KR20160072423A

KR20160072423A - Vaccine producing and antibody assaying methods, using PEDV envelope proteins

Info

Publication number: KR20160072423A
Application number: KR1020140179998A
Authority: KR
Inventors: 주한수; 정현규
Original assignee: 주식회사 한수양돈연구소
Priority date: 2014-12-15
Filing date: 2014-12-15
Publication date: 2016-06-23

Abstract

The present invention relates to a method for producing a vaccine and measuring an antibody with respect to the virus, using an outer skin protein of a porcine epidemic diarrhea virus and, more particularly, to a method for infecting a Vero cell with an outdoor isolate of PEDV, and separating and extracting the outer skin protein of a PED virus therefrom; a method for producing a vaccine of a PED virus produced by uniformly mixing the outer skin protein of the separated PED virus with an immune booster including mineral oil and an emulsifier or a suspension promoter; a method for measuring a blood antibody with respect to a PED virus for measuring a concentration of an antibody by a RIDEA method in which ELISA and the principle of radioimmunoassay diffusion are combined by absorbing the outer skin protein (antigen) of the separated PED virus in a substrate to be reacted with a pig serum; and a method for measuring an antibody a cell with respect to a PED virus measured in a delayed type hypersensitivity reaction (DTH) method by intradermally inoculating the outer skin protein of the separated PED virus to a testing pig. According to the present invention, the method for producing a vaccine and a method for measuring an antibody, using the outer skin protein of a porcine epidemic diarrhea virus is capable of preventing mortality of newborn piglets and economically and easily diagnosing whether of infection of a virus of a pig in which infection of a PED virus is suspicious.

Description

Technical Field [0001] The present invention relates to vaccine production methods and vaccines,

The present invention relates to a vaccine composition for preventing the proliferation and excretion of a virus from a pig vaccinated with a vaccine against parasitic diarrhea virus infection, And a measurement method.

The causative agents of porcine major infectious diseases caused by coronaviruses include Porcine epidemic diarrhea virus (PEDV), Transmissible gastroenteritis virus (TGEV) and Porcine respiratory coronavirus (PRCV) , And PEDV and TGEV, both of which have been occurring for a long time in Korea, have caused huge damage to the pig industry.

Porcine Epidemic Diarrhea (PED) was first reported in Europe and has been causing numerous damage to many Asian countries, including Korea, for many years. In particular, the disease has recently spread to the United States, Canada and some parts of South America, and has attracted worldwide attention.

In farms infected with PED virus, vomiting and severe diarrhea symptoms are observed in pigs within 1 week of age and almost 100% mortality rate is observed. The newborn pigs last for 4-8 weeks and can be prevented in pigs fed with antibody colostrum made from infected pigs.

Since the vaccine that prevents the disease is currently being made and sold, however, it can not provide sufficient protection effect, so pig farmers can produce fecal or intestinal fluid of piglets infected with PED virus and artificially infect the pregnant donor before delivery. And let them eat piglets through colostrum to defend against disease. However, there are many farmers who do not have sufficient defense effect because the artificial infection process is complicated and can not be performed uniformly.

In addition, PEDV can not be cured, so prevention and prevention of damage are alleviated through damage prevention. Due to the nature of the virus, laboratory diagnosis is not available and there are many problems in diagnosis due to the same relationship between clinical symptoms and infectious gastroenteritis virus infection. In addition, the most widely used method for the diagnosis of PEDV is reverse transcription polymerase chain reaction (RT-PCR, Kweon et al., JVM S, 59 (3): 231-232, 1997) However, this method is expensive because the equipment required for the diagnosis process is expensive, requires expertise, has a high diagnostic cost due to high cost of the enzyme and its cost, and is not suitable for outdoor application.

It is known that commercially available vaccine strains have long been isolated from seed viruses, are cultured for a long time in transplanted tissues, and have not been satisfactorily effective due to defective or defective antigens.

The present inventors isolated and propagated the PED virus outdoor isolate using Vero cells and then killed the Vero cells and extracted the PED virus envelope protein at a high concentration. In addition, the method of producing a vaccine using the PED virus envelope protein The present inventors completed the present invention by developing a method for measuring a blood antibody and an intracellular antibody against PED virus of a pig suspected of having PED virus infection using the PED virus envelope protein together.

It is an object of the present invention to provide a method for proliferating a porcine epidemic diarrhea virus isolate using Vero cells or the like and a method for extracting a high concentration of PED virus envelope protein therefrom.

Yet another object of the present invention is to provide an effective vaccine production method capable of inhibiting virus infection, proliferation and excretion, as well as prevention of death of pig pigs, that is, a method for producing a vaccine using the separated PED virus envelope protein . Conventional vaccine manufacturing techniques have been made by inactivating the virus itself in vivo using tissue-cultured cells and then adsorbing the virus on formalin and adsorbing it on an aluminum gel. However, in order to make a more effective vaccine, Is extracted by physicochemical method and mixed with a special combination of immunity enhancer to make the product more effective than the existing vaccine.

It is another object of the present invention to provide a method for measuring blood antibodies and intracellular antibodies against PED viruses in pigs suspected of infecting PED virus using the separated PED virus envelope proteins.

In order to achieve the above object, the present invention provides a method for producing PEDV, comprising the steps of: 1) infecting Vero cells in which PEDV has been removed from a cell culture medium, subculturing the PEDV in a culture medium containing serum and removing trypsin; 2) recovering cells at the early stage of cell degeneration during the subculture and centrifuging; And 3) treating the recovered cells with a chemical to separate the envelope protein of the PED virus from the cells; And 4) further isolating and concentrating the envelope protein of the PED virus from the cells through centrifugation, thereby providing a method for extracting the coat protein of high purity and high concentration of PED virus.

The present invention also relates to a vaccine for PED virus, which is prepared by uniformly mixing an envelope protein of PED virus isolated by the method for extracting the coat protein of the PED virus and an immunostimulant comprising mineral oil and an emulsifier or a suspension promoter And a manufacturing method thereof.

Also, the present invention provides a method for producing PED virus, comprising the steps of: 1) adsorbing a coat protein (antigen) of PED virus isolated by the method of extracting the coat protein of the PED virus on a substrate and then covering the agar; 2) isolating serum from pigs infected with PED virus; 3) reacting the serum in step 2) with the antigen in the agar of step 1); And 4) measuring the concentration of the antibody by the RIDEA method in which the antigen-antibody reaction is combined with an ELISA and a principle of radioactive immunodiffusion.

The present invention also provides a method for producing a PED virus, comprising the steps of: 1) inoculating a coat protein of PED virus isolated by the method of extracting the coat protein of the PED virus into a test pig; And 2) measuring the reaction diameter by a Delayed type hypersensitivity reaction (DTH) method 24-72 hours after the inoculation, to provide a method for measuring an intracellular antibody against PED virus.

Hereinafter, the present invention will be described in detail.

The academic background directly related to the present invention and the advantages of the present invention will be described as follows.

1) PEDV separation from outdoor farm samples

Since PEDV is very difficult to isolate and cultivate unlike other viruses, it mainly depends on PCR method to diagnose infection with this virus. Although there are no published papers on this virus isolation rate, it is known that when more than 100 PEDV PCR positive samples are cultured, one sample is successfully isolated. In fact, there are only two separate culture reports in Korea for more than 10 years (Kwon Chang-hee, Park Bong-gyun).

However, it can be confirmed from the present invention that separation and culture success rate can be improved by understanding the characteristics of the present virus and cultured cells. The present invention provides a highly successful separation method by preparing a fresh virus as a separation material, improving a cell culture method, and an infectious cell lineage method.

2) PEDV envelope protein extraction method

The PED virus isolates isolated from the outdoor farm samples are grown using Vero cells or the like, and further cultured in a medium containing serum-free trypsin. During this incubation period, a small amount of cell culture medium and cells attached to the bottom of the culture bottle are treated with a chemical agent such as Triton X-100 and centrifuged to provide a method for extracting high-purity PED virus coat protein .

3) Vaccine recipe for PEDV

The envelope proteins are extracted from the PEDV-infected cells, and the maximum amount of antibody can be prepared by mixing them with an immunopotentiator. As the immunostimulant, water and oil are mixed well using mineral oil and appropriate emulsifier. And provides a method for maintaining uniform suspension of these mixed solutions and obtaining a high immunity effect without side effects on the inoculation site.

4) Antibody test for PEDV-infected pigs

Antibodies from PEDV-infected pigs are tested by ELISA, Immunofluorescent antibody (IFA), or Serum neutralization (SN). Among these three methods, the neutralization test method is known to be an important index to measure defense ability by using live PEDV as an antigen. However, due to the nature of the virus, which requires the use of trypsin, the test procedure is complicated and poorly sensitive, resulting in low titers.

Pigs recovered after infection with PED virus form antibodies, and these antibodies can be largely classified into blood antibodies and intracellular antibodies. The present invention provides a method for measuring these antibodies.

4-1) Blood antibody test

The concentration of the antibody is measured by the RIDEA (Radial Immunodiffusion Enzyme Assay) method in which the PED virus envelope protein prepared as described above is adsorbed on a substrate and the test serum is reacted with the ELISA and the principle of radioactive immunodiffusion is combined.

4-2) Intracellular antibody measurement of newborn piglets

A small amount of the coat protein antigen prepared as described above can be intradermally injected into newborn piglets and immunity can be confirmed through a delayed type hypersensitivity reaction (DTH).

The present invention relates to: 1) infecting Vero cells from which PEDV has been removed from a cell culture-derived medium, culturing the PEDV in a culture medium containing serum-free and trypsin-containing medium; 2) recovering cells at the early stage of cell degeneration during the subculture and centrifuging; And 3) treating the recovered cells with a chemical to separate the envelope protein of the PED virus from the cells; And 4) further isolating and concentrating the envelope protein of the PED virus from the cells through centrifugation, thereby providing a method for extracting the coat protein of high purity and high concentration of PED virus.

In the method for extracting the coat protein of highly pure and concentrated PED virus according to the present invention, the chemical for separating the coat protein of PED virus from the cells of step 3) is preferably Triton X-100, Preferably, the envelope protein of the PED virus of step 4) is isolated and concentrated in a supernatant of centrifugation at 10,000 rpm.

In the vaccine preparation method of PED virus according to the present invention, the emulsifier or the suspension promoter is preferably mixed with 80 to 150 parts by weight of Tween 85 in 100 parts by weight of Span 85, and the immunostimulating agent is mixed with 100 parts by weight of mineral oil 100 And 5 to 20 parts by weight of an emulsifier or a suspension promoter are mixed in the weight portion.

By using special conditions and materials, the time required to read the test results is shortened to within 3 hours, and since blood material adsorbed on the filter paper is used, the blood collection process can be simplified to promptly and accurately diagnose PEDV-infected pigs . Hereinafter, the diagnostic process of the present invention will be described in detail.

(1) Agar type (2) Agar concentration (3) By adjusting the amount of agar, the result is read from 24 hours to 3 hours .

The entire life cycle of this diagnostic method is described as follows.

1) The blood is absorbed on the filter paper by a veterinarian or a training shorthair, and it is directly inspected on the farm or dried and sent to the laboratory.

2) Cut the filter paper with the blood size of about 6 mm in diameter with a paper hole cutter (punch)

3) Arrange the test vessel as shown in Fig.

4) The cells were cultured for 90 minutes at room temperature

5) After removing the agar

6) Wash three times with wash (0.5% Tween 85 buffer).

7) After incubation with enzyme-conjugated anti-porcine immunoglobulin for 45 minutes

8) Repeating the cleaning process as in 6)

9) When the 1% agar solution containing the coloring agent is covered

10) After 5 to 10 minutes, the positive specimens are dark purple and the negative specimens are colorless and transparent.

The preparation process and the inspection method of the present invention will now be described.

(1) Picking of the sword material

In the case of newborn piglets, the blood that is bleeding in the stomach or castration is adsorbed on the filter paper (1 ㅧ 5 cm) and dried, or the wet adsorbed filter paper is cut into circular (diameter 6 mm ) To be used for the test. The extra filter paper adsorbed on the sample can be stored in a refrigerator at 4 ° C for use in the test.

(2) Preparation of antigen

Details are described in the following examples.

(3) Manufacture of inspection boards

The test plate may be a bowl-shaped container capable of adhering the antigen. Polystyrene syringes (Petridish) are the easiest to use and appropriate antigen concentrations are diluted in Adhesion Buffer, then placed in Petri dishes and cultured at 4 ° C. The adhesive buffer solution is a solution mainly containing 0.01-0.1 M (pH 9-10) NaHCO ₃ and Na ₂ CO ₃ .

The antigen remaining after adhesion is removed and incubated with a solution of bovine serum albumin to fix the antigen binding and increase the specificity. Once the antigen-binding membrane is immobilized, the agar solution is added to harden it. Test vessels manufactured above can be kept for more than 6 months after closing the lid and maintaining proper humidity at 4 ℃ and preventing contamination.

(4) Inspection method

The filter paper on which the sample is adsorbed is adhered onto the agar of the test plate as shown in Fig. At this time, the scrubbed disc should be first wetted with distilled water. After adhering the filter paper disc and incubating at room temperature for 90 minutes, the antibody in the sperm penetrates under the agar and binds to the antigen. Carefully remove the agar and wash it three times with the washing solution. After three washes, the wash solution is completely removed and the cells are incubated at room temperature for 30-60 minutes with the appropriate concentration of immunoenzymes (eg goat anti swine peroxidase conjugate). Soak the agar in 1% buffer, dissolve in boiling water and store at 56 ℃. Rinse 3 times with wash solution again to remove unbound immunoenzymes. After washing, add a colored solution (1% agar solution containing Aminosaicylic acid and H ₂ O ₂ ).

As shown in FIG. 2, in the positive reaction, adhesion with an antigen such as an antibody or an immunological enzyme occurs, causing a reaction of a coloring agent to produce a dark purple color. Even though the antibody is washed out during washing, It shows colorless and transparent reaction because it can not attach. (Fig. 3)

These colors change in color as time goes by, and the reaction appears forever. The agar in which the reaction takes place has the advantage of preserving the test results forever because the color remains, although it is dry. In the case of positive reaction, 1) natural infection 2) vaccination 3) it can be solved by maternal antibody. If the vaccine is not allowed to be used as in Korea, a positive result indicates infection of pathogenic virus in the field. Opening for the manufacture of a kit for outdoor inspection (1) Test inspection (2) Concentrated washing solution (3) Immunoenzymes (4) Coloring agent (5) Positive serum filter paper (6) Negative serum filter paper, It is necessary to have an electric port to boil the water.

The vaccine production method and the antibody measurement method using the coat protein of the swine fever diarrhea virus according to the present invention as described above can prevent the death of newborn piglets and it is economically and economically feasible to infect pigs suspected of having PED virus infection It is easy to diagnose.

1 is an arrangement view of a claw material according to the present invention,
2 is a cross-sectional explanatory view of the positive-proof material according to the present invention,
3 is a cross-sectional explanatory view of the negative-tone screen material according to the present invention.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the drawings. However, it should be understood that the following embodiments are provided so that those skilled in the art may understand the present invention without departing from the scope and spirit of the present invention. It is not.

Example 1 PED virus isolation and culture method from outdoor materials

First, outdoor PEDV isolates should have live PED viruses with high titers. Usually qPCR measures the amount of PEDV RNA expressed in cT, but the exact method should be to directly infect susceptible pigs and test for Pig infective dose.

In the present invention, the PEDV RNA was detected in the outdoor samples, and the material was used to scrape the inner wall of the small intestine (intestinal fluid) at the early stage of diarrhea expression after oral ingestion into the piglets of 3 days old. Therefore, it can be regarded that the amount of viable virus in the sample is the highest. These small intestine materials were suspended in 1: 5 or 1:10 in 10 μg / ml trypsin-supplemented MEM (minimal essential medium) and infected with Vero cells cultured for 3-7 days. At this time, Vero cells should be prepared to be 3-7 days old by using MEM supplemented with 8% FCS and antibiotics on a 24-well cell culture plate. Washing medium (WM medium - MEM 450 ml + TPB 50 ml + trypsin 1.0 [mu] g). Vero cell monolayers inoculated with the emulsion (0.2 ml / well) were adsorbed for 1 hour at 37 ° C and cultured in a 5% CO ₂ incubator at 37 ° C in a 1.5 ml / well culture medium. At this time, culture medium containing 5-10 트 trypsin (CM medium - MEM 450 ml + TPB 50 ml + trypsin 5.0 - 10)) was changed daily and cultured for 5-7 days while maintaining the cells as healthy cells.

The TPB (Tryptose phosphate broth) was purchased from Teknova Cat. The composition consists of 2.0% tryptose, 0.2% glucose, 0.5% NaCl (sodium chloride), 0.25% dibasic sodium phosphate and 500 ml distilled water. Respectively.

In the primary culture, PEDV-specific cytotoxic effects (CPE) are usually not observed. During the subculture of these infectious materials, Vero cell monolayer of 3-7 days old was washed twice with a washing medium, and the supernatant (1 ml / well of the cell supernatant was removed and the bottom cells were scraped and suspended in 0.5 ml of the remaining cells and cells ) 0.2 ml / well. After incubation at 37 ° C for 1 hour, the culture medium was injected. When the PEDV CPE was not seen after 5 passages in this way, it was regarded as virus isolation. Once the samples showing PEDV CPE were confirmed positive by PCR, they were confirmed as PEDV by partial or full-length sequencing.

In the present invention, six samples were separated from one another (PEDV HS-1 - PEDV cT value 16.4), and another one (PEDV HS-2 - PEDV cT value 15.4) PEDV specific CPE was observed. The PEDV-1 strain sequenced 28,033 nucleotides of the entire RNA genome. In contrast, CPE was not observed in the four cT samples (32.6, 34.5, 36.9 and 38.2).

Example 2 Extraction of Coat Protein from PED Virus (Extraction from Dead Virus)

The isolated PED virus isolates were grown using Vero cells grown for 1-5 days. At this time, the cell culture medium was removed, PED virus was inoculated, adsorbed at 37 ° C for 1 hour, and the medium containing 2-10 μl of trypsin was added without serum and cultured at 37 ° C for 1-3 days. During the incubation period, a small amount of the cell culture medium and the cells attached to the bottom of the culture bottle were recovered and centrifuged at the early stage of cell degeneration. Twenty-fold times the amount of infected cells (Triton X-100) was added and centrifuged at 4-25 ° C for 2-15 hours. In this process, the envelope protein of PED virus was liberated and extracted, and relatively pure virus envelope proteins were obtained in the upper layer by centrifuging the cells and virus envelope protein at 4 ° C for about 60 minutes at about 10,000 rpm rotation. These proteins are in a concentrated state and can be appropriately diluted using buffered saline depending on the application.

&Lt; Example 3 > Preparation of vaccine using viral coat protein

Extracellular protein extraction from PEDV infected cells was described above. The combination of these viral proteins and immunostimulants was used to generate antibodies as much as possible. As the immune enhancer, mineral oil was used and a proper emulsifier was used to mix water and oil well. There is a need for a manufacturing technique that maintains homogeneous suspension properties in accordance with kinds, blending ratios, compounding methods, and the like, and obtains a high immunity effect on the inoculated site without side effects. As a result of various comparative tests, a suspension accelerator was prepared by mixing emulsifier Span 85 and Tween 85 at a ratio of 54 ml and 64 ml, and 10 ml of the suspension promoter and 90 ml of mineral oil (ratio of 1: 9) To prepare an optimal immunostimulant.

Diluted viral envelope proteins were mixed at a ratio of 1: 1 to these immunostimulants and buffered saline at appropriate concentrations, and mixed uniformly using an ultrasonic generator or a high-speed mixer. These mixed materials were shaken at room temperature, and it was confirmed that a uniform suspension without separation layer was maintained for up to 4 hours.

The vaccine prepared as described above was inoculated twice to 16 pigs of 4 weeks old at intervals of 2 weeks, and the test results as shown in Table 2 were obtained.

The dose-neutralizing antibody in pigs inoculated with the test vaccine Pig number During the first vaccination First inoculation
after 2 weeks Second inoculation
after 2 weeks One - 1: 2 1:32 2 - 1: 2 1: 256 3 - 1: 4 1:32 4 - 1: 4 1:32 5 1: 2 1: 4 1:64 6 - 1: 4 1:16 7 1: 8 1: 8 1:32 8 - 1: 2 1:64 9 - 1: 4 1:64 10 - 1: 2 1:64 11 - 1: 8 1: 128 12 1: 2 1: 4 1: 128 13 1: 2 1: 4 1: 8 14 1: 2 1:32 1:32 15 - 1: 4 1:32 16 - 1: 8 1: 256

-: PED virus neutralizing antibody <1: 2

Two inoculations were made every two weeks with 1 ㎖ of 4 - week - old pigs.

As shown in the table above, when the PEDV neutralizing antibody was tested 2 weeks after the vaccination, 16 cases showed high antibody level (1:32 - 1: 256) except for 2 cases.

Example 4: Antibody test in pigs after PED virus infection

In pigs recovered after infection with this virus, they form antibodies. These antibodies can be classified into blood antibodies and intracellular antibodies.

<4-1> Measurement of antibody in blood using diagnostic kit

Blood antibodies can be conveniently measured using a kit prepared as follows. First, the PED virus envelope protein prepared as described above is adsorbed in a test vessel, the agar is placed on the antigen, the test serum is placed in a hole formed in a hard agar, and the concentration of the antibody is measured by RIDEA, which is a combination of ELISA and radioimmunoprecipitation Were measured.

These detailed techniques are similar to the Pseudorabies antibody assay published by JOO et al. (1984). (See JOO et al., 1984 and Patent Publication No. 91-4121)

<4-2> Measurement of intracellular antibody using DTH

In order to measure the intracellular antibody of the newborn piglets, a small amount (0.1 ml) of the envelope protein antigen prepared as described above was injected intradermally (intraderminus between the navel of the pig and thigh anthers), and the cell immune response (Delayed type hypersensitivity reaction = DTH) were measured. At this time, the diameter of the inoculation reaction was different according to the amount of cell immunity received from the mother piglet. The piglets of well - immunized sows showed 0.4-6.5 ㎜ and the piglets from unimmunized sows were within 0.4 ㎜.

<References>

M. HOFMANN AND R. WYLER (1988) Propagation of the Virus of Porcine Epidemic Diarrhea in Cell Culture. J. CLINICAL MICROBIOL. 1988, p. 2235-2239 Vol. 26, No. 11

SHI Xin-fu, ZHANG Yu-jing, LIU Xiu-fan and WANG Xi-liang (2009) Generation and characterization of a cold-adapted attenuated live H3N2 subtype influenza virus vaccine candidate Chin Med J 122 (23): 2880-2885

Dan Zheng, Yinglei Yi 1, and Ze Chen (2012) Development of Live-Attenuated Influenza Vaccines against Outbreaks of H5N1 Influenza Viruses , 4: 3589-3605

Heightened adaptive immune responses following vaccination with a temperature-sensitive, live-attenuated influenza virus compared to adjuvanted, whole-inactivated virus in pigs. Crystal L. Lovinga, Amy L. Vincenta, Lindomar Penab, and Daniel R. Perezb, Vaccine. 2012 30: 5830-5838

(2009) Efficacy of an intranasal modified live bovine respiratory syncytial virus and temperature-sensitive parainfluenza type 3 virus vaccine in 3-week-old calves experimentally challenged with PI3V The Veterinary Journal 179: 101-108

J.R. Patel, J. Foildi, H. Bateman, J. Williams, S. Didlick, R. Stark (2003) Equid herpesvirus (EHV-1) live vaccine strain C147: efficacy against respiratory diseases following EHV types 1 and 4 Microbiology 92: 1-17

AN Wen-qi, YANG Peng-hui, DUAN Yue-qiang, LUO De-yan, TANG Chong, JIA Wei-hong, XING Li, J. VIROLOGY, 2011, p. 7225-7235 Vol. 85, No. 14. A Host-Specific, Temperature-Sensitive Translation Defect Determines the Attenuation Phenotype of a Human Rhinovirus / Poliovirus Chimera, PV1 (RIPO) Nusrat Jahan, Eckard Wimmer, and Steffen Mueller

Respiratory Syncytial Virus Modified by Deletions of the NS2 Gene and Amino Acid S1313 of the L Polymerase Protein Is a Temperature-Sensitive, Live-Attenuated Vaccine Candidate That Is Phenotypically Stable at Physiological Temperature. Cindy Luongo, Christine C. Winter, Peter L. Collins, Ursula J. Buchholz 2013, 87 Number 4 Journal of Virology p. 1985-1996

Joo HS. Molitor TW and Leman AD (1984) Radial immunodiffusion enzyme assay for detection of antibodies to pseudorabies virus. Am. J. Vet. Res. 45: 2096-2098

Comparison of enzyme-linked immunosorbent assay and RT-PCR for the detection of porcine epidemic diarrhoea virus. Enrica Sozzi *, Andrea Luppi, Davide Lelli, Ana Moreno Martin, Elena Canelli, Emiliana Brocchi, Antonio Lavazza, Paolo Cordioli Res Veterinary Sci 88 (2010) 166-168

Comparison of an enzyme-linked immunosorbent assay with serum neutralization test for serodiagnosis of porcine epidemic diarrhea virus infection. Jin Sik Oh, Dae Sub Song, Jeong Sun Yang, Ju Young Song, Hyoung Joon Moon, Tae Yung Kim, Bong Kyun Park * J. Vet. Sci. (2005), 6 (4), 349-352

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments, but, on the contrary, This is possible.

Claims

1) propagating PEDV by transfecting the Vero cells, which have removed the cell culture medium, from the field isolate of PEDV, and subculturing the cells in a culture medium containing serum and removing trypsin;
2) recovering cells at the early stage of cell degeneration during the subculture and centrifuging; And
3) treating the recovered cells with a chemical to separate the envelope protein of the PED virus from the cells; And
4) further isolating and concentrating the envelope protein of the PED virus from the cells through centrifugation, and extracting the envelope protein of the highly pure and concentrated PED virus.

[Claim 3] The method according to claim 1, wherein the chemical for separating the envelope protein of PED virus from the cells of step 3) is Triton X-100.

The method according to claim 1, wherein the envelope protein of the PED virus in step 4) is separated and concentrated in a supernatant of centrifugation at 10,000 rpm.

1) A vaccine for PED virus, which is prepared by uniformly mixing an envelope protein of PED virus isolated by the method of any one of claims 1 to 3 with an immunostimulant comprising mineral oil and an emulsifier or a suspension promoter Gt;

5. The method of claim 4, wherein the emulsifier or the suspension promoter is prepared by mixing 80 to 150 parts by weight of Tween 85 with 100 parts by weight of Span 85.

[Claim 5] The method according to claim 4, wherein the immunostimulant is prepared by mixing 5 to 20 parts by weight of an emulsifier or a suspension promoter with 100 parts by weight of mineral oil.

1) adsorbing the coat protein (antigen) of the PED virus isolated by the method of any one of claims 1 to 3 on a substrate and covering the agar on the substrate;
2) isolating serum from pigs infected with PED virus;
3) reacting the serum in step 2) with the antigen in the agar of step 1); And
4) measuring the concentration of the antibody by the RIDEA method in which the antigen-antibody reaction is combined with ELISA and the principle of radioactive immunodiffusion.

1) Inoculating the test animal with the coat protein of the PED virus isolated by the method of any one of claims 1 to 3; And
2) measuring the reaction diameter by a Delayed type hypersensitivity reaction (DTH) method 24-72 hours after the inoculation, and measuring the intracellular antibody against the PED virus.