WO2009038283A1

WO2009038283A1 - Vector for cell surface expression of pig igg fc domain, host cell transformed with the vector, and method of manufacturing vaccine against viruses related to pig diseases using the host cell

Info

Publication number: WO2009038283A1
Application number: PCT/KR2008/004424
Authority: WO
Inventors: Bang Hun Hyun; Hyun Mi Pyo; In Joong Kim; Soo Dong Cho; Hyung Kwan Jang; Sung Cheol Moon; Jae Young Song; Se Yeoun Cha
Original assignee: REPUBLIC OF KOREA(Management : Ministry of Agriculture and Forestry, National Veterinary Research & Quarantive Service; Komipharm International Co., Ltd.
Priority date: 2007-09-21
Filing date: 2008-07-30
Publication date: 2009-03-26

Abstract

Provided is a cell surface expression vector for a crystalline fragment (Fc) of porcine immunoglobulin G (IgG), a host cell transfected with the vector, and a method of producing a vaccine against a virus related to a pig disease. More particularly, provided is a vector having a gene encoding an Fc domain of porcine IgG ligased with a gene encoding a transmembrane (TM) gene of porcine transferrin receptor (TR) to fix the TM domain of porcine TR located at an N- terminus of a recombinant protein, and to express the Fc domain of porcine IgG having a hinge, CH2 and CH3 located at a C-terminus of the recombinant protein, a host cell transfected with the vector to express the Fc domain of porcine IgG on its surface, and a method of producing a vaccine against a virus related to a pig disease using the virus reproduced in the host cell. When a virus is reproduced in the host cell, Fc gene is inserted into a viral envelope, and thus vaccines providing excellent immunity to various viruses related to pig diseases can be easily produced.

Description

VECTOR FOR CELL SURFACE EXPRESSION OF PIG IGG FC

DOMAIN, HOST CELL TRANSFORMED WITH THE VECTOR,

AND METHOD OF MANUFACTURING VACCINE AGAINST

VIRUSES RELATED TO PIG DISEASES USING THE HOST

CELL Technical Field

[1] The present invention relates to a vector for cell surface expression of a crystallized fragment (Fc) domain of porcine immunoglobulin G (IgG), a host cell transfected with the vector, and a method of producing vaccines against viruses related to pig diseases using the host cell. More particularly, the present invention relates to a vector carrying a gene encoding an Fc domain of porcine IgG (hereinafter, a porcine IgG Fc domain) and a gene encoding a transmembrane (TM) domain of a porcine transferrin receptor (TR), which are connected by ligation, in such a way that the TM domain of the porcine TR (hereinafter, the porcine TR TM domain) located at the N-terminus of the recombinant protein is fixed to a cell membrane, and the porcine IgG Fc domain, which includes a hinge, CH2 and CH3, located at the C-terminus of the recombinant protein, is expressed on a cell surface. The present invention also relates to a host cell which is transfected with the vector and thus expresses the porcine IgG Fc domain on the cell surface, and a method of producing vaccines against viruses related to pig diseases by infecting the host cell with the viruses and reproducing the viruses in the infected host cell. Background Art

[2] Among all kinds of immunoglobulin, immunoglobulin G (IgG) plays the biggest role in helping to prevent infection by binding to bacteria or their toxins outside a blood vessel and preventing their penetration. Particularly, the IgG is bonded to a foreign bacterium or its toxin, resulting in opsonization, in such a way that a foreign substance is removed from a host by a microphase according to the antibody-dependant cell- mediated cytotoxicity (CDCC) mechanism. The IgG consists of heavy and light chains, formed in a "Y" shape, and is divided into a Fab unit and an Fc unit by Papain. A variable region of the Fab unit can change its morphology and thus bind to almost all kinds of antigen. The Fc unit stimulates cell activation by binding to receptors of a macrocyte and a lymphocyte.

[3] Meanwhile, it has been reported that a human IgG Fc domain expressed on a cell surface stimulates an Fc receptor on a surface of a phagocyte to activate the phagocyte (Stabilia et al., Nat Biotechnol 1998, 16(13): 1357-60). Also, it has been reported that an inactive vaccine is produced using a virus obtained by expressing mouse IgG Fc domains on cell surfaces derived from a porcine kidney cell line CPK and a rabbit kidney cell line RKl 3, infecting the cells with a virus having an envelope, and reproducing the virus in the infected cells. The animal is vaccinated with the resulting virus, which stimulates antibody production and yields excellent resistance to challenge inoculation (Takashima et al., Vaccine 2005, 23:3775-82).

[4] However, research into expression of a porcine antibody Fc domain has not yet been reported, current research is looking only at genetic information of the porcine antibody (Butler et al., Development and Comparative Immunology 2006, 30:199-221), and the DNA sequence of a porcine IgG-subclass antibody has been reported (Kacshovics et al., J Immunol 1994, 153:3566-73).

[5] Meanwhile, the TR is a glycoprotein serving as a receptor on a cell membrane for transferrin delivering iron from outside the cell. The TR exists on the cell surface and plays an important role in introducing iron ions into the cell by strongly binding to transferrin in response to receptor-mediated endocytosis. Most TM domains serving as a receptor on the cell surface are first-class TM domains which have an N-terminus facing outside the cell, and a C-terminus facing a cytoplasm, but the TM domain of the TR is a second-class TM domain which has an N-terminus facing a cytoplasm, and a C-terminus facing outside the cell. Disclosure of Invention

Technical Problem

[6] In the course of studying a method for cell surface expression of the porcine IgG Fc domain, the present inventors found that the porcine IgG Fc domain is expressed on a surface of a cell transfected with a vector constructed to express the porcine IgG antibody Fc domain on the cell surface using a gene encoding the porcine TR TM domain, and thus arrived at the present invention. Technical Solution

[7] The present invention provides a vector for cell surface expression of a porcine IgG

Fc domain.

[8] The present invention also provides a host cell transfected with the vector.

[9] The present invention also provides a method of producing vaccines against viruses related to pig diseases using the host cell.

[10] According to an aspect of the present invention, a vector for cell surface expression of a porcine IgG Fc domain having a gene encoding a porcine TR TM domain and a gene encoding the porcine IgG Fc domain is provided.

[11] According to another aspect of the present invention, a host cell transfected with the vector is provided.

[12] According to still another aspect of the present invention, a method of producing vaccines against viruses related to pig diseases is provided, which includes infecting the host cell with a virus related to a pig disease and reproducing the virus in the infected cell, and producing a live vaccine or an inactive vaccine using the virus reproduced in the previous step.

[13]

[14] Hereinafter, the present invention will be described in detail.

[15] To express a porcine IgG Fc domain on a cell surface, genes coding a porcine IgG Fc domain and a porcine TR TM domain are ligased and inserted into a vector in such a way that the porcine TR TM domain located at an N-terminus of a recombinant protein is fixed to a cell membrane, and the porcine IgG Fc domain, which includes a hinge, CH2 and CH3 or CH2 and CH3, located at a C-terminus of the recombinant protein, is expressed on the cell surface. That is, as shown in FIG. 1, the porcine TR TM domain becomes the N-terminus and is fixed to the cell membrane, and the porcine IgG Fc domain sequentially including a hinge, CH2 and CH3, or CH2 and CH3, becomes the C-terminus and is expressed on the cell surface.

[16] In addition, the vector includes a fluorescent protein-coding gene to readily estimate transfection efficiency, and an antibiotic resistance gene to select a transfectant.

[17] Thus, the vector includes a gene encoding the porcine TR TM domain, and a gene encoding the porcine IgG Fc domain.

[18] The vector may further include a replication origin, a promoter, a fluorescent protein- coding gene, and an antibiotic resistance gene, other than the gene encoding the porcine TR TM domain and the gene encoding the porcine IgG Fc domain, and a host cell transfected with the vector described above expresses the porcine IgG Fc domain on the cell surface.

[19] The gene encoding the porcine TR TM domain may be represented by SEQ. ID. NO:

3 or 16.

[20] The gene encoding the porcine IgG Fc domain may be a gene encoding a hinge, CH2 and CH3, which may be represented by SEQ. ID. NO: 7 or 20. Alternatively, the gene encoding the porcine IgG Fc domain may be a gene encoding CH2 and CH3, which may be represented by SEQ. ID. NO: 8.

[21] Also, the vector includes a fluorescent protein-coding gene. The fluorescent protein- coding gene, preferably, a green fluorescent protein (GFP), is used as a marker protein to readily estimate transfection efficiency with respect to a culture cell by a fluorescent microscope.

[22] The vector may further include an antibiotic resistance gene as a selection marker for a transfectant. The antibiotic resistance gene used in the present invention includes Geneticin (G418), Neomycin, Hygromycin, Zeocin and Puromycin resistance genes, but the present invention is not limited thereto. Preferably, a Zeocin or Puromycin resistance gene is used.

[23] The vector may further include a replication origin, i.e., a specific nucleotide sequence from which the replication is initiated. Preferably, the replication origin may be a eukaryotic or prokaryotic replication origin, for example, a Simian virus 40 (SV40), Epstein Barr virus (EBV) or pUC replication origin.

[24] Furthermore, the vector may include a promoter having transcription initiating activity of a target gene. The promoter may include a binding site for an RNA polymerase and be located upstream in an encoded region. The promoter that can be used in the present invention includes cytomegalovirus (CMV), SV40, thymidine kinase (TK), elongation factor- 1 alpha (EF- lα), β-casein, respiratory syncytial virus (RSV) and CMV5, but the present invention is not limited thereto.

[25] In an exemplary embodiment of the present invention, pEF-1 α is used as a promoter for the genes encoding the porcine TR TM domain and the porcine IgG Fc domain, and CMV is used as a promoter for the fluorescent protein-coding gene (see FIG. 2).

[26] In another exemplary embodiment of the present invention, CMV5 is used as a promoter for the genes encoding the porcine TR TM domain and the porcine IgG Fc domain, and the fluorescent protein-coding gene (see FIG. 20).

[27] In addition, the vector may include non-coding nucleotide sequences promoting or affecting transcription, translation or expression of a target gene, for example, a start codon, a termination codon, a polyadenyl signal, a Kozak, an enhancer, a signal sequence for membrane targeting or secretion, and an internal ribosome entry site (IRES).

[28] The vector having such a configuration in the present invention may have a genetic map shown in FIG. 12, 13 or 25.

[29] In an exemplary embodiment of the present invention, pBGFP-pIgGFc and pBGFP- plgGdHFc plasmids are constructed by cloning a gene encoding a porcine TR TM domain, a gene encoding a porcine IgG Fc domain and a GFP gene, binding the gene encoding the porcine TR TM domain with the gene encoding the porcine IgG Fc domain by ligation, and respectively inserting the ligased genes into pBudGFP carrying the GFP gene. That is, the pGpTRpIgGlFc and the pGpTRpIgG IdHFc are constructed by cutting pGpTRR2 plasmid carrying the gene encoding the porcine TR TM domain with BamHl and SacII, cutting pGpIgGlFcR and pGpIgGldHFcR plasmids having the gene encoding the porcine IgG Fc domain with the same restriction enzymes to obtain the gene encoding the porcine IgG Fc domain, and inserting the obtained genes into the pGpTRR2 plasmid. After that, pBGFP-pTR-Fc and pBGFP-pTR-dHFc plasmids in which a pEF-1 gene, the porcine TR gene, the porcine IgG Fc gene, a pCMV gene and the GFP gene are connected with each other to be sequentially operated are constructed by cutting the pGpTRpIgGlFc and the pGpTRpIgG IdHFc with Notl and Pmel to obtain gene fragments, and inserting the fragments into the same site in the pBudGFP plasmid carrying the CMV promoter, the GFP gene, the SV40p(A) and the Zeocin resistance gene (see FIG. 11).

[30] In another exemplary embodiment of the present invention, genes encoding a porcine

TR TM domain and a porcine IgG Fc domain are cloned into pT7 plasmids in such a way that pT7/TR-Fc plasmid and pT7/Fc plasmid are constructed, respectively. The cloned plasmids are cut with a restriction enzyme and then bound to each other by ligation in such a way that pT7/TR-Fc plasmid is constructed. Subsequently, the pT7/TR-Fc plasmid is cut with a restriction enzyme to obtain the TR-Fc fragment, and the fragment is ligased to pCMV5-IRES-GFP vector to construct pCMV5-IRES-GFP/TR-Fc plasmid in which a CMV5 gene, the TR-Fc gene, an IRES and a GFP gene are connected to be sequentially operated (see FIGS. 22, 24 and 25).

[31] The vector according to the present invention may be introduced to a host cell according to one of the methods that are well known to those skilled in the art. According to the present invention, the introduction of the vector to a cell may be achieved by any method for introducing a nucleotide to a cell, or by a suitable standard technique selected from those that are well known in this field, depending on the host cell. For example, the standard techniques include electroporation, calcium phosphate (CaPO₄) precipitation, calcium chloride (CaCl₂) precipitation, retroviral infection, microinjection, PEG, DEAE-dextran, cationic liposome, and lithium acetate-DMSO methods.

[32] In the present invention, the host cell that can be transfected with a vector may be a mammalian cell. Particularly, the host cells in which viruses related to pig diseases and having an envelope can be reproduced, may include PK- 15 (pig kidney), ST (pig testicles), Vero (monkey kidney), MA- 104 (monkey kidney), MARC (mouse B-cell), Neuro-2A (mouse neuroblastoma), BGK (black goat kidney), RK- 13 (rabbit kidney), BHK (hamster kidney), HRT- 18 (human colon), SK-N-AS (human neuroblastoma), and MDCK (dog kidney). In the present embodiment, the monkey kidney cell Vero and the porcine kidney cell PK- 15 are used.

[33] The host cell expresses the porcine IgG Fc domain on its surface.

[34] In an exemplary embodiment of the present invention, Vero cells are transfected with the vector described above, and the transfected cell lines are selected using Zeocin™ - containing medium. To be specific, one cell line transfected with the pBGFP-pTR-Fc, i.e., Vero-pIgGFcl, and three cell lines transfected with the pBGFP-pTR-dHFc, i.e., Vero-pIgdHFc(l), Vero-pIgGdHFc(2) and Vero-pIgGdHFc(3), are selected (see Experimental example 1.4). [35] Further, expression of the porcine IgG Fc domain in the transfectant selected above is analyzed using ELISA or a fluorescent antibody method (see Experimental example 1.5). As a result, the expression of the porcine IgG Fc domain is detected in the Vero- plgGFcl, the Vero-pIgGdHFc(2) and the Vero-pIgGdHFc(3), but not in the Vero- plgGdHFc(l) which exhibits Zeocin resistance and GFP expression (see FIGS. 15 to 17).

[36] Also, the tranfected cell lines Vero-pIgGFcl, Vero-pIgGdHFc(2) and Vero- pIgGdHFc(3) are screened by Western blot analysis to detect the presence of the porcine IgG Fc domain (see Experimental example 1.6). As a result, an approximately 50-kDa protein band corresponding to the porcine IgG Fc domain can be detected from each of the three cell lines expressing the porcine IgG Fc domain (see FIG. 18).

[37] Finally, the expressions of the porcine IgG Fc domain on cell membranes of the transfected Vero cell lines Vero-pIgGFcl, Vero-pIgGdHFc(2) and Vero-pIgGdHFc(3) and in envelopes of viruses reproduced in the cells are detected using a protein A antibody conjugated with a gold particle (see Experimental example 1.7). As a result, it can be seen from all three cell lines that the gold particle reacts with the cell membrane and the envelope of the Aujeszky's virus reproduced in the cell before being budded from the cell (see FIG. 19).

[38] According to the results described above, among the transfected cell lines, the Vero- pIgGFcl is deposited with the Korean Cell Line Research Foundation (KCLRF) in the Cancer Research Institute of Seoul National University under Accession No. KCLRF- BP-00165 (August 17, 2007).

[39] In another exemplary embodiment of the present invention, PK- 15 cells are transfected with the vector described above, and then the transfected cell lines are selected using Puromycin-containing medium, and specifically, three cell lines transfected with pCMV5-IRES-GFP/TR-Fc (Clones 3-1 to 3-3) are selectively selected (see Experimental example 2.7).

[40] Further, expression of a porcine IgG Fc domain in the selected transfectant is analyzed by ELISA or a fluorescent antibody method (see Experimental example 2.8). As a result, GFP expression is detected in Clones 3-1 and 3-2, and Puromycin resistance and the expression of the porcine IgG Fc domain are detected in all three Clones 3-1 to 3-3 (see FIGS. 27 and 28).

[41] According to the above results, among the selected cell lines, Clone 3-1, named

CPK/SFc, is deposited with the KCLRF in the Cancer Research Institute of Seoul National University under Accession No. KCLRF-BP-OO 154 (April 2, 2007).

[42] According to the present invention, when the cell line expressing the porcine IgG Fc domain on its surface is infected with a pig disease-related virus having an envelope, the virus is reproduced in and then budded from the cell, the porcine IgG Fc domain is contained in a surface of the viral particle. Then, when a live or inactive vaccine is produced using the virus carrying the porcine IgG Fc domain in its envelope, and then a pig is vaccinated with the vaccine, a receptor for the Fc domain present on a surface of an antigen-presenting cell consisting of various phagocytes is stimulated, in such a way that cellular immunity and humoral immunity are increased and thus enhance im- munostimulation in the pig. Accordingly, the cell line in which the porcine IgG Fc domain is expressed on the cell surface can facilitate the production of a variety of vaccines providing excellent immunity to various enveloped viruses that can be reproduced in the cell line.

[43] The present invention provides a method of producing vaccines for pig disease- related viruses, including (a) infecting a cell line expressing a porcine IgG Fc domain on its cell surface with a pig disease-related virus and reproducing the virus, and (b) producing a live or inactive vaccine using the virus reproduced in step (a).

[44] In step (a), the pig disease-related virus may be an enveloped virus, for example, porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), procine rota virus (PoRotaV), Aujeszky's disease virus (ADV), Japanese encephalitis virus (JEV), hog cholera virus (HCV), porcine reproductive and respiratory syndrome virus (PRRSV), simian immunodeficiency virus (SIV) or swine pox virus (SPV).

[45] In step (a), the process of infecting and reproducing viruses in a cell line may be performed by one of the methods well known to those skilled in the art, which will be described in more detail. When cells from the developed cell line are cultured to have a confluency of 50 to 80% to a culture vessel, the cells are infected with a specific virus. An infection time and a reproduction period may be varied according to the kind of the virus. Generally, the infection time is about 3 to 12 hours, and the reproduction period is about 3 days to one week. After infection and reproduction of the viruses, cell culture supernatant is collected. In the cell culture supernatant, the virus containing the Fc domain in its envelope is included. To eliminate the possibility of cell mutation, the cells are subcultured in a specific antibiotic-containing medium for a period of three generations depending on the antibiotic resistance genes inserted into the vector. Finally, the reproduced virus contains the Fc gene in its envelope.

[46] In step (b), the live or inactive vaccine may be produced using the reproduced virus obtained in step (a) by a method well known to those skilled in the art. Particularly, the inactive vaccine may be produced by inactivating the virus by treatment with formalin or a specific method provided by a manufacturer, purifying the inactive virus by a conventional method and performing a germfree test and virus content test to the purified virus, and adding a preservative and a saline solution as a buffer solution for dilution, or adding a stabilizer to the purified virus. The resulting solution may be subjected to a germfree test and a preservative content test. Finally, the vaccine may be processed into a lyophilized or liquid type.

Advantageous Effects

[47] According to the present invention, an expression vector may express a porcine IgG

Fc domain on a cell surface, and when a virus is reproduced in a host cell transfected with the vector, the porcine IgG Fc domain is included in an envelope of the virus.

Thus, a vaccine providing excellent immunity to various viruses related to pig diseases can be easily produced.

Brief Description of the Drawings [48] FIG. 1 shows schematic diagrams of a porcine IgG Fc domain expressed on a cell surface. [49] A— Common type of antigen-binding antibody, names of respective domains are shown.

[50] B and C— Schematic diagrams illustrating different types of the porcine IgG Fc expression. A transferrin domain of a porcine TR is combined with a cell membrane to become an NH-terminus, and the Fc domain sequentially including a hinge, CH2 and

CH3 (B), or CH2 and CH3 (C) of the porcine IgG is expressed to become a COOH- terminus. [51] FIG. 2 shows schematic diagrams of pBGFP-pIgGFc and pBGFP-pTR-pIgGdHFc plasmids for gene insertion, which express the porcine IgG Fc domain on the surface of a mammalian cell. [52] FIG. 3 shows electrophoresis results for a porcine TR gene and a porcine IgG Fc gene achieved by PCR amplification, cloning and digestion with EcoRI. [53] Lane 1— Cloned TR gene from porcine liver tissue

[54] Lane 2— Cloned TR gene from porcine kidney cell line (PK- 15)

[55] Lane 3— 1-kb DNA size marker

[56] Lane 4— Cloned Fc gene from porcine spleen tissue (H+CH2+CH3)

[57] Lane 5— Cloned Fc gene from porcine spleen tissue (CH2+CH3)

[58] FIG. 4 shows a DNA sequence of a porcine TR TM domain gene cloned by inserting a Notl site, a Kozak and a start codon (ATG) at the 5' end, and a BamHI site at the 3' end. [59] FIG. 5 shows a genetic map of pGpTRR2 plasmid carrying a cloned porcine TR gene. [60] FIG. 6 shows a DNA sequence of the porcine IgG Fc domain (plgG-Fc) including a hinge cloned by inserting a BamHI site at the 5' end and a Pmel site at the 3' end. [61] FIG. 7 shows a genetic map of pGpIgGlFcR plasmid carrying the cloned plgG-Fc gene encoding the porcine IgG Fc domain including a hinge. [62] FIG. 8 shows the DNA sequence of porcine IgG Fc domain gene (plgG-dHFc) excluding a hinge, which is cloned by inserting a BamHI site at the 5' end and a Pmel site at the 3' end. [63] FIG. 9 shows a genetic map of pGpIgGldHFcR plasmid carrying the cloned plgG- dHFc gene encoding the porcine IgG Fc domain excluding a hinge. [64] FIG. 10 shows a genetic map of pBugGFP plasmid constructed by inserting GFP gene into pBudCE4.1 plasmid to be expressed by aid of CMV promoter to easily estimate transfection efficiency on the basis of GFP expression. [65] FIG. 11 shows a schematic diagram illustrating processes of constructing pBGFP- pTR-pIgGFc and pBGFP-pTR-pIgGdHFc plasmids for gene insertion, which include insertion of a GFP gene, and insertion of pTR and plgG-Fc genes and pTR and plgG- dHFc genes, respectively. [66] FIG. 12 shows a genetic map of the final form of pBGFP-pTR-dHFc plasmid for insertion of pTR-pIgGdHFc gene designed to express both the GFP gene and the pTR- plgG-dHFc gene. [67] FIG. 13 shows a genetic map of the final form of pBGFP-pTR-Fc plasmid for insertion of pTR-pIgG-Fc gene designed to express both the GFP gene and the pTR- plgG-Fc gene. [68] FIG. 14 shows optical and fluorescent microscopic photographs to detect GFP gene expression after transfecting Vero cells with the pBGFP-pTR-Fc and pBGFP- pTR-dHFc plasmids, respectively.

[69] A— Vero cell transfected with pBGFP-pTR-Fc

[70] B— Vero cell transfected with pBGFP-pTR-dHFc

[71] FIG. 15 shows ELISA results using an anti-porcine IgG(H+L) antibody to detect porcine IgG antibody Fc expression in the transfected cell line according to the present invention.

[72] A— Cell line that does not react with anti-porcine IgG(H+L) antibody

[73] B— Cell line that reacts with anti- porcine IgG(H+L) antibody

[74] FIG. 16 shows fluorescent microscopic photographs using an FITC conjugated anti- porcine IgG antibody to detect porcine IgG Fc expression in the transfected cell line after fixation of the cell according to the present invention. [75] A— Vero-pIgGdHFc(2) cell line

[76] B— Vero-pIgGFcl cell line

[77] C— normal Vero cell line (Vero- WT) that is not transfected (Control group)

[78] FIG. 17 shows a fluorescent microscopic photograph using an FITC conjugated anti- porcine IgG antibody to detect porcine IgG Fc expression in the transfected cell line without fixation according to the present invention. [79] FIG. 18 shows results achieved by Western blot analysis to detect the presence of a porcine IgG Fc protein in the transfected cell line according to the present invention. [80] Lane 1— Vero-WT lysate

[81] Lane 2— Vero-pIgGdHFc(2) cell line lysate

[82] Lane 3— Vero-pIgGFc(3) cell line lysate

[83] Lane 4— Vero-pIgGFcl cell line lysate

[84] Lane 5— Pre-stained protein size marker

[85] FIG. 19 shows electron microscopic photographs of the cell line infected with an

Aujeszky's virus and reacting with a gold particle-binding protein A antibody to detect porcine IgG Fc surface expression in the transfected cell line according to the present invention . [86] A— Gold particle reacted on a cell membrane which becomes an envelope of the virus budded from the cell membrane

[87] B— Gold particle reacted with an envelope at an open round end of the virus

[88] C— Gold particle reacted outside the cell along the cell membrane

[89] FIG. 20 shows a structural diagram of a plasmid vector (pCMV5-IRES-GFP/TR-Fc) to express a porcine IgG Fc gene on the cell surface. [90] FIG. 21 shows a DNA sequence and amino acid sequence of a cloned porcine TR

TM domain gene to express the porcine IgG Fc gene on the cell surface. The gene includes a BamHI restriction enzyme site, a Kozak and a start codon (ATG) at the 5' end and a Kpnl restriction enzyme site at the 3' end to facilitate cloning. [91] FIG. 22 shows a genetic map of T7/TR plasmid obtained by TA cloning of the porcine TR TM domain gene, which is amplified by RT-PCR from total RNA extracted from a porcine kidney cell line, into pT7 blue vector (novagen, USA). [92] FIG. 23 shows a DNA sequence and amino acid sequence corresponding to the porcine IgG Fc domain including a hinge, CH2 and CH3, in which a kpnl restriction enzyme site is included at the 5' end and a BamHI restriction enzyme site is included at the 3' end to facilitate cloning. [93] FIG. 24 shows a genetic map of pT7/Fc plasmid obtained by TA cloning of the porcine IgG Fc domain gene, which is amplified by RT-PCR form total RNA extracted from the porcine spleen tissue, into pT7 blue vector (novagen, USA). [94] FIG. 25 shows a genetic map of pCMV5-IRES-GFP/TR-Fc plasmid which can express both the porcine TR TM domain gene and the porcine IgG Fc gene. [95] FIG. 26 shows a schematic diagram illustrating a series of process of expressing the porcine IgG Fc gene on a surface of a porcine kidney cell line (PK- 15) transfected with the cloned pCMV5-IRES-GFP/TR-Fc plasmid. [96] FIG. 27 shows optical (left) and fluorescent microscopic (right) photographs taken in a dark room to detect GFP gene expression after the transfection of the PK- 15 cell line with the pCMV5-IRES-GFP/TR-Fc plasmid. [97] FIG. 28 shows comparison of optical densities measured at 490nm to estimate the ex- pression of porcine IgG antibody Fc achieved by cell-based ELISA with respect to monoclonal PK- 15 cells selected on the basis of Puromycin sensitivity after the transfection of the PK- 15 cell line with the pCMV5-IRES-GFP/TR-Fc plasmid. Best Mode for Carrying Out the Invention

[98] [Experimental example 11

[99] 1. Cloning of genes encoding porcine TR and porcine IgG Fc domain

[100] <1-1> Isolation of total RNA from porcine live and spleen cells and PK-15 cell line

[101] To clone genes encoding a porcine TR and a porcine IgG Fc domain, RT-PCR was used. To this end, total RNA was isolated from mRNA-rich cells to synthesize cDNA. An actively proliferating cell has a large number of human transferrin receptors (hTR) (Larrick and Cresswell, J Supramol Struct. 1979, 11:579-86).

[102] Total RNA was extracted from a liver cell of a porcine liver tissue or a porcine kidney cell line [ATCC-CCL33] (hereinafter, referred to as PK-15 cell line) to clone porcine TR gene. Also, total RNA was extracted from a spleen cell of a porcine spleen tissue that is a blood-forming organ, to clone a gene encoding a porcine IgG Fc domain. The isolation of the total RNA was achieved using a RNeasy mini kit (Quagen) according to a manufacturer's instructions, and the isolated total RNA was stored at -7O⁰C before being used.

[103] <l-2> Cloning of gene encoding porcine TR TM domain

[104] For cloning of a porcine TR gene, a primer that can polymerize the porcine TR gene was prepared by the following way. The primer was designed to polymerize porcine TR TM domain gene on the basis of the base sequence of the porcine TR gene deposited under GenBank Accession No. AF416763 reported by Python et al. (J Anim Breed Genet, 2005, 122(sl):5-14) with reference to the literature reported by Schneider et al. (J Cell Sci Suppl., 1985, 3:139-49), the hTR base sequence deposited under GenBank Accession No. X01060 and the information of the primer for polymerizing hTR TM domain gene reported by Stabila et al. (Nat Biotechnol., 1998, 16(13):169-49).

[105] Porcine TR sense primer (pTRf) was designed by sequentially elongating a Notl site, a Kozak base sequence and a start codon at the 5' end

(5'-AGCGGCCGC-GCCACC-ATG-ATGGATCAAGCTAGA-S', SEQ.ID.NO:1), and pTR anti-sense primer (pTRr) was designed by elongating a BamHI site (5'-CGCGGATCC-ATCTGTTTTTGATTCTACACG-S', SEQ.ID.NO:2). The customized oligonucleotides were purchased from Bioneer in Korea.

[106] Single-stranded cDNA was synthesized by reaction of 2 g total RNA extracted from the liver cell derived of the porcine liver tissue or the PK-15 cell line in <1-1> with 2pmole pTRr primer (SEQ.ID.NO:2) and Superscript™ II reverse-transcriptase (Invitrogen) at 42⁰C for 50 minutes according to a manufacturer's instructions.

[107] 5ul cDNA of the porcine TR gene was screened using the Expand Long Template

PCR system (Roche, Germany) with IuI of lOpmole/ul pTRf (SEQ.ID.NO: 1) and IuI pTRr primer (SEQ.ID.NO: 2) according to the manufacturer's instructions. The PCR was performed by 10 cycles, each cycle including reactions at 94⁰C (2 minutes), 94⁰C (10 seconds), 54⁰C (30 seconds) and 68⁰C (4 minutes), and another 20 cycles, in which the first cycle included reactions at 94⁰C (15 seconds), 54⁰C (30 seconds) and 68⁰C (4 minutes) and each reaction in the other cycles was performed for 20 more seconds. After that, elongation was performed at 68⁰C for 8 minutes.

[108] PCR product was cut into fragments with EcoRI, and the fragments were separated by sizes using electrophoresis. As a result, as shown in FIG. 3, an about 320-bp band was detected from both Lanes 1 and 2 corresponding to the porcine liver tissues and the PK- 15 samples.

[109] The PCR products were TA-cloned into pGemTeasy (Promega) to obtain pGpTRR2 plasmid, whose base sequence was sequenced. The base sequence of the porcine TR (SEQ.ID.NO:3) and the genetic map of the pGpTRR2 plasmid are shown in FIGS. 4 and 5, respectively.

[110] <l-3> Cloning of gene encoding porcine IgG Fc domain

[111] For cloning of a gene encoding porcine IgG Fc domain, a primer for amplification of the porcine IgG Fc domain gene was designed by the following way. Three kinds of primers were designed to amplify the porcine IgG Fc domain gene with reference to the genetic information and base sequence of the gene deposited under GenBank Accession No. U03778 reported by Kacskovics et al. (J Immunol. 1994, 153(8):3565-73).

[112] A sense primer (plgG-Fcf) was used to clone the porcine IgG Fc domain gene (plgG-Fc) consisting of a hinge, CHl and CH2 domains, or a sense primer (plgG-dHFcf) was used to clone the porcine IgG Fc domain gene (plgG-dHFc) consisting of CHl and CH2 domains except a hinge. An anti-sense primer (plgGlr) was commonly used to amplify both plgG-Fc and plgG-dHFc genes. The primer plgG- Fcf was designed by inserting a BamHl site at 5' end (5'-CGCGGATCC - GTGGCCGGGCCCTCGGTCTTC-3', SEQ. ID. NO:4), the primer plgG-dHFcf was designed by inserting a BamHl site at 5'end (5'-CGCGGATCC - GGAATACACCAGCCGCAAACA-3', SEQ. ID. NO:5), and the anti-sense primer plgGlr was designed by inserting a Pmel site after a stop codon (5'-CGGTTTAAAC TCATTTACCCTGAGTCTTGGA-3', SEQ. ID. NO:6). The customized oligonucleotides were purchased from Bioneer in Korea.

[113] Single-stranded cDNA was synthesized by reaction of 2g of the total RNA extracted from the spleen cell derived from the porcine spleen tissue in procedure <1-1> with 2pmole of the primer plgGlr (SEQ. ID. NO:6) and Superscript™ II reverse- transcriptase (Invirtogen) at 42⁰C for 50 minutes according to a manufacturer's instructions.

[114] For amplification of the plgG-Fc gene, IuI each of the plgG-Fcf primer (lOpmole/ul) and the plgGlr primer were added, and for amplification of the plgG-dHFc gene, IuI each of the plgG-dHFcf and plgGlr primers were added. For each gene, 5ul cDNA of IgGl gene was added to perform PCR using the Expand Long Template PCR system (Roche, Germany) according to the manufacturer's instructions. The reaction was performed by 10 cycles, each cycle including reactions at 94⁰C (2 minutes), 94⁰C (10 seconds), 54⁰C (30 seconds) and 68⁰C (4 minutes), and another 20 cycles, in which the first cycle included reactions 94⁰C (15 seconds), 54⁰C (30 seconds) and 68⁰C (4 minutes), and each reaction in the other cycles performed for 20 more seconds. After that, elongation was performed at 68⁰C for 8 minutes.

[115] After the PCR was completed, PCR products were cut into fragments with EcoRl, the fragments were separated by sizes using electrophoresis. As a result, as shown in FIG. 3, an about 710-bp band was found from the plgGl-Fc gene in Lane 4, and an about 660-bp band was found from the plgGl-dHFc gene in Lane 5.

[116] These genes were TA-cloned into pGemTeasy vector (Promega) to obtain pGpIgGlFcR and pGpIgGldHFcR plasmids, respectively, whose base sequences were sequenced. The base sequences of the plgG-Fc and plgG-dHFc genes are shown in FIGS. 6 (SEQ. ID. NO: 7) and 8 (SEQ. ID.NO:8), and genetic maps of the pGpIgGlFcR and pGpIgGldHFcR plasmids are shown in FIGS. 7 and 9, respectively.

[117] 2. Cloning of GFP gene

[118] For use of GFP as a marker protein to detect transfection efficiency with respect to a culture cell, GFP gene was amplified and cloned, and a plasmid carrying the GFP gene was constructed.

[119] For the amplification of the GFP gene, first, primers were designed by the following way. Sense primer GFPnsiF (5'-ATGCATTAGTTATTAATAGT-S', SEQ. ID. NO:9) and anti-sense primer SV40mluR (5'-ACGCGTTAAGATACATTGAT-S', SEQ. ID. NO: 10) were designed to amplify a specific region from Nsil site before CMV promoter to MIuI site after SV40 polyadenylation signal (p(A)) by PCR with reference to the base sequence and genetic map of phrGFP-Nl plasmid (stratagene) provided from the manufacturer, and these customized oligonucleotides were purchased from Bioneer in Korea.

[120] To amplify the GFP gene by aid of CMV promoter, PCR was performed using lOOpM each of the sense primer GFPnsiF (SEQ. ID. NO:9) and the anti-sense primer SV40mluR (SEQ.ID.NO:10) and O.lug phrGFP-Nl plasmid by 30 cycles at an annealing temperature of 6O⁰C.

[121] PCR product was separated by electrophoresis, according to which an about 1.8-kb band was found, and the band was TA-cloned into pGemTeasy (Promega) to obtain pGGFPR plasmid.

[122] The pGGFPR plasmid was cut into fragments with Spel and EcoRI to obtain an about 1.3-kb gene fragment containing CMV promoter-GFP gene-SV40p(A), which was ligased with a 3.8-kb fragment obtained by digestion of pBudCE4.1 (Invtrogen) plasmid with Spel and EcoRI, thereby obtaining pBudGFP plasmid, whose genetic map is shown in FIG. 10.

[123] 3. Construction of plasmid carrying porcine TR TM gene, porcine IgG Fc domain gene, and GFP gene

[124] After binding of the porcine TR TM gene with plgG-Fc gene or plgG-dHFc gene, the resulting gene was inserted into pBudGFP to obtain pBGFP-pIgGFc or pBGFP- plgGdHFc.

[125] First, the pGpIgGlFcR or pGpIgGldHFcR plasmid carrying the plgG-Fc or plgG- dHFc gene obtained by PCR amplification and cloning in procedure <l-3> was cut into fragments with BamHI and SacII to obtain a 716- or 671-bp gene fragment, which was ligased with a 3.3-kb gene fragment obtained by digestion of pGpTRR2 constructed in procedure <l-2> with BamHI and SacII, thereby obtaining pGpTRpIgGlFc and pGpTRpIgG IdHFc plasmids. These plasmids were analyzed by restriction en- donuclease analysis. The analyzed pGpTRIgGlFc or pGpTRpIgG IdHFc plasmids was cut into with Notl and Pmel to obtain a 1,011- or 966-bp gene fragment, respectively, which was ligased with a 5.0-kb gene fragment to be obtained by digestion of pBudGFP plasmid with Notl and Pmel in Experimental example 2, thereby finally obtaining pBGFP-pTR-Fc and pBGFP-pTR-dHFc plasmid inducing the porcine IgGl antibody Fc on a cell surface (see FIG. 11). The genetic maps of the pBGFP-pTR-Fc and pBGFP-pTR-dHFc plasmids are shown in FIGS. 12 and 13, respectively.

[126] 4. Transfection and selection of cell line using pBGFP-pTR-Fc and pBGFP- pTR-dHFc plasmids

[127] <4-l> Transfection of cell line

[128] First, transfection for preparing a cell line expressing a Fc domain on a surface of a cell membrane was performed by the following way. Each of DNAs of the pBGFP- pTR-Fc and pBGFP-pTR-dHFc plasmids obtained in above procedure 3 was inserted into a Vero cell (ATCC CCL-81, USA) for transfection using FuGENE HD transfection reagent (Roche) according to the manufacture's manual. That is, a co- transfection mixture was prepared by mixing 2ug plasmid DNA with lOOul serum- free medium (α-MEM only supplemented with non-essential amino acids), adding 3ul of a FuGENE HD transfection reagent solution, and adding 800ul serum-free medium. The mixture was stored at room temperature for 15 minutes. Meanwhile, drops of the co- transfection mixture were added to the Vero cell line grown to have a confluency of 80% to a 6-well plate in a monolayer, and the cell line was incubated in a CO₂ incubator at 37⁰C for 24 hours. Then, GFP expression was detected using a fluorescent microscope to estimate transfection efficiency.

[ 129] <4-2> Selection of transfectants

[130] Transfected Fc expression cell lines were selectively selected from the transfected cell lines obtained in procedure <4-2> using Zeocin™-containing medium.

[131] After 24-hour transfection, the GFP expression was confirmed. The cells from each of the 6 wells were transferred to a 100mm petri dish, and cultured in general culture medium, i.e., 500ug/ml Zeocin™ (In vistrgen) -containing DMEM (Gibco) supplemented with 5% fetal bovine serum (Hybriserum, Austria), a non-essential amino acid (Gibco) and antibiotic- antifungal agent (Gibco). The cells were subcultured for a period of 2 to 3 generations, from which live Vero cell colonies were harvested and treated with trypsin (Gibco) to detach cells from one another in a cloning cylinder. The detached cells were cultured by colonies to establish independent cell line. Thus, one cell line, Vero-pIgGFcl, transfected with pBGFP-pTR-Fc, and three cell lines, Vero- plgGdHFc(l), Vero-pIgGdHFc(2) and Vero-pIgGdHFc(3), transfected with pBGFP- pTR-dHFc were selected due to their excellent cell proliferation ability.

[132] 5. Analysis of Fc expression in transfected cell line using ELISA or fluorescent antibody method

[133] <5-l> Analysis using ELISA

[134] Fc expression in the Vero cell lines selected as a monoclone having Zeocin resistance after the transfection in the procedure 4 was detected by ELISA. Here, as a control group, non-transfected Vero cell line was used.

[135] First, the cells were fixed in PBS containing 4% paraformaldehyde (Sigma) at room temperature for 10 minutes, treated with a 1:1 mixture of acetone (Sigma) and methanol cooled down to -2O⁰C in an ice bucket for 2 minutes to increase permeability of the cells, and reacted with horse radish peroxidase (HRP) conjugated anti-porcine IgG(H+L) (KPL) in PBS containing 3% bovine serum albumin (BSA) at room temperature for one hour. Subsequently, SureBlue™ TMB Microwell peroxidase substrate (KPL) was added for color development, and the optical density (OD) was read at 650nm by the ELISA reader (Bio-Rad).

[136] As a result, as shown in FIG. 15, all of the cell lines which did not react with anti- porcine IgG HRP exhibited low OD levels of 0.1 or less regardless of expression of Fc, and all of the Vero-pIgGFcl, Vero-pIgGdHFc(2) and Vero-pIgGdHFc(3) cell lines reacting with anti-porcine IgG HRP exhibited high OD levels of 0.75 or more to confirm the Fc expression. The Vero cell line which was not transfected (Vero- WT) also exhibit a low OD level of 0.1 or less. Meanwhile, the Vero-pIgGdHFc(l) was resistive to Zeocin, and exhibited the GFP expression, but did not exhibit the Fc expression. [137] <5-2> Analysis using fluorescent antibody method

[138] Each of the vero cell line selected as a monoclone having Zeocin resistance after transfection in above procedure 4 was subjected to a fluorescent antibody method to detect the Fc expression. That is, fixed or unfixed cell lines reacted with FITC conjugated porcine IgG(H+L) (KPL) antibodies, in such a way that fluorescence in the cells was detected using a fluorescent microscope.

[139] As a result, fluorescence was not detected from the Vero cell line (Vero-WT) which was not transfected regardless of fixation, but fluorescence was detected from all of the transfected Vero-pIgGFcl, Vero-pIgGdHFc(2) and Vero-pIgGdHFc(3) regardless of fixation, thereby confirming the Fc expression. However, in the fixed cells, strong fluorescence was detected at cytoplasm, for example, around nuclear envelope as shown in FIG. 16, and in the unfixed cells, fluorescence was all detected on the surfaces of the three transfected cells as shown in FIG. 17.

[140] 6. Detection of porcine Fc protein from transfected cell line using Western blot analysis

[141] The presence of Fc protein in the Vero-pIgGFc(l), Vero-pIgGdHFc(2) and Vero- pIgGdHFc(3) cell lines selected as a monoclone having Zeocin resistance after transfection in procedure 4 was detected by Western blot analysis. Here, as a control group, non-transfected Vero cell line (Vero-WT) was used.

[142] The cell lines were analyzed by sodium dodecyl sulphate-poly acrylamide gel electrophoresis (SDS-PAGE), and then subjected to the Western blot analysis using alkaline phosphatase conjugated anti-porcine IgG antibody (hereinafter, referred to as anti-porcine IgG AP) according to the following way.

[143] Each cell line was cultured in a 25cm² flask at 37⁰C in 5% CO² atmosphere for 3 days. Then, the cells were washed twice with PBS, and harvested using a scraper. The cells were precipitated by centrifugation at 12,000rpm for 5 minutes, and then a supernatant was removed. The cell pellet was dissolved in 500ul cell extraction buffer (BIOSOURCE, CA). After that, the resulting solution was subjected to sonication twice for 10 seconds and centrifugation at 14,000rpm for 10 minutes. A supernatant was transferred to a new eppendorf tube, and added with phenylmethylsulfonyl fluoride benzylsulfonyl fluoride (PMSF, Sigma) to give a total concentration of ImM.

[144] The prepared cell extracts were subjected to electrophoresis with a protein size marker through 10% SDS-PAGE gel at 190 volts for about 4 hours. The separated proteins were transferred to a polyvinylidene difluoride (PVDF) membrane by application of an electric field, and the membrane was blocked in a blocking solution, i.e., 5% nonfat dried milk-containing PBS-T solution (0.1% Tween 20 in PBS) at room temperature for one hour. After that, the PVDF membrane reacted with alkaline phosphatase conjugated goat-derived anti-porcine IgG(H+L) AP (BETHYL, Texas) in a blocking solution diluted 1,000 times at room temperature for one hour, and then washed with a PBS-T solution three times. The washed PVDF membrane was developed using a 1-STEP™ NBT-BCIP solution (Pierce, IL).

[145] As a result, as shown in FIG. 18, Fc protein bands corresponding to about 5OkDa can be detected only in the cell lines expressing Fc.

[146] 7. Detection of Fc cell surface expression in transfected cell line using gold particle

[147] Fc expression on a cell membrane of the Vero cell line selected as a monoclone having Zeocin resistance after transfection in procedure 4 and an envelope of a virus reproduced in the cell was detected using a gold particle conjugated protein A antibody.

[148] First, when the selected Vero cell line was grown to have a confluency of 80 to 90% to a 75cm² tissue culture flask in a monolayer, tissue culture medium was removed, and the cells were maintained for infection in an infectious solution prepared by diluting porcine Aujesky's viruses with culture medium to reach a multiplicity of infection of 0.1 to 1 m.o.i. with respect to the cell count at 37⁰C for one hour. Then, the infectious solution was replaced by fresh tissue culture medium to incubate the infected cells at 37⁰C in 5% CO2 atmosphere for 3 days, and the cell were harvested. The cells were washed with PBS and reacted with a IOnm gold particle conjugated protein A antibody that can be strongly bound to the porcine IgG Fc unit (British-Biocell International, PAGlO), and observed under an electron microscope.

[149] As a result, in the non-transfected Vero cell line (Vero-WT) the reaction of the gold particle with the cell membrane and the envelope of the porcine Aujeszky's virus reproduced in the cell cannot be detected. However, in the Vero-pIgGFcl, Vero- pIgGdHFc(2) and Vero-pIgGdHFc(3) cell lines, as shown in FIG. 19, it can be detected that the gold particles reacted with the cell membrane and the envelop of the porcine Aujesky's virus reproduced in the cell before being budded from the cell. Accordingly, it can be confirmed that the porcine Fc is expressed on surfaces of the transfected cell lines. Mode for the Invention

[150] [Experimental example 21

[151] 1. Isolation of total RNA from porcine spleen cell and PK- 15 cell line

[152] Total RNA was extracted from a porcine normal kidney cell line to clone porcine TR TM gene. An immunologically- active organ (spleen) was selected to clone porcine IgG Fc domain gene (Takasima, 2005), and a spleen was extracted from a pig raised by a domestic pig raiser and homogenized. From these spleens, total RNA was extracted using the QuickGene RNA cultured cell kit (Fujifilm) and the QuickGene-810 (Fujifilm) according to the manufacturer's manual. The extracted total RNA was stored at -8O⁰C before being used.

[153] 2. Amplification, cloning and sequencing of porcine TR TM gene from total RNA

[154] A primer was designed to amplify a gene encoding an N-terminus on the basis of the porcine TR complete conding sequence (TR complete cds) deposited under GenBank Accession No. AF416763 with reference to the primer reported by Takasima et al. (2005) to amplify only functional genes capable of expressing porcine IgG Fc gene on a cell membrane.

[155] A porcine TR sense primer (TR-F) was designed to have a cds starting point sequentially including BamHI site, Kozak base sequence and start codon (ATG) to obtain 5'-GGATCC-GCCACC- ATG- ATGGATC AAGCT AGA-3' (30mer; SEQ.ID.NO:14), and a porcine TR anti-sense primer (TR-R) was designed to have kpnl site to obtain 5'-GGTACCATCTGTTTTTGATTCTACACGT-S' (28mer; SEQ.ID.NO:15). These customized oligonucleotides were purchased from Espec oligo in Japan.

[156] lug of the total RNA extracted from the PK- 15 cell in procedure 1 reacted with

2.5uM Oligo dT primer and M-MLV reverse transcriptase (Invitrogen) at 42⁰C for 30 minutes, was maintained at 99⁰C (heat block) for 5 minutes, and cooled down to 4⁰C according to the manufacture's manual to obtain cDNA.

[157] A PCR cocktail was prepared by mixing 0.5ul each of TR-F (2OuM) primer and TR- R (2OuM) primer with lug of the porcine cDNA obtained using the PrimeScript™ RT- PCR kit according to the manufacturer's manual, and the reaction was performed by 30 cycles of pre-denaturation at 94⁰C (1 minute), denaturation at 94⁰C (30 seconds), annealing at 6O⁰C (30 seconds) and elongation at 72⁰C (30 seconds), and post- elongation at 72⁰C for 5 minutes in the PCR machine (MJ research). After the completion of the PCR reaction, IuI of the first PCR cocktail was taken as a template for second PCR, which was performed by 30 cycles of pre-denaturation at 94⁰C (1 minute), denaturation at 94⁰C (30 seconds), annealing at 63⁰C (30 seconds), and elongation at 72⁰C (30 seconds), and then post-elongation at 72⁰C (5 minutes) in the PCR machine (MJ research).

[158] After the second PCR, PCR product was separated by electrophoresis through 1% agarose gel to detect a 318-bp target gene band, and the amplified gene was analyzed by DNA sequencing using the BigDye Terminator Cycle Sequencing Kit Ver. 3.1 (ABI) and the 3130 Automatic Genetic Analyzer (ABI) to estimate homology to the corresponding gene registered in the Genbank data base. The amplified gene was cloned into TA cloning vector (Novagen, USA) to obtain pT7/TR plasmid vector. [159] The DNA sequence (SEQ.ID.NO: 16) and amino acid sequence (SEQ.ID.NO: 17) of the cloned porcine TR TM gene, and the genetic map of the pT7/TR plasmid are shown in FIGS. 21 and 22, respectively.

[160] 3. Amplification, cloning and sequencing of porcine IgG Fc gene from total RNA

[161] As porcine IgG genes disclosed so far were aligned to compare their homology to the target gene, a primer capable of amplifying porcine IgG Fc gene including a hinge, CH2 and CH3 units was designed on the basis of the gene deposited under GenBank Accession No. M81770.

[162] To amplify the porcine IgG Fc gene including a hinge, CH2 and CH3, a porcine IgG Fc sense primer (IgG-F) was designed to have Kpnl restriction enzyme site at the 5' end to obtain 5'-GGTACC-CGTGTTGGAACAAAGACCAAAC-3' (28mer; SEQ.ID.NO: 18), and a porcine IgG Fc anti-sense primer (IgG-R) was designed to have BamHI restriction enzyme site at 5' end to be 5'-GGATCC -

TCATTT ACCCTGAGTCTTGGAG-S' (28mer; SEQ.ID.NO: 19). These customized oligonucleotides were purchased from Espec oligo in Japan.

[163] lug of the total RNA extracted from the spleen of the domestic porcine in procedure 1 reacted with 2.5uM oligo dT primer and M-MLV reverse transcriptase (Invitrogen) at 42⁰C for 30 minutes, was maintained at 99⁰C (heat block) for 5 minutes, and cooled down to 4⁰C according to the manufacture's manual to obtain cDNA.

[164] A PCR cocktail was prepared by mixing 0.5ul each of slgG-F (2OuM) primer and slgG-R (2OuM) primer with lug of the porcine cDNA obtained using the PrimeScript T^M RT-PCR kit according to the manufacture's manual, and the reaction was performed by 35 cycles of pre-denaturation at 94⁰C (1 minute), denaturation at 94⁰C (30 seconds), annealing at 6O⁰C (30 seconds) and elongation at 72⁰C (30 seconds), and then post- elongation at 72⁰C for 5 minutes in the PCR machine (MJ research). After the PCR reaction, IuI of the first PCR cocktail was taken as a template for second PCR, which was performed by 35 cycles of pre-denaturation at 94⁰C (1 minute), denaturation at 94⁰C (30 seconds), annealing at 63⁰C (30 seconds), and elongation at 72⁰C (30 seconds), and then post-elongation at 72⁰C (5 minutes) in the PCR machine (MJ research).

[165] After the second PCR, PCR product was separated by electrophoresis through 1% agarose gel to detect a 711-bp target gene band, and the amplified DNA was sequenced using the BigDye Terminator Cycle Sequencing Kit Ver. 3.1 (ABI) and the 3130 Automatic Genetic Analyzer (ABI) to compare homology with respect to the corresponding gene registered in the Genbank data base. The amplified gene was cloned into TA cloning vector (Novagen, USA) to obtain pT7/Tc plasmid vector.

[166] The DNA sequence (SEQ.ID.NO.20) and amino acid sequence (SEQ.ID.NO. 21) of the cloned porcine IgG Fc gene, and the genetic map of the pT7/Fc plasmid are shown in FIGS. 23 and 24.

[167] 4. Construction of novel pT7blue/TR-Fc plasmid vector in connection of cloned Porcine TR TM gene and porcine IgG Fc gene

[168] The porcine IgG Fc gene (pT7/Fc) cloned into pT7 blue vector after the PCR amplification in procedure 3 was cut into fragments with Kpnl to obtain a 318-bp gene fragment by gel extraction, which was ligased with a 3.6-kb gene fragment (pT7-TR) obtained by digestion of the cloned porcine TR gene into pT7 blue vector after the PCR amplification in procedure 2 with Kpnl to obtain pT7/TR-Fc plasmid. Before ligation, these fragments were subjected to CIAP treatment to prevent self-ligation. The plasmid DNA was transformed into DH5α competent cell, and then analyzed by restriction fragment-length polymorphism to map the plasmid vector. The pT7/TR-Fc plasmid was cut into fragments with BamHI to obtain a 1029-bp porcine TR-IgG Fc (TR-Fc) gene fragment, which was pre-treated before being inserted into a novel plasmid (pCMV5-IRES-GFP). The TR-Fc was subjected to Klenow treatment to form blunt ends. Also, the pCMV5-IRES-GFP vector was cut into fragments with BgIII, and the fragments were subjected to Klenow treatment at 37⁰C for 10 minutes and CIAP treatment at 5O⁰C for 30 minutes. The prepared TR-Fc fragment and the pCMV5-IRES-GFP vector gene fragment were ligased, and thus pCMV5-IRES-GFP/TR-Fc plasmid was constructed. The plasmid DNA was transformed into a DH5α competent cell. Then, the DNA was extracted by the Alkalysis mini-prep method, and treated by RFLP to detect a final product pCMV5-IRES-GFP/TR-Fc. The genetic map of the plasmid is shown in FIG. 25.

[169] 5. Transfection of pCMV5-IRES-GFP/TR-Fc gene fragment into PK-15 cell line

[170] As shown in FIG. 26, the pCMV5-IRES-GFP/TR-Fc plasmid constructed in procedure 4 was transfected into the PK-15 cell using Lipofectamin (Invitrogen) by a conventional mammalian cell transfection into PK-15 derived from a porcine kidney cell in which porcine viruses can be reproduced according to a manufacturer's manual. IuI plasmid DNA was mixed with lOOul serum-free medium (OPTI-MEM, Gibco), and then 6ul Plus reagent solution was added. lOOul of serum- free medium (OPTO-MEM, Gibco) was dispensed into another tube, and 4ul Lipofectamin reagent was added thereto. The mixture was maintained at room temperature for 15 minutes. After the reaction was terminated, all the sample in serum-free medium containing Lipofectamin reagent was taken and mixed with the plasmid DNA (added with Plus reagent) to prepare DNA-liposome solution, which was maintained at room temperature for 15 minutes. 800ul serum- free medium (OPTI-MEM, Gibco) was dispensed into a selected well where the PK-15 cells having a confluency of 50 to 80% were present. After the reaction was completed, the DNA-liposome solution (transfected mixture) was added by drops to the well where the PK-15 cells are grown to have a confluency of 50 to 80% in a monolayer for uniform dispensation. The cells were incubated in a 5% CO₂ incubator at 37⁰C for 12 hours, and then transferred to serum-containing medium (7.5% FCS PRMI 1640) for 24-hour culture. Expression of GFP genes was observed using a fluorescent microscope. Also, Fc expression was observed by a direct immunostain method using HRP-Rabbit Anti-Porcine IgG(H+L) antibody (Zymed).

[171] 6. Selection of drug sensitive clone of transfected Fc expression cell line using Puromvcin-containing medium

[172] Cell culture medium was prepared by adding Puromycin (Sigma) to serum- containing PRMI 1640 (7.5% FCS, Gibco) to give a concentration of 400ug/ml. The cells in the 6-well plate were subjected to the detection of the expression of the GFP genes using the fluorescent microscope, and the cells of each well were transferred to 100mm petri dishes, and cultured in Puromycin-containing medium. After transfection, limited dilution cell cloning was performed to a Puromycin resistance cell line at least three times. Finally, a plurality of stocks of the cell line clones resistive to drugs were obtained, in which the IgG Fc expression was detected.

[173] 7. Detection of porcine Fc expression of Puromycin resistance cell line using ELISA or fluorescent antibody method

[174] The expression of the Fc protein in the clone of each cell line was achieved by detecting the expression of GFP gene using the fluorescent microscope in a dark room (see FIG. 27). Cellular protein fractions were obtained by conventional SDS-PAGE to estimate a molecular weight of the expressed protein, and transferred to a nitrocellulose membrane (Bio-Rad) by a conventional technique to estimate the molecular weight of the expressed protein using mouse anti-swine IgG Fc-specific horseradish peroxidase-conjugated antibody (Sigma).

[175] ELISA was performed to detect Fc expression and measure expression level by the cell line clones. Briefly speaking, 100,000 Fc expression cells per well were seeded in a 96- well tissue culture plate (Corning), and reacted with HRP-Rabbit Anti-Porcine IgG(H+L) antibody (Zymed) for antigen-antibody reaction. SAT -blue was added to the plate, which was maintained for 30 minutes. The OD was read at 490nm using the ThermoMax plate reader (Molecular Devices).

[176] Consequently, as shown in FIG. 12, normal cells exhibited a low OD level of 0.1 or less at 490nm, but the selected clones exhibited stable and high OD levels but different from clones, through the same cellular ELISA. According to the results, it can be confirmed that the Fc gene was expressed in the clones (see FIG. 28).

[177] While the invention has been shown and described with reference to certain exemplary embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Industrial Applicability

[178] According to the present invention, an expression vector may express a porcine IgG Fc domain on a cell surface, and when a virus is reproduced in a host cell transfected with the vector, the porcine IgG Fc domain is included in an envelope of the virus. Thus, a vaccine providing excellent immunity to various viruses related to pig diseases can be easily produced.

[179]

[180]

Claims

[I] A vector for cell surface expression for a crystallized fragment (Fc) domain of porcine immunoglobulin G (IgG) comprising a gene encoding a transmembrane domain of porcine Transferrin receptor and a gene encoding an Fc domain of porcine IgG.

[2] The vector according to claim 1, wherein the gene encoding the transmembrane domain is represented by SEQ. ID. NO: 3 or 16. [3] The vector according to claim 1, wherein the gene encoding Fc domain is a gene encoding a hinge, CH2 and CH3 or a gene encoding CH2 and CH3. [4] The vector according to claim 3, wherein the gene encoding the hinge, CH2 and

CH3 is represented by SEQ. ID. NO: 7 or 20. [5] The vector according to claim 3, wherein the gene encoding the CH2 and CH3 is represented by SEQ. ID. NO: 8. [6] The vector according to claim 1 having a genetic map shown in FIGS. 12, 13 or

25.

[7] A host cell transfected with the vector of claim 1.

[8] The host cell according to claim 7 derived from a mammalian cell.

[9] The host cell according to claim 7 expressing an Fc domain of porcine IgG on a surface of the host cell. [10] The host cell according to claim 7 whose base sequence is deposited under

Accession No. KCLRF-BP-OO 165.

[I I] The host cell according to claim 7 whose base sequence is deposited under Accession No. KCLRF-BP-OO 154.

[12] A method of producing vaccines against viruses related to pig diseases, comprising:

(a) infecting a host cell transfected with a cell surface expression vector carrying a gene encoding a transmembrane domain of porcine transferrin receptor and a gene encoding an Fc domain of porcine IgG with a virus related to a pig disease, and reproducing the virus; and

(b) producing a live or inactive vaccine using the virus reproduced in step (a). [13] The method according to claim 12, wherein the gene encoding the transmembrane domain is represented by SEQ.ID. NO: 3 or 16.

[14] The method according to claim 12, wherein the gene encoding the Fc domain is a gene encoding a hinge, CH2 and CH3 or a gene encoding CH2 and CH3.

[15] The method according to claim 14, wherein the gene encoding the hinge, CH2 and CH3 is represented by SEQ. ID. NO: 7 or 20.

[16] The method according to claim 14, wherein the gene encoding the CH2 and CH3 is represented by SEQ.ID.NO: 8.

[17] The method according to claim 12, wherein the vector has a genetic map shown in FIGS. 12, 13 or 25.

[18] The method according to claim 12, wherein the host cell is derived from a mammalian cell.

[19] The method according to claim 18, wherein base sequence of the host cell is deposited under Accession No. KCLRF-BP-OO 165.

[20] The method according to claim 18, wherein base sequence of the host cell is deposited under Accession No. KCLRF-BP-OO 154.

[21] The method according to claim 12, wherein the Fc domain of porcine IgG is expressed on a surface of the host cell.

[22] The method according to claim 12, wherein the viruses related to pig diseases in step (a) are selected from the group consisting of porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), procine rota virus (PoRotaV), Aujeszky's disease virus (ADV), Japanese encephalitis virus (JEV), hog cholera virus (HCV), porcine reproductive and respiratory syndrome virus (PRRSV), simian immunodeficiency virus (SIV) or swine pox virus (SPV).