Porcine epidemic diarrhea vaccine composition, preparation method and application

Abstract

The application discloses a porcine epidemic diarrhea disease vaccine composition and a preparation method and application of the vaccine composition. The inactivated vaccine can effectively protect against the attack of epidemic virulent strains, has good cross-protection, and can effectively protect a pig group against porcine epidemic diarrhea and improve the productivity of the pig group.

Description

TECHNICAL FIELD

[0001] The present application relates to a porcine epidemic diarrhea vaccine composition and a preparation method and application thereof, and belongs to the field of biological medicine. BACKGROUND

[0002] Porcine epidemic diarrhea (PED) is an acute, highly contagious enteric disease of pigs caused by porcine epidemic diarrhea virus (PEDV), which is characterized by diarrhea, vomiting, dehydration and high mortality rate in suckling piglets. Pigs of all ages are susceptible, especially suckling piglets, with a mortality rate and morbidity rate close to 100%. The disease has become a serious problem in the development of pig farming industry.

[0003] There is no specific drug for treating porcine epidemic diarrhea at present, and the conventional treatment effect is poor, so the prevention by vaccine is still the main method. However, the antigen content required by the existing inactivated vaccine is generally high, and high-titer virus liquid needs to be prepared, which has high production cost. Therefore, there is an urgent need for an inactivated vaccine with controllable cost and high immunization efficacy to solve the above problems. SUMMARY

[0004] The technical problem to be solved by the present application is to provide a porcine epidemic diarrhea virus vaccine composition, which has high immunization efficacy and can provide effective cross-protection to epidemic wild strains under a lower antigen content, showing significant cross-immunity characteristics.

[0005] One aspect of the present application is to provide a porcine epidemic diarrhea virus vaccine composition, which comprises an immunizing amount of porcine epidemic diarrhea virus strain antigen and a pharmaceutically acceptable carrier; the porcine epidemic diarrhea virus strain antigen is an inactivated whole virus antigen.

[0006] As an embodiment of the present application, the porcine epidemic diarrhea virus strain of the present application is porcine epidemic diarrhea virus strain HN1303, and the biological preservation number of the HN1303 strain is CCTCC NO. V201514, the preservation unit is China Center for Type Culture Collection, the preservation address is Wuhan University, Wuhan, China, the preservation time is March 4, 2015, and it is disclosed in Chinese patent application CN106148287A.

[0007] As a preferred embodiment of the present application, in the vaccine composition of the present application, the antigen of the porcine epidemic diarrhea virus strain comprises inactivated whole virus of the porcine epidemic diarrhea virus strain; the content of the inactivated whole virus antigen of the porcine epidemic diarrhea virus is 10 6.0TCID 50 / ml 7.0 TCID 50 / ml.

[0008] The term "vaccine composition" as used herein refers to a pharmaceutical composition comprising immunogenic porcine epidemic diarrhea virus. The pharmaceutical composition can induce, stimulate or enhance an immune response in a swine against porcine epidemic diarrhea virus. The vaccine composition is an inactivated vaccine of an immunizing amount of a porcine epidemic diarrhea virus strain.

[0009] The term "inactivated vaccine", also known as killed vaccine, as used herein refers to a suspension of inactivated virus used as an antigen to produce immunity. Examples of inactivated vaccines include whole virus vaccines and split vaccines. Inactivated vaccines can be readily produced using known methods. For example, whole virus inactivated vaccines can be obtained by treating the virus with a formaldehyde solution. Split vaccines can be prepared from the viral envelope after treatment with an ether. For example, the virulent strain HN1303 strain of the present application can be inactivated to produce an inactivated vaccine.

[0010] The amounts of the components or ingredients of the compositions of the present application are preferably therapeutically effective amounts. By a therapeutically effective amount is meant an amount necessary to perform their immunological function in the host to which the composition is administered without causing undue side effects. The precise amount of each ingredient and the composition to be administered will vary according to factors such as the type of disease being treated, the type and age of the animal being treated, the mode of administration, and other ingredients in the composition.

[0011] According to the present application, in the vaccine composition, the porcine epidemic diarrhea virus strain antigen is present in an amount of > 10 6.0 TCID 50 / ml.

[0012] Preferably, in the vaccine composition, the porcine epidemic diarrhea virus strain antigen is present in an amount of 10 6.0 TCID 50 / ml 7.0 TCID 50 / ml.

[0013] As a preferred embodiment of the present application, in the vaccine composition of the present application, the porcine epidemic diarrhea virus strain antigen comprises inactivated whole virus antigen of the porcine epidemic diarrhea virus HN1303 strain; the inactivated HN1303 strain whole virus antigen is present in an amount of > 10 6.0 TCID 50 / ml.

[0014] As a preferred embodiment of the present invention, in the vaccine composition of the present invention, the inactivated HN1303 strain whole virus antigen content in the vaccine composition is 10 6.0 TCID 50 / ml~10 7.0 TCID 50 / ml.

[0015] The vaccine composition of the present invention can be formulated using available technology, preferably formulated together with a veterinary pharmaceutically acceptable carrier. For example, oil can help stabilize the formulation and additionally serve as a vaccine adjuvant. Therefore, in the present invention, the pharmaceutically acceptable vaccine adjuvant includes an oil adjuvant selected from white oil, squalane or squalene, Drake oil, and other animal oils, vegetable oils, or mineral oils. The above-mentioned oil adjuvant can be of natural origin or obtained through artificial synthesis. In the present invention, the vaccine composition is an oil-in-water emulsion, a water-in-oil emulsion, or a double emulsion, and the double emulsion is generally a water-in-oil-in-water emulsion.

[0016] In one embodiment of the present invention, the vaccine composition further comprises a suspending agent, a surfactant, an antigen inactivating agent or a preservative. The suspending agent may include, for example, aluminum stearate, and other suspending agents available in the art. The surfactant may include, for example, sorbitan monooleate (TWEEN series), SPAN, and other surfactants available in the art. The antigen inactivating agent includes (but is not limited to), for example, formalin, β-propiolactone, etc. The preservative includes, for example, thimerosal. The use and dosage of the above substances are well known to those skilled in the art.

[0017] Since oil adjuvants may cause certain side effects on animals, other adjuvants in the art, including aluminum hydroxide, aluminum phosphate and other metal salts, may also be selected to prepare suspensions to reduce immune stimulation.

[0018] Preferably, the adjuvant selected in the present invention is an aluminum gel adjuvant, which includes aluminum salt, polyacrylic acid, and dextran sulfate.

[0019] Preferably, the aluminum salt is selected from aluminum hydroxide and aluminum phosphate.

[0020] As an embodiment of the present invention, the aluminum gel adjuvant of the present invention is prepared by the following method:

[0021] (1) A 5% w / v aluminum sulfate solution was added to the solution at 500 rpm with stirring, and the solution was washed with a 5% w / v sodium hydroxide solution at a volume 0.4 times the volume of the aluminum sulfate solution. The precipitate was washed with normal saline by centrifugation and then suspended in normal saline to a volume equal to that of the aluminum sulfate solution.

[0022] (2) Add polyacrylic acid and dextran sulfate in sequence and stir evenly;

[0023] (3) Homogenize in a homogenizer; preferably, homogenize at 800 bar and 50° C. for 4 times.

[0024] Dextran sulfate is usually sold in the form of its sodium salt, and therefore, the term "dextran sulfate" also includes its salt form, including its sodium salt form. Preferably, the average molecular weight of dextran sulfate ranges from 15,000 to 35,000, and the average molecular weight can be 15,000, 16,000, 17,000, 18,000, 19,000, 20,000, 21,000, 22,000, 23,000, 24,000, 25,000, 26,000, 27,000, 28,000, 29,000, 30,000, 31,000, 32,000, 33,000, 34,000 or 35,000.

[0025] Preferably, the average molecular weight of the polyacrylic acid is in the range of 1000-3500, and the average molecular weight can be 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400 or 3500.

[0026] Preferably, the added amount of polyacrylic acid is 2 mg / ml, and the added amount of dextran sulfate is 0.4 mg / ml.

[0027] Another aspect of the present invention is to provide a method for preparing the vaccine composition, wherein the method comprises:

[0028] (1) culturing and multiplying the porcine epidemic diarrhea virus;

[0029] (2) inactivating the porcine epidemic diarrhea virus proliferated in step (1); and

[0030] (3) Add adjuvant and emulsify.

[0031] Another aspect of the present invention is to provide the use of the vaccine composition in the preparation of a medicament for preventing and / or treating porcine epidemic diarrhea.

[0032] The term "prevention" as used in the present invention refers to all actions that inhibit porcine epidemic diarrhea or delay the onset of the disease by administering the vaccine composition according to the present invention. The term "treatment" refers to all actions that alleviate or improve the symptoms caused by porcine epidemic diarrhea virus infection by administering the vaccine composition according to the present invention.

[0033] Advantages of the invention

[0034] The prepared porcine epidemic diarrhea virus inactivated vaccine has good immunogenicity and stability, and after inoculation of piglets and replacement gilts, the body can be stimulated to produce immunity quickly, and a higher neutralizing antibody level is produced, and the antibody is maintained at a higher level for a long time; the inactivated vaccine is safe for piglets and pregnant sows. The inactivated vaccine can effectively protect the attack of epidemic virulent strains, has good cross protection, and can effectively protect the pig herd against porcine epidemic diarrhea and improve the productivity of the pig herd. DETAILED DESCRIPTION

[0035] The advantages and characteristics of the present application will become more apparent with the description of specific embodiments. However, these embodiments are only exemplary and do not constitute any limitation on the scope of the present application. Those skilled in the art should understand that the details and forms of the technical solutions of the present application can be modified or replaced without departing from the spirit and scope of the present application, and such modifications and replacements fall within the protection scope of the present application.

[0036] In order to make the present application easier to understand, the present application will be further described in combination with specific examples. The experimental methods described in the present application are all conventional methods unless otherwise specified; the biological materials described in the present application can be obtained from commercial channels unless otherwise specified.

[0037] Example 1: Propagation culture of porcine epidemic diarrhea virus HN1303 strain

[0038] 1. Virus propagation

[0039] The isolated porcine epidemic diarrhea virus HN1303 strain virus liquid was diluted 100 times, 1 ml of which was inoculated into Vero cells (25T), adsorbed at 37°C for 2 h, then the maintenance solution was added to the appropriate volume, and placed in a 37°C, 5% CO2 culture, cultured for 36-48 h, and then the virus liquid was harvested when the cell lesion reached more than 80%. The virus content was not less than 10 7.0 TCID 50 / ml.

[0040] 2. Virus TCID 50 determination

[0041] After the Vero cells were passaged, 3 million cells / well were plated in a 96-well cell plate, 100 μl per well. Place in a 37°C, 5% CO2 incubator for 1 day, and the cells grow into a good monolayer. The harvested virus liquid was diluted 10 times by gradient dilution (10 -1 , 10 -2 …10 -8 ), 10 -4 , 10 -5 , 10 -6, 10 -7 , 10 -8 Five dilutions were added to a 96-well cell plate grown as a monolayer of cells. Eight replicates were made for each dilution, with 100 μl per well. The cells were cultured in a 37°C, 5% CO2 incubator for 4 days, and the cytopathic effect was observed daily. The viral titer was calculated according to the Reed-Muench method based on the number of cytopathic wells. 50 Up to 10 7.0 TCID 50 / ml or above.

[0042] Example 2 Preparation of adjuvant

[0043] 1. Preparation of Adjuvant A

[0044] Take 2500 mL of 5% W / V aluminum sulfate solution, add 1000 mL of 5% W / V sodium hydroxide solution while stirring at 500 rpm, wash the precipitate twice with normal saline by centrifugation, and then suspend it in normal saline to make the total amount reach 2500 mL.

[0045] 2. Preparation of Adjuvant B

[0046] (1) Take 2500 mL of 5% w / v aluminum sulfate solution, add 1000 mL of 5% w / v sodium hydroxide solution while stirring at 500 rpm, wash the precipitate twice with normal saline by centrifugation, and resuspend it in normal saline to a total volume of 2500 mL;

[0047] (2) Add polyacrylic acid and dextran sulfate (polyacrylic acid 2 mg / ml, dextran sulfate 0.4 mg / ml) in sequence and stir evenly;

[0048] (3) Homogenize the mixture 4 times in a homogenizer (Canada ATS homogenizer) at 800 bar and 50°C.

[0049] Example 3 Preparation of HN1303 PEDV inactivated vaccine

[0050] The virus fluid harvested from Example 1 was inactivated with β-propiolactone and tested for inactivation. The antigen that passed the inactivation test was emulsified with the adjuvant prepared in Example 2 at a volume ratio of 9:1 to prepare an inactivated porcine epidemic diarrhea vaccine. The specific ratios are shown in Table 1.

[0051] Table 1 Composition ratio of HN1303 strain vaccine composition

[0052] Vaccine 1 Vaccine 2 Vaccine 3 Vaccine 4 HN1303 strain antigen 10 6.0 TCID 50 / ml]]> <![CDATA[10 7.0 TCID 50 / ml]]> 10 6.0 TCID 50 / ml]]> <![CDATA[10 7.0 TCID 50 / ml]]> Adjuvant A 10％ 10％ - - Adjuvant B - - 10％ 10％

[0053] Example 4 Safety Test of HN1303 PEDV Inactivated Vaccine

[0054] 1. Safety test for piglets

[0055] Twenty-five piglets, 8-10 days old and negative for PEDV antigen and antibodies, were randomly divided into five groups of five. Groups 1, 2, 3, and 4 were vaccinated intramuscularly with approved vaccines 1, 2, 3, and 4, respectively (see Table 2). A second vaccination was administered 14 days after the first vaccination. Both the first and second vaccinations were administered at a double dose of 4 ml per piglet. The piglets were observed for 14 days after vaccination to monitor the inoculation site and clinical symptoms. A control group, Group 5, was established without any treatment. The results are shown in Table 2.

[0056] Table 2 Piglet safety test results

[0057]

[0058] Five piglets in each immunization group were observed for 14 days after inoculation with the inactivated PEDV vaccine. They were in good spirits, had normal diet, maintained a normal body temperature, and showed no clinical symptoms of diarrhea. Fourteen days after the second vaccination, the piglets were sacrificed, and pathological sections of the inoculation site were taken and examined, revealing no abnormalities. This demonstrates the safety of all vaccines in piglets.

[0059] 2. Safety test for pregnant sows

[0060] Twenty pregnant sows negative for PEDV antigen and antibodies were randomly divided into four groups of five. Groups 6, 7, 8, and 9 were vaccinated intramuscularly with approved vaccines 1, 2, 3, and 4, respectively (see group designation in Table 3). The first vaccination was administered seven weeks before farrowing at a dose of 4 ml per sow. The second vaccination was administered four weeks before farrowing at a double dose of 4 ml per sow. The inoculation site was observed, and the number and quality of piglets born were analyzed. A control group of two pigs in Group 10 was assigned and received no treatment. The results are shown in Table 3.

[0061] Table 3 Results of safety test on pregnant sows

[0062]

[0063]

[0064] Five pregnant sows in each immunization group showed no obvious clinical symptoms after vaccination with the inactivated PEDV vaccine, and no abnormalities were observed at the vaccination site. During farrowing, one weak piglet was born in each of the immunization and control groups, with no cases of abortion or stillbirth. There was no significant difference in average litter size between the immunization and control groups, demonstrating the safety of the vaccine in sows.

[0065] The safety test results show that the porcine epidemic diarrhea virus HN1303 strain of the present invention is safe for piglets and pregnant sows and has no side effects.

[0066] Example 5 Immune efficacy test of HN1303 PEDV inactivated vaccine

[0067] 1. Piglet immune efficacy test

[0068] Twenty-five piglets, 8-10 days old and negative for PEDV antigen and antibodies, were randomly divided into five groups of five. Groups 11, 12, 13, and 14 were vaccinated intramuscularly with approved vaccines 1, 2, 3, and 4, respectively (see group designation in Table 4), at 2 ml per piglet. Group 15 served as a control group and received no treatment. A second vaccination was administered 14 days after the first vaccination. The piglets were observed for 14 days following the vaccination, including the site of inoculation and clinical symptoms. Starting two weeks after the second vaccination, blood samples were collected weekly for serum PEDV neutralizing antibody titers. The results are shown in Table 4.

[0069] Table 4 Neutralizing antibody results after immunization of piglets with inactivated PEDV vaccine

[0070]

[0071]

[0072] None of the immunized piglets showed any inflammatory reactions, such as redness, swelling, lumps, or scabs, at the inoculation site, nor did they exhibit any obvious abnormal clinical symptoms. Serum neutralizing antibody results for PEDV showed that neutralizing antibodies in all five piglets in groups 2, 3, and 4 were greater than 1:32 by week three after vaccination. Neutralizing antibodies peaked in week four and remained for one week, gradually declining after week five. In group 1, neutralizing antibodies in all five piglets reached 1:32 by week four, remained there until week five, and fell below 1:32 by week six. Vaccine 3, despite its lower antigen content, achieved neutralizing antibodies equal to or exceeding those in Vaccine 2, which had a higher antigen content. These results indicate that for 8-10 day-old piglets, the inactivated PEDV vaccine containing adjuvant B has a lower antigen content, better immune efficacy, and longer-lasting immunity.

[0073] 2. Sow immune efficacy test

[0074] Twenty PEDV antigen- and antibody-negative gilts were randomly divided into five groups of four. Groups 16, 17, 18, and 19 were vaccinated intramuscularly with approved vaccines 1, 2, 3, and 4, respectively (see Table 5 for group designation). 2 ml per gilt was administered intramuscularly (2 ml / gilt). Group 20 served as a blank control group and received no treatment. The first vaccination was administered seven weeks before farrowing, followed by two vaccinations four weeks before farrowing. Post-vaccination, the gilts were observed for inflammation at the vaccination site and clinical symptoms. Starting two weeks after the second vaccination, blood was collected weekly to measure serum PEDV neutralizing antibody titers. The results are shown in Table 5.

[0075] Table 5 Neutralizing antibody results after immunization of gilts with inactivated vaccine

[0076]

[0077]

[0078] No inflammatory reactions such as redness, swelling, lumps, or scabs were observed at the inoculation sites of the immunized sows, and no obvious abnormal clinical symptoms were observed. The results of serum PEDV neutralizing antibodies showed that in Vaccine Groups 2, 3, and 4, the neutralizing antibodies of all sows were greater than 1:32 in the third week after vaccination. They reached their peak in the fourth week after vaccination and were maintained for one week, gradually declining after the fifth week after vaccination. In Vaccine Group 1, the neutralizing antibodies of the four reserve sows only reached 1:32 in the fourth week after vaccination, and were maintained until the fifth week. After the sixth week, they had dropped below 1:32. At a lower antigen content, the neutralizing antibodies of Vaccine 3 reached or exceeded those of Vaccine 2, which had a higher antigen content. The test results show that after sows were vaccinated with the inactivated PEDV vaccine containing adjuvant B of the present invention, the antigen content was lower, and it was able to produce higher neutralizing antibodies, which could be maintained at a relatively high level, and had good immune efficacy.

[0079] Example 6 Comparative test of challenge protection of HN1303 strain PEDV inactivated vaccine

[0080] Three healthy sows with the same due date and negative for PEDV antibody antigen were selected. They were divided into three groups. The 21st group of sows were vaccinated with vaccine 1, the 22nd group of sows were vaccinated with vaccine 3, and the 23rd group of sows were vaccinated with PBS. All sows were first vaccinated 7 weeks before delivery, with 2ml / head injected into the neck muscle, and the second vaccination was performed in the same way 4 weeks before delivery. Ten piglets in good condition were selected from each group for the challenge test, and each group was randomly divided into two groups of 5 piglets each; 5 were breastfed; 5 did not eat breast milk and were artificially raised. When the piglets were 7 days old, they were orally challenged with the virus (10 5.667 TCID 50 / ml, 2ml / head). The piglet groups and challenge conditions are shown in Table 6.

[0081] Table 6 Comparative test results of challenge protection of HN1303 PEDV inactivated vaccine

[0082]

[0083] The results showed that the inactivated vaccine containing adjuvant B of the present invention had good protection against the attack of the epidemic strain of porcine epidemic diarrhea virus at a lower antigen content, while the inactivated vaccine containing adjuvant A had a protection rate of only 2 / 5 against the attack of the epidemic strain at a lower antigen content, and all the patients in the attack control group became ill.

[0084] In summary, the inactivated vaccine containing adjuvant B prepared by the present invention has a good immune protection effect on piglets and can have a good immune protection effect against prevalent strains.

[0085] Example 7 Broad-spectrum protection test of HN1303 PEDV inactivated vaccine

[0086] Two healthy sows with the same due date were selected, and the PEDV antibody antigen was negative. They were divided into two groups, the sows in group 24 were vaccinated with vaccine 3, and the sows in group 25 were vaccinated with PBS. All sows were first vaccinated 7 weeks before delivery, with 2ml / head injected into the neck muscle, and the second vaccination was performed in the same way 4 weeks before delivery. Ten piglets in good condition were selected from each group for the challenge test, and each group was randomly divided into two groups of 5 piglets each; 5 piglets were breastfed; 5 piglets did not eat breast milk and were artificially raised. When the piglets were 7 days old, they were orally challenged with the virus (10 5.667 TCID 50 / ml, 2ml / head). The piglet groups and challenge conditions are shown in Table 7.

[0087] Table 7 Results of broad-spectrum challenge protection test of HN1303 PEDV inactivated vaccine

[0088]

[0089] The results showed that the inactivated vaccine containing adjuvant B of the present invention had good protection against the challenge of the classical strain of porcine epidemic diarrhea virus at a lower antigen content, and all the mice in the challenge control group became ill.

[0090] This indicates that the inactivated vaccine containing adjuvant B prepared by the present invention has a good immune protection effect on piglets, and not only has a good immune protection effect on prevalent strains, but also has a good cross-immune protection effect on classic strains.

[0091] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as a preferred embodiment, it is not intended to limit the present invention. Any technician familiar with the present profession can make some changes or modifications to equivalent embodiments of the technical contents disclosed above without departing from the scope of the technical solution of the present invention. However, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A vaccine composition, characterized in that The vaccine composition comprises an immunizing amount of inactivated whole virus antigen of porcine epidemic diarrhea virus HN1303 strain and an aluminum gel adjuvant; The porcine epidemic diarrhea virus strain is HN1303 strain, the biological deposit number of the HN1303 strain is: CCTCC NO.V201514, the depository is the China Center for Type Culture Collection, the deposit address is Wuhan University, Wuhan, China, and the deposit date is March 4, 2015; The aluminum gel adjuvant is aluminum salt, polyacrylic acid and dextran sulfate; The preparation method of the aluminum gel adjuvant comprises: (1) Take a 5% aluminum sulfate solution, add 0.4 times the volume of the aluminum sulfate solution to a 5% sodium hydroxide solution under strong stirring, wash the precipitate with normal saline by centrifugation, and then suspend it in normal saline; (2) Add polyacrylic acid and dextran sulfate in sequence, with the amount of polyacrylic acid added to a final concentration of 2 mg / ml and the amount of dextran sulfate added to a final concentration of 0.4 mg / ml, and stir evenly; (3) Homogenize with a homogenizer.

2. The vaccine composition according to claim 1, characterized in that The inactivated HN1303 strain whole virus antigen content is ≥10 6.0 TCID 50 / ml.

3. The vaccine composition according to claim 2, characterized in that The inactivated HN1303 strain whole virus antigen content in the vaccine composition is 10 6.0 TCID 50 / ml ~10 7.0 TCID 50 / ml.

4. The vaccine composition according to claim 1, characterized in that The aluminum salt is selected from aluminum hydroxide and aluminum phosphate.

5. A method for preparing the aluminum gel adjuvant according to claim 1, characterized in that: The method comprises: (1) Take a 5% aluminum sulfate solution, add 0.4 times the volume of the aluminum sulfate solution to a 5% sodium hydroxide solution under strong stirring, wash the precipitate with normal saline by centrifugation, and then suspend it in normal saline; (2) Add polyacrylic acid and dextran sulfate in sequence, with the amount of polyacrylic acid added to a final concentration of 2 mg / ml and the amount of dextran sulfate added to a final concentration of 0.4 mg / ml, and stir evenly; (3) Homogenize with a homogenizer.

6. A method for preparing the vaccine composition according to any one of claims 1 to 4, characterized in that: The method comprises: (1) Proliferating and culturing the porcine epidemic diarrhea virus strain according to claim 1; (2) inactivating the porcine epidemic diarrhea virus strain propagated in step (1); and (3) Add aluminum gel adjuvant and emulsify.

7. The method for preparing a vaccine composition according to claim 6, wherein The adjuvant is prepared by the method according to claim 5.

8. Use of the vaccine composition according to any one of claims 1 to 4 in the preparation of a medicament for preventing and / or treating porcine epidemic diarrhea.

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