KR100984745B1 - A vector for cell surface expression of poultry IgG Fc domain, a host cell transformed with the vector and a manufacturing method of vaccine against viruses related to poultry diseases using the host cell - Google Patents

Abstract

The present invention is a vector for expressing the cell surface of the Crystallized Fragment (hereinafter referred to as "Fc") domain of immunoglobulin G (hereinafter referred to as "IgG") of poultry, a host transformed by the vector The present invention relates to a method for producing a vaccine against poultry disease related viruses using the cells and the host cells. In the case of propagating the virus in the host cell according to the present invention, by including the Fc in the viral envelope, it is possible to easily produce a vaccine having excellent immunity against various poultry disease-related viruses.

Poultry, Chicken IgG, Fc Gene, Surface Expression, Vector

Description

A vector for cell surface expression of poultry IgG Fc domain for cell surface expression of the F. C. domain of poultry immunoglobulin, a host cell transformed with the vector and a poultry disease related virus using the host cell. , a host cell transformed with the vector and a manufacturing method of vaccine against viruses related to poultry diseases using the host cell}

The present invention is a vector for expressing the cell surface of a Crystallized Fragment (hereinafter referred to as "Fc") domain of poultry immunoglobulin G (hereinafter referred to as "IgG"), a host cell transformed by the vector And it relates to a method for producing a vaccine against poultry disease-related viruses using the host cell.

More specifically, the present invention relates to a gene encoding the Fc domain of poultry IgG and a gene encoding a transmembrane (hereinafter referred to as "TM") domain of the poultry transferrin receptor (hereinafter referred to as "TR"). By ligating and inserting it into the vector, the TM domain of poultry TR located at the N-terminus of the recombinant protein is fixed to the cell membrane, and the joint region constituting the Fc domain of poultry IgG located at the C-terminus of the recombinant protein. a vector prepared to express a joint region and a constant region on a cell surface, a host cell transformed with the vector to express the Fc domain of poultry IgG on the cell surface, and a poultry disease related virus to the host cell. The present invention relates to a method for preparing a vaccine against poultry disease-related viruses using viruses that have been infected and propagated.

IgG plays the largest role among immunoglobulins. It helps to fight infections, primarily by binding to bacteria or their toxins outside the blood vessels to prevent their invasion. In particular, IgG binds to macrophage according to antibody-dependent cell-mediated cytotoxicity (CDCC) mechanism by binding and opsonizing with an externally invading bacterium or its toxin. Remove foreign material from the host. IgG is composed of heavy and light chains and forms a Y-shape, which is divided into pap and Fc portions by papain. Variable regions of the Fab region vary in various ways to bind most antigens. The Fc portion binds to receptors on macrophages and lymphocytes to promote cell activation.

On the other hand, according to the literature, the Fc domain of human IgG expressed on the cell surface has been reported to activate the phagocytic cells by stimulating the Fc receptor on the phagocytic surface (Stabila et al., Nat Biotechnol 1998, 16: 1357-1360) ), Expressing the Fc domain of mouse IgG on the cell surface of RK-13, a rabbit kidney cell line, infecting and propagating a virus having an envelope on the cell, to prepare an inactivated vaccine using the virus obtained therefrom, Immunization with animals has been reported to increase the induction of antibody production and to provide excellent protection against challenge (Takashima et al., Vaccine 2005, 23: 3775-3782).

However, no expression studies on poultry antibody Fc domains have been reported to date. However, IgG (IgY) in birds has a predominant distribution in serum and is functionally and structurally related to IgG or IgG in mammals (Warr et al., Immunol. Today, 1995, 16: 392-398). In addition, base sequences of chicken IgG antibodies have been reported (9 parvari et al., EMBO J 1998, 7: 739-744).

TR, on the other hand, is a type of glycoprotein as a cell membrane receptor for transferrin that carries iron extracellularly. The TR, which is present on the cell surface, binds strongly to transferrin and plays an important role in allowing iron ions to enter the cell through receptor mediated endocytosis. Most TM domains that act as receptors at the cell surface are type 1 TM domains with the N-terminus towards the outside of the cell and the C-terminus towards the cytoplasm, while the TM domain of TR is the N-terminus towards the cytoplasm and C The end is characterized by being a type 2 TM domain facing out of the cell.

The inventors of the present invention, while studying the cell surface expression method of the Fc domain of poultry IgG, using a gene encoding the TM domain of the poultry TR to prepare a vector to express the Fc domain of the poultry IgG antibody on the cell surface, using The present invention was completed by confirming that the Fc domain of poultry IgG is expressed on the surface of the transformed cell line.

Accordingly, it is an object of the present invention to provide a vector for cell surface expression of the Fc domain of poultry IgG.

Another object of the present invention is to provide a host cell transformed with the vector.

In addition, another object of the present invention to provide a method for producing a vaccine against poultry disease-related viruses using the host cell.

In order to achieve the above object of the present invention, the present invention provides a vector for cell surface expression of poultry IgG Fc domain comprising a gene encoding the TM domain of poultry TR and a gene encoding the Fc domain of poultry IgG. .

The present invention also provides a host cell transformed with the vector.

In addition, the present invention comprises the steps of (a) infecting the host cell poultry disease-related virus to multiply; And (b) provides a method for producing a vaccine for poultry disease-related viruses comprising the step of preparing a live poison vaccine or inactivated vaccine using the virus propagated in step (a).

The expression vector according to the present invention can express the Fc domain of poultry IgG on the cell surface, and when the virus is propagated in the host cell transformed with the vector, the viral envelope contains the Fc domain of the poultry IgG, thereby various Easily produce vaccines with high immunity against poultry disease-related viruses.

Hereinafter, the present invention will be described in detail.

In the present invention, the term "poultry" includes, but is not limited to, chicken, quail, turkey, guinea chicken, duck, mallard, flower tank, pigeon, and the like.

The inventors ligated a gene encoding the Fc domain of chicken IgG and a gene encoding the TM domain of chicken TR and inserted it into a vector so that the Fc domain of poultry, in particular chicken IgG, was expressed on the cell surface. The TM domain of chicken TR located at the N-terminus of the recombinant protein was immobilized on the cell membrane and the joint and constant regions constituting the Fc domain of chicken IgG located at the C-terminus of the recombinant protein were expressed on the cell surface. That is, as shown in Fig. 1, the TM domain of chicken TR forms the N-terminus and binds to the cell membrane, and the joint region and the constant region constituting the Fc domain of chicken IgG are sequentially expressed to form the C-terminus.

In addition, the vector of the present invention included a fluorescent protein gene so as to immediately confirm the transformation efficiency, and to include the antibiotic resistance gene for selection of the transformant.

Thus, in one embodiment, the vector according to the invention comprises a gene encoding the TM domain of chicken TR and a gene encoding the Fc domain of chicken IgG.

In addition, the vector according to the present invention may further include a replication origin, a promoter, a fluorescent protein gene and an antibiotic resistance gene in addition to the gene encoding the TM domain of chicken TR and the gene encoding the Fc domain of chicken IgG. The host cell transformed with the constructed vector expresses the Fc domain of chicken IgG on the cell surface.

The gene encoding the TM domain of the chicken TR is preferably represented by SEQ ID NO: 3, and the amino acid sequence is represented by SEQ ID NO: 7.

The gene encoding the Fc domain of the chicken IgG may be a gene encoding a joint region and a constant region, preferably these genes are represented by SEQ ID NO: 6, the amino acid sequence is represented by SEQ ID NO: 8.

In addition, the vector of the present invention contains a fluorescent protein gene. The fluorescent protein gene was used as a marker protein for immediately confirming the transformation efficiency of the culture cells with a fluorescence microscope, and preferably, green fluorescent protein (hereinafter referred to as "GFP") can be used.

In addition, the vector of the present invention may include an antibiotic resistance gene as a selection marker of the transformant. Antibiotic resistance genes that can be used in the present invention include, but are not limited to, geneicin (G418), neomycin, hygromycin, zeocin, and puromycin resistance genes. Preferably, the furomycin resistance gene can be used.

In addition, the vector of the present invention may include a replication origin, which is a specific nucleic acid sequence from which replication is initiated. Preferred origins of replication may be eukaryotic or prokaryotic origins of replication, and include, for example, Simian virus 40 (SV40), Epistein Barr Virus (EBV), pUC origin, and the like.

Furthermore, the vector of the present invention may include a promoter having a transcription initiation activity of a target gene. The promoter includes a binding site for the RNA polymerase and may be located upstream of the coding region. Promoters that can be used in the present invention include CMV (Cytomegalovirus), SV40 (Simian virus 40), TK (Thymidine kinase), EF-1α (Elongation factor-1 alpha), β-casein, RSV (Respiratory syncytial virus), CMV5 Etc., but is not limited thereto.

In a preferred embodiment of the present invention, CMV5 was used as a promoter of the gene encoding the TM domain of chicken TR, the gene encoding the Fc domain of chicken IgG, and the fluorescent protein gene (see FIG. 2).

In addition, the vector of the present invention is a non-translated nucleic acid sequence that helps or enhances the transcription, translation or expression of a gene of interest, such as initiation codon, termination codon, polyadenylation signal, Kozak, enhancer, membrane. Signal sequences for targeting or secretion, an Internal Ribosome Entry Site (IRES), and the like.

The vector of the present invention having such a structure may have a genetic map as shown in FIG. 7.

In a preferred embodiment of the present invention, the gene encoding the TM domain of chicken TR and the gene encoding the Fc domain of chicken IgG are cloned into the "pT7" plasmid, respectively, so that the "pT7 / c-TR" plasmid and "pT7 / c-Fc" are cloned. "Plasmids were prepared, and the two cloned plasmids were digested with restriction enzymes and ligated to produce the" pT7 / cTR-Fc "plasmid. Next, a TR-Fc fragment was obtained by treating a restriction enzyme from the “pT7 / cTR-Fc” plasmid, and then ligated to the “pCMV5-IRES-GFP” vector to obtain genes of CMV5, TR-Fc, IRES and GFP. Sequentially operably linked “pCMV5-IRES-GFP / cTR-Fc” plasmids were prepared (see FIGS. 4, 6 and 7).

The vector according to the present invention can be introduced into a host cell according to methods known in the art. The method of introducing a vector of the present invention into a cell includes any method of introducing a nucleic acid into a cell, and may be performed by selecting a suitable standard technique as known in the art depending on the host cell. For example, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, retroviral infection, microinjection, PEG, DEAE-dextran, Cationic liposome method, lithium acetate-DMSO method and the like.

The host cell which can be transformed with the vector of the present invention is preferably an avian cell or a mammalian cell, and is suitable for the purpose of the present invention, that is, as a cell capable of proliferating an enveloped virus that is related to poultry disease. For example, QT35 (quail fibroblasts), LMH (chicken hepatocytes), MDCK (dog kidney cells), Vero (monkey kidney cells), MDBK (th kidney cells), PK-15 (pig kidney cells), ST ( Porcine testicular cells), MA-104 (monkey kidney cells), TF-104 (monkey kidney cells), MARK (mouse B-cells), Neuro-2A (mouse neuroblasts), BGK (black goat kidney cells), RK-13 (Rabbit kidney cells), BHK (hanster kidney cells), HmLu-1 (hamster lung cells), HRT-18 (human colon cells), SK-N-AS (human neuroblasts), and the like. In the embodiment of the present invention, Vero, a kidney cell of monkey, was disclosed.

In a preferred embodiment, the host cell of the present invention expresses the Fc domain of chicken IgG on the surface of the host cell.

In a preferred embodiment of the present invention, after transforming Vero cells with the vector of the present invention, a transformed cell line was selected using a medium containing puromycin, specifically, "pCMV5-IRES-GFP / cTR-". Cells transformed with Fc ″ were selected selectively (see Example 6.).

Furthermore, the present inventors analyzed the Fc domain expression ability of the chicken IgG of the transformant selected above by using ELISA or fluorescent antibody method (see Example 7.). As a result, expression of GFP in "Clone 1", "Clone 3", "Clone 4", "Clone 6", and "Clone 7" was confirmed, and "Clone 1", "Clone 3", "Clone 4" , "Clone 6" and "Clone 7" exhibited furomycin resistance and were found to express the Fc domain of chicken IgG (see Figures 9 and 10).

From the above results, the present inventors named "Clone 3" among the transformed cell lines as "Vero / cFc" and deposited the KCLRF-BP- on February 27, 2008 to the Korea Cell Line Research Foundation in the Cancer Research Institute of Seoul National University. Deposited as 00181.

Therefore, in one preferred embodiment of the present invention, when a cell line expressing the Fc domain of chicken IgG according to the present invention on the cell surface is infected and propagated by a chicken disease-related virus having the envelope of the virus, the virus is spread out of the cell. In the process of release, in the case of a live venom vaccine or inactivated vaccine prepared with a virus containing the Fc domain of chicken IgG in the viral envelope on the particle surface, these vaccines are inoculated into the chicken Immune stimulating effects are enhanced by increasing cell immunity and humoral immunity, such as stimulating receptors for Fc domains expressed on the surface of antigen presenting cells consisting of various phagocytes. Therefore, the cell line expressing the Fc domain of the chicken IgG according to the present invention on the cell surface makes it easy to produce a variety of vaccines excellent in immunity against a variety of viruses with an envelope capable of proliferating in this cell line.

Therefore, the present invention comprises the steps of (a) infecting and propagating poultry disease related virus to a cell line expressing the Fc domain of poultry IgG on the cell surface; And (b) provides a method for producing a vaccine for poultry disease-related viruses comprising the step of preparing a live poison vaccine or inactivated vaccine using the virus propagated in step (a).

The poultry disease related virus in step (a) is preferably an enveloped virus, for example Newcastle Disease Virus (NDV), Infectious Bronchitis Virus (IBV), Avian Influenza Virus (AIV), Avian Pneumovirus (APV), Infectious Laryngitis virus (ILTV), poultry virus (FPV), Marek's disease virus (MDV), chicken leukemia virus (ALLV), retinopathy virus (REV), duck enteritis virus (DEV), turkey coronavirus enteritis virus ( TCoV), West Nile Virus, Pigeon Herpes Virus (PHV), Chicken Sarcoma Virus (ASV), Chicken Myoblastoma Virus (Avian Myeloblastosis Virus), Varicella Zoster Virus (VZV), and the like.

The process of infecting and propagating the virus in the cell line in step (a) may be performed by a method known by those skilled in the art, which will be described in more detail as follows. When a cell line develops 50-80%, it infects certain viruses. The duration of infection and duration of infection can vary depending on the type of virus. In general, the duration of infection is usually about 1 to 2 hours, and the growth period is 3 to 7 days. When the infection and propagation due to the virus is finished, the culture supernatant is collected. The culture supernatant contains a virus containing Fc in the viral envelope. In order to remove the mutant cells, cells are cultured at three intervals in a medium containing a specific antibiotic according to the antibiotic resistance gene inserted into the vector to eliminate the possibility of cell mutation. Viruses propagated in this way will contain Fc in the viral envelope.

In the step (b), as a method for producing a live venom vaccine or inactivated vaccine from the propagated virus obtained in step (a), methods known in the art can be used. In particular, when preparing an inactivated vaccine, the virus is treated by a specific method according to the formalin or the manufacturer's instructions to inactivate the virus, and the inactivated virus is purified by a conventional method, thereby aseptic testing and virus content of the virus stock solution. Experiments are carried out, and a preservative and a buffer saline solution are added, and a dilution mixture or stabilizer is added. The final stock can be subjected to aseptic experiments and preservative content tests. The final vaccine can be prepared in a state such as lyophilization, liquid phase, or the like as desired.

Hereinafter, the specific method of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited to these Examples.

[ Example ]

1. Chicken liver cells and From splenocytes  gun RNA Separation of

The RT-PCR method was used to clone genes encoding the Fc domains of chicken TR and IgG. To this end, total RNA (total RNA) was isolated from cells enriched with mRNA for each to prepare cDNA.

Total RNA was extracted from hepatocytes of chicken liver tissue to clone the chicken TR gene. In addition, to clone a gene encoding the Fc domain of chicken IgG, he selected a chicken spleen with a hematopoietic group and strong immunological activity, followed by homogenization after extraction of the spleen of domestically raised chickens, and a QuickGene RNA Cultured Cell Kit ( Fujifilm) and QuickGene-810 (Fujifilm) were used to extract each total RNA according to the manufacturer's manual. The extracted total RNA was used stored at -80 ℃.

2. Gun RNA Chickens from TR of TM  Amplification of genes, Cloning  And sequence analysis

In order to clone the chicken TR gene, a primer for amplifying the chicken TR gene was prepared by the following method. Based on the cTR sequence of GenBank accession number X55348 as reported by Gerhardt et al. (Unpublished, title: Structure and organization of the chicken transferrin receptor and its cDNA sequence), Stabila et al. (Nat. Biotechnol. 1998, 16: 1357-1360). The primer for amplifying the TM domain gene of chicken TR was designed with reference to the reported primer information for TM domain gene amplification of hTR.

The chicken TR sense primer of SEQ ID NO: 1 (hereinafter referred to as "c-TRf") was designed to be 5'-AGCGGCCGCGCCACCATGGATCATGCCAGAGCA-3 '(33mer) by extending to the 5' end in the order of NotI site, Kozak sequence and initiation codon. The c-TR antisense primer of SEQ ID NO: 2 (hereinafter referred to as "c-TRr") extends the KpnI site to design 5'-GGTACCCCTGGCAGCCAGCTCGATTCGTCC-3 '(30mer) and to synthesize oligonucleotides (Espec oligo, Japan) Was used.

1 μg of total RNA extracted from hepatocytes of chicken liver tissue and Oligo dT primer of 2.5 μM using PrimeScriptTM RT-PCR Kit (Takara) for 5 minutes at 65 ° C. according to the manufacturer's method for sufficient denaturation / After the annealing, the reaction was carried out at 42 ℃ for 30 minutes, the reaction at 95 ℃ (heat block) for 5 minutes, cooled to 4 ℃ to prepare a cDNA.

The PCR reaction solution was prepared according to the manufacturer's method by adding 0.5 μl of 20 pmole / μl of c-TRf primer and c-TRr primer to 1 μg of chicken cDNA obtained using PrimeScriptTM RT-PCR Kit (Takara). For the reaction conditions, after 94 ℃ 1 minute reaction, 35 cycles of 94 ℃ 30 seconds, 58 ℃ 30 seconds, 72 ℃ 30 seconds, and then 72 ℃ 5 minutes reaction using a PCR machine (Takara PCR thermal cycler dicer) Was carried out.

After completion of the PCR reaction, the target gene band of 321 bp was confirmed by electrophoresis on 2% agarose, and the amplified gene was cloned into the TA cloning vector (Novagen, USA) to prepare a “pT7 / cTR” plasmid. DNA sequences were analyzed using Kit Ver. 3.1 (ABI) and 3130 Automatic Genetic Analyzer (ABI), and the degree of homology with the genes registered in the conventional GenBank data base. Gene maps of No. 3) and the “pT7 / cTR” plasmid are shown in FIGS. 3 and 4, respectively.

3. Gun RNA Chickens from IgG of Fc  Amplification of genes, Cloning  And sequence analysis

In order to clone the gene encoding the Fc domain of chicken IgG, a primer for amplifying the Fc domain gene of chicken IgG was designed by the following method. GenBank accession number reported by Parvari et al. (EMBO J. 1988, 7: 739-744) based on structural features by constant region with reference to Suzuki et al. (Glycobilogy 2004, 14: 275-292). A primer for amplifying the Fc domain gene of chicken IgG was designed by referring to the genetic information and the nucleotide sequence of the gene of X07174.

In order to amplify the gene of the Fc domain corresponding to the joint region and constant region domain of chicken IgG, the chicken Fc sense primer (hereinafter referred to as "c-IgGf") of SEQ ID NO: 4 is KpnI on the 5 'side. The restriction enzyme recognition sequence was linked to 5'-GGTACCCAAATCGACGCATGGGGCCACGGGAC-3 '(32mer), and the chicken IgG Fc antisense primer (SEQ ID NO. Was designed to 5'-GCGGCCGCTCATTTACCAGCCTGTTTCTGCA-3 '(31mer) and oligonucleic acid was used by order synthesis (Espec oligo, Japan).

Using 1 μg of total RNA extracted from the spleen of the Korean chicken and 2.5 μM Oligo dT primer in 1, using PrimeScriptTM RT-PCR Kit (Takara) for 5 minutes at 65 ° C. according to the manufacturer's method to fully denaturation / annealing After the reaction, the reaction was carried out at 42 ° C. for 30 minutes, the reaction at 95 ° C. (heat block) for 5 minutes, and cooled at 4 ° C. to prepare cDNA.

The PCR reaction solution was prepared according to the manufacturer's method by adding 0.5 μl of 20 pmole / μl of c-IgGf primer and c-IgGr primer to 1 μg of chicken cDNA obtained using PrimeScriptTM RT-PCR Kit (Takara). As the reaction condition, after 94 ° C 1 minute reaction, 35 cycles at 94 ° C 30 seconds, 62 ° C 30 seconds, 72 ° C 30 seconds, and 72 ° C for 5 minutes, the reaction was performed using a PCR machine (Takara PCR thermal cycler dicer). Was carried out.

After completion of the PCR reaction, the target gene band of 1,340 bp was confirmed by electrophoresis on 1% agarose, and the amplified gene was cloned into the TA cloning vector (Novagen, USA) to obtain the “pT7 / c-Fc” plasmid. DNA sequences were analyzed using BigDye Terminator Cycle Sequencing Kit Ver.3.1 (ABI) and 3130 Automatic Genetic Analyzer (ABI), and the degree of homology with the genes registered in the GenBank data base. Genetic maps of the nucleotide sequence of the chicken Fc (SEQ ID NO: 6) and the "pT7 / cFc" plasmid are shown in FIGS. 5 and 6, respectively.

4. Cloned  chicken TR of TM  Genes and Chicken IgG of Fc  Construction of new “pT7 / cTR-Fc” plasmid vectors by combining genes

After the PCR amplification in 3, the chicken IgG Fc (pT7 / cFc) gene cloned into the pT7 blue vector was treated with KpnI to take a 1,357 bp gene fragment by gel extraction, and after PCR amplification in pT7 blue The chicken transferrin receptor (pT7 / cTR) gene cloned into the vector was also treated with KpnI to obtain a fragment of 3.2 kb of gene, followed by CIAP treatment to prevent self-ligation. Two gene fragments (pT7 / cTR and pT7 / cFc) were ligated to prepare a “pT7 / cTR-Fc” plasmid, and the plasmid DNA thus formed was transformed into DH5α competent cells, followed by an RFLP method using restriction enzymes. (Restriction fragment length polymorphism) was used to confirm the construction of the plasmid vector. The identified “pT7 / cTR-Fc” plasmid was treated with NotI to treat a 1,661 bp chicken TR-IgG Fc gene (cTR). After acquiring -Fc), pretreatment was performed to insert this gene fragment into a new plasmid (pCMV5-IRES-GFP). The pCMV5-IRES-GFP vector was also treated with NotI and subjected to CIAP treatment at 50 ° C for 30 minutes. The prepared cTR-Fc fragment and the pCMV5-IRES-GFP vector gene fragment were ligated to prepare a “pCMV5-IRES-GFP / cTR-Fc” plasmid, and the plasmid DNA thus formed was transformed into DH5α competent cells. Afterwards, DNA was extracted using the Alkalysis mini-prep method and the final product (pCMV5-IRES-GFP / cTR-Fc) ultimately obtained by RFLP method using restriction enzyme was identified. Shown in

5. “ pCMV5 - IRES - GFP Of cTR - Fc "Of the plasmid gene Vero  Cell line Transfect Sean

As shown in Fig. 8, the “pCMV5-IRES-GFP / cTR-Fc” plasmid prepared in step 4 according to the conventional mammalian cell transfection method for Vero derived from monkey kidney cells capable of virus propagation of chickens. Transfection was carried out using Lipofectamin (Invitrogen) on Vero cells according to the manufacturer's manual 1 μg of plasmid DNA was mixed with 100 μl of serum-free medium (OPTI-MEM, Gibco), and 6 μl of Plus reagent solution was added and mixed, 100 μl of serum-free medium (OPTI-MEM, Gibco) was added to another tube, and 4 μl of Lipofectamin reagent was added and left at room temperature for 15 minutes. All samples containing Lipofectamin reagent were added to the serum medium and mixed with plasmid DNA (plus reagent) to prepare a DNA-liposome solution, which was left at room temperature for 15 minutes. Jung of Wells with a confluency of 50-80% were selected to dispense 800 μl of serum-free medium (OPTI-MEM, Gibco) Vero cells that formed a monolayer of 50-80% after the reaction was completed. DNA-liposomal solution (transfection mixture) was added dropwise to the wells in which the wells were present, evenly distributed throughout the wells, reacted for 12 hours in a 5% CO2 incubator at 37 ° C, and serum-added medium (7.5% FCS RPMI 1640), cultured for 24 hours, and examined the expression of GFP gene using a fluorescence microscope.The affinity purified antibody peroxidase-labeled goat anti-chicken IgG (H + L) antibody (hereinafter referred to as “HRP- Anti-chicken IgG ", KPL) was used to confirm the expression of Fc by direct immunostain method.

6. Puromycin  Transformation with Addition Medium Fc  Selection of drug-sensitive clones of expressing cell lines ( selection )

Cell culture medium was prepared so that the concentration of puromycin (sigma) in the serum-added PRMI 1640 (7.5% FCS, Gibco) was 400 μg / ml. 6-well cells whose expression of the GFP gene was confirmed by fluorescence microscopy were transferred to 100 mm petri dish, and then cultured in a medium containing puromycin. After transfection, restriction dilution cell cloning for medicinal resistant cell lines in puromycin was repeated at least three times. Finally, a plurality of stocks of cell line clones showing drug resistance were obtained to confirm IgG Fc expression.

7. Cell ELISA  Or fluorescent antibody method Puromycin  Chickens in resistant cell lines Fc  Expression confirmation

Expression test of the Fc protein for each cell line clone was observed in the dark field environment by using a fluorescent microscope to observe the expression of the GFP gene (see Fig. 9), and in order to check the molecular weight of the expression protein cell protein fraction conventional Obtained through the SDS-PAGE method, the cell fraction was transferred to a cellulose membrane (nitrocellulose membrane, Bio-Rad) according to a conventional technique, and then the molecular weight of the expression protein was confirmed using HRP-anti-chicken IgG antibody (KPL). .

Fc expression using ELISA was also confirmed for each cell line clone. Briefly, the cells are seeded with 80% Fc expressing cells per well in 96-well tissue culture plates, followed by antigen-antibody with HRP-anti-chicken IgG antibody (KPL). The reaction was carried out. The colorant solution SAT blue was added and left for 30 minutes, and then absorbance was measured using a ThermoMax plate reader (Molecular Devices) at O.D490 nm.

As a result, as shown in FIG. 10, the same cell ELISA showed a low value of 0.15 or less at 490 nm through the same cellular ELISA process, and selected clones showed individual differences for each clone, but showed stable high OD values, indicating that Fc was expressed. (See FIG. 10).

8. Radioactive cell proliferation assay Using Puromycin  Chickens in resistant cell lines Fc  Molecules to antigen presenting cells Recognition  Confirm

The spleens of the chickens were extracted and re-dispensed to 2 × 10 ⁶ cells / ml in RPMI1640 (containing 10% FBS) medium. 100 μl of lymphocytes were dispensed on a 96-well cell culture plate and 5 × 10 ⁶ cells / ml of Fc-expressing cell lines were injected into Vero cells and Vero cells, respectively. After 42 hours of incubation at 37 ° C. in a 5% CO 2 incubator, 1 μl Ci [ ³ H] thymidine was added to each well. After 6 hours of incubation, cells were harvested to measure isotope activity.

As a result, as shown in Fig. 11, the normal cell showed the level of 1,500 CPM through the same radioactive cell proliferation assay, and the selected clone showed the level of 5,500 CPM, and the Fc molecule expressed in the selected clone was expressed in the antigen-presenting cells. It was confirmed that it is recognized (see Fig. 11).

1 is a schematic diagram expressing the Fc domain portion of chicken IgG on the cell surface.

A --- In general, antibodies bind to antigen.

B --- In general, antibodies are expressed on the membrane surface of B cells by the type I transmembrane domain.

The C --- inverted potential, the Fc domain is expressed on the cell surface by the type II transmembrane domain.

Fig. 2 is a schematic diagram of a plasmid vector (pCMV5-IRES-GFP / cTR-Fc) for expressing the Fc gene of chicken IgG on the surface of cells.

Figure 3 shows the nucleotide sequence and amino acid sequence of the TM domain of the chicken TR cloned to express the Fc gene of chicken IgG on the surface of the cell. Cloning was facilitated by inserting the NotI restriction enzyme recognition site, Kozak and initiation codon (ATG) at the 5 'end, and the KpnI restriction enzyme recognition site at the 3' end.

Figure 4 shows a schematic diagram of the gene map of the "pT7 / cTR" plasmid TA-cloned the TM domain gene of TR amplified by RT-PCR in the total RNA extracted from the monkey kidney cell line in pT7 blue vector (Novagen, USA).

Figure 5 shows the nucleotide sequence and amino acid sequence of the joint region, constant region of the Fc portion of chicken IgG. Cloning was facilitated by inserting the kpnI restriction enzyme recognition site at the 5 'end and the NotI restriction enzyme recognition site at the 3' end.

Figure 6 shows a schematic diagram of the gene map of the "pT7 / cFc" plasmid TA-cloned the gene of the IgG Fc portion amplified by RT-PCR in the total RNA extracted from chicken spleen tissue in the pT7 blue vector (Novagen, USA).

7 shows a schematic diagram of the gene map of the "pCMV5-IRES-GFP / cTR-Fc" plasmid capable of double expression of the TM domain gene of chicken TR and the Fc gene of chicken IgG.

FIG. 8 is a schematic diagram showing a series of processes in which the cloned “pCMV5-IRES-GFP / cTR-Fc” plasmid is transfected into a monkey kidney cell line (Vero) to express the Fc gene of chicken IgG on the surface of cells.

Figure 9 is a photograph of transfection of the "pCMV5-IRES-GFP / cTR-Fc" plasmid into the Vero cell line, and the expression of the GFP gene using a fluorescent microscope in a dark field environment. A is clone 4, B is clone 6, the left side of the picture is an optical microscope picture, and the right side is a fluorescence microscope picture.

Figure 10 shows the Fc portion of IgG antibodies by transfecting the "pCMV5-IRES-GFP / cTR-Fc" plasmid into Vero cell lines, followed by cellular ELISA on selected monoclonal Vero cells based on puromycin drug sensitivity. Is a graph measuring the absorbance at 490nm.

FIG. 11 is a graph showing the proliferation rate of cells whether the Vero cell line of monoclonal 6 expressing chicken IgG Fc domain is recognized by chicken antigen presenting cells.

<110> Komipharm International Co., LTD. <120> A vector for cell surface expression of poultry IgG Fc domain, a          host cell transformed with the vector and a manufacturing method          of vaccine against viruses related to poultry diseases using the          host cell <160> 8 <170> KopatentIn 1.71 <210> 1 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> c-TRf <400> 1 agcggccgcg ccaccatgga tcatgccaga gca 33 <210> 2 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> c-TRr <400> 2 ggtacccctg gcagccagct cgattcgtcc 30 <210> 3 <211> 321 <212> DNA <213> Chichen <400> 3 agcggccgcg ccaccatgga tcatgccaga gcagcattgt ctaacttgtt cagcgtcgag 60 ccgatgtcgt acacacgttt cagcattgct cggcaaacag atggagacaa cagccacgtg 120 gagatgaagc tgtctgctga tgatgaggaa ggaggggaca ttgaaaggcc agagcacatg 180 catgtcagta tggctcagcc acagagaaat ggcaagagac tctgcttctt ggtcattgca 240 gctgttctcc tccttttgat tgggtttctt attggctact tgagttatcg tggacgaatc 300 gagctggctg ccaggggtac c 321 <210> 4 <211> 32 <212> DNA <213> Artificial Sequence <220> C-IgGf <400> 4 ggtacccaaa tcgacgcatg gggccacggg ac 32 <210> 5 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> c-IgGr <400> 5 gcggccgctc atttaccagc ctgtttctgc a 31 <210> 6 <211> 1340 <212> DNA <213> Chichen <400> 6 ggtacccaaa tcgacgcatg gggccacggg accgaagtca tcgtctcctc cgcgagcccc 60 acatcgcccc cccgattgta ccctctatcc gcctgttgtt ccgactcggc tgtcccgccg 120 gccgtgggct gcctgttgtc cccttcgtcc gccggcggca tctcctggga gggctccgga 180 ggtacggcgg tggccggcag agtttcgggg acccccgtga agctcagctt cgtccgcctc 240 agccccggcg agaagaggaa aagcttcgtc tgcagcgccg cccccggggg ggcgctgctc 300 aaaaaggagg tgcaggtctg ccgggtagat cccgtaccgc ctgtagcccc agaggtgcag 360 gtcctccacc cctcctcctg caccccgagc caatccgagt cggtggagct gttgtgtttg 420 gtgacggggt tctccccggc gtcggcggag gtcgaatggt tggtggacgg agtgggggga 480 cttttggtgg cctcccaaag cccggcggtc cgcagcggat ccacctacag cctgagcagc 540 cgcgtcaacg tcagcggcac cgattggagg gaagggaaga gttacagctg tagggtgagg 600 caccccgcaa ccaacaccgt ggtggaggat cacgtcaagg gatgcccgga cggcgctcag 660 agctgcagcc ccatccagct gtacgccatc ccacccagcc cgggcgagct gtacatcagc 720 ttagacgcca aactgaggtg cctggtggtc aacctgccca gcgattccag cctcagcgtc 780 acctggacca gggagaagag tgggaacctc cggcccgacc cgatggtcct ccaagaacac 840 ttcaacggca cctacagcgc cagcagcgcc gtccccgtca gcacccagga ttggttatcc 900 ggggagaggt tcacctgcac cgtgcagcac gaggagctgc ccctgccgct cagcaagagc 960 gtctacagga acacgggacc caccacccca cctctgatct accccttcgc cccccacccg 1020 gaagagctgt ccctctcccg cgtcaccctg agctgcctgg tccgcggctt ccgcccacgt 1080 gacatcgaga tccggtggct ccgcgaccac cgcgccgttc ccgccaccga attcgtcacc 1140 accgccgtcc tcccggaaga gagaaccgca aacggcgccg gcggtgacgg cgacaccttc 1200 ttcgtgtaca gtaagatgag cgtggagacc gccaagtgga acggcgggac ggtgttcgcc 1260 tgcatggcgg tgcacgaggc gctgcccatg cgcttcagcc agcgcacgct gcagaaacag 1320 gctggtaaat gagcggccgc 1340 <210> 7 <211> 100 <212> PRT <213> chichen <400> 7 Met Asp His Ala Arg Ala Ala Leu Ser Asn Leu Phe Ser Val Glu Pro   1 5 10 15 Met Ser Tyr Thr Arg Phe Ser Ile Ala Arg Gln Thr Asp Gly Asp Asn              20 25 30 Ser His Val Glu Met Lys Leu Ser Ala Asp Asp Glu Glu Gly Gly Asp          35 40 45 Ile Glu Arg Pro Glu His Met His Val Ser Met Ala Gln Pro Gln Arg      50 55 60 Asn Gly Lys Arg Leu Cys Phe Leu Val Ile Ala Ala Val Leu Leu Leu  65 70 75 80 Leu Ile Gly Phe Leu Ile Gly Tyr Leu Ser Tyr Arg Gly Arg Ile Glu                  85 90 95 Leu Ala Ala Arg             100 <210> 8 <211> 441 <212> PRT <213> chichen <400> 8 Gln Ile Asp Ala Trp Gly His Gly Thr Glu Val Ile Val Ser Ser Ala   1 5 10 15 Ser Pro Thr Ser Pro Pro Arg Leu Tyr Pro Leu Ser Ala Cys Cys Ser              20 25 30 Asp Ser Ala Val Pro Pro Ala Val Gly Cys Leu Leu Ser Pro Ser Ser          35 40 45 Ala Gly Gly Ile Ser Trp Glu Gly Ser Gly Gly Thr Ala Val Ala Gly      50 55 60 Arg Val Ser Gly Thr Pro Val Lys Leu Ser Phe Val Arg Leu Ser Pro  65 70 75 80 Gly Glu Lys Arg Lys Ser Phe Val Cys Ser Ala Ala Pro Gly Gly Ala                  85 90 95 Leu Leu Lys Lys Glu Val Gln Val Cys Arg Val Asp Pro Val Pro Pro             100 105 110 Val Ala Pro Glu Val Gln Val Leu His Pro Ser Ser Cys Thr Pro Ser         115 120 125 Gln Ser Glu Ser Val Glu Leu Leu Cys Leu Val Thr Gly Phe Ser Pro     130 135 140 Ala Ser Ala Glu Val Glu Trp Leu Val Asp Gly Val Gly Gly Leu Leu 145 150 155 160 Val Ala Ser Gln Ser Pro Ala Val Arg Ser Gly Ser Thr Tyr Ser Leu                 165 170 175 Ser Ser Arg Val Asn Val Ser Gly Thr Asp Trp Arg Glu Gly Lys Ser             180 185 190 Tyr Ser Cys Arg Val Arg His Pro Ala Thr Asn Thr Val Val Glu Asp         195 200 205 His Val Lys Gly Cys Pro Asp Gly Ala Gln Ser Cys Ser Pro Ile Gln     210 215 220 Leu Tyr Ala Ile Pro Pro Ser Pro Gly Glu Leu Tyr Ile Ser Leu Asp 225 230 235 240 Ala Lys Leu Arg Cys Leu Val Val Asn Leu Pro Ser Asp Ser Ser Leu                 245 250 255 Ser Val Thr Trp Thr Arg Glu Lys Ser Gly Asn Leu Arg Pro Asp Pro             260 265 270 Met Val Leu Gln Glu His Phe Asn Gly Thr Tyr Ser Ala Ser Ser Ala         275 280 285 Val Pro Val Ser Thr Gln Asp Trp Leu Ser Gly Glu Arg Phe Thr Cys     290 295 300 Thr Val Gln His Glu Glu Leu Pro Leu Pro Leu Ser Lys Ser Val Tyr 305 310 315 320 Arg Asn Thr Gly Pro Thr Thr Pro Pro Leu Ile Tyr Pro Phe Ala Pro                 325 330 335 His Pro Glu Glu Leu Ser Leu Ser Arg Val Thr Leu Ser Cys Leu Val             340 345 350 Arg Gly Phe Arg Pro Arg Asp Ile Glu Ile Arg Trp Leu Arg Asp His         355 360 365 Arg Ala Val Pro Ala Thr Glu Phe Val Thr Thr Ala Val Leu Pro Glu     370 375 380 Glu Arg Thr Ala Asn Gly Ala Gly Gly Asp Gly Asp Thr Phe Phe Val 385 390 395 400 Tyr Ser Lys Met Ser Val Glu Thr Ala Lys Trp Asn Gly Gly Thr Val                 405 410 415 Phe Ala Cys Met Ala Val His Glu Ala Leu Pro Met Arg Phe Ser Gln             420 425 430 Arg Thr Leu Gln Lys Gln Ala Gly Lys         435 440  

Claims (18)

Accession number KCLRF-BP-00181 transformed with a vector for cell surface expression of the Fc domain of chicken immunoglobulin G comprising a gene encoding the transmembrane domain of a chicken transferrin receptor and a gene encoding the Fc domain of chicken immunoglobulin G Host cells. The host cell according to claim 1, wherein the gene encoding the transmembrane domain is represented by SEQ ID NO: 3. The host cell of claim 1, wherein the gene encoding the Fc domain is a gene encoding a gene encoding a joint region and a constant region. 4. The host cell according to claim 3, wherein the gene encoding the joint region and the constant region is represented by SEQ ID NO: 6. The host cell of claim 1, wherein the cell surface expression vector has a genetic map of FIG. 7. delete delete delete delete (a) Accession No. KCLRF-, transformed with a vector for cell surface expression of an Fc domain of chicken immunoglobulin G comprising a gene encoding a transmembrane domain of a chicken transferrin receptor and a gene encoding a Fc domain of chicken immunoglobulin G Infecting and propagating a poultry disease related virus to a host cell of BP-00181; And (b) a method for producing a vaccine against poultry disease-related viruses, comprising the step of preparing a live poison vaccine or inactivated vaccine using the virus propagated in step (a). The method of claim 10, wherein the gene encoding the transmembrane domain is represented by SEQ ID NO: 3. 12. The method of claim 10, wherein the gene encoding the Fc domain is a gene encoding a joint region and a constant region. 13. The method of claim 12, wherein the gene encoding the joint region and the constant region is represented by SEQ ID NO: 6. The method of claim 10, wherein the cell surface expression vector of step (a) has the gene map of FIG. 7. delete delete delete The poultry disease-associated virus of step (a) is Newcastle Disease Virus (NDV), Infectious Bronchitis Virus (IBV), Avian Influenza Virus (AIV), Avian Pneumovirus (APV), Infectious Laryngitis Virus (ILTV). ), Poultry virus (FPV), Marek's disease virus (MDV), chicken leukemia virus (ALLV), reticuloendothelial virus (REV), duck enteritis virus (DEV), turkey coronavirus enteritis virus (TCoV), West Nile Virus (West Nile Virus), pigeon herpes virus (PHV), chicken sarcoma virus (ASV), chicken myeloblastosis virus (Avian Myeloblastosis Virus) and varicella zoster virus (VZV) .

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