KR20170022705A

KR20170022705A - Expression Vector to express pAPN of porcine epidemic diarrhea virus and transgenic mouse

Info

Publication number: KR20170022705A
Application number: KR1020150118028A
Authority: KR
Inventors: 신현진; 노재랑; 양동군; 현방훈; 조수동
Original assignee: 충남대학교산학협력단; 대한민국(농림축산식품부 농림축산검역본부장)
Priority date: 2015-08-21
Filing date: 2015-08-21
Publication date: 2017-03-02

Abstract

The present invention relates to a recombinant vector to express pAPN of a porcine epidemic diarrhea virus, a transgenic mouse, and a use thereof. More specifically, the present invention relates to a recombinant vector to express pAPN protein which comprises a promoter and an APN gene of pigs, a transgenic mouse, and a method for diagnosing PEDV. By using the transgenic mouse according to the present invention, it is possible to confirm an infection mechanism of the porcine epidemic diarrhea virus and develop an effective vaccine and medicine using the same.

Description

A recombinant vector expressing pAPN of a porcine epidemic diarrhea virus, a transgenic mouse, and a use thereof [Expression Vector to express pAPN of porcine epidemic diarrhea virus and transgenic mouse]

The present invention relates to a recombinant vector expressing pAPN of a porcine epidemic diarrhea virus, a transgenic mouse and uses thereof.

Porcine Epidemic Diarrhea (PED) occurs regardless of age and is characterized by vomiting and hydrosoluble diarrhea and is one of the leading causes of diarrhea. The PED is currently one of the most important viral diseases that causes enormous damage to the pig industry in Asian countries including Korea.

This disease occurs especially in winter, especially in the pre - weaning piglets, and is caused by dehydration with diarrhea. However, the disease is now being invented in other seasons than in winter, and the onset is also becoming more diverse.

The incubation period of PED is usually 26 to 36 hours for new piglets and 2 days for finishing pigs. The symptom is judged as a child is younger. In the newborn piglet, anorexia and vomiting occur immediately after diarrhea. Suddenly, diarrhea develops in the newborn piglet. Dehydration is severe in the newborn pig within one week of age and diarrhea occurs for 3 ~ 4 days. The mortality rate varies depending on the age, but usually the mortality rate is 50% within 1 week of life, and 90% in severe cases.

Although Korea is quarantining and importing many species from the United States and European countries, interestingly, there are no reported cases in North America or South American countries such as the United States, and the pig farming industry in Europe after the outbreak, It is almost disappearing trend. However, Asian countries, especially in colder months in winter, Korea, Japan, and China are characterized by many outbreaks. These reasons have not yet been clarified.

Currently, virulence vaccines developed in Korea have been produced and distributed to farmers. However, reports on the onset of this disease have not been much reduced. This is similar to the situation in other countries.

Porcine Epidemic Diarrhea Virus (PEDV), a causative agent of PED disease, is an RNA virus belonging to Coronavirdae , which is replicating very rapidly due to the nature of RNA viruses.

Currently, there is no treatment for PEDV.

Korean Patent Registration No. 10-0513168 (Probiionic Co., Ltd., Aug. 31, 2005) discloses a method for preventing or treating diarrhea caused by the virus by inhibiting coronavirus infection of livestock using probiotic Enterococcus faecalis Probio-056 And Korean Patent Registration No. 10-0884085 (Ministry of Agriculture, Forestry and Livestock, Ministry of Agriculture, Forestry and Livestock Quarantine Headquarters, Feb. 10, 2009) discloses a single chain variable fragment antibody that specifically neutralizes PEDV An expression vector containing a gene constructing the gene, and a recombinant protein containing the same.

In addition, a variety of patents have been proposed for feed compositions containing specific additives, but commercially available therapeutic agents are only a part of them, and the effect as a therapeutic agent for PEDV is unsatisfactory. To prevent secondary bacterial infection after PEDV infection, antibiotics and antimicrobial drugs are administered to provide enough glucose or electrolyte to relieve dehydration, to keep water at all times, to maintain a pleasant environment The treatment of reducing the mortality of piglets is mainly done.

In addition to these treatments, efforts to prevent PEDV are also needed.

Prevention of PEDV should be carried out thoroughly on prevention, hygiene, vaccination, and prompt action should be taken.

To prevent pandemic diarrhea due to epidemic diarrhea, the vaccine was first vaccinated 5 ~ 6 weeks before pregnancy and second vaccination 2 ~ 3 weeks before delivery. By implementing the transit antibody, diarrhea of the piglets can be prevented.

In addition, in a farm where diarrhea occurs, a method of preventing piglets by transferring high transit antibody to piglets through artificial infection by feeding fresh intestinal contents of infected money to sows whose delivery schedule is more than 2 weeks have. However, if the artificial infection is carried out improperly as the intestinal contents of the infected money, it may cause spread of other pathogens on the farm. If the sow is scheduled to be delivered within 2 weeks, thoroughly disinfect the sow's money, move it to the delivery room, and thoroughly manage the pest control to prevent transmission of pathogens, so that the piglets are not infected with viruses until at least 3 weeks after delivery do. In addition, as a countermeasure against PED in the farm, it is necessary to give birth in a clean place to sows having a faster delivery period and completely isolate piglets until 3 weeks after birth

The diagnosis of PED is very difficult to diagnose based on clinical symptoms alone. In the case of young piglets, there is no symptoms or mild clinical symptoms in young piglets. On the other hand, when acute diarrhea is seen in the cause and the breeding pond, PED may be suspected but it is difficult to confirm.

The definitive diagnosis of PED is made by fluorescent antibody test using frozen tissue sections of infected pigs' plant and ileum or by enzyme chain reaction (PCR) which detects PEDV-specific genes in intestines or feces. However, the detection rate of fluorescent antigens is high in the early stage of diarrhea, but decreases markedly after mid - stage when the regeneration of villi is progressing. In addition, since the antigen is not detected in the small intestine with marked villi atrophy, the detection rate of the antigen depends on the sampling time and site of the material. Therefore, It is a difficult situation.

In addition, Korean Patent Laid-Open Publication No. 2005-0057750 (BioDom Co., Ltd., June 16, 2005) was used to easily serologically diagnose TGE and PED infection serologically without special test equipment or trained personnel. Although the rapid diagnosis method for simultaneous diagnosis of PEDV infection using immuno-chromatography (Immunochromatography) has been disclosed, this method can not be considered to have high accuracy and reliability.

Korean Patent Registration No. 10-0513168 (Probiionic Co., Ltd., Aug. 31, 2005) Korea Patent Registration No. 10-0884085 (Republic of Korea (Management Division: Ministry of Agriculture, Forestry and Livestock, Ministry of Agriculture, Forestry and Livestock Quarantine Headquarters, 2009.02.10) Korean Patent Laid-Open Publication No. 2005-0057750 (Biodom Co., 2005.06.16)

The present applicant has conducted various studies to diagnose and treat PEDV with high reliability. As a result, a recombinant vector capable of expressing pAPN protein, which is known as a receptor protein of PEDV, is produced and used to obtain a transgenic mouse, It is confirmed that pAPN is expressed in the small intestine, lung and kidney of this mouse, and thus it can be applied particularly useful for the diagnosis of PEDV, thus completing the present invention.

Accordingly, an object of the present invention is to provide a recombinant vector capable of expressing a pAPN protein.

It is another object of the present invention to provide a transgenic mouse transformed with the recombinant vector.

It is still another object of the present invention to provide a method for diagnosing porcine epidemic diarrhea virus (PEDV) using the transgenic mouse.

To achieve the above object, there is provided a recombinant expression vector for expressing pAPN protein comprising a promoter and a pAPN protein gene.

More preferably, the promoter is a proximal promoter represented by SEQ ID NO: 2.

More preferably, the promoter is operably linked to the pAPN protein gene of SEQ ID NO: 3 and the BGH-polyA signal sequence of SEQ ID NO: 4.

More preferably, the recombinant expression vector further comprises a flag tag of SEQ ID NO: 5.

More preferably, the recombinant expression vector is characterized by having a cleavage map of Figure 2-A.

More preferably, the recombinant expression vector is characterized by being represented by SEQ ID NO: 1.

The present invention provides a transgenic mouse transformed with a recombinant expression vector for expression of pAPN protein comprising a promoter and a pAPN protein gene.

The present invention provides a method for producing a recombinant expression vector, comprising the steps of: preparing a recombinant expression vector for expressing pAPN protein comprising a promoter and a pAPN protein gene; And transforming the recombinant vector into a mouse embryo.

The present invention provides a method for diagnosing porcine epidemic diarrhea virus (PEDV) using the transgenic mouse.

More preferably, the diagnosis is selected from the group consisting of small intestine cells, lung cells, and kidney cells of a mouse.

The genetic modification according to the present invention is minimized and the transgenic mouse capable of expressing APN can be used to identify the infection mechanism of the swine viral diarrhea virus and to develop effective vaccines and therapeutic agents using the same.

Figure 1 is a schematic diagram showing the structure and activity of the mouse proximal APN promoter.
Figure 2 is a diagram showing the structure and characteristics of a porcine APN transformation vector.
Figure 3 is a diagram related to the development of porcine APN transgenic mice.
Figure 4 shows pig APN expression in the small intestine of porcine APN transgenic mice.
Figure 5 is an image showing pig APN expression in various tissues of porcine APN transgenic mice.
Figure 6 is an image showing immunohistochemical analysis of the small intestine of PEDV infected pig APN transgenic mice.
Figure 7 is a graph showing PEDV replication in the small intestine of porcine APN transgenic mice.

As used herein, the term "promoter " refers to a DNA sequence capable of regulating the transcription of a specific nucleotide sequence into mRNA when linked to a specific sequence, preferably a proximal promoter or a distal promoter.

As used herein, the term "recombinant vector" refers to a gene construct containing an essential regulatory element operatively linked to express a gene construct in a suitable host cell, which is capable of expressing the desired protein or RNA of interest.

The term "pAPN" protein referred to in the present specification refers to porcine aminopeptidase N protein, which means a protein known as a receptor protein of PEDV. This means that a subject, that is, a protein detected when a pig is infected with PEDV, can be diagnosed as having been infected with PEDV by expressing it in a cell.

The proximal promoter refers to a portion within about 250 bases forward from the start of transcription and is a major part directly affecting the regulation of transcription and binding sites for specific transcriptional regulatory factors.

The distal promoter is located at a distance from the start of transcription and is generally less influential and secondary than the proximal promoter in regulating transcription and is a site where specific transcription factors bind.

As used herein, the term "transformation" refers to altering the genetic properties of an organism by exogenously given DNA. Examples of the transformation method include various known methods such as microinjection, electroporation, particle bombardment, sperm-mediated gene transfer, viral infection A method using viral infection, direct muscle injection, insulator, and trnasposon may be appropriately selected and applied. Preferably, in the present invention, an expression vector can be transformed into a mouse embryo by microinjection.

Hereinafter, the present invention will be described in more detail.

The present invention provides a recombinant expression vector for expression of pAPN protein comprising a promoter and a pAPN protein gene.

First, we evaluated the expression of pAPN protein by a promoter. Using a proximal promoter and a distal promoter, a recombinant expression vector for expression of pAPN protein containing a promoter and pAPN protein gene was prepared and proved that the pAPN protein was expressed well . Since expression of pAPN protein by the proximal promoter was higher than that of the distal promoter, further experiments were demonstrated using the proximal promoter.

Wherein the promoter is a recombinant expression vector operatively linked to the pAPN protein gene and the BGH-polyA signal sequence. The recombinant expression vector may be a recombinant expression vector having a cleavage map of Figure 2-A.

The inventors also developed a transgenic mouse (TRANSGENIC mouse MODEL) expressing the porcine aminopeptidase N (pAPN) protein, which is ultimately known as a receptor protein of PEDV, and evaluated its efficacy.

According to a preferred experimental example of the present invention, a recombinant expression vector for pAPN protein expression comprising a promoter and a pAPN protein gene is used to transform a porcine aminopeptidase N (pAPN) protein, known as a receptor protein of PEDV, Mouse.

Cloning was carried out with a proximal promoter expressing pAPN to make Tg mice expressing pAPN. We demonstrated the expression of pAPN in mouse embryos produced and succeeded in producing two lines (CNU-1 and CNU-2).

PCR and Western blotting were used to demonstrate that the two pAPN Tg mouse lines actually expressed pAPN, and pAPN expression in the organs (small intestine) was confirmed by immunohistochemistry .

PCR was used to verify the expression of the pAPN Tg mouse by organs, and it was proved that the Tg mouse developed by the present inventors expressed pAPN in the small intestine, lung, and kidney.

To confirm whether the pAPN Tg mice actually infected with PEDV, PEDV was infected and their infection was confirmed. No specific clinical symptoms were observed, but slight Yanbyeol (dilute stool) was observed and PdV infection could be detected in Tg mice. Through this, we demonstrated that pAPN Tg mice developed by the present inventors successfully infected PEDV.

Also, we investigated how much PEDV was infected by date. Wild-type mice showed no infectivity to PEDV, but pAPN Tg mice demonstrated an increase in the amount of PEDV infection over time.

Also, the transgenic mice according to the present invention can be used to identify the infection mechanism of swine diarrheal virus and to develop effective vaccines and therapeutic agents using the transgenic mice.

The present invention provides a method for producing a recombinant expression vector comprising the steps of: preparing the recombinant expression vector of claim 1; And transforming the recombinant vector into a mouse embryo.

The present invention provides a method for diagnosing porcine epidemic diarrhea virus (PEDV) using the transgenic mouse of claim 5.

More preferably, the diagnosis is selected from the group consisting of small intestine cells, lung cells, and kidney cells of a mouse

Hereinafter, the present invention will be described in more detail with reference to various experimental examples. The following examples are intended to illustrate the present invention and the scope of the present invention is not limited to these examples.

(Experimental Example)

2.1 Cells and viruses

Vero monkey kidney cells were placed in cell culture medium containing 10% fetal bovine serum (FBS), 100 U / ml penicillin G, 100 / / ml streptomycin, and 250 / / ml amphotericin B, ℃, and held for 5% CO ₂ condition. 293T cells were cultured in dibelco modified Eagle's medium (DMEM, Gibco) supplemented with 10% FBS, 100 U / ml penicillin G, 100 쨉 g / ml streptomycin and 250 쨉 g / ml amphotericin B.

All tissue culture reactants were purchased from Gibco (Carlsbad, CA, USA) and PEDV cell-adapted vaccine strains were purchased from Cruz and Shin, 2007; Grown and titrated according to Hofmann and Wyler, 1988, and stored at 80 ° C.

2.2 Reagents and Antibodies

PEDV-specific polyclonal antibodies and pig APN were generated in KPEDV-9-immunized BALB / c mice, respectively (Cruz et al., 2008). Antibody specificity was confirmed by enzyme-linked immunosorbent assays (ELISA) analysis. Anti-Flag M2 Monoclonal Antibody and Anti-Flag M2 Affinity Gels (gel beads) were purchased from Sigma-Aldrich (St. Louis, Mo., USA).

FITC (Fluorescein isothiocyanate) -conjugated anti-mouse IgG antibody was purchased from Santa Cruz Biotechnology Company (Santa Cruz, CA, USA).

2.3 Production of pig APN transgenic mouse model

C57BL / 6J genomic DNA was amplified by PCR using the following primers containing the proximal APN promoter regions (starting at nucleotide-1044) of the mouse proximal NotI and HindIII restriction sites, respectively.

primer:

5'-CCCGCGGCCGCAAGATTTGAAACAGTGGA-3 '

5'-CCCAAGCTTGATGCCGGTGGACAGGGA-3 '

The PCR product was cloned into pBluescript KS (+) vector and pGL3-Basic vector (Promega, Madison, WI, USA), which contains the promoterless luciferase reporter gene. The pig APN gene was amplified from total RNA isolated from porcine intestinal cells by RT-PCR using specific primers and cloned into pBluescript KS (+) vector. The sequence encoding the flag epitope (DYKDDDDK) was fused to 5'-CCCAAGCTTACCATGGCCAAGGGATTCTAC-3 'and 5'- CCCCTCGAGTCACTTGTCGTCATCGTCTTTGTAGTCGCTGTGCTCTATGAACCA- 3' primer using PCR to the 3'-end of porcine APN and the primers were flanked by contiguous NotI and HindIII restriction enzymes Site.

The BGH-polyA sequence was amplified from pcDNA3.1 vector (Invitrogen, Carlsbad, Calif., USA) by PCR using 5'-CCCCTCGAGCGACTGTGCCTTCTAGTT-3 'and 5'-CCCGGTACCCCATAGAGCCCACCGCAT-3' primers wherein the primers were flanking XhoI And KpnI restriction enzyme sites.

The PCR product was cloned into the pBluescript KS (+) vector. All PCR products were identified using automated sequencing.

To obtain the porcine APN transgene, pig APN-flag and mouse proximal APN promoter were ligated with pBluescript KS-BGH-polyA and then digested with HindIII / XhoI and NotI / HindIII, respectively.

Finally, a recombinant expression vector represented by SEQ ID NO: 1 was completed. The recombinant expression vector comprises a mouse APN proximal promoter (ninth nucleotide to 1111th nucleotide in SEQ ID NO: 1), a porcine APN gene sequence (SEQ ID NO: 1 in 1121 Nucleotide → 4009th nucleotide, ATG start codon, TGA termination codon), a flag tag (4010th nucleotide to 4033th nucleotide in SEQ ID NO: 1) and a poly A BGH (SEQ ID NO: 1 in SEQ ID NO: 4043 < / RTI > nucleotides to 4270 < th > nucleotides). In addition, cloning enzyme sites

gcggccgc Note I

aagctt Hin dIII

CTCGAG Xho I

GGTACC Kpn I.

2.4 Promoter luciferase assay

24-well plates were inoculated in 293T cells ^{(2.5 × 10 5 cells / ml} ). After 24 hours, each cell was infected with pGL3-Basic-mouse proximal APN promoter (mAPN-luc) (0.2 ug or 1 ug). Each cell was infected three times using Lipofectamine 2000 (Lipofectamine 2000, Invitrogen) according to the manufacturer's instructions. After 24 hours, each cell was lysed according to the manufacturer's protocol via a luciferase assay system (Promega) analyzer. All luciferase activity was regulated for [beta] -galactosidase activity.

2.5 Generation and measurement of porcine APN transgenic mice

The pig APN transgenes were linearized by cleavage using NotI and purified by gel extraction (Qiagen, Valencia, Calif., USA). Gain-of-function transformation was performed by microinjection of the purified DNA into the pronuclei of the ICR mouse conjugate and then transplanted into the fallopian tubes of female recipient mice. Transgenic mice were identified by PCR analysis of the endogenous DNA using the following primers:

primer:

PCR1-F: 5'-CCCAAGCTTACCATGGCCAAGGGATTCTAC-3 '

PCR1-R: 5'-GAAGTTGGAGAGCATCCT-3 ';

PCR2-F: 5'-GGCGTCCTACTTGCATGC-3 '

PCR2-R: 5'-CCCTCGAGTCACTTGTCGTCATCGTCTTTGTAGTCGCTGTGCTCTATGAACCA-3 '.

F1 mouse founder mice were backcrossed to have C57BL / 6J background for 5 generations. All mice used in this study were kept in a specific aseptic facility within the Biomedical Research Center of KAIST and TecPEDhnology (Daejeon, Korea).

2.6 PEDV Infection of Swine APN Transgenic Mice

Pig APN transformed or untransformed wild type mice were orally inoculated with 5 × 10 ⁶ TCID ₅₀ concentrations of KPEDV-9 and inoculated with phosphate buffer (PBS, pH 7.2) as negative control. After monitoring their clinical signals, feces were collected for 5 days. Two mice in each group were killed by marking the day of virus infection. Tissues were aseptically collected and prepared for RT-PCR and immunohistopathological analysis.

2.7 RNA Extraction and RT-PCR

Total RNAs were extracted from feces and tissue samples using TRIzol® reagent (Invitrogen) and then transcribed into cDNA using the Power cDNA synthesis kit (iNtRON Biotechnology, Korea) according to the manufacturer's protocol.

Pig APN-flag cDNA was PCR amplified using specific primers used for geno-typing PCR. As a control, the specific primers used to amplify? -Actin are as follows (mouse? -Actin-F: 5'-CGGTTCCGATGCCCTGAGGCTCTT-3 'and mouse? -Actin-R: 5'-CGTCACACTTCATGATGGAATTGA-3').

With respect to the cyclic parameter, the initial denaturation was carried out at 94 ° C for 2 minutes, followed by 30 cycles of denaturation at 94 ° C for 15 seconds, annealing at 60 ° C for 40 minutes, extension at 72 ° C for 90 seconds, Was performed at 72 degrees for 5 minutes. To determine the viral genome, viral RNA was extracted from the tissue homogenate using the Viral Gene-spin Viral DNA / RNA Extraction Kit (iNtRON Biotechnology) according to the manufacturer's instructions. M-MLV reverse transcriptase (iNtRON Biotechnology) was used for first-strand cDNA synthesis with 10 10 μl of viral RNA 10 extracted in 50 μl random main reaction. The specific primers used for the measurement of APN genes were PEDVN forward (5'-GGTACCATGGCATCTGTCAGCTTT-3 ') and PEDV-N reverse direction (5'-GGATCCTTAATTTCCTGTTCGAA-3').

RT-PCR was performed at 94 ° C for 2 minutes, followed by 30 cycles at 94 ° C for 20 seconds, 54 ° C for 10 seconds, and 72 ° C for 2 minutes, and an extension reaction was performed at 70 ° C for 10 minutes . PCR products were analyzed by gel electrophoresis and visualized by ethidium bromide staining and UV irradiation.

2.8 Immunoblotting

293T cells were cultured in a cell lysis buffer (25 mM Tris.Cl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM NaF, 1 mM sodium orthovanadate, 1 mM PMSF, 5% glycerol, 0.5% 100, and a protease inhibitor (Roche, Indianapolis, IN, USA). The small intestine was washed with PBS and homogenized in lysis buffer (1% NP40, 150 mM Tris.Cl, 50 mM NaCl, 1 mM EDTA). Cell or tissue lysates were separated by 8.15% gradient SDSPAGE and transferred to PVDF membrane (Amersham-Pharmacia Biotech, Piscataway, NJ, USA). Protein was labeled using anti-beta-actin antibody diluted 1: 10,000 with anti-flag monoclonal antibody diluted 1: 4000. Immunoconjugates were measured with peroxidase-conjugated goat anti-mouse IgG antibody (Santa Cruz Biotechnology) and visualized using Amersham ECL Western blotting assay reagent (Amersham-Pharmacia Biotech).

2.9 Flow cytometry

293T cells were transformed with a porcine APN transgene. After 24 hours, the cells were washed with PBS and separated by treatment with Non-Enzymatic Cell Dissociation Solution (Sigma-Aldrich) for 2 minutes. The isolated cells were collected and redispersed in a fluorescence activated cell sorting (FACS) staining buffer (PBS containing 2% FBS). Cells were stained with anti-flag M2 monoclonal antibody (1: 200) for 15 min at 4 ° C. Antibody-stained cells were analyzed by flow cytometry (FACSCalibur, Becton-Dickinson Biosciences, Franklin Lakes, NJ, USA).

Kidneys and liver tissues were collected from the mice, washed with PBS, and kidney cells or hepatocytes were separated from the tissues with a 70 μm mesh filter (Becton-Dickinson Biosciences). Then redispersed in FACS staining buffer and analyzed by the method described above.

2.10 Immunochemistry (IHC)

Small intestine samples were fixed in 10% buffered formalin and embedded in paraffin. A 5 탆 section of the row was mounted on a silane coated slide. The sections were paraffin removed for immunohistochemical analysis and endogenous peroxidase blocked in 0.6% H ₂ O ₂ / methanol for 30 minutes. Antigens were recovered by heating with electromagnetic waves in citrate buffer for 10 minutes, and the slides were incubated with 1.5% horse serum for 30 minutes at room temperature.

The polyclonal anti-porcine APN antiserum, monoclonal anti-flag antibody, or polyclonal anti-PEDV antiserum was applied overnight at 4 degrees. Biotinylated anti-mouse IgG secondary antibodies were measured using a vidin-biotin-peroxidase kit (Vectastain ABC kit, Vector Laboratories, Burlingame, CA, USA). Antibody binding was measured using chromogen diaminobenzidine (Vector Laboratories), and cells were stained with hematocylin and eosin.

2.11 PEDV replication in small intestine of pig APN transgenic mice

Both wild type and porcine APN transgenic mice were infected with PEDV on the first day. The viral titer in the inoculum was 5 × TCID ₅₀ 10 ⁶ . Two of the mice in each group were sacrificed on day 5, and small intestine samples were collected from all mice and homogenized. Each sample was centrifuged and the supernatant was stored until it was titrated at 80 ° C. Optimization was performed using Vero cells. The PEDV titers were measured and described as logarithmic values.

3.1 Production and evaluation of mouse APN proximal promoter

Figure 1 is a schematic diagram showing the structure and activity of the mouse proximal APN promoter. 1-A is a Proximal and Distal APN promoter region, wherein a black circle means a transcription start point. Figure 1-B shows the activity of the proximal APN promoter in 293T human embryonic kidney cells. 293T cells were transfected with the mouse procimal APM promoter (mAPN-luc, left panel) and serially diluted with pGL3-Basic vector. After 24 hours, the cells were lysed and luciferase activity assay was performed. Promoter activity was expressed relative to luciferase activity measured in infected cells with the pGL3-Basic vector. The error bars were expressed as standard deviations obtained after 3 repetitions.

As shown in Figure 1-A (Bhagwat et al ., 2001), mouse APN transcription is regulated by two different promoters. The distal promoter is located 8 kb upstream from the transcription start site and regulates APN expression in bone marrow and fibroblasts. The proximal promoter regulates APN expression in epithelial cells (Olsen et al., 1991; Shapiro et al., 1991). The present inventors allowed the mouse proximal APN promoter to express porcine APN in mouse epithelial cells. To test the activity of the promoter, the sequence encoding the promoter region was amplified and cloned into a vector lacking the pGL3-Basic promoter (Fig. 1B), and the promoter activity was measured by luciferase assay. As shown in Figure IB, luciferase activity was 8-fold higher in 293T human embryonic kidney cells infected with the vector with the mouse proximal APN promoter (mAPN-luc) than in cells transplanted into the empty vector.

3.2 Structure and Characterization of Porcine APN Transformants

Figure 2 is a diagram showing the structure and characteristics of a porcine APN transformation vector. 2-A is a schematic diagram of the foreign gene of pig APN. Figure 2-B shows the expression of the porcine APN foreign gene in epithelial cells. 293T cells were infected with a porcine APN foreign gene. After 24 hours, the cells were lysed and flag-labeled pig APN was immunoprecipitated using Anti-Flag M2 Affinity Gels (gel beads) and immunoblotting with anti-flag monoclonal antibody was performed: 1: pcDNA3.1 vector; 2: Pig APN foreign gene. FIG. 2-C is a diagram showing extracellular expression of a foreign gene of a porcine APN. 293T cells were infected with a porcine APN foreign gene. After 24 hours, the cells were isolated and stained with anti-flag monoclonal antibody and FITC-conjugated anti-mouse IgG secondary antibody and analyzed by flow cytometry.

A vector was prepared that coats the pig APN, which is regulated by the mouse proiximal APN promoter (porcine APN transformation) (Fig. 2-A). Pig APN cDNA was amplified from porcine intestinal cells and labeled at the C-terminus with a flag epitope to allow exogenous discrimination between expressed murine APN and expressed pig APN. The 2.9-kb flag epitope-tagged porcine APN cDNA was cloned into the pBluescript KS (+) vector under a 1.1-kb genome sequence control with the mouse proximal APN promoter.

The splice position of the cDNA and the polyadenylation signal were provided by the polyadenylation signal (BGH-PolyA) at the 0.2-kb small growth surface. The expression of the porcine APN protein was detected by immunoprecipitation in 293T cells infected with porcine APN Immunoblotting method. The flag-labeled recombinant protein was identified by immunoprecipitation with a molecular weight of about 150 kDa (a predicted molecular weight of porcine APN) (Fig. 2-B).

The present inventors measured the surface expression of porcine APN using flow cytometry using an anti-flag antibody, and confirmed that 21.3% of the cells exhibited flag-positive (Fig. 2-C). From these data, it is meant that the mouse proximal APN promoter proposed by the present invention effectively induces the expression of porcine APN.

3.3 Production of Transgenic Mouse Expressing Swine APN

Figure 3 is a diagram related to the development of porcine APN transgenic mice, wherein (A) shows the location of the PCR primers of the genomes used to detect foreign gene insertion.

Black arrow: PCR primer set, One; Gray arrow: PCR primer set.

(B) is a screening result of a pig APN transgenic mouse. The genomic DNA of CNUPEDm-1 and CNUPEDm-3 mice was isolated from the ends, and the foreign gene of pig APN was PCR amplified with two sets of genomic PCR primers.

WT: C57BL / 6 J untranslated mouse mouse; (+): Positive control group

The pig APN transgenes were linearized with NotI and DNA was microinjected into mouse conjugates. To screen for swine APN expression, two genomic PCR primers were designed as shown in Figure 3-A. The presence of the porcine APN transgene in mouse feces was monitored using PCR analysis of the terminal genomic DNA (Fig. 3-B). Microinjection of porcine APN transgenes produced two transgenic founders, CNUPEDm-1 and CNUPEDm-3. The transgenic mice were healthy and did not show any adverse effects on the expression of the transformants.

3.4 Characteristics of pig APN transgenic mice

Figure 4 shows pig APN expression in the small intestine of porcine APN transgenic mice. 4-A shows the result of measurement of porcine APN RNA in the small intestine of transgenic mice by RT-PCR. Small intestine samples were collected from non-transformed mouse wild type (1 and 2), CNUPEDm-1 (3 and 4), and CNUPEDm-3 mice (5 and 6). Pig APN RNAs were amplified using specific primers targeting pig APN. beta -actin was measured according to the loading control.

Figure 4-B is an image showing the expression of the porcine APN protein in the small intestine. Small intestine samples were collected from CNUPEDm-1 and CNUPEDm-3 mice. Pig APN protein was detected in intestinal homogenate by immunoblotting using anti-flag monoclonal antibodies (2 and 4). No recombinant proteins were detected in a small intestinal sample derived from non-transformed mouse wild type mice (1 and 3). beta -actin was used as a loading control.

4-C is an image showing immunohistochemical analysis of a swine APN transgenic mouse. The small intestine was prepared from a porcine APN transgenic mouse (ac: CNUPEDm-1; df: CNUPEDm-3) and immunized with anti-flag monoclonal antibodies (anti- (Anti-pAPN) (b and e), or normal mouse sera (c and f). The immune complexes were visualized with avidin-biotin-peroxidase, and the cells were counted after staining with hematoxylin and eosin. (Magnification x 40).

Because major pathological changes in porcine coronaviruses (eg, TGEV and PEDV) involve bowel disease, we measured expression of porcine APN in the small intestine using RTPCR, immunoblotting and IHC. The swine APN mRNA was expressed from feces derived from CNUPEDm-1 or CNUPEDm-3 (Fig. 4-A). Recombinant proteins with molecular weights predicted for porcine APN were expressed in the small intestine (Fig. 4-B). Immunohistochemical analysis with anti-flags and anti-swine APN antibodies clearly confirmed the expression of pig embryonic APN in the small intestine of the mouse model (Fig. 4-C). These results indicate that pig APN transgenic mice express the porcine APN in the small intestine.

On the other hand, the present inventors also screened for RT-PCR to confirm the expression characteristics of porcine APN in various tissues.

Figure 5 is an image showing pig APN expression in various tissues of porcine APN transgenic mice. FIG. 5-A shows the results of measurement of pig APN RNA in various tissues of transgenic mice using RT-PCR. Various tissue samples were obtained from CNUPEDm-3 mice. Pig APN RNA was amplified using specific primers targeting pig APN. beta -actin was used as a loading control.

FIG. 5-B is a graph showing the results of measurement of kidney of transgenic mice and intra-hepatic porcine APN-positive cells. At this time, kidney and liver cells were collected from CNUPEDm-3 mouse, and the cells were separated by a mesh filter. Pig APN-positive cells were stained with anti-flag monoclonal antibody and FITC-conjugated anti-mouse IgG secondary antibody and analyzed by flow cytometry. WT: C57BL / 6J untranslated mouse; APN-TG: pig APN transgenic mouse

Specifically, pig APN mRNA was strongly expressed in lung, kidney and intestine, and weakly expressed in liver (Fig. 5-A). The extracellular expression of these pig APNs was tested in kidney and liver using FACS analysis (Fig. 5-B). RT-PCR results showed that the number of flag-positive kidney cells in pig APN transgenic mice was significantly increased (2 to 4.3-fold) and slightly increased in flag-positive liver cells compared to untransformed wild-type mice 1.6 to 3 times). As a result, the present inventors confirmed the expression of porcine APN in the small intestine, lung, liver and kidney of a swine APN transgenic mouse.

3.5 PEDV infection of pig APN transgenic mice

Sensitivity to PEDV, one of the enteropathogenic porcine coronaviruses, was evaluated for porcine APN transgenic mice. Pig APN transgenic mice or non-transformed wild type mice were inoculated into the oral cavity using Vero-cell-adapted Korean strain of KPEDV-9, PEDV.

Figure 6 is an image showing immunohistochemical analysis of the small intestine of PEDV infected pig APN transgenic mice. 6-A is a photograph showing the excrement collected from a mouse at the indicated date after KPEDV-9 inoculation. Figure 6-B is an image of pig APN-transgenic mice (APN-TG) and non-transformed wild-type mice (WT) when KPEDV-9 was administered orally. Three days after inoculation, small intestine was collected and inoculated with mouse anti-PEDV polyclonal antiserum to tissue sections. (Magnification x 40).

Specifically, watery feces (dilute) were observed in the infected transgenic mice three days after inoculation (Fig. 6-A). On the fifth day, diarrhea, vomiting, fever, weight loss or death No clinically relevant changes were observed.

Virus replication was observed in various tissues extracted by RT-PCR and the results are shown in Table 1 below. Immunohistochemistry analysis of PEDV antigen in the small intestine revealed swine APN (FIG. 6-B), which is similar to that described in PEDV infected piglets (Debouck and Pensaert, 1980; Pensaert et al ., 1981) Lt; / RTI >

Day (Sun) Intestine lights liver kidney spleen WT TG WT TG WT TG WT TG WT TG One - + - - + - - - - + 2 - + - - + - - - - + 3 - + - - - - - - - + 5 - + ND ND ND ND ND ND ND ND WT: non-transgenic wild type mouse
TG: Transgenic mouse
ND: Not Determined

In Table 1 above, viral RNA in pig APN transgenic mice was observed in the small intestine for at least 5 days, in the spleen for at least 2 days, for at least 3 days, and not in the non-transgenic mice.

Figure 7 is also a graph showing PEDV replication in the small intestine of porcine APN transgenic mice. Both wild-type and porcine APN transgenic mice infected PEDV (5X TCID ₅₀ 10 ⁶ ) with the same day oral cavity. Two mice from each group were sacrificed by day 5, and small intestine samples were collected and titrated. Titration was performed using Vero cells. 7, the Y value is the PEDV activity (log value) within the TCID ₅₀ .

7, no histopathological changes in the PEDV-infected piglets were observed in the small intestine. As a result, these data indicate that pig APN transgenic mice are susceptible to PEDV infection, although general bowel disease symptoms have not been observed in PEDV-infected piglets (FIG. 7).

3.6 PEDV replication in small intestine of pig APN transgenic mice

Within the small intestine, PEDV replication in both wild type and porcine APN transgenic mice was confirmed and both were compared by VIRAL LOAD in the small intestine.

As a result, PEDV was not measured anywhere in the wild-type mouse sample. By comparison, the average figures in PEDV Day 1 of pigs APN transgenic mice was measured by TCID ₅₀ -10 ^3. ^1. TCID ₅₀ -10 ^3.8 , TCID ₅₀ -10 ^4.8 , TCID ₅₀ -10 ^5.0 , and TCID ₅₀ -10 ^{5.1 in the} order of days 2, 3, 4 and 5, respectively. However, no significant changes were observed from PI 3 to 5 days. PEDV replication in the small intestine of pig APN transgenic mice was clearly identified.

<110> Chung Nam Univ Foundation of Research & Business <120> Expression Vector to express pAPN of porcine epidemic diarrhea virus and transgenic mouse <130> 15P1188 <160> 5 <170> Kopatentin 2.0 <210> 1 <211> 4276 <212> DNA <213> Artificial Sequence <220> <223> Expression Vector to express pAPN of porcine epidemic diarrhea virus <400> 1 gcggccgcaa gatttgaaac agtggaatgt accggcgacg cgtgatgagt agttttaagg 60 ggtctgctgg aagcagggaa ttcatgagac tacagcaggg tctgctagag gctgtgagga 120 gaagggggtg agctggatca acgtcgggat gagcatcagc cagggtggct gtctgggagg 180 caggggccgg tgaaggagaa gggaaagggc atgtttgaca gagctggtta agtaatgtgt 240 tcatgtctac ccaggacaga gagccgaggg cggtgctgtg ctgtggggag gagggcttag 300 ctgtaagtgt ccaccatggg acggctcatg tgccaggtaa acctgaatgg gaaacactgt 360 catttacttc tgagaggtac gagagagaga gagagagaga gagagagaga gagagagaga 420 gggagagagg gagagagaga gagagaaggg ttcatttcgc cttctttgga gagctgaggg 480 gaaagaagtt ggtcaggagg aaaagggggc gaatctgaag actgtcccaa ggggcccctg 540 gcatctgttg aggaacccag aggtggagct ggtatgcttt tgatggcctg ctgggaactg 600 gcttcattct gctctgcctg ccttacctct cgagcctggg gaatctttcc ctgagtctaa 660 cctctgtcct catgagtctg ttctctccat ttctccttgc aggtagatta ccgtcactgc 720 cttcccccct gcctctaggt gccaagtctg cagcctgccc ttcagccccc gctctagctg 780 atttcccacc cccactcccc cctctccatc caggccacct gcagtcgtaa ccacacactg 840 tgttggttac agcaggcatc tcccctgccc acccccatcc cccatccctt accccaagcc 900 agctctgcac actgtttcat ttctgatctc tccagagcct gggcagagcg tacccctgtc 960 cagcctagtg accttcgcct gagctctggt taatatttgt ccgacccaaa ggcagtgggg 1020 ctccaccccc tgtgaggata taagctggcc ccggggctgc tgttctttcc tcttggcctg 1080 agctattccg agctccctgt ccaccggcat caagcttacc atggccaagg gattctacat 1140 ttccaaggcc ctgggcatcc tgggcatcct cctcggcgtg gcggccgtgg ccaccatcat 1200 cgctctgtct gtggtgtacg cccaggagaa gaacaagaat gccgagcatg tcccccaggc 1260 ccccacgtcg cccaccatca ccaccacagc cgccatcacc ttggaccaga gcaagccgtg 1320 gaaccggtac cgcctaccca caacgctgtt gcctgattcc tacaacgtga cgctgagacc 1380 ctacctcact cccaacgcgg atggcctgta catcttcaag ggcaaaagca tcgtccgctt 1440 catctgccag gagcccaccg atgtcatcat catccatagc aagaagctca actacaccac 1500 ccaggggcac atggtggtcc tgcggggcgt gggggactcc caggtcccag agatcgacag 1560 gactgagctg gtagagctca ctgagtacct ggtggtccac ctcaagggct cgctgcagcc 1620 cggccacatg tacgagatgg agagtgaatt ccagggggaa cttgccgacg acctggcagg 1680 cttctaccgc agcgagtaca tggagggcaa cgtcaaaaag gtgctggcca cgacacagat 1740 gcagtctaca gatgcccgga aatccttccc atgctttgac gagccagcca tgaaggccac 1800 gttcaacatc actctcatcc accctaacaa cctcacggcc ctgtccaata tgccgcccaa 1860 aggttccagc accccacttg cagaagaccc caactggtct gtcactgagt tcgaaaccac 1920 acctgtgatg tccacgtacc ttctggccta catcgtgagc gagttccaga gcgtgaatga 1980 aacggcccaa aatggcgtcc tgatccggat ctgggctcgg cctaatgcaa ttgcagaggg 2040 ccatggcatg tatgccctga atgtgacagg tcccatccta aacttctttg ccaatcatta 2100 taatacaccc tacccactcc ccaaatccga ccagattgcc ttgcccgact tcaatgccgg 2160 tgccatggag aactgggggc tggtgaccta ccgggagaac gcgctgctgt ttgacccaca 2220 gtcctcctcc atcagcaaca aagagcgagt tgtcactgtg attgctcacg agctggccca 2280 ccagtggttt ggcaacctgg tgaccctggc ctggtggaat gacctgtggc tgaatgaggg 2340 ctttgcctcc tatgtggagt acctgggtgc tgaccacgca gagcccacct ggaatctgaa 2400 agacctcatc gtgccaggcg acgtgtaccg agtgatggct gtggatgctc tggcttcctc 2460 ccacccgctg accacccctg ctgaggaggt caacacacct gcccagatca gcgagatgtt 2520 tgactccatc tcctacagca agggagcctc ggttatcagg atgctctcca acttcctgac 2580 tgaggacctg ttcaaggagg gcctggcgtc ctacttgcat gcctttgcct atcagaacac 2640 cacctacctg gacctgtggg agcacctgca gaaggctgtg gatgctcaga cgtccatcag 2700 gctgccagac actgtgagag ccatcatgga tcgatggacc ctgcagatgg gcttccccgt 2760 catcaccgtg gacaccaaga caggaaacat ctcacagaag cacttcctcc tcgactccga 2820 atccaacgtc acccgctcct cagcgttcga ctacctctgg attgttccca tctcatctat 2880 taaaaatggt gtgatgcagg atcactactg gctgcgggat gtttcccaag cccagaatga 2940 tttgttcaaa accgcatcgg acgattgggt cttgctgaac atcaacgtga caggctattt 3000 ccaggtgaac tacgacgagg acaactggag gatgattcag catcagctgc agacaaacct 3060 gccggtcatc cctgtcatca atcgggctca ggtcatctac gacagcttca acctggccac 3120 tgcccacatg gtccctgtca ccctggctct ggacaacacc ctcttcctga acggagagaa 3180 agagtacatg ccctggcagg ccgccctgag cagcctgagc tacttcagcc tcatgttcga 3240 ccgctccgag gtctatggcc ccatgaagaa atacctcagg aagcaggtcg aacccctctt 3300 ccaacatttc gaaactctca ctaaaaactg gaccgagcgc ccagaaaatc tgatggacca 3360 gtacagtgag attaatgcca tcagcactgc ctgctccaat ggattgcctc aatgtgagaa 3420 tctggccaag acccttttcg accagtggat gagcgaccca gaaaataacc cgatccaccc 3480 caacctgcgg tccaccatct actgcaatgc catagcccag ggcggccagg accagtggga 3540 ctttgcctgg gggcagttac aacaagccca gctggtaaat gaggccgaca aactccgctc 3600 agcgctggcc tgcagcaacg aggtctggct cctgaacagg tacctgggtt acaccctgaa 3660 cccggacctc attcggaagc aagacgccac ctccactatt aacagcattg ccagcaatgt 3720 catcgggcag cctctggcct gggattttgt ccagagcaac tggaagaagc tctttcagga 3780 ctatggcggt ggttccttct ccttctccaa cctcatccag ggtgtgaccc gaagattctc 3840 ctctgagttt gagctgcagc agctggagca gttcaagaag aacaacatgg atgtgggctt 3900 cggctccggc acccgggctc tggagcaagc cctggagaag accaaggcca acatcaagtg 3960 ggtgaaggag aacaaggagg tggtgttgaa ttggttcata gagcacagcg actacaaaga 4020 cgatgacgac aagtgactcg agcgactgtg ccttctagtt gccagccatc tgttgtttgc 4080 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa 4140 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg 4200 gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg 4260 ggctctatgg ggtacc 4276 <210> 2 <211> 1103 <212> DNA <213> Artificial Sequence <220> <223> mouse APN proximal promoter <400> 2 aagatttgaa acagtggaat gtaccggcga cgcgtgatga gtagttttaa ggggtctgct 60 ggaagcaggg aattcatgag actacagcag ggtctgctag aggctgtgag gagaaggggg 120 tgagctggat caacgtcggg atgagcatca gccagggtgg ctgtctggga ggcaggggcc 180 ggtgaaggag aagggaaagg gcatgtttga cagagctggt taagtaatgt gttcatgtct 240 acccaggaca gagagccgag ggcggtgctg tgctgtgggg aggagggctt agctgtaagt 300 gtccaccatg ggacggctca tgtgccaggt aaacctgaat gggaaacact gtcatttact 360 tctgagaggt acgagagaga gagagagaga gagagagaga gagagagaga gaggagagaga 420 gggagagaga gagagagaag ggttcatttc gccttctttg gagagctgag gggaaagaag 480 ttggtcagga ggaaaagggg gcgaatctga agactgtccc aaggggcccc tggcatctgt 540 tgaggaaccc agaggtggag ctggtatgct tttgatggcc tgctgggaac tggcttcatt 600 ctgctctgcc tgccttacct ctcgagcctg gggaatcttt ccctgagtct aacctctgtc 660 ctcatgagtc tgttctctcc atttctcctt gcaggtagat taccgtcact gccttccccc 720 ctgcctctag gtgccaagtc tgcagcctgc ccttcagccc ccgctctagc tgatttccca 780 cccccactcc cccctctcca tccaggccac ctgcagtcgt aaccacacac tgtgttggtt 840 acagcaggca tctcccctgc ccacccccat cccccatccc ttaccccaag ccagctctgc 900 acctgtttc atttctgatc tctccagagc ctgggcagag cgtacccctg tccagcctag 960 tgaccttcgc ctgagctctg gttaatattt gtccgaccca aaggcagtgg ggctccaccc 1020 cctgtgagga tataagctgg ccccggggct gctgttcttt cctcttggcc tgagctattc 1080 cgagctccct gtccaccggc atc 1103 <210> 3 <211> 2889 <212> DNA <213> Artificial Sequence <220> <223> porcine APN <400> 3 atggccaagg gattctacat ttccaaggcc ctgggcatcc tgggcatcct cctcggcgtg 60 gcggccgtgg ccaccatcat cgctctgtct gtggtgtacg cccaggagaa gaacaagaat 120 gccgagcatg tcccccaggc ccccacgtcg cccaccatca ccaccacagc cgccatcacc 180 ttggaccaga gcaagccgtg gaaccggtac cgcctaccca caacgctgtt gcctgattcc 240 tacaacgtga cgctgagacc ctacctcact cccaacgcgg atggcctgta catcttcaag 300 ggcaaaagca tcgtccgctt catctgccag gagcccaccg atgtcatcat catccatagc 360 aagaagctca actacaccac ccaggggcac atggtggtcc tgcggggcgt gggggactcc 420 caggtcccag agatcgacag gactgagctg gtagagctca ctgagtacct ggtggtccac 480 ctcaagggct cgctgcagcc cggccacatg tacgagatgg agagtgaatt ccagggggaa 540 cttgccgacg acctggcagg cttctaccgc agcgagtaca tggagggcaa cgtcaaaaag 600 gtgctggcca cgacacagat gcagtctaca gatgcccgga aatccttccc atgctttgac 660 gagccagcca tgaaggccac gttcaacatc actctcatcc accctaacaa cctcacggcc 720 ctgtccaata tgccgcccaa aggttccagc accccacttg cagaagaccc caactggtct 780 gtcactgagt tcgaaaccac acctgtgatg tccacgtacc ttctggccta catcgtgagc 840 gagttccaga gcgtgaatga aacggcccaa aatggcgtcc tgatccggat ctgggctcgg 900 cctaatgcaa ttgcagaggg ccatggcatg tatgccctga atgtgacagg tcccatccta 960 aacttctttg ccaatcatta taatacaccc tacccactcc ccaaatccga ccagattgcc 1020 ttgcccgact tcaatgccgg tgccatggag aactgggggc tggtgaccta ccgggagaac 1080 gcgctgctgt ttgacccaca gtcctcctcc atcagcaaca aagagcgagt tgtcactgtg 1140 attgctcacg agctggccca ccagtggttt ggcaacctgg tgaccctggc ctggtggaat 1200 gacctgtggc tgaatgaggg ctttgcctcc tatgtggagt acctgggtgc tgaccacgca 1260 gagcccacct ggaatctgaa agacctcatc gtgccaggcg acgtgtaccg agtgatggct 1320 gtggatgctc tggcttcctc ccacccgctg accacccctg ctgaggaggt caacacacct 1380 gcccagatca gcgagatgtt tgactccatc tcctacagca agggagcctc ggttatcagg 1440 atgctctcca acttcctgac tgaggacctg ttcaaggagg gcctggcgtc ctacttgcat 1500 gcctttgcct atcagaacac cacctacctg gacctgtggg agcacctgca gaaggctgtg 1560 gatgctcaga cgtccatcag gctgccagac actgtgagag ccatcatgga tcgatggacc 1620 ctgcagatgg gcttccccgt catcaccgtg gacaccaaga caggaaacat ctcacagaag 1680 cacttcctcc tcgactccga atccaacgtc acccgctcct cagcgttcga ctacctctgg 1740 attgttccca tctcatctat taaaaatggt gtgatgcagg atcactactg gctgcgggat 1800 gtttcccaag cccagaatga tttgttcaaa accgcatcgg acgattgggt cttgctgaac 1860 atcaacgtga caggctattt ccaggtgaac tacgacgagg acaactggag gatgattcag 1920 catcagctgc agacaaacct gccggtcatc cctgtcatca atcgggctca ggtcatctac 1980 gacagcttca acctggccac tgcccacatg gtccctgtca ccctggctct ggacaacacc 2040 ctcttcctga acggagagaa agagtacatg ccctggcagg ccgccctgag cagcctgagc 2100 tacttcagcc tcatgttcga ccgctccgag gtctatggcc ccatgaagaa atacctcagg 2160 aagcaggtcg aacccctctt ccaacatttc gaaactctca ctaaaaactg gaccgagcgc 2220 ccagaaaatc tgatggacca gtacagtgag attaatgcca tcagcactgc ctgctccaat 2280 ggattgcctc aatgtgagaa tctggccaag acccttttcg accagtggat gagcgaccca 2340 gaaaataacc cgatccaccc caacctgcgg tccaccatct actgcaatgc catagcccag 2400 ggcggccagg accagtggga ctttgcctgg gggcagttac aacaagccca gctggtaaat 2460 gaggccgaca aactccgctc agcgctggcc tgcagcaacg aggtctggct cctgaacagg 2520 tacctgggtt acaccctgaa cccggacctc attcggaagc aagacgccac ctccactatt 2580 aacagcattg ccagcaatgt catcgggcag cctctggcct gggattttgt ccagagcaac 2640 tggaagaagc tctttcagga ctatggcggt ggttccttct ccttctccaa cctcatccag 2700 ggtgtgaccc gaagattctc ctctgagttt gagctgcagc agctggagca gttcaagaag 2760 aacaacatgg atgtgggctt cggctccggc acccgggctc tggagcaagc cctggagaag 2820 accaaggcca acatcaagtg ggtgaaggag aacaaggagg tggtgttgaa ttggttcata 2880 gagcacagc 2889 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Flag tag <400> 4 gactacaaag acgatgacga caag 24 <210> 5 <211> 228 <212> DNA <213> Artificial Sequence <220> <223> BGH polyA <400> 5 cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga 60 ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt 120 gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg 180 attgggaaga caatagcagg catgctgggg atgcggtggg ctctatgg 228

Claims

A recombinant expression vector for expressing pAPN protein comprising a promoter and a pAPN protein gene.

2. The recombinant expression vector according to claim 1, wherein the promoter is a proximal promoter represented by SEQ ID NO: 2.

2. The recombinant expression vector according to claim 1, wherein the promoter is operably linked to the pAPN protein gene of SEQ ID NO: 3 and the BGH-polyA signal sequence of SEQ ID NO: 4.

The recombinant expression vector according to claim 3, further comprising a flag tag (Flag taq) shown in SEQ ID NO: 5

2. The recombinant expression vector of claim 1, wherein said recombinant expression vector has a cleavage map of Figure 2-A.

2. The recombinant expression vector according to claim 1, wherein the recombinant expression vector is represented by SEQ.

A transgenic mouse transformed with the recombinant expression vector of claim 1.

Preparing a recombinant expression vector of claim 1; And
And transforming the recombinant vector into a mouse embryo.

A method for diagnosing porcine epidemic diarrhea virus (PEDV) using the transgenic mouse of claim 7.

10. The method according to claim 9, wherein the diagnosis is selected from the group consisting of small intestinal cells, lung cells, and kidney cells of a mouse.