Porcine epidemic diarrhea virus egg yolk antibody as well as preparation method and application thereof

Abstract

The invention discloses a preparation method of an egg yolk antibody, which comprises the following steps of: preparing a vaccine composition from a porcine epidemic diarrhea virus strain with the microbial preservation number of CGMCC (China General Microbiological Culture Collection Center) NO.46190, inoculating 90-180-day-old laying hens with the vaccine composition to prepare hyper-immune eggs, and extracting the egg yolk antibody in the hyper-immune eggs by using an octanoic acid extraction method. The egg yolk antibody can effectively prevent porcine epidemic diarrhea, is good in specificity, safe and effective, and has a clinical application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of veterinary biological products, and relates to a porcine epidemic diarrhea virus yolk antibody and a preparation method and application thereof. BACKGROUND

[0002] Porcine epidemic diarrhea (PED) is an acute, highly contagious, viral enteric disease caused by porcine epidemic diarrhea virus (PEDV), with vomiting, diarrhea, dehydration and death as the main characteristics in piglets.

[0003] PEDV belongs to the alpha coronavirus genus. PEDV is a single-stranded RNA virus with a capsid, and the full-length genome is about 28 kb. The genome includes a 5' non-coding region, a 3' UTR, and 7 open reading frames (ORF1a, ORF1b, S, ORF3, E, M, N). The replication process of PEDV in cells involves multiple key stages, including virus adsorption and invasion, translation of replicase, transcription and replication of genes, synthesis of structural proteins, and assembly and release of virus particles.

[0004] PED was first reported in the United Kingdom in 1971, and then the disease spread in Europe and Asia. China had related reports of PED in 1973, and PEDV was identified in 1984. Since then, the PEDV epidemic strain in China has been a low pathogenic G1a subtype classic strain, and the use of CV777 vaccine can better control the further spread of the strain.

[0005] PEDV is a highly infectious virus that poses a serious threat to the global pig industry. Therefore, the development of a safe and efficient PED vaccine has been a key link in the prevention and control of PED. In recent years, with the continuous progress of science and technology, significant progress has been made in the research of PED vaccine. Currently, the research of PED vaccine mainly focuses on two types of vaccines: traditional vaccines and new type vaccines. Traditional vaccines include inactivated vaccines and attenuated live vaccines, which have been widely used in the prevention and control of various viral diseases. New type vaccines mainly include subunit vaccines, recombinant virus vector vaccines and nucleic acid vaccines, etc., which have higher safety and immunogenicity.

[0006] The main disadvantage of the foregoing vaccines is that they need to be prevented in advance and cannot treat or alleviate PED symptoms after infection, and it is not easy to increase the immune dose at any time, and it takes a period of time to produce antibodies after vaccination. However, there are few mature commercial PEDV yolk antibodies reported. SUMMARY

[0007] To solve the problems in the prior art, the first aspect of the present application provides a porcine epidemic diarrhea virus strain selected from S1, S2 and S3:

[0008] S1: the porcine epidemic diarrhea virus strain with a microbial preservation number of CGMCC NO. 46190;

[0009] S2: a passage virus strain of the porcine epidemic diarrhea virus strain with a microbial preservation number of CGMCC NO. 46190;

[0010] The passage virus strain of the porcine epidemic diarrhea virus strain with a microbial preservation number of CGMCC NO. 46190 does not change the transcription regulation activity of all genes in the genome, the transcription initiation and termination of all genes in the genome, the amino acid sequence of the protein encoded by all genes in the genome, the virus clinical pathogenicity, the virus immunogenicity and the virus reproduction ability relative to the porcine epidemic diarrhea virus strain with a microbial preservation number of CGMCC NO. 46190 during the passage process;

[0011] S3: a porcine epidemic diarrhea virus strain with the same genome sequence as the porcine epidemic diarrhea virus strain in S1 or S2.

[0012] The second aspect of the present application provides a vaccine composition for preventing, slowing down or controlling porcine epidemic diarrhea, wherein the vaccine composition takes the porcine epidemic diarrhea virus strain in the first aspect of the present application as an immunogen; or

[0013] The raw material of the vaccine composition includes the porcine epidemic diarrhea virus strain in the first aspect of the present application as an immunogen and an excipient.

[0014] In some embodiments, the excipient includes an oil-based adjuvant, a first emulsifier and a second emulsifier.

[0015] The components of the vaccine composition include the following in terms of the amount ratio:

[0016] The porcine epidemic diarrhea virus strain: oil-based adjuvant: first emulsifier: second emulsifier = 10 10.0 TCID 50 : 100-300g: 5-15g: 3-9g; wherein the amount of the porcine epidemic diarrhea virus strain is measured and calculated according to the amount of live virus.

[0017] In some embodiments, any one selected from the following A1, A2, A3, A4, A5, A6 and A7:

[0018] A1: the porcine epidemic diarrhea virus strain is an inactivated porcine epidemic diarrhea virus strain;

[0019] A2: the oil adjuvant is selected from white oil;

[0020] A3: the first emulsifier is selected from Span-80;

[0021] A4: the second emulsifier is selected from Tween-80;

[0022] A5: the components of the vaccine composition are included in the following ratio:

[0023] Porcine epidemic diarrhea virus strain: oil adjuvant: first emulsifier: second emulsifier = 94 x 10 8.0 TCID 50 : 190 g: 10 g: 6 g;

[0024] A6: the porcine epidemic diarrhea virus strain is an immunogen at a concentration of 0.1-10 x 10 8.0 TCID 50 / ml to prepare the vaccine composition;

[0025] A7: the TCID 50 of the porcine epidemic diarrhea virus strain is calculated according to the Reed-Muench method based on Vero cell culture.

[0026] The third aspect of the present application provides a preparation method of yolk antibody, which comprises the following steps:

[0027] In some embodiments, the preparation method comprises the following steps:

[0028] S1: the porcine epidemic diarrhea virus strain of the first aspect of the present application is prepared into a vaccine composition;

[0029] S2: the vaccine composition is used to inoculate avian animals to obtain hyperimmune eggs;

[0030] S3: yolk antibody is extracted from the hyperimmune eggs.

[0031] In some embodiments, any one of B1, B2 and B3 is selected:

[0032] B1: in step S1, the vaccine composition is the vaccine composition of the second aspect of the present application;

[0033] B2: in step S2, the preparation method of the hyperimmune eggs comprises the following steps:

[0034] Primary immunization: the vaccine composition is used to inoculate avian animals in the laying period;

[0035] Second immunization: the laying birds are inoculated with the vaccine composition 7-28 days after the primary immunization;

[0036] Third immunization: the laying birds are inoculated with the vaccine composition 7-28 days after the second immunization;

[0037] Fourth immunization: the laying birds are inoculated with the vaccine composition 7-28 days after the third immunization, and the hyperimmune egg is obtained;

[0038] B3: in step S3, the extraction method of the yolk antibody is to obtain the yolk antibody through the following two steps, three steps or four steps:

[0039] S3-1: obtaining the yolk of the hyperimmune egg;

[0040] S3-2: extracting the yolk to obtain the crude yolk antibody;

[0041] S3-3: concentrating the purified yolk antibody to obtain the concentrated yolk antibody;

[0042] S3-4: sterilizing the concentrated yolk antibody to obtain the sterile yolk antibody.

[0043] In some embodiments, any one selected from C1, C2, C3, C4, C5, C6, C7, C8, C9 and C10;

[0044] C1: in step S1, the preparation method of the vaccine composition comprises the following steps:

[0045] S1-1: mixing the oil-based adjuvant with the first emulsifier to obtain an oil phase adjuvant;

[0046] S1-2: mixing the inactivated porcine epidemic diarrhea virus with the second emulsifier to obtain an aqueous phase;

[0047] S1-3: mixing the oil phase adjuvant with the aqueous phase to obtain the vaccine composition;

[0048] C2: after the fourth immunization, when the yolk antibody titer level of the avian animal against the porcine epidemic diarrhea virus strain of the first aspect of the application is lower than 1:256, the avian animal is injected with the vaccine composition of the second aspect of the application in an amount of 2-5x10 7.0 TCID 50 of the inactivated porcine epidemic diarrhea virus strain of the first aspect of the application by intramuscular injection in the chest or intramuscular injection in the leg;

[0049] C3: in step S2, the poultry animal is selected from the group consisting of chicken, duck, goose;

[0050] C4: in step S2, the egg laying period is 90-180 days old;

[0051] C5: in step S2, the inoculation route is intramuscular injection in chest or leg;

[0052] C6: the inoculation dose of the primary immunization is 1-6x10 7.0 TCID 50 ; the inoculation dose of the second immunization is 1-10x10 7.0 TCID 50 ; the inoculation dose of the third immunization is 1-15x10 7.0 TCID 50 ; the inoculation dose of the fourth immunization is 1-15x10 7.0 TCID 50 ;

[0053] C7: the high immune egg is used after being disinfected before step S3-1;

[0054] C8: in S3-2, the step of preparing the crude egg yolk antibody is as follows:

[0055] S3-2-1: the egg yolk is mixed with acidified water with pH value of 4-5, the volume ratio of the egg yolk to the acidified water is 1:4-8, and stirring is performed to obtain a first mixture;

[0056] S3-2-2: the first mixture is mixed with an aqueous n-octanoic acid solution, the final concentration of the n-octanoic acid is 0.1-0.3v / v %, and centrifugation is performed to collect the supernatant to obtain the crude egg yolk antibody;

[0057] C9: in step S3-3, the purified egg yolk antibody is concentrated by using an ultrafiltration membrane stack with a molecular weight cut-off of 25-50KD, and the concentration multiple is 2-10;

[0058] C10: in step S3-4, the concentrated egg yolk antibody is filtered and sterilized by using a 0.22μm sterilization filter element.

[0059] The fourth aspect of the present application provides an egg yolk antibody, which is prepared by the preparation method of the third aspect of the present application.

[0060] The fifth aspect of the present application provides the use of the porcine epidemic diarrhea virus strain according to the first aspect of the present application, the vaccine composition according to the second aspect of the present application or the egg yolk antibody according to the fourth aspect of the present application in the preparation of a medicament for use alone, for use in conjunction with other immunological agents and / or drugs, or as a component of a combined preparation with other immunological agents and / or drugs for the treatment, prevention, alleviation and / or control of porcine epidemic diarrhea. BRIEF DESCRIPTION OF DRAWINGS

[0061] Figure 1 Figure 1 is a photograph of Vero cell cytopathic effect infected by F0 generation of PEDV isolates, in which the left is normal cells and the right is cytopathic effect.

[0062] Figure 2 Figure 2 is an electron microscope photograph of PEDV isolates, in which the entire scale length represents 100 nm.

[0063] Figure 3 Figure 3 is an egg yolk antibody curve.

[0064] Figure 4 Figure 4 is a photograph of anal morphology of immunized pigs.

[0065] Figure 5 Figure 5 is a photograph of anal morphology of challenge control pigs.

[0066] Figure 6 Figure 6 is a photograph of organ morphology of immunized pigs.

[0067] Figure 7 Figure 7 is a photograph of organ morphology of challenge control pigs. DETAILED DESCRIPTION

[0068] In order to make the purpose, technical solutions and advantages of the present application clearer, the embodiments of the present application will be further described in detail below with reference to the drawings.

[0069] The materials and instruments not described in the present application are conventional materials and instruments in the art. The operation details not described in the present application are conventional operations in the art. The software used in the present application is operated by conventional methods according to the instructions provided by the software provider. The kits used in the present application are operated by conventional methods according to the instructions provided by the kit manufacturer. The nucleic acid sequences listed in the present application are all from 5' end to 3' end from left to right. The protein sequences listed in the present application are all from N-terminal to C-terminal from left to right.

[0070] The passage "the passage virus strain of the porcine epidemic diarrhea virus strain with the microbial preservation number of CGMCC NO. 46190 does not change in the transcriptional regulation activity of all genes in the genome, the transcription initiation and termination of all genes in the genome, the amino acid sequence of the protein encoded by all genes in the genome, the virus clinical pathogenicity, the virus immunogenicity and the virus propagation ability relative to the porcine epidemic diarrhea virus strain with the microbial preservation number of CGMCC NO. 46190" includes but is not limited to the following cases:

[0071] The passage "the virus propagation ability does not change in the passage process of the YK strain" mainly refers to that the genome sequence does not have any mutation and maintains the original propagation ability (no mutation lethality, etc.), or the slight change in the propagation ability is within the system error range of the conventional technical detection system or the change degree that cannot be detected according to the conventional technology. According to the common sense in the art, even if two sub-generation viruses of a single clone plaque of the YK strain are separated, the determination results of the propagation ability indicators also have system errors (there are also certain differences in determination of different batches or different operators). Obviously, slight gene mutations inevitably occur in the virus passage process, and the coding sequence has a synonymous mutation or the non-coding region of the genome has a slight mutation which does not affect the virus propagation ability and the biological activity of the virus, and therefore, the passage virus strain belongs to the substantial technical contribution range of the preserved virus strain (the YK strain) and is the inevitable reasonable change range of the direct technical contribution of the present application, which will make the content of the patent protection, the actual use and the content defined in the claims not to be obviously separated. Similarly, the virus clinical pathogenicity and the virus immunogenicity not changing also refer to that the difference is not significant in the statistical sense, or the difference value is slightly higher than, about equal to or less than the standard deviation of the measurement method.

[0072] The passage "the transcriptional regulation activity of all genes does not change in the passage process of the YK strain" mainly refers to that the mode of accepting the endogenous or host-derived gene expression regulation of all transcriptional regulation elements does not change, the mode of accepting the external signal response does not have a detectable change, and the mode of the virus participating in the host signal transduction pathway and metabolism regulation does not have a detectable change. The slight mutation not participating in the regulation (for example, the slight change of the non-coding sequence base does not affect the expression regulation mode of the corresponding gene) belongs to the range of the transcriptional regulation activity not changing.

[0073] The passage "the transcription initiation and termination of all genes do not change in the passage process of the passage virus of the YK strain" mainly refers to that the mode of the gene being expressed and regulated does not change, and the transcription regulation of the gene does not have a detectable significant change.

[0074] The "amino acid sequences of all proteins in the YK strain passage virus do not change during the passage process" mainly refers to that during the passage process of the strain, the amino acid sequences of all proteins do not change, and synonymous mutations of genes do not affect the function of genes, and mutations during virus passage are inevitable, so the sub-virus with unchanged amino acid sequences of all proteins is still within the technical contribution range of the preserved strain.

[0075] The "porcine epidemic diarrhea virus strain with the same genome sequence as the porcine epidemic diarrhea virus strain described in S1 or S2" mainly refers to a porcine epidemic diarrhea virus strain that is not derived from the porcine epidemic diarrhea virus strain with the microbial preservation number CGMCC NO. 46190 or its passage strain, but is a porcine epidemic diarrhea virus strain independently discovered and isolated by anyone after the filing date of the present patent application. Obviously, anyone who obtains this strain by coincidence after the filing date of the present patent application should also be considered to fall within the protection scope of the present application.

[0076] Example 1: Acquisition, isolation and identification of porcine epidemic diarrhea virus YK strain

[0077] I. Sample isolation and processing

[0078] Newborn piglets in a pig farm in Liaoning Province showed symptoms of watery diarrhea, vomiting and death. Autopsy showed that the small intestine wall was thin and filled with yellow liquid, suggesting that porcine epidemic diarrhea virus infection had occurred. The duodenum of the 3-day-old newborn piglets that died of the disease was collected. The collected duodenum was cut longitudinally in a sterile tray, and the small intestinal mucosa layer was scraped with a sterile glass slide. The intestinal contents were homogenized with sterile PBS (pH 7.2) at a weight ratio of 1:5, frozen and thawed 3 times at -20°C, centrifuged at 10,000 rpm for 10 minutes at 4°C, and the supernatant was filtered with a 0.22 μm microporous filter to remove bacteria, aliquoted, and the filtrate was used as the virus isolation sample and stored in a -80°C refrigerator for use.

[0079] II. Virus isolation and passage culture

[0080] The digested Vero cells were adjusted to a cell concentration of 1×10 6The cell suspension was added to a T25 cell flask and incubated at 37°C and 5% CO2 for 3 days. The cells were washed twice with PBS and the prepared inoculum (DMEM medium containing a final concentration of 10 μg / ml trypsin) was added. The treated sample was inoculated into the cell flask at a ratio of 1% (v / v) and observed for cytopathic effect (CPE) at 37°C and 5% CO2. After 3-5 days, the virus culture was harvested, frozen and thawed 3 times at -20°C, centrifuged at 10,000 rpm for 10 minutes at 4°C, and the supernatant was collected. The culture was further passaged according to the above method until CPE appeared. When the characteristic CPE appeared stably, the harvested cell culture was used as the original seed virus, which was designated as the F0 generation. The seed virus was named as the PEDV YK strain, abbreviated as YK strain.

[0081] Typical CPE appeared when the blind passage reached the 5th generation, and the cell culture was collected and recorded as the 0th generation. CPE appeared in the virus culture at 48 hours, the infected cells swelled, rounded, granular degeneration, some cells fused to form syncytia, the nuclei aggregated, and finally the cells gradually detached, and more than 80% of the cells showed CPE at 72 hours. The photographs of the Vero cell CPE infected by the F0 generation are shown in Figure 1. Figure 1 , in which the left is the blank control group of Vero cells without virus inoculation, and the right is the cell CPE group.

[0082] III. Determination of the virus content (TCID 50 ) of the inoculum

[0083] A 96-well cell culture plate with Vero cells growing into a dense monolayer was prepared, and the cell culture medium was aspirated and the plate was washed once with PBS. The F0 generation of the PEDV strain was serially diluted 10-fold with the inoculum (DMEM medium containing a final concentration of 20 μg / ml trypsin + 10% TPB), and 10 -2 ~10 -8 μl of each dilution was added to 8 wells, and 100 μl of the inoculum was added to each well. At the same time, 8 wells of cell control were set up, and 200 μl of the inoculum was added to each well. The plate was incubated at 37°C and 5% CO2 for 96 hours. The CPE was recorded, and the TCID 50 was calculated according to the Reed-Muench method. The virus content of the F0 generation of the YK strain was 10 8.00 TCID 50 / ml.

[0084] IV. Observation of virus morphology

[0085] The PEDV isolate F0 generation infected Vero cells were collected by centrifugation, resuspended with PBS, repeatedly frozen and thawed at -20°C for 3 times, centrifuged at 10000 r / m for 10 min at 4°C, and the supernatant was taken on a 200-mesh copper screen. After the liquid was naturally dried, it was stained with 2% phosphotungstic acid for 2 min, and then dried completely and observed under a JEM-1400 transmission electron microscope (Japan Electron Corporation). The electron microscope photos are shown in Figure 2 As can be seen, the virus infected cells can release mature virions. The virions observed by electron microscope are spherical, with a size of 90-190 nm, and the virions have radially arranged filaments. The center of the virion is an electron opaque area. It is consistent with the morphological characteristics of porcine viral diarrhea virus.

[0086] V. Virus nucleic acid identification

[0087] According to the primers designed in the national standard (GB / T 34757-2017 and GB / T 36871-2018), the primers F1 (SEQ ID NO. 1) and R1 (SEQ ID NO. 2) were designed for the M gene of PEDV, the primers F2 (SEQ ID NO. 3) and R2 (SEQ ID NO. 4) were designed for the N gene of porcine transmissible gastroenteritis virus TGEV, the primers F3 (SEQ ID NO. 5) and R3 (SEQ ID NO. 6) were designed for the VP7 gene of porcine rotavirus PoRV, and the primers F4 (SEQ ID NO. 7) and R4 (SEQ ID NO. 8) were designed for sequencing the S1 gene of PEDV.

[0088] F1: 5'-TATGGCTTGCATCACTCTTA-3'

[0089] R1: 5'-TTGACTGAACGACCAACACG-3'

[0090] F2: 5'-TTACAAACTCGCTATCGCATGG-3'

[0091] R2: 5'-TCTTGTCACATCACCTTTACCTGC-3'

[0092] F3: 5'-CCCCGGTATTGAATATACCACAGT-3'

[0093] R3: 5'-TTTCTGTTGGCCACCCTTTAGT-3'

[0094] F4: 5'-GGTAAGTTGCTAGTGCGTA-3'

[0095] R4: 5'-CACAGAAAGAACTAAACCC-3'

[0096] The total RNA of the F0 generation venom was extracted with EasyPure Viral DNA / RNA Kit according to the reagent instruction. The total RNA was subjected to PCR amplification with the four pairs of primers, and the gel electrophoresis was performed on the amplification products of the first three pairs of primers. The results showed that the M gene amplification product of PEDV was positive, and the amplification products of the other two genes were negative. The S1 gene sequence was obtained by Sanger sequencing according to the amplification results of the fourth pair of primers, and then compared with the gene sequence in Genbank. The results showed that the homology with the S1 gene sequence of the LNHD-2018 strain of porcine epidemic diarrhea virus (Genbank number MT294133.1) was as high as 98.77%. It can be seen that the F0 generation virus is PEDV, not TGEV or PoRV which can also cause piglet diarrhea.

[0097] Six, virulence determination

[0098] The F0 generation of PEDV YK strain was diluted to 10 4.0 TCID 50 / ml with physiological saline, and 1-day-old piglets were orally inoculated at a dose of 1 ml per piglet, with 5 piglets in each group. Five negative controls (without inoculation) were set up at the same time. After inoculation, the piglets were observed for 7 consecutive days, and the clinical conditions of the piglets were observed every day. The morbidity and mortality of the piglets were calculated. The dead piglets were dissected to observe and record the pathological changes. At the end of the experiment, anal swabs were collected for nucleic acid detection.

[0099] Results: Vomiting, yellowish diarrhea, chills and huddling were observed in infected piglets 24 hours after inoculation. The feces became watery 48 hours later, and the piglets were obviously emaciated, dehydrated and died. All died 120 hours later. Dissection showed that the intestinal wall of the sick pigs was thin and full of gas, the stomach was full of undigested milk blocks, and the anal swab nucleic acid detection was positive. The negative controls had no above-mentioned symptoms and no abnormal state was found.

[0100] Seven, virus-specific specificity

[0101] The F0 generation of YK strain was diluted to 100 TCID 50After 0.1 ml, 0.5 ml was taken and mixed with an equal amount of self-made PEDV-specific positive serum, self-made TGEV-specific positive serum, and self-made PoRV-specific positive serum, and neutralized at 37°C for 1 hour. Then, 100 μl of the mixture was inoculated into each of 8 wells of Vero cells that had grown into a good and dense monolayer on a 96-well cell culture plate. 100 μl of cell maintenance medium was added to each well. At the same time, negative controls (using porcine epidemic diarrhea virus antibody-negative serum plus YK strain), virus controls (adding YK strain without serum), and normal cell controls (without virus solution and without serum) were set up, with 4 wells for each. They were cultured at 37°C under 5% CO2 conditions, and the cytopathic effect was observed daily for 4 days, and the cytopathic effect was recorded.

[0102] Results: There was no cytopathic effect in the PEDV positive serum group and the normal cell group; there was cytopathic effect in the negative control, virus control group, TGEV positive serum group, and PoRV positive serum group. Therefore, the YK strain has serological specificity for porcine epidemic diarrhea virus.

[0103] VIII. Microbial Preservation

[0104] The isolated porcine epidemic diarrhea virus YK strain of the present invention was submitted to a preservation institution recognized by the patent procedure for preservation; the preservation unit is: General Microbiology Center of China Committee for Culture Collection of Microorganisms; address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Its microbial preservation number is CGMCC No. 46190; the Chinese classification name is: Porcine Epidemic Diarrhea Virus; the Latin classification name is: Porcine Epidemic Diarrhea Virus; the name of the biological material is: PEDV-YK strain; the preservation time is: September 20, 2024; the proof of survival time is: September 20, 2024.

[0105] Example 2: Preparation of Vaccine Composition

[0106] The following operations were carried out in three batches in this example.

[0107] I. Virulent seeds for production

[0108] The F10 generation of virulent seeds obtained by culturing the F0 generation of the YK strain of porcine epidemic diarrhea virus in Vero cells.

[0109] II. Preparation of antigen solution

[0110] Vero cells which have been well grown in DMEM medium containing 10% fetal calf serum were digested with 0.25% trypsin-EDTA solution and subcultured for F10 generation. The Vero cells were expanded in a transfer bottle and subcultured until a dense monolayer was formed. The growth medium was discarded and cell inoculum (DMEM medium containing 10 μg / ml trypsin) was added. The F10 generation of porcine epidemic diarrhea virus YK strain was inoculated into the production inoculum YK strain at a 1% (v / v) inoculation amount and incubated at 37°C in 5% CO2. The cells were observed daily and harvested when the cytopathic effect reached more than 90%. The virus was stored at -20°C or below. After the virus was frozen and thawed twice, a few milliliters of the sample were removed for titer determination (the method was the same as that in Example 1, Section 3). Three batches of virus antigen were prepared using the F10 generation of the production inoculum by the same method.

[0111] The TCID of the three batches of virus antigen prepared was 50 10 8.20 TCID 50 / ml, 10 8.12 TCID 50 / ml, and 10 8.29 TCID 50 / ml, respectively, and was not less than 10 8.0 TCID 50 / ml.

[0112] III. Preparation of the vaccine

[0113] (1) Inactivation of the antigen and inactivation test

[0114] The three batches of virus antigen which passed the test were added to an aqueous solution of formaldehyde at a final concentration of 0.1% and stirred to mix well. The mixture was inactivated at 37°C for 24 hours with shaking. After inactivation, samples were taken for inactivation test. Vero cells which had formed a dense cell monolayer were inoculated with the inactivated virus antigen, and no cytopathic effect was observed, indicating that the inactivation was complete.

[0115] (2) Concentration of the antigen

[0116] The inactivated virus antigen solution which passed the test was concentrated by 2-10 times the volume through a 30 kDa ultrafiltration membrane.

[0117] (3) Preparation of the inactivated vaccine

[0118] (1) Preparation of the oil phase: 95 parts of injection white oil were heated and 5 parts of Span-80 was added and mixed well. The mixture was sterilized by autoclaving and stored for use.

[0119] (2) Preparation of the water phase: 94 parts of the concentrated antigen were added to 6 parts of sterilized Tween-80. The mixture was shaken well until the Tween-80 was completely dissolved.

[0120] (3) Emulsification: The oil phase was poured into the colloid mill first, stirred at 2500 r / min, and the water phase was slowly added, emulsified at 10000 r / min for 3-5 minutes at a ratio of 2:1 (V / V) of the oil phase and the water phase.

[0121] Three batches of vaccines prepared from three batches of virus liquid were named as B1, B2 and B3, respectively.

[0122] (4) Safety test

[0123] For B1, B2 and B3, 10 SPF chickens of 28 days old were subcutaneously injected with 2.0 ml of inactivated vaccine each, and observed for 10 days.

[0124] Results: All the inoculated chickens were healthy and alive, and no swelling, nodules, abnormal feeding or drinking, or any local and systemic adverse reactions were observed at the injection sites.

[0125] (5) Efficacy test

[0126] Three batches of inactivated vaccines (B1, B2 and B3) were inoculated into 28-day-old SPF chickens, each subcutaneously injected with 1.0 ml, and blood was collected for determination of neutralizing antibodies 21 days after immunization. The control group was SPF chickens without inoculation.

[0127] Results: The titer of the control group was <1:2, and the serum neutralizing antibody titers after inoculation of the three batches of vaccines were 1:94.12, 1:91.80 and 1:89.31, respectively. The neutralizing antibody titers were not less than 1:64, meeting the clinical use standard.

[0128] Example 3. Study of immunization program and preparation of egg yolk antibodies

[0129] I. Study of immunization program

[0130] Group A: 100 laying hens (120 days old, Hyline Brown commercial egg laying hens, the same below) were used and inactivated vaccine B1 was used for the basic immunization at 0.8 ml per chicken, injected intramuscularly in the chest; the second immunization was performed 14 days after inoculation at 1.0 ml per chicken, injected intramuscularly in the leg; the third immunization was performed 14 days after the second immunization at 1.0 ml per chicken, injected intramuscularly in the chest; the fourth immunization was performed 14 days after the third immunization at 1.0 ml per chicken, injected intramuscularly in the leg.

[0131] Group B: 100 laying hens were used and inactivated vaccine B1 was used for the basic immunization at 0.8 ml per chicken, injected intramuscularly in the chest; the second immunization was performed 14 days after inoculation at 1.0 ml per chicken, injected intramuscularly in the leg; the third immunization was performed 14 days after the second immunization at 1.0 ml per chicken, injected intramuscularly in the chest; the fourth immunization was performed 21 days after the third immunization at 1.0 ml per chicken, injected intramuscularly in the leg.

[0132] Group C: 100 laying hens were used and inactivated vaccine B1 was used, 1.0 ml per hen was injected in the chest muscle for the basic immunization; 14 days after the inoculation, the second immunization was performed, 1.5 ml per hen was injected in the leg muscle; 14 days after the second immunization, the third immunization was performed, 1.5 ml per hen was injected in the chest muscle; 21 days after the third immunization, the fourth immunization was performed, 1.5 ml per hen was injected in the leg muscle.

[0133] Control group: 50 healthy egg laying hens were not inoculated and were isolated for feeding.

[0134] The high immune egg of each group was collected randomly at the time of the four immunizations and 1-17 weeks after the fourth immunization, 20 eggs were collected, the egg yolk was separated, the yolk liquid was extracted aseptically, was diluted with 3 times the volume of the maintenance liquid (DMEM medium containing 25 μg / ml trypsin at the final concentration, the same below), was filtered aseptically, was diluted by 2 times the volume again to the appropriate titer, 4 dilution samples were taken, were mixed with 100 TCID 50 / 0.1 ml of the porcine epidemic diarrhea virus YK strain F10 in the same volume, 100 TCID 50 / 0.1 ml of the virus liquid was added to the same volume of cell maintenance liquid as the virus control, was placed in a 5% CO2 incubator at 37°C for 1 hour. The cell plate culture medium was discarded, the neutralizing liquid was adsorbed for 2 hours, after the liquid was discarded, the maintenance liquid 200 μl / well was added, was placed in a 5% CO2 incubator at 37°C for 5 days. The number of cells with CPE was recorded for each group, when all the virus control wells appeared CPE and the normal cell control wells did not appear CPE, the test was established. The neutralizing antibody titer was calculated according to the Reed-Muench method.

[0135] The yolk antibody titer statistical results are shown in Table 2. Figure 3 It can be seen that the yolk antibody of each group of laying hens reached 1:256 from 1 week after the fourth immunization. Compared with groups A and B, the antibody of group C was effective quickly, reached the standard after the fourth immunization, and maintained for a long time, and still reached the standard at 16 weeks after the fourth immunization. According to the test results, the immunization program of the high immune egg laying hen is as follows. The first inoculation is performed on the egg laying hen at 120 days old or above, 1.0 ml per hen is injected in the chest muscle; 14 days after the first inoculation, the second inoculation is performed, 1.5 ml per hen is injected in the leg muscle; 14 days after the second inoculation, the third inoculation is performed, 1.5 ml per hen is injected in the chest muscle; 21 days after the third immunization, the fourth immunization is performed, 1.5 ml per hen is injected in the leg muscle, and after that, the antibody titer reaches 1:256, the high immune egg is collected, and the yolk antibody is prepared. When the antibody level is lower than 1:256, the maintenance immunization is performed, 1.5 ml per hen is injected in the leg muscle.

[0136] II. Preparation of yolk antibody

[0137] (1) Collection of egg yolk

[0138] The high immune chicken eggs obtained 21 days after the fourth immunization of group C were immersed in 0.1% aqueous neojel disinfectant solution for 15 minutes. The egg white and yolk were separated by hand, and the egg white (white), germinal disc and chalaza were removed thoroughly, and the yolk was collected in a container and stirred thoroughly to make the yolk into a uniform paste. Sampling was performed for neutralizing antibody determination (the method was the same as that in section one of this example), which was recorded as the initial titer of yolk liquid antibody. Three batches were repeated.

[0139] The PEDV YK strain F10 virus seed was diluted with cell maintenance solution to 100 TCID 50 / 0.1ml. The diluted virus solution was added to the 96-well plate with 2-fold diluted samples, 6 wells for each dilution, and 100 μl per well. Neutralization was performed at 37°C, 5% CO2 for 1 hour. The Vero cell plate with dense monolayer cells was washed with PBS for 2 times. The neutralization liquid was added to the cell plate. Adsorption was performed at 37°C, 5% CO2 for 2 hours. Meanwhile, 6 wells of normal cell holes (without virus and antibody), virus control holes (with virus without egg antibody), and positive control holes (with PEDV positive serum instead of egg antibody) were set. The adsorbed cell plate was washed with PBS for 1 time, and all sample holes (normal cell holes, tested samples and positive control) were supplemented with maintenance solution 200 μl / well. Virus control holes were added with 100 TCID 50 / 0.1ml, 100 μl / well, supplemented with maintenance solution 100 μl / well, and incubated at 37°C, 5% CO2 for 48 hours.

[0140] The antibody titers of the three batches were 1:362, 1:351 and 1:413, respectively.

[0141] (II) Extraction of yolk antibody

[0142] Acidified water was prepared (pure water was adjusted to pH 4.2-4.8 with 1 mol / L hydrochloric acid), and the yolk was stirred thoroughly to make it into a uniform paste. According to the amount of egg yolk, 6 times the volume of the egg yolk was added to the acidified water, and the temperature was lowered to 2-8°C. After stirring uniformly, stirring was continued for 3 hours. It was placed at 4°C for 20 hours, and n-octanoic acid solution was added to a final concentration of 0.2% (v / v) while stirring for 10 minutes. It was placed at room temperature for 20 hours, and centrifuged at 10000 r / min using a continuous centrifuge. The supernatant was collected, which was the initial antibody. The neutralizing antibody titer was determined.

[0143] The determination method was the same as that in section (I).

[0144] Virus regression control: 100 TCID 50 / 0.1ml of virus solution was diluted by 10 times to 10 TCID 50 / 0.1ml and 1 TCID 50 / 0.1ml, 0.1 TCID 50 / 0.1ml, 100μl of each was added to the corresponding 96-well plate, and 100μl of maintenance solution was added.

[0145] All the virus control wells were lesioned, the normal cell control wells were not lesioned, and the virus regression 10TCID 50 All the 0.1ml wells were lesioned, and 0.1TCID 50 The 0.1ml wells should not be lesioned, and the regression was established. After inoculation, the number of cell wells with CPE was recorded for each group, and then the half-protective dose (PD 50 ) was calculated according to the Reed-Muench method.

[0146] Results: The antibody titers of the three batches were 1:49, 1:52, and 1:59, respectively.

[0147] (Three) Concentration

[0148] Concentration was performed by using an ultrafiltration membrane stack with a molecular weight cutoff of 30KD, and the concentration ratio was 2-10 times. After concentration, the antibody titer was determined.

[0149] The determination method was the same as that in (One), and the antibody titers of the three batches were 1:140, 1:143, and 1:152, respectively.

[0150] (Four) Sterilization

[0151] A 0.22μm sterilization filter was applied to filter and sterilize the concentrated egg yolk antibodies.

[0152] The obtained three batches of egg yolk antibodies were respectively named Y1, Y2, and Y3.

[0153] Example 4: Safety test of egg yolk antibodies

[0154] I. Safety test of egg yolk antibodies on 30-day-old piglets

[0155] Thirty 30-day-old susceptible healthy piglets produced by a porcine epidemic diarrhea virus antibody-negative non-immune healthy sow were divided into three groups, 10 piglets in each group. The piglets were orally administered with egg yolk antibodies Y1, Y2, and Y3, respectively, at 4.0ml (2 doses) per piglet, and a blank control group of 5 piglets (the same as not orally administered pigs) was set up. The piglets were raised under the same conditions without treatment. After immunization, the piglets were continuously observed for 10 days, and the body temperature and clinical manifestations were recorded. Necropsy was performed on the 10th day after immunization to observe whether there were abnormalities in the internal organs. The body weight before the test and on the 10th day after immunization was recorded.

[0156] (Clinical observation: The mental state, behavior activity, feces condition, feed (milk), and water drinking of the piglets after immunization were recorded, and the observation was performed daily for 10 consecutive days.

[0157] Results: After immunization, piglets had no abnormal feeding (milk), drinking water, good mental state, normal feces without diarrhea, no abnormal reactions, and all were healthy.

[0158] (II) Body temperature: The body temperature of 5 pigs in each group (3 in the control group) was recorded before immunization and for 3 days after immunization.

[0159] Results: The body temperature ranges of Y1, Y2, Y3 and the control group were 38.5-39.2℃, 38.5-39.4℃, 38.4-39.4℃ and 38.5-39.1℃, respectively. Therefore, inoculation of egg yolk antibodies had no significant effect on the body temperature of pigs.

[0160] (III) Growth performance: The body weight before the test was recorded, and the body weight was measured again at the end of the test to calculate the daily gain.

[0161] Results: The average daily gain of Y1, Y2, Y3 and the control group was 0.20 kg, 0.18 kg, 0.19 kg and 0.21 kg, respectively, 10 days after immunization. Therefore, inoculation of egg yolk antibodies had no significant effect on the growth performance of pigs.

[0162] (IV) Systemic autopsy: At the end of the observation period, 5 pigs in each group (3 in the control group) were randomly selected and killed by intravenous bloodletting, and systemic autopsy was performed to observe and record the lesions of solid organs and hollow organs.

[0163] Results: In Y1, Y2, Y3 and the control group, there were no abnormalities in liver, spleen, kidney, heart, gallbladder, stomach and intestinal tract.

[0164] The results showed that the safety of egg yolk antibodies against porcine epidemic diarrhea virus was good for super-dose inoculation in 30-day-old piglets.

[0165] II. Single-dose safety test of egg yolk antibodies on 1-day-old piglets

[0166] The difference between this experiment and the first section of this embodiment is that 1-day-old susceptible healthy piglets were used for inoculation of 2.0 ml per pig, and other steps and observation indicators were the same.

[0167] (I) Clinical observation: After immunization, piglets had no abnormal feeding (milk), drinking water, good mental state, normal feces without diarrhea, no abnormal reactions, and all were healthy.

[0168] (II) Body temperature: The body temperature ranges of Y1, Y2, Y3 and the control group were 38.5-39.7℃, 38.5-39.4℃, 38.5-39.8℃ and 38.5-39.3℃, respectively. Therefore, inoculation of egg yolk antibodies had no significant effect on the body temperature of pigs.

[0169] (Three) Growth performance: In Y1, Y2, Y3 and the control group, the average daily weight gain of 10 days after immunization was 0.14 kg, 0.15 kg, 0.15 kg, 0.14 kg, respectively. It can be seen that the inoculation of egg yolk antibody has no obvious effect on the growth performance of pigs.

[0170] (Four) Systemic autopsy: In Y1, Y2, Y3 and the control group, the liver, spleen, kidney, heart, gallbladder, stomach, and intestinal tract were all normal.

[0171] The test results show that the safety performance of porcine epidemic diarrhea virus egg yolk antibody for single-dose inoculation of 10-day-old piglets is good.

[0172] Three, single-dose repeated safety test of egg yolk antibody on 1-day-old piglets

[0173] Take 30 1-day-old susceptible healthy piglets produced by porcine epidemic diarrhea virus antibody-negative non-immune sows, divide them into three groups, 10 in each group. Each group is orally administered egg yolk antibody Y1, Y2, Y3, 2.0 ml per piglet, and each is repeated once with the same dose at an interval of 10 days. Another 5 pigs are taken as a blank control group, raised under the same conditions, and not inoculated. After the second immunization, observe for 10 days, record the body temperature and clinical manifestations; 10 days after the second immunization, perform an autopsy to observe whether there are abnormalities in the internal organs. Record the body weight before the test and 10 days after the second immunization.

[0174] The observation method is the same as that in the first section of this embodiment.

[0175] (I) Clinical observation: After immunization, piglets have no abnormal feeding (milk) and water drinking, good mental state, normal feces without diarrhea, no abnormal reactions, and all are healthy.

[0176] (II) Body temperature (record the body temperature of piglets before immunization and continuously measure the body temperature for 3 days after immunization): In Y1, Y2, Y3 and the control group, the body temperature range of different pigs in four days is 38.5-39.8℃, 38.5-39.8℃, 38.4-39.8℃, 38.5-39.8℃, respectively. It can be seen that the inoculation of egg yolk antibody has no obvious effect on the body temperature of pigs.

[0177] (Three) Growth performance: In Y1, Y2, Y3 and the control group, the average daily weight gain of 10 days after immunization was 0.14 kg, 0.15 kg, 0.15 kg, 0.15 kg, respectively. It can be seen that the inoculation of egg yolk antibody has no obvious effect on the growth performance of pigs.

[0178] (Four) Systemic autopsy: In Y1, Y2, Y3 and the control group, the liver, spleen, kidney, heart, gallbladder, stomach, and intestinal tract were all normal.

[0179] The test results show that the porcine epidemic diarrhea virus yolk antibody has good safety performance for single-dose repeated inoculation of 1-day-old piglets.

[0180] Example 5: Protection test of yolk antibody

[0181] I. Determination of immunoprotective efficacy

[0182] Take 30 1-day-old healthy susceptible piglets produced by porcine epidemic diarrhea virus antibody-negative non-immune sows, and divide them into 3 groups, 5 in each group. Take 3 batches of diarrhea yolk antibodies Y1, Y2, and Y3 prepared in the laboratory, respectively, and dilute them to 1:64 with sterilized normal saline. Each batch of yolk antibody is orally inoculated into 5 1-day-old healthy susceptible piglets, 2.0 ml per piglet. At the same time, 5 piglets for each are set up as an attack control (not inoculated with yolk antibody). Five days after the above immunization, 1.0 ml of test virus (PEDV YK strain) is orally administered to each piglet (containing 10 4.0 TCID 50 / ml), and 5 piglets for each are set up as a blank control group (not inoculated with yolk antibody and not inoculated), and no treatment is performed. After the attack, observe for 7 days, record the clinical symptoms and death of the piglets, and record the lesions by necropsy and collect anal swabs for nucleic acid detection.

[0183] Results: During the 7-day observation period, the three test groups and the blank control group had no abnormal drinking water, good mental state, normal feces without diarrhea, and no abnormal reactions. The attack control group died on the 4th to 6th day, and had different degrees of vomiting and diarrhea symptoms before death.

[0184] The primers for anal swab nucleic acid detection are F5 (SEQ ID NO. 9) and R5 (SEQ ID NO. 10).

[0185] F5: 5'-TATGGCTTGCATCACTCTTA-3'

[0186] R5: 5'-TTGACTGAACGACCAACACG-3'

[0187] The three test groups and the blank control group were negative, and the attack control group was positive.

[0188] The test results show that 1-day-old piglets inoculated with 3 batches of porcine diarrhea antibodies prepared in the laboratory by oral route can achieve 100% prevention of porcine diarrhea virus, and the oral administration is convenient and safe in clinical application.

[0189] II. Test of parallel relationship between neutralizing antibody titer and attack protection

[0190] Three batches of porcine diarrhea antibodies Y1, Y2, Y3 were diluted to 1:128, 1:64, 1:32 and 1:16 using sterilized normal saline, and 70 one-day-old piglets were randomly divided into 14 groups, with 5 piglets in each group. The immunization groups were orally administered with 2.0 ml of each group of yolk antibodies per piglet. The blank group and the challenge control group were not treated. Five days after immunization, the immunization groups and the challenge control group were orally administered with 1.0 ml of test virus (PEDV YK strain) per piglet (containing 10 4.0 TCID 50 / ml), the blank group was not treated, and the piglets were isolated and fed under the same conditions for 7 days. The clinical symptoms of the piglets were recorded, and necropsy and nucleic acid detection were performed at the end of the experiment (the same as in the first subsection of this embodiment).

[0191] Results: In the Y1 immunization group, the protection rates corresponding to dilutions of 1:128, 1:64, 1:32 and 1:16 were 100%, 100%, 60% and 40%, respectively. In the Y2 immunization group, the protection rates corresponding to dilutions of 1:128, 1:64, 1:32 and 1:16 were 100%, 100%, 60% and 20%, respectively. In the Y3 immunization group, the protection rates corresponding to dilutions of 1:128, 1:64, 1:32 and 1:16 were 100%, 80%, 40% and 40%, respectively. The protection rate of the challenge control group was 0, and the protection rate (survival rate) of the blank control group was 100%. Virus was detected in the dead animals, and no virus was detected in the surviving animals.

[0192] Figure 4 A photograph of the anus of a healthy piglet in the immunization group, Figure 5 A photograph of the anus of a piglet in the challenge control group.

[0193] Figure 6 A photograph of the small intestine of a healthy piglet in the immunization group. Figure 7 A photograph of the small intestine of a piglet in the challenge control group.

[0194] The challenge protection rates of the 1:128 group, the 1:64 group, the 1:32 group and the 1:16 group were 100%, 80%-100%, 40%-60% and 20%-40%, respectively. The challenge control group had 100% morbidity, and the blank control had 100% survival, indicating that the minimum preventive dose of porcine diarrhea yolk antibody was 1:64, orally administered at 2.0 ml per piglet.

[0195] It is to be understood by those skilled in the art that the present application can be implemented by other embodiments that do not depart from the spirit or essential characteristics thereof. Therefore, the above-disclosed embodiments, in all aspects, are only illustrative and not the only. All changes within the scope of the present application or within the scope equivalent to the present application are included in the present application.

Claims

1. A porcine epidemic diarrhea virus strain, wherein the porcine epidemic diarrhea virus strain is selected from S1, S2 and S3: S1: Porcine epidemic diarrhea virus strain with microbial preservation number CGMCC NO.46190; S2: A passaged viral strain of porcine epidemic diarrhea virus with microbial accession number CGMCC NO. 46190; During the passage of the porcine epidemic diarrhea virus strain with microbial accession number CGMCC NO. 46190, the transcriptional regulatory activities of all genes in the genome, the transcription initiation and termination of all genes in the genome, the amino acid sequences of the proteins encoded by all genes in the genome, the clinical pathogenicity of the virus, the immunogenicity of the virus, and the viral replication capacity remained unchanged compared with the porcine epidemic diarrhea virus strain with microbial accession number CGMCC NO. 46190. S3: A porcine epidemic diarrhea virus strain with the same genome sequence as the porcine epidemic diarrhea virus strain described in S1 or S2.

2. A vaccine composition for preventing, mitigating, or controlling porcine epidemic diarrhea (PED), wherein the vaccine composition uses the porcine epidemic diarrhea virus strain of claim 1 as an immunogen; or The raw materials of the vaccine composition include the porcine epidemic diarrhea virus strain of claim 1, which is used as an immunogen, and excipients.

3. The vaccine composition according to claim 2, characterized in that, The excipients include oil-based adjuvants, a first emulsifier, and a second emulsifier; The components of the vaccine composition, in the indicated proportions, include: The porcine epidemic diarrhea virus strain: oil-based adjuvant: first emulsifier: second emulsifier = 10 10.0 TCID 50 : 100-300g: 5-15g: 3-9g; wherein, the dosage of the porcine epidemic diarrhea virus strain is measured and calculated according to the dosage of live virus.

4. The vaccine composition according to claims 2 and 3, characterized in that, Choose from any one of the following: A1, A2, A3, A4, A5, A6, and A7; A1: The porcine epidemic diarrhea virus strain mentioned is an inactivated porcine epidemic diarrhea virus strain; A2: The oil-based adjuvant is selected from white oil; A3: The first emulsifier is selected from Span-80; A4: The second type of emulsifier is selected from Tween-80; A5: The components of the vaccine composition, in the indicated proportions, include: The porcine epidemic diarrhea virus strain: oil-based adjuvant: first emulsifier: second emulsifier = 94 × 10 8.0 TCID 50 : 190g : 10g : 6g; A6: With a concentration of 0.1-10×10 8.0 TCID 50 The vaccine composition was prepared using the porcine epidemic diarrhea virus strain at a concentration of / ml as an immunogen. A7: The TCID of the porcine epidemic diarrhea virus strain. 50 It was calculated based on Vero cell culture using the Reed-Muench method.

5. A method for preparing egg yolk antibodies, wherein the method comprises: immunizing poultry with the porcine epidemic diarrhea virus strain described in claim 1 as an immunogen to prepare the egg yolk antibodies.

6. The preparation method according to claim 5, characterized in that, The preparation method includes the following steps: S1: Prepare a vaccine composition from the porcine epidemic diarrhea virus strain described in claim 1; S2: Inoculate poultry animals with the vaccine composition to obtain highly immune poultry eggs; S3: Extract yolk antibodies from the highly immune poultry eggs.

7. The preparation method according to claim 6, characterized in that, Choose from any one of B1, B2, and B3 below; B1: In step S1, the vaccine composition is the vaccine composition according to any one of claims 2-4; B2: In step S2, the steps of the method for preparing the high-immunity poultry egg are as follows: Basic immunization: Egg-laying poultry are vaccinated with the vaccine composition described above; Second immunization: 7-28 days after the primary immunization, the laying poultry are vaccinated with the vaccine composition. Third immunization: 7-28 days after the second immunization, the laying poultry are vaccinated with the vaccine composition. Fourth immunization: 7-28 days after the third immunization, the laying poultry are inoculated with the vaccine composition to obtain the highly immune poultry eggs; B3: In step S3, the egg yolk antibody is extracted by the following first two, first three, or four steps: S3-1: Obtain the yolk of the highly immune poultry egg; S3-2: Extract the egg yolk to obtain crude egg yolk antibody; S3-3: Concentrate the purified egg yolk antibody to obtain concentrated egg yolk antibody; S3-4: Sterilize the concentrated egg yolk antibody to obtain sterile egg yolk antibody.

8. The preparation method according to claim 7, characterized in that, Selected from any one of C1, C2, C3, C4, C5, C6, C7, C8, C9, and C10; C1: In step S1, the method for preparing the vaccine composition includes the following steps: S1-1: The oil-based adjuvant is mixed with the first emulsifier to obtain an oil-phase adjuvant; S1-2: The inactivated porcine epidemic diarrhea virus is mixed with the second emulsifier to obtain an aqueous phase; S1-3: Mix the oil phase adjuvant with the aqueous phase to obtain the vaccine composition; C2: After the fourth immunization, when the yolk antibody titer of the poultry against the porcine epidemic diarrhea virus strain of claim 1 is lower than 1:256, the poultry shall be injected intramuscularly into the chest or leg with a dose calculated based on the amount of non-inactivated porcine epidemic diarrhea virus strain of claim 1, which is 2-5 × 10⁻⁶. 7.0 TCID 50 The vaccine composition according to any one of claims 2-4; C3: In step S2, the poultry animals are selected from: chickens, ducks, and geese; C4: In step S2, the laying period is 90-180 days; C5: In step S2, the route of administration is intramuscular injection in the chest or intramuscular injection in the leg; C6: The dosage of the basic immunization dose, calculated based on the amount of the non-inactivated porcine epidemic diarrhea virus strain according to claim 1, is 1-6 × 10⁻⁶. 7.0 TCID 50 The dose for the second immunization is 1-10 × 10⁻⁶. 7.0 TCID 50 The dosage for the third immunization is 1-15 × 10⁻⁶. 7.0 TCID 50 The dosage for the fourth immunization is 1-15 × 10⁻⁶. 7.0 TCID 50 ; C7: The highly immune poultry eggs are disinfected before use, prior to step S3-1; In C8:S3-2, the steps for preparing the crude egg yolk antibody are as follows: S3-2-1: Mix the egg yolk with acidified water with a pH of 4-5, wherein the volume ratio of the egg yolk to the acidified water is 1:4-8, and stir to obtain a first mixture; S3-2-2: Mix the first mixture with an aqueous solution of caprylic acid, wherein the final concentration of caprylic acid is 0.1-0.3 v / v%, centrifuge, collect the supernatant, and obtain crude egg yolk antibody; C9: In step S3-3, the purified egg yolk antibody is concentrated using an ultrafiltration membrane stack with a molecular weight cutoff of 25-50KD, and the concentration factor is 2-10 times. C10: In steps S3-4, a 0.22μm sterilization filter cartridge is used to filter and sterilize the concentrated egg yolk antibody.

9. An egg yolk antibody, said egg yolk antibody being prepared by the preparation method according to any one of claims 6-8.

10. Use of the porcine epidemic diarrhea virus strain of claim 1, the vaccine composition of any one of claims 2-4, or the egg yolk antibody of claim 9 in the preparation of a formulation for use alone, for use in combination with other immunizing agents and / or drugs, or as a component of a compound formulation composed of other immunizing agents and / or drugs for the treatment, prevention, mitigation, and / or control of porcine epidemic diarrhea.

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