KR100432977B1 - A method for culturing porcine epidemic diarrhea virus and live vaccine thereof by using porcine aminopeptidase n - Google Patents

Abstract

The present invention relates to a method of culturing swine epidemic diarrheal virus (PEDV), comprising: culturing animal cells used to propagate swine epidemic diarrheal virus (PEDV); And inoculating and propagating the cultured animal cells with the swine epidemic diarrheal virus (PEDV) using a virus propagation medium to which the swine epidemic diarrheal virus (PEDV) cell receptor is added. The present invention also relates to a swine pandemic diarrheal virus (PEDV) live venom vaccine, characterized in that it comprises a swine epidemic diarrheal virus (PEDV) and the cellular receptor of the swine epidemic diarrheal virus (PEDV).

According to the present invention, a swine epidemic diarrheal virus (PEDV) can be cultured at high titer, and a vaccine much better immunogenicity than a conventional swine epidemic diarrheal virus live venom vaccine can be prepared.

Description

A method for cultivating swine pandemic diarrheal virus using swine aminopeptidase ene and its live venom vaccine {A METHOD FOR CULTURING PORCINE EPIDEMIC DIARRHEA VIRUS AND LIVE VACCINE THEREOF BY USING PORCINE AMINOPEPTIDASE N}

The swine pandemic diarrheal virus has an average size of 130 nm, including projection, and contains glycosylated peplomer protein (85-135 kDa), glycosylated envelope protein (20-32 kDa), There is an unglycosylated RNA-binding nucleocapsid protein (58 kDa) without sugar addition. It belongs to group I of coronaviruses and, according to genetic analysis, is in the middle stage of human coronavirus 229E strain and swine infectious gastritis virus.

The damage caused by the swine epidemic diarrheal virus was first reported in 1971 in the United States and in finishing pigs in the United Kingdom, and in 1992 in Korea. The incidence of 100% across all ages, especially in piglets, causes mortality of more than 50%, with clinical symptoms of acute severe diarrhea, vomiting, anorexia, and hypogonadism.

Vaccination to prevent swine pandemic diarrheal virus infection is given to sows. When the vaccinated sows produce enough immune antibodies, they feed colostrum born from the sows and feed the colostrum with the antibodies in the colostrum, thus preventing the pig epidemic diarrheal virus infection in piglets.

In Korea, a vaccine for preventing swine epidemic diarrheal virus infection is already on the market, but the effect is not satisfactory, so it is not trusted by pig farmers. The reason is that the virus inoculated to sows did not grow enough to generate enough antibodies.

In order to solve the above problems, the present invention is to identify the cell receptor of the swine epidemic diarrheal virus, and to provide a method of culturing the swine epidemic diarrheal virus at high titer by adding this receptor, and provides a swine epidemic diarrheal live vaccine more excellent immunogenicity It is for that purpose.

1 is a photograph showing the results of a virus overlay protein binding assay (Virus Overlay Protein Binding Assay) to confirm that the cell receptor of the swine epidemic diarrheal virus is a pig aminopeptidase enzyme,

2 is a diagram showing the result of propagating swine epidemic diarrheal virus in Vero cells by adding porcine aminopeptidase ene,

3 is a diagram showing a proliferation curve when the pig epidemic diarrheal virus is propagated in Vero cells by adding pig aminopeptidase ene,

Figure 4 is a schematic diagram of the process of producing a swine epidemic diarrheal virus live venom vaccine by adding pig aminopeptidase yen,

5 is a diagram showing the change of the antibody in the guinea pig of swine epidemic diarrheal virus live venom vaccine to which pig amino peptidase ene was added,

6 is a diagram showing changes in neutralizing antibodies in rabbits of the swine epidemic diarrheal virus live venom vaccine to which swine aminopeptidase ene was added,

7 is a diagram showing changes in neutralizing antibodies in rabbits of the swine epidemic diarrheal virus live venom vaccine to which pig aminopeptidase ene was added,

Fig. 8 shows the change in antibody in sows of the swine epidemic diarrheal virus live venom vaccine to which swine aminopeptidase ene was added.

The method of culturing the swine epidemic diarrheal virus (PEDV) of the present invention comprises the steps of culturing animal cells used to propagate swine epidemic diarrheal virus (PEDV); And inoculating and propagating the cultured animal cells with the swine epidemic diarrheal virus (PEDV) using a virus propagation medium to which the cell epidemic of the swine epidemic diarrheal virus is added.

The animal cell may be any animal cell used to propagate swine epidemic diarrheal virus (PEDV). These animal cells include, for example, vero cells.

The cellular receptor of the swine epidemic diarrheal virus (PEDV) can be, for example, the receptor of porcine enterocytes. Examples of these porcine enterocyte receptors include porcine aminopeptidase N.

The virus propagation medium may be used in any embryo used in the art for propagating swine pandemic diarrheal virus (PEDV), for example α-MEM, 3% tryptose, phosphate broth phosphate broth, 0.3% yeast extract, and an antibiotics-antimycotic mixture.

In addition, the present invention relates to a swine epidemic diarrheal virus (PEDV) live venom vaccine, characterized in that it comprises a swine epidemic diarrheal virus (PEDV) and the cellular receptor of the swine epidemic diarrheal virus (PEDV).

In the above-described swine pandemic diarrheal virus (PEDV) live venom vaccine, the cellular receptor of swine pandemic diarrheal virus (PEDV) includes, for example, the receptor of porcine enterocytes. Examples of these porcine enterocyte receptors include porcine aminopeptidase N.

To this end, the present inventors first identify the cellular receptor of the swine epidemic diarrheal virus, using the cell receptor as described above to reveal a method for propagating swine epidemic diarrheal virus to high titer and a method for producing a swine epidemic diarrheal disease virulent vaccine The high immunogenicity of these live venom vaccines was confirmed by inoculation into guinea pigs, rabbits and sows.

Hereinafter, the present invention will be described in detail through examples. However, these embodiments are for illustrative purposes only and the present invention is not limited to these embodiments.

Example

Example 1

[Identification of Cell Receptor of Porcine Pandemic Diarrheal Virus]

The swine epidemic diarrheal virus used in the present invention is a KPED-9 strain distributed by the National Veterinary Research and Quarantine Service, and is currently used as a swine epidemic diarrheal virus live poison vaccine vaccine in Korea. Virus Overlay Protein Binding Assay (VOPBA) was used to identify intracellular receptors of the swine epidemic diarrheal virus. As VOPBA cells, porcine enterocytes most susceptible to swine pandemic diarrheal virus and Vero cells and swine testicle cells were used as control cells that were not sensitive.

The pig enterocytes were used for 10 days old pig enterocytes. After the autopsy, the small intestine was isolated and washed seven times with saline. Afterwards, the mucus of the small intestine was scraped off with slide glass, and 3g was cut out. And 9 ml mannitol buffer (2 mM Tris-HCl, 50 mM mannitol, leupeptin 1 g / ml), pepstatin A (0.7 µg / ml), trypsin inhibitor (2.5 µg / ml) ML) and PMSF (0.1 mM). Thereafter, the tissue was emulsified using a homogenizer and then put back into 45 ml of manitol buffer. MgCl ₂ was adjusted to 10 mM in the last emulsion and reacted on ice for 20 minutes. Thereafter, the mixture was centrifuged at 3,000xg for 15 minutes at 4 ° C. The precipitated precipitate was discarded and the supernatant was collected again and centrifuged at 27,000xg at 4 ° C for 30 minutes. Subsequently, the precipitate was settled again after being suspended in manitol buffer and used at -20 ° C.

The Vero cells and porcine testicular cells used as control cells were MM containing 5% fetal bovine serum and 2% antibiotic-antimycotic agent mixture (Gibco Laboratories, Grand Island, NY). The cells were cultured in a medium essential medium (MEM). After the cell monolayer was formed, washed with phosphate buffered saline (PBS 7.2, PBS), trypsinized, and after all the cells were suspended, the cells were centrifuged at 1,500 xg for 5 minutes at 4 ° C. It was used while suspended in phosphate buffer and stored at -20 ℃.

The porcine enterocytes, Vero cells, and pig testicular cell suspensions are added to nonreducing sample buffer (4% sodium dodecyl sulfate, 10% glycerol, 0.625M Tris-HCl, pH 6.8) and boiled for 10 minutes. Cell lysates were made and added to 8.5% polyacrylamide gel and electrophoresed at 120V. Electrophoresis buffer was used to dissolve 25mM Tris, 192mM glycine, 0.1% SDS in 1 liter of distilled water. After electrophoresis the gel was dried and the molecular marker (rainbow marker) (Amersham RPN 756) was used.

The dried gel was transferred to PVDF membrane at 45V for 17 hours (transfer buffer was 25mM Tris, 192mM glycine, 0.1% SDS, 20% methanol dissolved in 1 liter of tertiary distilled water).

Add orbital shaker for 1 hour with PVDF membrane transferred to PBST (dissolved 0.05% Tween 20 in PBS) into blocking buffer made with 5% skim milk and 1% BSA. Reaction was performed in an orbital shaker and washed three times with PBST solution for 5 minutes.

Then, 10 ml of a dilution of 10 ^5.5 TCID ₅₀ / ml swine epidemic diarrheal virus with MEM was added 10 ml, and the PVDF membrane was reacted in an orbital shaker for 1 hour, followed by 5 ml of PBST solution. Wash three times for a minute.

100-fold dilution of PEDV monoclonal antibody (provided by the National Veterinary Research and Quarantine Service) to PBST, add PVDF membrane to 10 ml of this solution and react for 1 hour in an orbital shaker The solution was washed three times for 5 minutes.

After diluting 5,000-fold of anti-radial peroxidase (HRP) -conjugated anti mouse Immunoglobulin (HPL) -conjugated anti mouse Immunoglobulin (HPL) bound to PBST, 10 ml was added for 1 hour. React in an orbital shaker and wash three times with PBST solution for 15 minutes.

Then, the reaction was performed for 1 minute in an ECL solution (Amersham RPN 2106) and developed with an ECL film to identify a specific binding protein portion of PEDV.

As a result, as shown in Figure 1, the specific binding portion of the swine epidemic diarrheal virus at 150 KDa position was confirmed, this site coincides with the molecular weight of aminopeptidase ene known as the cell receptor of the coronavirus, swine epidemic diarrhea It was confirmed that the pig enterocyte receptor of the virus was an aminopeptidase ene.

Example 2

[How to grow pig epidemic diarrhea virus to high titer by adding pig aminopeptidase ene]

In this example, in order to propagate the swine epidemic diarrheal virus to high titer, the swine epipeptidase N (Sigma Cat. No. L0632) identified as a cell receptor of the swine epidemic diarrheal virus identified in Example 1 was swine epidemic diarrheal virus When swine propagation was added to the virus propagation medium, the swine epidemic diarrheal virus was allowed to grow to high titer. In order to confirm the proliferation with high titer, the virus titer was confirmed when the cell degeneration effect was 100% in Vero cells, and the intracellular and the extracellular viral titer were measured at each incubation time.

The swine pandemic diarrheal virus used in this example was KPED-9, which was distributed by the National Veterinary Research and Quarantine Service, and Vero cells (Catalog No .: CCL81) purchased from ATCC in the United States were used. Roller bottle (Roller bottle, 850cm ² , Corning) Evero cells were formed more than 90% monolayer in 24 hours, washed three times with PBS, swine epidemic diarrheal virus 10 ^2.5 TCID ₅₀ /0.1ml to pig aminopep Diluted with virus proliferation medium (α-MEM, 3% Tryptose Phosphat Broth, 0.3% Yeast Extract, Antibiotics-antimycotic mixture, pH 7.2) to which Tidase N (Sigma Catalog No. L0632) was added, Incubated at 37 ° C. for 1 hour.

The concentration of porcine aminopeptidase ene was added to the virus propagation medium at 2.4 pg / ml, 0.24 pg / ml, and 0.024 pg / ml to investigate the effects of the swine epidemic diarrheal virus. After 1 hour of inoculation time, it was washed 3 times with PBS again, and virus propagation medium containing porcine aminopeptidase was added at each concentration of 100ml per rotary culture bottle, and 0.1-0.2rpm was added to the rotary culture machine to make 37- ℃. The virus was propagated for 3-4 days at. When the cytopathic effect of the swine pandemic diarrheal virus was observed more than 90%, freeze thawing was repeated three times and centrifuged at 1,500 rpm for 7 minutes to make the supernatant the swine epidemic diarrheal virus culture.

In addition, as a control, swine epidemic diarrheal virus was propagated by the same method using a virus propagation medium added with only 10 µg / ml of trypsin, a swine epidemic diarrheal virus propagation method.

The titer of swine pandemic diarrheal virus was measured using a known swine pandemic diarrheal virus assay. That is, each of the swine epidemic diarrheal viruses grown in swine aminopeptidase ene and trypsin-containing virus propagation medium was diluted 10-fold with trypsin-containing virus-producing medium, and the cells were 96-well microplates. Vero cells in which monolayers were formed) were used. After the Vero cells with monolayers were washed three times with PBS, the virus solution was added at 100 μl / well for each dilution step and inoculated at 37 ° C. for 1 hour and washed three times with PBS. After washing, the culture medium for trypsin was added again to 200 μl / well each well, followed by incubation at 37 ° C. for 5 days, and the titer was calculated by the Reed and Munch method.

As a result, both the virus culture medium containing swine aminopeptidase ene and the virus culture medium containing trypsin had clear borders, formed co-forms, and showed the cytopathic effect of the typical swine epidemic diarrheal virus virus, which became nucleus and dropped from the bottom. . However, virus cultures ^grown with the addition of porcine aminopeptidase ene showed titers of 10 ^4.9-5.3 TCID ₅₀ /0.1 ^ml depending on the concentration of porcine aminopeptidase ene contained in the virus propagation medium. Virus cultures grown by addition showed a titer of 10 ^4.1 TCID ₅₀ /0.1ml, which means that the virus growth medium containing pig aminopeptidase yen is up to 10 ^0.8 TCID ₅₀ /0.1mL (6.3 times) than the virus growth medium containing trypsin. It can be seen that the virus proliferation of the () (see Figure 2).

In addition, in order to confirm the process of increasing the proliferation of the virus, a one-step growth curve of the swine epidemic diarrheal virus was prepared to reconfirm that the virus of high titer was multiplied. That is, after forming Vero cells in 6-well microplates in 24 hours, monolayers were washed three times with PBS, inoculated with 1 ml of 10 ^1.1 TCID ₅₀ /0.1 ml of virus and incubated at 37 ° C for 1 hour. It was. After 1 hour of inoculation, the cells were washed three times with PBS, followed by 2 ml of virus propagation medium containing trypsin (10 µg / ml) and 2 ml of virus propagation medium containing pork aminopeptidase ene (2.4 pg / ml). / well was added and incubated at 37 ℃. The titers of intracellular and extracellular viruses were measured at 0, 2, 4, 6, 8, 10, 12, 15, 18, 21, 24, 48 and 72 hours after incubation. The extracellular virus titer was centrifuged at 1,500 rpm for 7 minutes for each hour, and the supernatant was used. Intracellular virus titer was collected by cell scraper at each time, suspended in each new virus growth medium, and mixed with frozen sediment when frozen for extracellular virus titer. After freezing and thawing, the cells were centrifuged at 1,500 rpm for 7 minutes and the virus titer was measured using the supernatant.

As a result, as shown in Figure 3, the intracellular virus began to be detected 6 hours after the inoculation and showed the highest titer from 24 hours to 48 hours after the inoculation, this phenomenon was the virus cultured in the virus growth medium with trypsin added There was no difference between the culture medium and the virus culture cultured with the virus propagation medium supplemented with pig aminopeptidase ene. However, the extracellular virus was detected in the virus culture medium incubated with a pig aminopeptidase-enriched virus growth medium at 12 hours and showed the highest titer at 48 hours. Virus cultures cultured with solvent medium began to be detected from 15 hours late 15 hours and showed the highest titer at 24 hours and then decreased. In addition, the extracellular virus titers showed higher titers in porcine aminopeptidase ene media than in trypsin-added media, confirming that the swine epidemic diarrheal virus proliferated to high titers. Therefore, the composition of virus propagation medium during cell culture of swine pandemic diarrheal virus was adjusted to 2.4% of porcine aminopeptidase N (Sigma Cat. No. L0632) and 3% of total medium. Trypose Phosphate Broth is added, yeast extract is added to 0.3% of the total medium, and the pH 7.2 α-MEM is added with the antibiotic-Antiycotic mixture. Caused the highest titer of viral propagation. In addition, as a virus culture method, Vero cells having a 90% or more monolayer formed after washing with PBS three times were inoculated with the virus, adsorbed at 37 ° C. for 1 hour, washed three times with PBS again, and incubated at 37 ° C. for 3-5 days. After repeated freeze-thawing of the virus-proliferated cell cultures three times, centrifugation at 1,500 rpm for 7 minutes showed the best result.

Example 3

[How to prepare swine pandemic diarrheal disease live vaccine by adding pig aminopeptidase ene]

Porcine epidemic diarrheal disease live vaccine was prepared using the swine epidemic diarrheal virus incubated at high titer by adding pig aminopeptidase ene. The manufacturing process is briefly shown in FIG.

Freeze-dried pigs were prepared by adding 5 µg of pig aminopeptidase yen to 1.5 ml of a swine pandemic diarrheal virus virus titer having a titer of 10 ^5.0 TCID ₅₀ /0.1 ml or more grown using pig aminopeptidase yen. Pandemic diarrhea virus bulk. 1.5 ml of this bulk and the same amount of lyophilized LPGG (0.217M Lactose, 0.0038M KHPO ₄ , 0.0072MK ₂ HPO ₄ , 0.0049M monosodium glutamate, 1% Gelatin) were mixed at room temperature for 3 hours and then 10ml after adding 3㎖ by the bottle for 72 hours and freeze-dried two minutes byeongdang 5 sows (mother pig) vaccinated porcine epidemic diarrhea virus saengdok (1 per person 2㎖ muscle inoculation, two 1 minute 10 ^4.5 TCID ₅₀ or more of porcine epidemic diarrhea virus for Titer containing) was made so that the pig aminopeptidase ene was inoculated 1 μg per sow. For experimental animal experiments, pig aminopeptidase N was prepared by adding 2.4 pg for guinea pigs and 24 pg for rabbits.

Example 4

[Immunogenicity Confirmation in the Guinea Pig of Porcine Epidemic Diarrheal Disease with Pig Aminopeptidase En]

A swine pandemic diarrheal virulent vaccine prepared by adding pig aminopeptidase ene was inoculated into guinea pigs to observe changes in immunogenicity.

The 200g guinea pigs were divided into three groups of five dogs. Group A was freeze-dried swine epidemic diarrheal virulence vaccine with 2.4 pg / ml of pig aminopeptidase (10 ^4.5 TCID ₅₀ / ml when diluted with 5 ml of PBS). Titer of 1 ml), and group B had a potency of 10 ^4.5 TCID ₅₀ / ml when diluted with 5 ml of PBS. ) Was inoculated 1 ml each, and C group was inoculated 1 ml each of a virus growth medium containing pig aminopeptidase ene. Each guinea pig was raised for 6 weeks and collected for each group each week to measure serum neutralizing antibodies.

Serum neutralizing antibodies were measured using a 96 well microplate. That is, the serum was inactivated at 56 ° C. for 30 minutes, diluted in binary virus-promoting medium with trypsin, and neutralized for 1 hour by adding the same amount of 200 TCID ₅₀ /0.1 ml of KPED-9. Thus reacted virus-serum mixture was added to 100 μl / well of Vero cells formed with monolayers washed three times with PBS, and inoculated at 37 ° C. for 1 hour, followed by three times washing with PBS and 10 μg / ml of trypsin. Serum neutralization titers were examined by measuring the effect of cell degeneration by adding 200 μl / well of the virus growth medium added to each of the cells and culturing at 37 ° C. for 5 days.

As a result, as shown in Fig. 5, group A guinea pigs inoculated with 2.4 pg of pig aminopeptidase yen and swine epidemic diarrheal virus per guinea pig continued to decrease until 6 weeks after showing the highest antibody value in the first and second weeks. It was. The antibody titers of guinea pigs peaked at 2 ^5.2 at 1 and 2 weeks of inoculation and gradually decreased to 2 ^4.8 at 3 weeks, 2 ^4.3 at 4 weeks, 2 ^{3.7 at} 5 weeks, and 2 ^2.8 at 6 weeks. Group B guinea pigs were vaccinated with Swine Pandemic Diarrheal Virus only. Antibody titers gradually decreased after peaking at 2 weeks (2 ^3.4 ), 2 ^3.2 at 3 weeks, 2 ^2.5 at 4 weeks, 2 ^2.8 , 6 at 5 weeks. when the State Supreme antibodies of me a group showed a 2 ^2.7 is compared with two ^2.8-fold showed a lower potency, vaccines, antibodies go antibodies of group B vaccination after 1 week a group from 1 to 4 weeks were able more significant ( p <0.01).

Example 5

[Immunogenicity Identification in Rabbits of Porcine Epidemic Diarrheal Disease with Pig Aminopeptidase En]

Rabbits were inoculated with a live pandemic diarrheal virus vaccine prepared by adding pig aminopeptidase ene to observe changes in immunogenicity.

Two rabbits weighing 1.5 kg were divided into three groups, each group lyophilized swine epidemic diarrhea virulent vaccine with 24 pg / ml of pig aminopeptidase (10 ^4.5 TCID ₅₀ / ml when diluted with 5 ml of PBS). Titer of 1 ml), and group B had a potency of 10 ^4.5 TCID ₅₀ / ml when diluted with 5 ml of PBS. ) Was inoculated 1 ml each, and C group was inoculated 1 ml each of a virus growth medium containing pig aminopeptidase ene. Three weeks after the first inoculation, the vaccine was boosted in the same manner. Each rabbit was reared for six weeks, and blood was collected from each group every week to measure antibody titers by serum neutralizing antibody and indirect enzyme immunoassay.

Indirect enzyme-linked immunoassay was performed using 50 mM carbonate buffer (1 μg per well) of swine epidemic diarrheal virus antigens concentrated in ammonium sulfate in 96 well micotiter plates (Nunc, USA). Coated with buffer (pH9.6) at 4 ° C. for 18 hours and washed three times with PBS-tween 20 (0.05%) (PBS-T). After blocking with 3% gelatin (gelatin) for 1 hour at 37 ℃. Thereafter, rabbit serum was diluted 1: 500 with PBS-T, and 100 μl / well was added thereto, followed by reaction at 37 ° C. for 1 hour. After washing three times again with PBS-T, HBS radish peroxidase-conjugated goat anti-rabbit IgG (Horse radish peroxidase-conjugated goat anti-rabbit IgG) antibody with PBS-T was 1: 5,000. Dilution was carried out, and 100 μl of each well was added, followed by reaction at 37 ° C. for 1 hour. After washing three times with PBS-T, 100 μl of ABTS was added to each well, followed by color development at room temperature for 20 minutes, followed by stopping with 0.5 MH ₂ SO _4, and then absorbance at 405 nm.

As shown in FIG. 6, the change in serum neutralizing antibody titers showed low neutralizing antibody titers in both groups prior to booster vaccination. However, group A rabbits inoculated with 24 pg of pig aminopeptidase N and pig epidemic diarrheal virus per rabbit after booster vaccination continued to increase during the test period (p <0.01). The results of antibody titers measured by indirect enzyme-immunoassay showed similar results to serum neutralized antibody titers (see FIG. 7). However, group A rabbits inoculated with swine aminopeptidase yen and swine epidemic diarrheal virus at the first two, five and six weeks were significantly higher (p <0.01) than group B rabbits inoculated with only the swine epidemic diarrheal virus. The antibody was shown.

Example 6

[Immunogenicity Identification in Sows of Porcine Epidemic Diarrheal Vaccine Added with Pig Aminopeptidase En]

Swine epidemic diarrheal live vaccines prepared by adding pig aminopeptidase ene were inoculated into sows to observe changes in immunogenicity.

Divided by the weight sows 5 rats Group 150kg or more A groups, pig amino peptidase yen per minute, the first two (2㎖) the 1㎍ be added freeze-dried porcine epidemic diarrhea saengdok vaccine (when diluted with a PBS of 10㎖ 10 ^5.0 TCID ₅₀ / having a titer of 2㎖) muscles were inoculated by 2㎖, when B group pig amino peptidase yen hayeoteul not added, diluted in PBS of porcine epidemic diarrhea saengdok vaccine (10㎖ 10 ^5.0 TCID ₅₀ / 2 ml) was inoculated with 2 ml each, and C group was inoculated with 2 ml each with a virus propagation medium containing pig aminopeptidase ene. Primary vaccination was given 5 weeks before delivery and secondary vaccination was performed 2 weeks before delivery. The antibody titers in blood before and after the first vaccination, before the second vaccination, and in colostrum at delivery were measured. Antibody titration was performed by neutralizing antibody assay.

As a result, as shown in FIG. 8, group A sows inoculated with 1 μg of pig aminopeptidase yen and swine epidemic diarrheal virus per pig had an average antibody titer of 2 ^6.2 after 3 weeks of the first inoculation. Group B sows inoculated with virus showed 2 ^6.0 antibody titer, showing no significant difference (p <0.01). However, at the time of delivery following group 2 inoculation, group A sows inoculated with pig aminopeptidase yen and the swine epidemic diarrheal virus had an average of 2 ^7.0 blood neutralizing antibodies and 2 ^7.8 colostrum neutralizing antibodies. Group B sows inoculated with epidemic diarrheal virus alone showed significant differences between 2 ^5.4 blood neutralizing antibodies and 2 ^6.0 colostrum neutralizing antibodies, and the immunity of the swine pandemic diarrheal virus live venom vaccine with pig aminopeptidase. Originality was better than the existing swine pandemic diarrheal virus live venom vaccine (p <0.01).

According to the present invention, a vaccine that is much more immunogenic than the swine epidemic diarrheal virus live venom vaccine incubated with a virus propagation medium containing a conventional trypsin can be prepared using swine aminopeptidase en, and swine epidemic diarrheal virus Two doses of live poison vaccines in sows can more effectively prevent swine epidemic diarrhea in piglets.

Claims (8)

Culturing the animal cells used to propagate swine pandemic diarrheal virus (PEDV); Inoculating and propagating the cultured animal cells with the swine epidemic diarrheal virus (PEDV) using a virus propagation medium to which porcine aminopeptidase N is added as a cell receptor for the swine epidemic diarrheal virus ; Porcine epidemic diarrheal virus (PEDV) cultivation method comprising a. [Claim 2] The method of claim 1, wherein the animal cells are vero cells. delete delete The method of claim 1, wherein the virus growth medium is α-MEM, 3% tryptose, phosphate broth, 0.3% yeast extract, antibiotic-antimycotic mixture (antibiotics-antimycotic) mixture, and pH 7.2, and the cellular receptor of the swine epidemic diarrheal virus (PEDV) is porcine aminopeptidase N as a propagation virus at a concentration of 0.024 pg / ml ~ 2.4 pg / ml Method for culturing swine pandemic diarrheal virus (PEDV), characterized in that it is added to the medium. Porcine epidemic diarrheal virus (PEDV) and porcine epidemic diarrheal virus (PEDV) livepox vaccine comprising porcine aminopeptidase N as the cell receptor of said pig epidemic diarrheal virus (PEDV). delete delete