain 1 1 × 10 ⁶ to ^{1 × 10 10 (colony forming unit} ) cfu / ㎖ preferably 1 day 1 × 10 ⁶ to It may range from 1 × 10 ⁸ cfu / ml.

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. Provide a method of treatment.

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. Preferably 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.

Advantages and features of the present invention and methods for achieving them will be apparent with reference to the embodiments described below in detail. However, the present invention is not limited to the embodiments disclosed below, but will be implemented in various forms, and only the embodiments are intended to complete the disclosure of the present invention, and the general knowledge in the technical field to which the present invention pertains. It is provided to fully convey the scope of the invention to those skilled in the art, and the present invention is defined only by the scope of the claims.

실시예 1: tailless pCD163 제조Example 1: Preparation of tailless pCD163

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. First, 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. As a result, 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. 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.

To construct a recombinant tailless pCD163 gene, 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. After culturing the colonies generated the next day and extracting the plasmid DNA using it, 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.

Example 2 Preparation of Vector Inserting Tailless pCD163 Gene and Wild-type pCD163 Gene

As a vector for preparing a plasmid containing a cell receptor tailless pCD163 gene, 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). Supernatants containing the recombinant retrovirus packaged with each target gene were inoculated into BHK-21 cell line (PRRS virus insoluble animal cell line), and 48 hours later, neomycin (Invitrogen) was added at 500 / concentration. MRNA expression of wild-type pCD163 and tailless pCD163 was detected via RT-PCR in each cell clone (BHK-pCD163 and BHK-tailless pCD163) selected by antibiotic treatment, resulting in the successful insertion of each pCD163 gene into cells. It was confirmed.

In the above, 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. In other words, 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.Molony Murine Leukemia virus (MMLV) gag / pol gene is a retrovirus capsid and viral enzymes (reverse). transcriptase, integrase, and protease), 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.

Example 4 Analysis of Expression of Tailless pCD163 and Wild-type pCD163 in Transgenic Cell Lines

<4-1> 형광항체법 이용 <4-1> Fluorescent antibody method

First, a fluorescent antibody method was performed to verify cell surface expression of CD163 protein. To this end, after culturing each cell, 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. As a result, 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). ).

<4-2> 웨스턴 블랏 (western blot)<4-2> western blot

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).

Example 5 Identification of Increased Porcine Respiratory Syndrome Virus (PRRS Virus) Infection and Proliferation in Tailless pCD163 Expressing Cell Lines

Susceptibility testing of 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).

As a result of counting and analyzing the number of virus-infected cells, it was also confirmed that the BHK-tailed pCD163 increased up to about 3 times compared to BHK-pCD163 cells (control) (see FIG. 7).

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).

In addition, the comparative analysis of the expression rate of the newly synthesized PRRS viral nonstructural (NSP2) and structural (N) proteins after virus inoculation showed a significantly elevated increase in the BHK-tailless pCD163 cell line, whereas The production rate of the corresponding CD163 did not show a big difference (see FIG. 9).

Finally, comparing the 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).

Claims

porcine respiratory syndrome virus (PRRS virus) sensitive cell line, wherein the tailless pCD163 polypeptide is expressed.
infecting porcine respiratory syndrome virus (PRRS virus) sensitive cell line expressing tailless pCD163 polypeptide (PRRS virus); And

A method for producing swine respiratory syndrome virus (PRRS virus) comprising culturing cells infected with the swine respiratory syndrome virus (PRRS virus).
Transforming porcine respiratory syndrome virus (PRRS virus) insoluble animal cell line with a recombinant vector comprising a tailless pCD163 gene;

Inducing expression of tailless pCD163 in said transformant to increase susceptibility to PRRS virus infection of porcine respiratory syndrome virus (PRRS virus) proliferative insoluble animal cell lines;

Infecting the cell line with increased porcine respiratory syndrome virus (PRRS virus) susceptibility with PRRS virus ex vivo or in vitro;

Culturing the cells infected with the porcine respiratory syndrome virus (PRRS virus); And

PRRS virus antigen production method comprising the step of separating the porcine respiratory syndrome virus (PRRS virus) antigen from the cell culture.
The method of claim 3,

The tailless pCD163 gene is a PRRS virus antigen production method, characterized in that the base sequence of SEQ ID NO: 1.
The method of claim 3,

The porcine respiratory syndrome virus (PRRS virus) insoluble animal cell line BHK-21 (small hamster kidney), PK15 (pig kidney), PAM (ATCC CRL-2843, -2844, -2845), ST (pork ring), Production of PRRS virus antigens, characterized in that it is one selected from the group consisting of Vero (monkey kidney cells), Marc-145 (from monkey kidney cells), Madin-Darby bovine kidney (MDBK) and Madin-Darby canine kidney (MDCK) cells Way.
The method of claim 3,

The vector for producing a recombinant vector containing the tailless pCD163 gene is one selected from the group consisting of a pFB-Neo retroviral vector, a pFB-Zeo retroviral vector, and a mammalian expression vector. PRRS virus antigen production method.
An immunogenic composition comprising a PRRS virus antigen produced in a porcine respiratory syndrome virus (PRRS virus) sensitive cell line, wherein a tailless pCD163 polypeptide is expressed.
The method of claim 7, wherein

Immunogenic composition, characterized in that the porcine respiratory syndrome virus (PRRS virus) antigen is a killed PRRS virus, attenuated live PRRS virus or any immunogenic fragment of PRRS virus.
The method of claim 7, wherein

Immunogenic composition, characterized in that the immunogenic composition is a death vaccine.
The method of claim 7, wherein

Immunogenic composition, characterized in that the immunogenic composition is an attenuated live vaccine.
To prevent or treat 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. Way.
The method of claim 11,

And said animal is a pig. A method for preventing or treating infection with porcine respiratory syndrome virus (PRRS virus).