KR20200093899A - Immortalized porcine alveolar macrophage cell line and method for detecting antigenic peptide using the same - Google Patents

Abstract

The present invention provides an immortalized porcine lung macrophage cell line prepared by transfecting an expression vector into which a human telomerase reverse transcriptase (hTERT) gene is inserted and an expression vector into which a simian vacuolating virus40 large T (SV40LT) gene is inserted, a preparing method thereof, and a detection method of the antigenic peptide using the same. Since the immortalized porcine lung macrophage cell line iPAM61 and iPAM303 prepared from the present invention is very similar in shape, growth rate, and antigen presentation function to the primary porcine lung macrophage line, it can be used to evaluate the interaction between virus-derived antigen peptides and SLA molecules so that it can be usefully used as a method for selecting antigenic peptides for developing excellent vaccines in the field of veterinary medicine.

Description

Immortalized porcine alveolar macrophage cell line and method for detecting antigenic peptide using the same}

The present invention relates to a method for detecting an immortalized porcine lung macrophage cell line and an antigenic peptide using the same, and more specifically, a gene expressing human telomerase reverse transcriptase (hTERT) in pig primary lung macrophage and Immortalized porcine lung macrophage cell line prepared by transfecting a vector into which a gene expressing monkey virus 40 large T antigen (simian vacuolating virus 40 large T, SV40LT) is inserted, and a peptide derived from porcine circovirus type 2 and porcine main tissue It relates to a method for detecting an antigenic peptide for vaccine development by measuring the binding force of a conformation complex class II molecule.

Peptide presentation to helper T (CD4 ⁺ ) lymphocytes and cytotoxic T (CTL, CD8 ⁺ ) lymphocytes by major histocompatibility complexes (MHC) is essential for adaptive immunity in vertebrates. MHC class I and II molecules represent intracellular and extracellular peptides, respectively. Studying the formation of peptide-MHC complexes is important to expand knowledge about epitope selection for vaccine development, evolutionary mechanisms of pathogens for host immune evasion, and the impact of MHC polymorphisms on disease resistance and susceptibility.

It has been suggested that the absolute binding ability of a peptide to MHC is a good indicator of immunogenicity. Accordingly, various bioinformation tools and resources have been reported, including MHC-PPM, NetMHCpan-3.0, NetMHCII, and MHCIIpan, which can predict the binding affinity of peptides to MHC molecules. However, it is necessary to experimentally verify the interaction of the predicted MHC molecule and peptide and its effect on the immune system. To this end, cell-free systems using purified MHC molecules, artificial antigen production systems transforming E. coli or yeast with recombinant MHC class II α and β chains and natural MHC molecules on the cell surface Several methods have been developed, including the mammalian cell system.

For non-human species such as swine, studies dealing with the interaction between MHC and epitopes have been limited. Diseases lead to serious losses in terms of productivity, so the development of disease resistant and effective vaccines against major infectious diseases of pigs is an important issue in large-scale pig breeding. However, the interaction between swine leukocyte antigen (SLA) and epitopes of viral pathogens has not been extensively studied.

To improve antigen presentation ability, MHC genes have evolved to expand their diversity. For example, alleles have been reported for 2165 HLA-DRB1, 1196 HLA-DQB1 and 94 HLA-DQA1 as human leukocyte antigen (HLA). On the other hand, as SLA, alleles have been reported for 99 SLA-DRB1, 53 SLA-DQB1, and 26 SLA-DQA, suggesting that more research is needed to improve the understanding of the animal's immune system. .

Monocytes and macrophages play an important role in the immune response through phagocytosis, antigen presentation and cytokine secretion. At the same time, macrophages include porcine respiratory and reproductive syndrome virus (PRRSV), African swine fever virus (ASFV), classical swine fever virus (CSFV), and swine circo It acts as a target cell for the replication of major pathogenic viruses in pigs such as virus 1 (porcine circovirus 1, PVC-1) and porcine circovirus 2 (PVC-2).

Pig alveolar macrophage (PAM) cell lines are porcine adenovirus (PAV), vaccine virus (VV), bovine adenovirus (BAV), parainfluenza virus, and simple Herpes simplex virus (HSV), swine poxvirus, African swine fever virus (ASFV), typical swine cholera virus (CSFV), pseudo-viral virus (pseudorabies virus, PRV) and vesicular stomatitis virus ( vesicular stomatitis virus (VSV). Thus, the PAM cell line can be a good model for studying virus-host interactions, including the formation of peptide-SLA complexes. However, due to the difficulties associated with cell preparation, limited lifespan, and experimental variation from other source animals, stable cell lines with the phenotypic properties of primary porcine lung macrophages are considered potential alternatives.

Korean Patent Publication No. 10-2012-0077858 (2012.07.10) Korean Patent Publication No. 10-2016-0041148 (2016.04.18) PCT Patent Publication 2015/086739 A1 (2015.06.18)

Takato Takenouchi et al., Front Vet Sci. 2017, 4: 132.

The simian vacuolating virus40 large T (SV40LT) antigen and human telomerase (hTERT) have been demonstrated as a simple and reliable method of immortalizing primary cells. The inventors of the present invention have established two stable immortalized PAM cell lines with known MHC haplotype information, and measure the binding affinity between the MHC class II molecule and the peptide from porcine circovirus 2 (PCV-2). The two cell lines (iPAM303 and iPAM61) were used for this purpose.

Thus, the present invention can be used to evaluate the binding affinity of various pathogenic peptides to SLA class II molecules, which can be useful for immunogenic applications in pigs.

According to an aspect of the present invention, an expression vector into which a human telomerase reverse transcriptase (hTERT) gene is inserted; And an expression vector into which the monkey fear virus 40 large T antigen (simian vacuolating virus 40 large T, SV40LT) gene is inserted; an immortalized porcine lung macrophage cell line prepared by transfection.

In one embodiment, the immortalized porcine lung macrophage cell line is a homozygous or heterozygous in the pig histocompatibility complex (MHC) class I, and may be a heterozygous in the porcine MHC class II. The MHC class I may be SLA1 or SLA2, and the MHC class II may be one selected from the group consisting of SLA-DQA, SLA-DQB1 and SLA-DRB1.

According to another aspect of the invention, (a) obtaining lung cells from a pig; And (b) obtaining a transfected cell by transfecting the obtained vector with the hTERT gene inserted expression vector and the SV40LT gene inserted expression vector.

In one embodiment, the expression vector in which the hTERT gene of step (b) is inserted may be one selected from the group consisting of pBABE-hTERT-Puro, pBABE-Hygro-hTERT, pLV-hTERT and pLenti-hTERT, and the SV40LT The gene-inserted expression vector may be one selected from the group consisting of pBABE-SV40LT-Puro, pZip-neo-SV40LT and pEPI-1 SV40LT. In addition, the transfected cells may be selected by a method of culturing with puromycin.

According to another aspect of the present invention, (i) reacting a sample with the immortalized porcine lung macrophage cell line of claim 1 to obtain a reactant; And (ii) measuring the MHC binding affinity of the obtained reactant.

In one embodiment, in step (ii), the MHC binding may be a binding between an antigenic peptide and an MHC class II molecule, and the MHC binding affinity is flow cytometry, enzyme-linked immunosorbent analysis (enzyme-linked) It may be performed by one or more selected from the group consisting of immunosorbent assay, ELISA, pull-down assay, and gel shift assay.

In one embodiment, the antigenic peptide is African swine fever virus, typical swine cholera virus, swine genital and respiratory syndrome virus, swine adenovirus, foot and mouth disease virus, hepatitis E virus, parvovirus, swine pandemic diarrheal virus, swine respiratory Viruses consisting of corona virus, rotavirus, swine delta corona virus, swine influenza virus, swine vesicular virus and swine infectious gastroenteritis virus; Bacteria consisting of Brachyspira, Lawsonia intracellularis, pneumonia mycoplasma, mold Mycobacterium tuberculosis, Pasteurella and Salmonella; And it may be derived from one or more selected from parasites consisting of Cryptosporidium, Toxoplasmosis and Trichinosis.

According to another aspect of the present invention, (i) reacting a sample with the immortalized porcine lung macrophage cell line of claim 1 to obtain a reactant; And (ii) measuring the MHC binding affinity of the obtained reactant.

According to the present invention, the immortalized swine lung macrophage cell line is transfected with a vector in which a gene expressing human telomerase (hTERT) and a gene expressing monkey virus 40 vs. T antigen (SV40LT) are inserted into pig primary lung macrophages. It was found that it was possible to confirm the binding affinity between the antigen peptide of the porcine circovirus type 2 and the molecule of SLA using this cell line.

Therefore, the method for detecting an immortalized porcine lung macrophage cell line of the present invention and an antigenic peptide using the same can be used as a peptide selection method for excellent vaccine development in the field of veterinary medicine since the interaction between the antigenic peptide and the SLA molecule can be evaluated. have.

1 is a morphological comparison of immortalized porcine lung macrophages in different cell cycles.
2 is a graph showing the growth curve of immortalized porcine lung macrophages, early stage primary porcine lung macrophages and 3D4/21. The primary porcine lung macrophages used 4 cell cycles, and the immortalized porcine lung macrophages used 35 cell cycles. The incubation period was indicated on the x-axis.
Figure 3 is a photograph showing the non-adherent growth of porcine lung macrophages immortalized in semi-solid agar.
4 is a photograph comparing SLA-DR expression levels using immunohistochemical staining. The first PAMs are PAM61(A) and PAM303(B); The immortalized PAMs are iPAM61(A) and iPAM303(B). Control cells are 3D4/21 (C) and PK15 (C). The first column shows the result of DAPI staining, and the second column shows the result of staining with anti-SLA-DR specific antibody. The third column shows an image in which the first column and the second column are merged (scale bar: 100 μm).
5 is a photograph showing the results of PCR amplification of hTERT from genomic DNA of immortalized PAM cells. The results of genomic PCR (A) and reverse transcription (RT) PCR (B) are shown. The name of the cell line is indicated at the top, "+", positive control plasmid; "-", a negative control, GAPDH is used as a control for RT-PCR. The size of the PCR product is indicated by the arrows.
6 is a photograph showing the results of PCR amplification of SV40LT from genomic DNA of immortalized PAM cells (iPAM303 and iPAM61). The plasmid containing SV40LT was used as a positive control, and the expected size (858 bp) is indicated by arrows.
7 is a result of comparing the efficiency of the PCV-2 ORF2 peptide that binds to SLA class II molecules among various cells. The fluorescence expression level of the SLA-peptide complex was compared in iPAM61, iPAM303 and PK15, and the left (A, C, E) and right (B, D, F) columns were biotin-labeled peptide-free group and biotin, respectively. Represents a group with labeled peptides. (G) is a graph comparing fluorescence signals from SLA-peptide complexes in iPAM61, iPAM303 and PK15. The result of comparison with respect to the parenthesis is shown (P <0.001).
8 is a result of comparing the efficiency of the PCV-2 ORF2 peptide binding to SLA class II molecules in PAM303 and PAM61. Comparison of the fluorescence levels of SLA-peptide complexes in PAM61 and PAM303, the left (A, C) and right (B, D) columns indicate a group without a biotin-labeled peptide and a group with a biotin-labeled peptide, respectively. Shows. (E) shows the difference in fluorescence expression level from SLA-peptide complex in PAM61 and PAM303 (P <0.001).

The present invention is a human telomerase reverse transcriptase (human telomerase reverse transcriptase, hTERT) gene expression vector is inserted; And an expression vector into which the monkey fear virus 40 large T antigen (simian vacuolating virus 40 large T, SV40LT) gene is inserted; to provide an immortalized porcine lung macrophage cell line prepared by transfection.

In one embodiment, the immortalized porcine pulmonary macrophage cell line is a homozygous or heterozygous in SLA 1 and SLA2 of porcine major histocompatibility complex (MHC) class I, SLA-DQA, SLA-DQB1 of MHC class II And heterozygotes in SLA-DRB1. Swine MHC class I belongs to the SLA1 and SLA2 genes, and may have homozygous or heterozygous in each. In addition, MHC class II belongs to the SLA-DQA, SLA-DQb1, and SLA-DRB1 genes, and may have homozygous or heterozygous properties (see Table 2).

SLA genomic sequence-based genotyping method (GSBT) was performed from the immortalized porcine lung macrophages used in the present invention (iPAM61, iPAM303 and ATCC 3D4/21) to perform MHC class I gene ( SLA1 4 alleles for SLA2) , 5 alleles for SLA-DRB1 of the MHC class II genes, and 4 alleles for SLA-DQA and SLA-DQB1 . In particular, unlike ATCC 3D4/21, it can be confirmed that the MHC class II gene in iPAM61 and iPAM303 is a heterozygote, and the MHC class I gene in iPAM303 is also a heterozygous conjugate, and based on this gene diversity, the antigenic candidate peptide for the MHC molecule The binding affinity can be evaluated.

The immortalized porcine lung macrophage line (iPAM303, iPAM61) has the size and shape (spindle shape) of the 5th generation primary porcine lung macrophage cell line, unlike commercially available porcine lung macrophage ATCC 3D4/21 having a small cell size and agglomerated form. ). In addition, unlike ATCC 3D4/21, which showed remarkably rapid proliferation in the growth curve results from 24 to 96 hours after cell adhesion, the primary porcine lung macrophage line and the immortalized porcine lung macrophage line of the present invention exhibit a similar pattern. In the results of confirming that it is possible to grow without a solid surface (non-adhesive growth) using a soft agar clonogenic assay, ATCC 3D4/21 forms a number of colonies, whereas the primary porcine lung macrophage cell line It shows that the immortalized porcine lung macrophage cell line of the present invention does not form colonies even after 2 weeks.

In particular, as another important characteristic in the immortalized porcine lung macrophage cell line of the present invention, the antigen-presenting function, that is, the expression of the MHC class II gene, SLA-DR, is strongly demonstrated, so that the similarity with the primary porcine lung macrophage can be additionally confirmed. This feature is not found in ATCC 3D4/21 and porcine fibroblast PK15.

In addition, the present invention (a) obtaining lung cells from pigs; And (b) transfecting the obtained expression vector with the hTERT gene inserted into the cell and the expression vector inserted with the SV40LT gene to obtain transfected cells.

In step (a), the lungs are separated from the pig, and then the obtained solution obtained by performing a method of obtaining lung cells (eg, bronchoalveolar lavage method, etc.) from the separated lungs is filtered and centrifuged to separate lung cells. Can get The obtained lung cells can be stored in liquid nitrogen by incubating or freezing in a cell culture medium supplemented with fetal bovine serum (FBS) and antibiotics for a period of time at 37° C. in a CO ₂ cell incubator.

Telomerase is an enzyme protein that catalyzes the addition of a telomer repeat sequence (TTAGGG) to the 3'end of the telomere, and serves to repair telomer lost due to cell aging. In most normal adult cells, telomerase is not expressed, and the length of telomeres is gradually decreased with each cell replication, and the decrease in telomer repeat sequence induces cell aging. In humans, cells expressing human telomerase reverse transcriptase (hereinafter referred to as'hTERT') have been shown to bypass normal cellular aging pathways.

The monkey virus fear virus 40 large T antigen (simian vacuolating virus 40 large T, hereinafter referred to as'SV40LT') functions as a carcinogenic gene, and the expression of the T antigen moves the cell to the S phase to allow this gene to host DNA. The transformation caused in this way has been shown to disrupt the balance of host cell growth division and the like and to repeat the cell cycle. SV40LT is a gene encoding a viral T-antigen isolated from SV40, a DNA cancer-causing virus, and is a multi-functional transformation protein that binds to p53 and Rb gene products.

Here, the'expression vector' is a vector capable of expressing a protein in a suitable host cell, which facilitates manipulation of a DNA sequence and other genetics linked to a control sequence capable of regulating the expression of a protein or other genetic manipulation, or of a protein. Refers to a DNA construct (DNA construct) containing sequences necessary for optimizing expression or replicating of vectors. The regulatory sequence includes a promoter for regulating transcription, an operator selectively added to regulate transcription, an appropriate mRNA ribosomal binding site, and sequences for regulating transcription/translation termination.

The expression vector usable in step (b) may be a commercially available vector comprising a gene containing hTERT and a commercially available vector comprising a SV40LT gene, respectively. In the case of hTERT, it may be one selected from the group consisting of pBABE-hTERT-Puro, pBABE-Hygro-hTERT, pLV-hTERT and pLenti-hTERT, and in the case of SV40LT pBABE-SV40LT-Puro, pZip-neo-SV40LT and pEPI -1 It may be one selected from the group consisting of SV40LT.

The transfected lung cells can be cultured together with a certain amount of puromycin to select for 1 week to 10 weeks, preferably 3 weeks to 6 weeks, and most preferably 4 weeks.

In addition, the present invention comprises the steps of (i) reacting the sample with the immortalized pig lung macrophage cell line of claim 1 to obtain a reactant; And (ii) measuring the MHC binding affinity of the obtained reactant.

In step (ii), the MHC binding may be an antigenic peptide and a binding between MHC class II molecules, and the MHC binding affinity may be determined by flow cytometry, enzyme-linked immunosorbent assay, ELISA. ), pull-down assay (pull-down assay) and gel shift assay (gel shift assay) is performed by one or more methods selected from the group consisting of, or in addition to any method known in the art can measure the MHC binding force.

In one embodiment, the antigenic peptide is African swine fever virus, typical swine cholera virus, swine genital and respiratory syndrome virus, swine adenovirus, foot and mouth disease virus, hepatitis E virus, parvovirus, swine pandemic diarrheal virus, swine respiratory Viruses consisting of corona virus, rotavirus, swine delta corona virus, swine influenza virus, swine vesicular virus and swine infectious gastroenteritis virus; Bacteria consisting of Brachyspira, Lawsonia intracellularis, pneumonia mycoplasma, mold Mycobacterium tuberculosis, Pasteurella and Salmonella; And it may be derived from one or more selected from parasites consisting of Cryptosporidium, Toxoplasmosis and Trichinosis.

In addition, the present invention comprises the steps of (i) reacting the sample with the immortalized pig lung macrophage cell line of claim 1 to obtain a reactant; And (ii) measuring the MHC binding affinity of the obtained reactant.

As a result of confirming the binding affinity between the peptide derived from PCV-2 and the SLA class II molecule in the immortalized porcine lung macrophages of the present invention (iPAM61 and iPAM303), iPAM61 (33.7% ± 3.4) in iPAM303 (73.3% ± 5.4) ), indicating that iPAM303 showed higher affinity than peptide bound to SLA class II molecule (SLA-DR or SLA-DQ) than iPAM61. In addition, these results showed a similar pattern to the primary porcine lung macrophages (PAM61 and PAM303).

Hereinafter, the present invention will be described in more detail through examples. However, the following examples are intended to illustrate the present invention, and the scope of the present invention is not limited thereto.

1. Materials and Methods

(1) Collection of pig lung macrophages and cell culture

PAM was isolated from 10 clinically healthy Yorkshire pigs 10 weeks of age grown on a local pig farm. Animals were humanely euthanized and lungs were collected for cell isolation. Phosphate buffer was used for bronchoalveolar lavage in the traditional method. The bronchial alveolar lavage fluid was then filtered. PAM was collected by centrifugation at 400 x g for 5 minutes, and Roswell Park Memorial supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin-gentamycin antibiotic mixture (Gibco, NY, USA) It was cultured in a 6-well plate in an institute (RPMI) 1640 (Hyclone, UT, USA) medium at 37° C. and a 5% CO ₂ incubator. The attached lung macrophages were cultured for 48 hours and the cell freezing medium in an isopropanol box at -80°C [RPMI 1640; 20% FBS; 10% dimethyl sulfoxide (DMSO)] overnight, then stored in liquid nitrogen until use.

The PAM 3D4/21 cell line (ATCC CRL-2843) was purchased from the American Type Culture Collection (ATCC) and cultured in RPMI 1640 supplemented with 10% FBS and antibiotic mixture. The fibroblast PK15 cell line (ATCC CCL-33) was cultured in DMEM supplemented with 10% FBS and antibiotic mixture.

All animal testing procedures were approved by the Institute of Animal Care and Use Committee (IACUC).

(2) SLA typing

Genomic DNA was prepared from cells using RNA/DNA minu kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. Typing of the five SLA genes, SLA1 , SLA2 , SLA-DQB1 , SLA-DRB1 and SLA-DQA , is preceded by Le M, et al., Gene, 2015, 564:228, Park K, et al., Tissue Antigens , 2010, 76:301, Thong LM, et al., Tissue Antigens, 2011, 77:572, Le MT, et al., Tissue Antigens, 2012, 80:528, Choi H, et al., Tissue Antigens, 2015 , 86:255) using genomic sequence-based typing (GSBT). Sequence analysis of PCR products for each locus was performed according to the manufacturer's protocol using the ABI PRISM BigDyeTM Terminator Cycle Sequencing Kit (Applied Biosystem, USA). The sequence analysis results were analyzed using CLC Workbench (CLC Bio, Arhus N, Denmark).

(3) Immortalization of PAM

Primary lung macrophages were transfected with pBABE-SV40LT-Puro and pBABE-hTERT-Puro using a PolyMag kit (Chemicell, Berlin, Germany) according to the manufacturer's protocol. After 48 hours, cells were selected for 4 weeks using 2 μg/ml puromycin (Sigma-Aldrich, lnc, Germany). Cells were continuously cultured up to 35 passages. Cells were continuously frozen in cell freeze medium without antibiotics in cryogenic vials (cryogenic bial, Nalgene, NY, USA) at -80°C for 24 hours prior to storage in liquid nitrogen.

(4) Confirmation of fusion of hTERT and SV40LT by PCR

Genomic DNA was isolated from 5×10 ⁵ cells using RNA/DNA minu kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. PCR includes 100 ng of genomic DNA, 0.5 μM hTERT or SV40LT primer pair (see Table 1), 1.2 U Super-Therm DNA polymerase (JMR Holdings, Kent, UK) in 1.2× PCR bufer (1.5 mM MgCl ₂ ) and 20 μl reaction with 0.1 mM dNTP was performed using T3000 Termocycler (Biometra, Gottingen, Germany).

hTERT-F 5'-GCCGAGACCAAGCACTTCCTCTACT-3' (SEQ ID NO: 1) hTERT-R 5'-GCAACTTGCTCCAGACACTCTTCCG-3' (SEQ ID NO: 2) SV40LT-For 5'-GATGGCTGGAGTTGCTTGGCTACAC-3' (SEQ ID NO: 3) SV40LT-Rev 5'-GCCTGAAATGAGCCTTGGGACTGTG-3' (SEQ ID NO: 4)

Primer pairs hTERT-F and hTERT-R produce 778 bp amplicons, and primer pairs SV40LT-For and SV40LT-Rev produce 858 bp amplicons.

The PCR profile was 95° C., followed by an initial denaturation step of 5 minutes, followed by 94° C. (30 seconds) [denaturation step] → 63° C. (30 seconds) [annealing step] → 72° C. (45 seconds) [synthesis (Extension) step] was repeated 35 cycles, and then was composed of a final extension step of 5 minutes at 72°C. PCR products were analyzed on a 1% agarose gel via electrophoresis.

(5) Cell growth curve and soft agar growth evaluation

Immortalized PAM (2×10 ⁵ cells/well) was seeded into a 6-well plate containing RPMI 1640 with 15% FBS and 5 μg/ml gentamicin. After the cells were injected, trypsin treatment was performed at 24, 48, 72, and 96 hours, and then counted using trypan blue exclusion. The cell number at each time point was plotted. 1×10 ⁴ cells in a 6-well semi-solid agar dish prepared with 0.8% agar and 0.7% agarose at the bottom and upper layers to measure cell anchorage-independent proliferation, a typical characteristic of immortalized cells/ Wells of iPAM303 and iPAM61 were incubated for 2 weeks, respectively. Cells were fed cell culture medium every other day. Colony formation was confirmed under an optical microscope (Model BX51TF, Olympus, Tokyo, Japan).

(6) Immunohistochemistry

Cells were seeded into glass cover slips (Waner Instruments, LLC, Hamden, USA) in 6-well plates at a density of 1.2 x 10 ⁵ cells/well and incubated for 24 hours. The cover slip was then washed with 1× phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde for 15 minutes at room temperature. Cells were washed with 1× PBS and then incubated in 1× PBS containing 5% bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, MO, USA) for 30 minutes. Cells were then reacted with 1× PBS containing 2.5% BSA with mouse anti-SLA-DRB1 monoclonal antibody (1: 250 dilution, Bio-Rad, California, USA) for 2 hours. For visualization, cells were reacted with Alexa Fluor 568 conjugated goat anti-mouse antibody (1/500 dilution, Invitrogen, Massachusetts, USA) in 1× PBS containing 2.5% BSA for 1 hour. To cover with a glass cover slip, VECTASHIELD Mounting Medium with DAPI (Vector, Burlingame, CA, USA) was used. Samples were analyzed under a fluorescence microscope (model BX51TF, Olympus, Tokyo, Japan).

(7) Analysis of peptides that bind SLA class II molecules

In order to analyze peptide affinity using immortalized PAM, the present inventors have developed a 15-amino acid peptide (RSHLGQILRRRPWLV) derived from the open reading frame (ORF) 2 of porcine circovirus type 2 (PCV-2) Representing the binding region, SEQ ID NO: 5) was selected, which is known to encode the major capsid protein and activate the SLA class II-limited immune response. The selected sequence is synthesized through solid phase peptide synthesis using a commercial service (GeneScript, Piscataway Township, NJ, USA), conjugated with biotin at the N-terminus, and then subjected to high performance liquid chromatography (HPLC). Purified.

For analysis via flow cytometry, cells were harvested using trypsin-EDTA and pellets were resuspended in 100 μl of RPMI 1640 medium containing 5% FBS and pH adjusted to 7.2. Cells (1×10 ⁵ ) were reacted with biotinylated peptide (50 μM) in a microcentrifuge tube at 37° C. and 5% CO ₂ for 5 hours. Cells were centrifuged at 400×g for 5 minutes and then washed twice with fluorescence-activated cell sorting (FACS) buffer (1× PBS, 1% FBS, 0.05% sodium azide). The cell pellet was resuspended in a 1:40 dilution of streptavidin-FITC conjugate (Thermo Fisher Scientifc, Massachusetts, USA) in wash buffer and then reacted on ice for 30 minutes. The cells were then washed twice with wash buffer and finally resuspended in 500 μl FACS buffer. The fluorescence signal of the peptide-SLA class II complex was quantified by FACScalibur (BD Bioscience, San Jose, CA, USA) using CellQuest software. The results were calculated and analyzed by one-way analysis of variance (ANOVA) and Student's t-test.

2. Results

(1) Generation of immortalized porcine lung macrophages (PAM)

The phenotype at passage 35 of PAM transfected with hTERT and SV40LT was evaluated. The round-shaped PAM at passage 0 became spindle-shape when attached to the culture plate at the initial passage 5 (see FIG. 1 ). The morphology of immortalized PAM (iPAM) was very similar to that of early PAM. The results were consistent across both iPAM61 and iPAM303. However, ATCC 3D4/21 was different in shape and smaller than iPAM61 and iPAM303. ATCC 3D4/21 adhered to the surface of the culture tube after 24 hours. The number of healthy cells was counted every 24 hours. Unlike the first PAM, which stopped cell division at 15 passages, the cells proliferated exponentially without showing signs of aging. The growth curves of the 35 passage iPAM with the initial PAM and 3D4/21 are shown in FIG. 2. The pattern was similar among all compared cells except 3D4/21, which showed markedly rapid proliferation, supporting consistent properties between early PAM and iPAM in the early stages (see FIG. 2).

Non-adhesive growth is the ability of a transformed cell to grow without the need for a solid surface, and this ability in iPAM61, iPAM303 and 3D4/21 cells using a soft agar clonogenic assay. Tested. While 3D4/21 cells formed multiple colonies, iPAM303, iPAM61 and primary PAM cells did not form colonies after 2 weeks in semi-solid agar (see FIG. 3). This means that the characteristics of iPAM61 and iPAM303 are closer to those of the initial PAM.

Expression of the MHC class II gene is an important characteristic of professional antigen-presenting cells (APCs) such as macrophages. The presence of SLA-DR molecules in iPAM61, iPAM303 and 3D4/21 cells was investigated using anti-SLA-DRB1 antibody (FIGS. 4A and B). A positive signal indicating strong expression of SLA-DR was observed in iPAM303 and iPAM61 and was similar to that observed in primary PAM prior to passage 5; On the other hand, the signal was hardly detected in 3D4/21 cells other than APC and porcine fibroblast PK15 (Fig. 4C). These results indicated that iPAM61 and iPAM303 were more similar to the primary PAM than the publicly available immortalized PAM cells, including 3D4/21.

Integration and expression of hTERT and SV40LT in iPAM61 and iPAM303 were evaluated by PCR. Both iPAM61 and iPAM303 produced 778-bp amplicons corresponding to hTERT (see FIG. 5). However, no amplicon for SV40LT was observed in both cells. Consistently, it was also unable to amplify SV40LT from the plasmid construct itself (see Figure 6), indicating that the inability to amplify the gene may be due to structural constraints in the sequence. Similar phenomena have also been reported in previous literature (Weingartl HM et al., J Virol Methods, 2002, 104:203-216).

(2) Gene polymorphism confirmation of SLA gene from 3 immortalized PAM cells

SLA GSBT was performed on SLA1, SLA2, SLA-DQA, SLA-DQB1 and SLA-DRB1 on 3D4/21, iPAM61 and iPAM303. For the MHC class I gene, four alleles for SLA1 and SLA2 Each was confirmed; For the MHC class II gene, 5 alleles were identified for SLA-DRB1 , whereas 4 alleles were identified for SLA-DQA and SLA-DQB1 , respectively (see Table 2).

Cell line type Class I Class II SLA1 SLA2 DQA DQB1 DRB1 3D4/21 040101 040201 0203 0201 0501 iPAM61 0402 0801 0201/0303 0201/0901 0201/1301 iPAM303 0801/0810 0502/1201 0106/0201 0202/0701 0402/0602

iPAM61 was homozygous for both SLA1 and SLA2 , and heterozygous for all three MHC class II genes typed in the present invention. iPAM303 was a heterozygote for all 5 genes and showed high genetic diversity in the MHC gene. The allele overlap between the three immortalized PAM cells was low, indicating that the results of the MHC-peptide binding assay using these cells can be compared and evaluated for differences in the binding affinity of the candidate peptide to the MHC molecule.

(3) Confirmation of binding affinity between PCV-2 derived peptide and SLA class II molecule in immortalized PAM cells whose haplotype is determined

Understanding the binding affinity between pathogenic peptides and MHC class II molecules is important in predicting the intensity of adaptive immune responses to peptides derived from specific pathogens. To develop a method for measuring the binding affinity of a candidate peptide antigen to a SLA-DR or SLA-DQ molecule in pigs, established immortalized PAM cells iPAM61 and iPAM303, which characterize the MHC haplotype information Then, a synthetic biotinylated peptide (Biotin-RSHLGQILRRRPWLV) matching the amino acid region of 16-30 digits of PCV2 ORF2 was exposed to these cells. It has been reported in the prior literature (Stevenson LS., Viral Immunol, 2007, 20:389) that the peptide induces a specific immune response against PCV-2 without knowledge of the binding affinity for SLA-DQ or SLA-DR . . Since SLA class II-deficient APC is not currently available, pig fibroblasts (PK15) were used as a negative control to indirectly compare signals from specific binding of peptides to MHC class II molecules. Since the expression of SLA-DR was rarely detected in PK15 (FIG. 4C ), it was proved that the signal observed in PAM was derived from a peptide bound to the SLA-DR molecule.

The fluorescence level corresponding to the number of peptides bound to PAM was measured by flow cytometry after reacting iPAM with biotin-labeled peptide for 5 hours (see FIG. 7 ). Unlike the specific signal observed in iPAM and PAM cells, the specific signal was very low for PK15 (7.0%±1.5).

In addition, the signal strength was significantly higher than iPAM61 (33.7% ± 3.4) in iPAM303 (73.3% ± 5.4), indicating a difference in the number of bound peptides. The signal strength of the initial PAM was 69.9±0.6% and 39.5±0.9% for PAM303 and PAM61, respectively, which was similar to that of iPAM (see FIG. 8). These results suggest that the peptide bound to the SLA-DR molecule of iPAM303 ( SLA-DRB1 *0402/0602) or the SLA-DQ molecule ( SLA-DQA *0106/0201, SLA-DQB1 *0202/0701) is the SLA-DR of iPAM61 It suggests a higher affinity or higher efficiency than the peptide bound to the molecule ( SLA-DRB1 *0201/1301) or SLA-DQ molecule ( SLA-DQA *0201/0303, SLA-DQB1 *0201/0901) .

(4) Conclusion

The present inventors conducted a small-scale study to demonstrate successfully confirming the binding affinity between the PCV-2 ORF2 peptide and the MHC class II molecule in the porcine immune system. Differences in the degree of binding of peptides to various MHC class II haplotypes were shown, and differences in the degree of binding of these peptides may probably reflect the efficiency of antigen presentation. Therefore, for the first time through the present invention, the results of highlighting the difference in the binding affinity of a specific epitope to the SLA molecule were shown.

The binding of the antigenic peptide to the MHC molecule is saturable and exhibits low affinity interactions (dissociation constants [K _d ] ~ 10 ^-6 M) with very slow peptide binding and separation. Once bound, the peptide can be bound for hours to days. The more stable the peptide-MHC complex, the longer it remains on the surface of antigen presenting cells to ensure productive interaction with antigen-specific T cells, rather than a lower affinity complex.

Several methods have been proposed to confirm the affinity of peptides for MHC molecules, including purified MHC proteins and whole-cell peptide binding assays. The whole-cell peptide binding assay presents a problem in that it is difficult to distinguish peptide degradation by cell surface protease and non-specific peptide signals that bind other cell surface molecules in addition to binding with MHC itself. However, the whole-cell assay can produce cell panels without the need for replication and expression for multiple MHC alleles, and at the same time may have advantages over other methods in the estimation of peptide-MHC complexes for various MHC alleles. have. In addition, peptide-treated cells can be further used to measure the efficacy of T cell activation, which is a critical indicator of T cell-mediated immune responses. The panel of immortalized PAMs with sufficient cell lines containing various MHC alleles is a useful tool to improve disease resistance by regulating the frequency of MHC alleles in animal populations, as well as screening peptides against superior epitopes for vaccine development Can be

In a full-cell assay, accurate determination of peptide-MHC binding affinity to assess the contribution of non-specific signals results from peptide-MHC binding reactions as well as from peptide-MHC class II-deficient mutant cells and reactions with blocking antibodies. It is necessary to evaluate. However, it was not possible to obtain an antibody for blocking peptide-MHC complex formation, and thus it was not possible to directly measure a non-specific signal due to the lack of currently available tools for SLA. Therefore, the degree of nonspecific peptide binding to pig cells was indirectly estimated using the fibroblast cell line PK15. The signal intensity from the peptide-SLA complex was similar to the expression level of SLA-DR (FIGS. 7E and 7F ), indicating that the non-specific contribution to the peptide-SLA complex can be neglected. Although the possibility of non-specific binding to PAM-specific receptors rather than fibroblasts cannot be neglected, the difference in signal between iPAM61 and iPAM303 indicates that it is not. Thus, the strategy used in the present invention indicates that it is applicable to confirm the binding affinity of a specific peptide to an SLA molecule. However, it is necessary to establish more cell lines with various MHC haplotypes.

The 15 amino acid peptides used in this study are not suitable for presentation by MHC class I molecules, because the binding capacity of MHC class I molecules is limited to peptides 8 to 10 amino acids long; Therefore, only the effect of MHC class II binding was considered. In the SLA system, there are two major MHC class II molecules, SLA-DQ and SLA-DR. However, it was not known at this point whether the binding of the peptide was limited to either DQ or DR or to two molecules. To fully explain this, more research and development of related tools are needed.

Both hTERT and SV40LT were transformed into cells, but the inability to amplify SV40LT from iPAM indicates that overcoming replication aging of iPAM may be due to hTERT alone. However, the possibility of SV40LT as a role of causality could not be excluded because SV40LT was not amplified even in the plasmid structure itself (see FIG. 6 ). Evaluating the molecular pathways associated with SV40LT overexpression in mammalian cells can help to reveal this.

Different MHC alleles are associated with peptide binding repertoires of different sizes, affinity and immunogenicity. The purpose of this invention was to establish and evaluate the potential of our strategy to estimate the binding affinity of a peptide to SLA molecule. From the results of using a larger number of immortalized PAMs currently in progress, it is possible to gain a deeper understanding of the properties of the SLA class II gene and other alleles of important disease-related peptides in pigs. This approach can also be applied to other livestock species.

(5) Summary

Porcine alveolar macrophage (PAM) is useful for studying viral infections and immune responses in pigs; However, these cells have limited long-term use due to their short life span. The present inventors transfected the hTERT gene and SV40LT gene to immortalize the primary PAM and established two immortalized porcine alveolar macrophage (iPAM) cells that actively proliferate even after passage 35. These cells possessed the characteristics of primary PAM, including the strong expression of porcine leukocyte antigen (SLA) class II gene and the inability to multiply by non-adhesion. After characterizing their SLA gene, a peptide-SLA binding assay was performed using the peptide of type 2 porcine circovirus open reading frame 2 to experimentally measure the binding affinity of the peptide to SLA class II. The number of peptides bound to cells measured by fluorescence was iPAM61 (33.7% ± 3.4; SLA-DQA *0201/0303, DQB1 *0201/0901, DRB1 *0201/1301) and iPAM303 (73.3% ± 5.4; SLA- Unlike DQA *0106/0201, DQB1 *0202/0701, DRB1 *0402/0602), it was very small in PK15 cells (7.0%±1.5), not antigen-presenting cells. The difference in peptide binding between the two iPAMs seemed to be due to the difference in alleles between the expressed SLA class II molecules. The development of a panel of immortal PAM cells with various SLA haplotypes and the use of the methods established in the present invention can be a useful tool for evaluating the interaction between antigenic peptides and SLA molecules, and in veterinary medicine including vaccine development. It is also important for many applications.

<110> Konkuk University Industrial Cooperation Corp <120> Immortalized porcine alveolar macrophage cell line and method for detecting antigenic peptide using the same <130> P181128 <160> 5 <170> KoPatentIn 3.0 <210> 1 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> hTERT-F <400> 1 gccgagacca agcacttcct ctact 25 <210> 2 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> hTERT-R <400> 2 gcaacttgct ccagacactc ttccg 25 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> SV40LT-For <400> 3 gatggctgga gttgcttggc tacac 25 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> SV40LT-Rev <400> 4 gcctgaaatg agccttggga ctgtg 25 <210> 5 <211> 15 <212> PRT <213> Porcine circovirus <220> <221> PEPTIDE <222> (1)..(15) <223> Porcine circovirus type 2 open reading frame <400> 5 Arg Ser His Leu Gly Gln Ile Leu Arg Arg Arg Pro Trp Leu Val 1 5 10 15

Claims (13)

An expression vector in which a human telomerase reverse transcriptase (hTERT) gene is inserted; And an immortalized porcine lung macrophage cell line prepared by transfecting an expression vector into which a monkey fear virus 40 large T antigen (simian vacuolating virus 40 large T, SV40LT) gene is inserted. The immortalized pig according to claim 1, wherein the immortalized porcine lung macrophage cell line is a homozygous or heterozygous in the pig histocompatibility complex (MHC) class I, and a heterozygous in porcine MHC class II. Pulmonary macrophage cell line. The immortalized porcine lung macrophage cell line according to claim 2, wherein the MHC class I is any one of SLA1 and SLA2. The immortalized porcine lung macrophage cell line according to claim 2, wherein the MHC class II is one selected from the group consisting of SLA-DQA, SLA-DQB1 and SLA-DRB1. (a) obtaining lung cells from a pig; And
(b) obtaining a transfected cell by transfecting the obtained lung cell with an expression vector into which the hTERT gene has been inserted and an expression vector into which the SV40LT gene has been inserted.
Method of producing immortalized pig lung macrophage cell line comprising a. The expression vector in which the hTERT gene of step (b) is inserted is pBABE-hTERT-Puro, pBABE-Hygro-hTERT, pLV-hTERT. And pLenti-hTERT method for producing immortalized pig lung macrophage cell line, characterized in that the one selected from the group consisting of. The immortalized pig according to claim 5, wherein the expression vector into which the SV40LT gene of step (b) is inserted is one selected from the group consisting of pBABE-SV40LT-Puro, pZip-neo-SV40LT and pEPI-1 SV40LT. Method of manufacturing lung macrophages. 6. The method of claim 5, wherein the transfected cells of step (b) are selected by culturing with puromycin. (i) reacting a sample with the immortalized porcine lung macrophage cell line of claim 1 to obtain a reactant; And
(Ii) measuring the MHC binding affinity of the obtained reactant
Method for detecting an antigenic peptide comprising a. 10. The method according to claim 9, wherein the MHC binding in step (ii) is between an antigenic peptide and an MHC class II molecule. The method according to claim 9, wherein the MHC binding affinity of step (ii) is flow cytometry, enzyme-linked immunosorbent assay (ELISA), pull-down assay and gel. A method for detecting an antigenic peptide, characterized in that it is performed by one or more selected from the group consisting of a gel shift assay. 10. The method of claim 9, wherein the antigenic peptide is African swine fever virus, typical swine cholera virus, porcine genital and respiratory syndrome virus, swine adenovirus, foot and mouth disease virus, hepatitis E virus, parvovirus, swine pandemic diarrhea virus, swine Viruses consisting of respiratory coronavirus, rotavirus, swine delta corona virus, swine influenza virus, swine blep virus and swine infectious gastroenteritis virus; Bacteria consisting of Brachyspira, Lawsonia intracellularis, pneumonia mycoplasma, mold Mycobacterium tuberculosis, Pasteurella and Salmonella; And Cryptosporidium, Toxoplasma gondii, and a parasite consisting of trichinosis. (i) reacting a sample with the immortalized porcine lung macrophage cell line of claim 1 to obtain a reactant; And
(Ii) measuring the MHC binding affinity of the obtained reactant
The screening method of the virus containing.

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