Porcine epidemic diarrhea virus and porcine delta coronavirus bivalent attenuated live vaccine and preparation method thereof

Abstract

The application discloses a porcine epidemic diarrhea virus and porcine delta coronavirus double vaccine and a preparation method thereof. The double attenuated live vaccine is prepared from a PEDV G2a genotype weak strain CH / HBXT-P240 / 2018 (the preservation number is CGMCC NO:45213), a PEDV G2b genotype weak strain CH / HNPJ-P240 / 2017 (the preservation number is CGMCC NO:5212) and a PDCoV weak strain CH / XJYN-P240 / 2016 (the preservation number is CGMCC NO:45211) as seed viruses for vaccine preparation, virus proliferation is carried out in combination with a cell suspension culture process, and the double vaccine is prepared through vaccine preparation and freeze-drying. The vaccine has strong immunogenicity, good cross-protection effect and high safety, can prevent piglet diarrhea caused by two kinds of coronaviruses at the same time, and is suitable for immune prevention and control in large-scale farms.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, specifically to a bivalent attenuated live vaccine of porcine epidemic diarrhea virus and porcine delta coronavirus and its preparation method. Background Technology

[0002] Porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PDCoV) are the two main pathogens causing viral diarrhea in piglets. PEDV and PDCoV both belong to the family Coronaviridae, but to the genera α-coronavirus and δ-coronavirus, respectively. They are highly similar in viral morphology and the clinical symptoms of the diseases they cause, both causing typical symptoms such as diarrhea, vomiting, and dehydration in piglets. Pathological changes in both involve atrophy and loss of intestinal villi, but their antigenicity is fundamentally different. Studies have shown that there is a lack of antigenic cross-reactivity between the two at the level of neutralizing antibodies; a neutralizing immune response against one virus cannot effectively protect against infection by the other, and a single vaccine cannot simultaneously control both diseases. However, epidemiological surveys show that co-infection with PEDV and PDCoV is common in pig herds. Co-infection not only exacerbates the severity of diarrhea and mortality in sick piglets but also increases the difficulty of etiological diagnosis and immunization strategy development. Using two separate monovalent vaccines for immunization not only increases the number of injections and animal stress but also leads to increased labor costs and more complex immunization procedures.

[0003] PEDV has been widespread since 2010, with the G2 genotype becoming the dominant circulating strain. The G2a and G2b subtypes alternate or co-circulate in different regions of China, making it difficult for single-subtype vaccines to provide effective cross-protection. PDCoV, a newly discovered porcine enteric coronavirus in 2014, currently has no commercially available approved vaccines domestically or internationally. While there are research reports on bivalent inactivated vaccines combining PEDV and PDCoV, inactivated vaccines primarily induce humoral immune responses, with weaker cellular immune induction capabilities, and require adjuvants, resulting in a relatively limited immunization route. In contrast, live attenuated vaccines, by mimicking the natural infection process, can effectively activate a triple response of mucosal, humoral, and cellular immunity. They offer advantages such as lower immunization doses, longer protection periods, and flexible immunization routes, making them more suitable for emergency preventative vaccination against porcine diarrhea.

[0004] Therefore, developing a safe, reliable, highly protective, and broad-spectrum attenuated PEDV / PDCoV bivalent vaccine is of great practical significance and has broad application prospects for effectively controlling the severe situation of mixed infection of the two coronaviruses in pig herds and reducing economic losses in the pig farming industry. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a live attenuated bivalent vaccine of porcine epidemic diarrhea virus and porcine delta coronavirus with high immune protection rate, good safety and suitable for emergency preventive vaccination, and a method for preparing the same.

[0006] To achieve its purpose, the present invention adopts the following technical solution: The present invention provides a bivalent attenuated live vaccine of porcine epidemic diarrhea virus and porcine delta coronavirus, comprising viral fluids of attenuated strains of porcine epidemic diarrhea virus and attenuated strains of porcine delta coronavirus; The attenuated strain of porcine epidemic diarrhea virus contains a viral titer ≥10. 7.0 TCID 50 / mL of the G2a genotype strain PEDV CH / HBXT-P240 / 2018, with a viral titer ≥10 7.0 TCID 50 / mL of the G2b genotype strain PEDV CH / HNPJ-P240 / 2017; the porcine delta coronavirus attenuated strain had a viral titer ≥10. 9.0 TCID 50 / mL of PDCoV CH / XJYN-P240 / 2016 strain.

[0007] Specifically, the G2a genotype attenuated strain CH / HBXT-P240 / 2018 is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at the Institute of Microbiology, Chinese Academy of Sciences, Beijing, China, 100101, with accession number CGMCC NO.45213 and deposit date of May 9, 2022.

[0008] The G2b genotype attenuated strain CH / HNPJ-P240 / 2017 is deposited at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, Beijing, China 100101, with accession number CGMCC NO.45212 and deposit date of May 9, 2022.

[0009] The attenuated porcine delta coronavirus strain, PDCoV CH / XJYN-P240 / 2016, is deposited at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, Beijing, China 100101, China, accession number CGMCC NO.45211, deposited on May 9, 2022.

[0010] As a further preferred embodiment of the technical solution of the present invention, the attenuated strains PEDV CH / HBXT-P240 / 2018, PEDV CH / HNPJ-P240 / 2017, and PDCoV CH / XJYN-P240 / 2016 were all obtained by continuous passage of cells to attenuate to the 240th generation.

[0011] Preferably, the bivalent attenuated live vaccine further includes a pharmaceutically acceptable lyophilized protectant.

[0012] Preferably, the freeze-drying protectant is an aqueous solution, comprising, by 100% by mass: 3% gelatin, 5% sucrose, 2% trehalose, 0.5% L-arginine, 0.5% mannitol, 2% monosodium glutamate, 5% polyvinylpyrrolidone, with the remainder being pharmaceutical water.

[0013] Preferably, the vaccine is a freeze-dried preparation, which, after reconstitution, forms a homogeneous suspension.

[0014] The method for preparing a bivalent attenuated live vaccine of porcine epidemic diarrhea virus and porcine delta coronavirus provided by the present invention includes the following steps: (1) Seed virus propagation: The attenuated strains PEDV CH / HBXT-P240 / 2018, PEDVCH / HNPJ-P240 / 2017 and PDCoV CH / XJYN-P240 / 2016 were inoculated into sensitive cells for seed virus propagation; (2) Preparation of virus solution: Three attenuated strains were cultured using cell suspension culture technology, and the virus solution was harvested when the cytopathic effect reached more than 80%. (3) Virus purification and concentration: The virus supernatant after natural precipitation was taken, filtered and concentrated, the virus titer was determined, and the purity test was passed before it was used as the virus solution for vaccine preparation. (4) Vaccine preparation: Mix the three virus solutions, add freeze-drying protectant, and mix well; (5) Packaging and freeze-drying: After quantitative packaging, freeze-drying is carried out to obtain the bivalent live attenuated vaccine.

[0015] Preferably, in step (1), the sensitive cells inoculated with the attenuated strains PEDV CH / HBXT-P240 / 2018 and PEDV CH / HNPJ-P240 / 2017 are Vero cells, and the sensitive cells inoculated with the attenuated strain PDCoV CH / XJYN-P240 / 2016 are LLC-PK1 cells.

[0016] Preferably, in step (2), the conditions for cell suspension culture are: temperature 37°C, pH 7.2, dissolved oxygen 50%, and stirring speed 60 r / min.

[0017] Preferably, the suspended cells used are ST cells, and the culture medium used for culturing ST cells in suspension is CD ST323 medium.

[0018] Preferably, step (4) involves mixing the three types of viral solutions for seed preparation, PEDV G2a CH / HBXT / 2018, PEDV G2b CH / HNPJ / 2017 and PDCoV CH / XJYN / 2016, at a volume ratio of 1:1:2, and then mixing them with a freeze-drying protectant at a volume ratio of 1:1.

[0019] By adopting the above technical solution, the beneficial effects of the present invention are as follows: (1) The bivalent attenuated vaccine prepared in this invention covers two major genotypes of PEDV (G2a and G2b) and emerging PDCoV. It has a high degree of matching with the currently prevalent strains in the field and has good cross-protection effect. It can effectively cope with the complex epidemic situation of mixed infection of multiple coronaviruses and alternating epidemic of different strains in the current pig herd.

[0020] (2) The present invention uses a multivalent attenuated live vaccine to prevent multiple diseases at the same time with a single immunization, which reduces the stress response and labor costs caused by separate administration of monovalent vaccines and improves breeding efficiency.

[0021] (3) The present invention adopts a suspension culture process, which has a high virus yield and is suitable for large-scale production. Attached Figure Description

[0022] Figure 1 The appearance of the bivalent attenuated live vaccine prepared according to the present invention; Figure 2 The results of necropsy of the test piglets after challenge with the virus; Figure 3 The results of immunohistochemical examination of the jejunum of the tested piglets after challenge with the virus are shown. Detailed Implementation

[0023] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0024] Unless otherwise specified, all materials or instruments used in the following embodiments of the present invention can be obtained commercially.

[0025] Example 1: A bivalent attenuated live vaccine of porcine epidemic diarrhea virus and porcine delta coronavirus and its preparation method 1. Toxin 1.1 Source of the toxin (1) The attenuated strain of porcine epidemic diarrhea virus G2a, PEDV CH / HBXT-P240 / 2018, was obtained by attenuating the wild-type PEDV CH / HBXT / 2018 strain isolated from a pig farm in Xingtai, Hebei Province, through 240 passages in Vero cells. It is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC NO.45213. It was prepared, preserved, and provided by the applicant in the previous stage.

[0026] (2) The attenuated porcine epidemic diarrhea virus (PEDV) G2b genotype strain CH / HNPJ-P240 / 2017 was obtained by attenuating the wild-type PEDV CH / / HNPJ / 2017 strain isolated from a pig farm in Pingjiang, Hunan Province, through 240 passages in Vero cells. It is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.45212. It was prepared, preserved, and provided by the applicant.

[0027] (3) The porcine delta-coronavirus cell attenuated strain PDCoV CH / XJYN-P240 / 2016 was obtained by attenuating the wild-type PDCoV CH / XJYN / 2016 strain isolated from a pig farm in Yining City, Xinjiang Uygur Autonomous Region, through continuous passage 240 times in LLC-PK1 cells. It is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.45211. It was prepared, preserved, and provided by the applicant.

[0028] 1.2 Propagation of Seed Viruses (1) Propagation of PEDV seed virus: Take out one P240 generation cell culture of PEDV G2a CH / HBXT / 2018 strain and one P240 generation cell culture of PEDV G2b CH / HNPJ / 2017 strain from the liquid nitrogen tank, thaw at room temperature, centrifuge at 3500 rpm for 10 minutes at 4℃ to remove cell pellet, take 500 μL of virus supernatant, add it to 4.5 mL of DMEM cell culture medium, and add trypsin to make the final concentration in this 5 mL mixture reach 20 µg / mL, shake to mix well and let stand for later use; take T25 monolayer Vero-CCL81 cells with a density of 80%, discard the culture medium in the bottle, wash with DPBS 3 times, and then add the above-prepared virus solution, place in a 37℃ 5% CO2 constant temperature incubator for culture, observe the cell cytopathic effect every day, and when the cell cytopathic effect reaches 80% to 90%, collect the virus solution. After repeated freeze-thaw cycles (3 times), store at -80°C for later use.

[0029] (2) PDCoV virus propagation: Take one P240 cell culture of PDCoV CH / XJYN / 2016 strain from the liquid nitrogen tank, thaw it at room temperature, centrifuge at 3500 rpm for 10 minutes at 4℃ to remove cell pellet, take 500 μL of virus supernatant, add it to 4.5 mL of DMEM cell culture medium, and add trypsin to make the final concentration in this 5 mL mixture reach 20 µg / mL, shake to mix well and let stand for later use. Take a bottle of T25 monolayer LLC-PK1 cells with a density of 80%, discard the culture medium in the bottle, wash 3 times with DPBS, then add the virus solution prepared in the previous step, and place it in a 37℃ 5% CO2 constant temperature incubator for culture. Observe the cytopathic effect every day. When the cytopathic effect reaches 80% to 90%, collect the virus solution. Repeat the freeze-thaw cycle 3 times and store at -80℃ for later use.

[0030] 2. Preparation of virus solution for vaccine production 2.1 Cell Expansion Culture: ST cells, after suspension acclimatization and stored in the laboratory, were seeded in a bioreactor and cultured in serum-free medium CD ST323 (purchased from Gansu Jianshun Biotechnology Co., Ltd.). The culture conditions were optimized through single-factor experiments, and the initial seeding density (0.5 × 10⁻⁶) was investigated. 6 cells / mL, 0.8×10 6 cells / mL, 1.2×10 6 cells / mL and 1.5×10 6 The effects of parameters such as cell density (cells / mL), temperature (35℃, 37℃, and 39℃), pH (6.8, 7.0, 7.2, and 7.4), dissolved oxygen (DO) (20%, 30%, 50%, and 80%), and rotation speed (20 r / min, 40 r / min, 60 r / min, and 80 r / min) on cell growth were investigated. The optimal culture conditions were determined to be: ST cell seeding density of 0.8 × 10⁻⁶ cells / mL. 6 1.2 × 10⁻⁶ cells / ml ~ 1.2 × 10⁻⁶ cells / ml 6 The culture conditions were: cell density / ml, culture temperature 37℃, pH 7.2, dissolved oxygen (DO) 50%, and rotation speed 60 r / min. Under these conditions, after 72 hours of culture, the viable cell count reached 8 × 10⁶ cells / ml. 6 Cells / ml, with a viability of over 95%.

[0031] 2.2 Virus inoculation: When the density of ST cells in suspension culture in the bioreactor reaches 6.5 × 10⁻⁶... 6 ~8.0×10 6 When the cell density was 1 × 10⁶ cells / mL, the cell density was diluted to 1 × 10⁶ cells / mL with fresh culture medium. 6 ~2×10 6Cells were cultured at a concentration of 10 μg / mL, followed by the addition of an appropriate amount of trypsin to achieve a final concentration of 20 μg / mL in the diluted cell suspension. PEDV 2a, PEDV 2b, and attenuated PDCoV strains were then inoculated at an MOI of 0.1. After inoculation, the cells were cultured at 37°C with the pH maintained at 7.2, DO at 50%, and the stirring speed set at 40 rpm.

[0032] 2.3 Virus harvesting: After inoculation, the virus was cultured for 18–24 hours until cytopathic effects reached 80% or higher. The viral supernatant, after natural precipitation, was filtered through a 0.45 μm filter and concentrated 10–20 times using a membrane with a 100 kDa molecular weight cutoff to obtain three types of viral solutions, which were stored below -80°C for later use. Simultaneously, samples were taken for purity testing and virus content determination.

[0033] (1) Purity test: The sterility test, mycoplasma test and exogenous virus test were carried out in the order of the appendices of Part III of the 2020 edition of the Chinese Veterinary Pharmacopoeia. The results are shown in Table 1 and Table 2.

[0034] Table 1. Results of sterility and mycoplasma testing of viral fluid. Table 2 Results of Exogenous Virus Detection in Viral Fluids The results in Tables 1 and 2 show that none of the three viral solutions were contaminated with bacteria or mycoplasma, and all tests for exogenous viruses were negative.

[0035] (2) Virus content determination Take three types of viral solutions and serially dilute them 10-fold with serum-free DMEM medium (containing 20 μg / mL trypsin) to a final volume of 10. -9 Diluted virus solution was added to 96-well cell culture plates containing a confluent monolayer of Vero-CCL81 cells (for PEDV detection) or LLC-PK1 cells (for PDCoV detection), with 8 replicates per dilution. 0.1 mL was added to each well and incubated at 37°C in a 5% CO2 cell culture incubator for 1 h. Then, 0.1 mL of serum-free DMEM medium (containing 20 μg / mL trypsin) was added to each well. All inoculated cells were cultured at 37°C for 3 days. Cytopathic effects were observed, and the number of wells with cytopathic effects was recorded. Viral titers were calculated using the Reed-Muench method. The results are shown in Table 3.

[0036] Table 3 Results of Virus Content Measurement 3. Preparation of freeze-drying protectant The freeze-drying protectant formula is as follows: by 100% by mass, it contains 3% gelatin, 5% sucrose, 2% trehalose, 0.5% L-arginine, 0.5% mannitol, 2% monosodium glutamate, 5% polyvinylpyrrolidone, and the remainder is pharmaceutical water; it is sterilized by filtration through a 0.22μm filter membrane and stored at 4℃ for later use.

[0037] 4. Vaccine preparation and freeze-drying Take PEDV G2a, PEDV G2b and PDCoV virus solutions from 2.3 and mix them in a volume ratio of 1:1:2 to obtain a mixture. Then, mix the mixture with a freeze-drying protectant in a volume ratio of 1:1 and freeze-dry it in a low-temperature freeze dryer to obtain a bivalent attenuated live vaccine of porcine epidemic diarrhea virus and porcine delta coronavirus, and store it at -20℃.

[0038] Example 2: Vaccine Testing Three batches of vaccines were prepared according to the above method, with batch numbers 20241001, 20241002, and 20241003, respectively.

[0039] 1. Physical characteristics: The prepared attenuated live vaccine appears as a light red, spongy, loose mass. Figure 1 Dissolve rapidly after adding diluent (0.01 mol / L, pH 7.2-7.4 sterile PBS).

[0040] 2. Sterility test: The sterility test was conducted according to the appendix of Part III of the 2020 edition of the Chinese Veterinary Pharmacopoeia, and no bacterial growth was observed (Table 4).

[0041] 3. Mycoplasma test: The test was conducted according to the appendix of Part III of the 2020 edition of the Chinese Veterinary Pharmacopoeia. No significant pH changes were found in the liquid culture medium, and no "fried egg" shaped mycoplasma colonies were found on the solid culture medium after transplantation (Table 4).

[0042] Table 4 Results of vaccine sterility and mycoplasma testing 4. Exogenous virus testing: Tested according to the appendix of Part III of the 2020 edition of the Chinese Veterinary Pharmacopoeia, no exogenous virus contamination was found (Table 5).

[0043] Table 5. Results of Exogenous Virus Testing in Vaccines 5. Safety Test: Twenty healthy, susceptible piglets aged 3-5 days from a pig farm in Gansu Province (negative for classical swine fever virus, porcine circovirus type 2, and porcine reproductive and respiratory syndrome virus; neutralizing antibody titers for PEDV and PDCoV not exceeding 1:2) were selected. They were randomly divided into four groups of five piglets each. Piglets were observed for two days prior to vaccination, with their body temperature measured daily and the average value taken as the baseline temperature. Group 1 received a single-dose vaccination, specifically an intramuscular injection in the neck of one dose of the bivalent attenuated live vaccine prepared in Example 1 (containing PEDV: 10...). 5.5 TCID 50 / Toufen, PEDV G2a:PEDV G2b=1:1, PDCoV: 10 6.0 TCID 50 Group 1 received a single dose of PBS (1 dose intramuscularly, followed by a booster dose 2 weeks later); Group 2 received a super-dose vaccination (10 doses intramuscularly); and Group 4 served as a control group (intramuscularly injected with PBS). After 21 days of continuous observation, no adverse reactions caused by the vaccine were observed in any of the vaccinated piglets. Compared with the control group, no abnormalities were observed in body temperature, feed intake, water consumption, or mental state. No inflammatory reactions were observed at the injection sites, and all pigs were 100% healthy (Table 6).

[0044] Table 6. Clinical observation results of safety trials 6. Efficacy Test: Thirty healthy, susceptible piglets aged 3-5 days that were negative for both PEDV and PDCoV antigens and antibodies were randomly divided into two groups: an immunization group (n=15) and a negative control group (PBS, n=15). The immunization group received one dose of the bivalent attenuated live vaccine prepared in Example 1 via intramuscular injection in the neck, while the control group received 1 mL of PBS per piglet via intramuscular injection in the neck. Serum was collected from each piglet before immunization and 21 days after immunization for neutralizing antibody detection. Specifically, the neutralizing antibody titer was determined using the fixed virus-dilution serum method. After inactivating the serum at 56℃ for 30 min, it was serially diluted 2-fold in a 96-well plate (4 wells per dilution) and mixed with an equal volume of virus solution (200 TCID50). 50 Incubate at 37℃ for 1 hour; simultaneously set up virus controls (200, 20, 2, 0.2 TCID). 50(And normal cell control). The mixture was added to 96-well plates pre-coated with 70%-80% Vero cells (LLC-PK1 cells for PDCoV), incubated at 37°C for 1 h, washed 3 times with PBS, and then replaced with maintenance medium (containing 20 μg / ml trypsin). CPE was observed daily for 5-7 days. The neutralizing titer was expressed as the reciprocal of the highest dilution that inhibited 50% of viral lesions in the serum. The results are shown in Table 7. Neutralizing antibodies against three strains of PEDV—G2a (CH / HBXT / 2018), G2b (CH / HNPJ / 2017), and PDCoV (CH / XJYN-P6 / 2016)—were detectable in the serum of piglets immunized with the bivalent attenuated live vaccine prepared in Example 1 21 days later.

[0045] Table 7 Neutralizing antibody detection results Furthermore, 21 days after immunization, the immunized piglets were randomly divided into 3 groups of 5 piglets each, and each group was given 10 doses of antibiotics. 4.33 Oral administration of TCID50 / head to virulent porcine PEDV G2a strain CH / HBXT-P6 / 2018 and virulent PEDV G2b strain CH / HNPJ-P6 / 2017, and at a dose of 10 4 The virulent PDCoV strain CH / XJYN-P6 / 2016 was administered orally at a dose of TCID50 / pig. Similarly, 15 piglets in the PBS group were randomly divided into 3 groups and challenged with the virus simultaneously with the immunized group. The clinical manifestations of the piglets after challenge were observed daily. The results are shown in Table 8. In the immunized group, the 15 piglets showed no abnormalities in suckling, mental state, or fecal condition, with a survival rate of 100% and a protection rate of 100%. In contrast, all 15 piglets in the PBS control group exhibited typical diarrhea symptoms, lethargy, decreased feed intake, and even death, with a protection rate of 0%.

[0046] Table 8. Virus challenge protection rate in immunized piglets After a 7-day observation period, the piglets were necropsed, and the results were as follows: Figure 2 As shown, the intestines and organs of piglets in the immunized protection group were normal. In contrast, piglets in the PBS control group exhibited pathological changes such as thin, transparent intestinal walls filled with yellow contents, small intestinal mucosal hemorrhage, and mesenteric lymph node edema. Jejunum was collected from the tested piglets, fixed with 10% neutral formaldehyde solution, and subjected to immunohistochemical examination. The fixed tissue was dehydrated, cleared, paraffin-embedded, and embedded according to pathological histological methods to prepare routine sections. PEDV S protein monoclonal antibody or PDCoV N protein monoclonal antibody prepared and preserved in our laboratory was used as the primary antibody, HRP-labeled goat anti-mouse IgG as the secondary antibody, and DAB chromogenic reagent kit was used for staining. Images were acquired using a microscopic imaging system. Results are shown below. Figure 3In the PBS control group, a large number of PEDV or PDCoV positive antigen signals were observed on the intestinal epithelial cells of the jejunum of piglets, while no corresponding antigen signals were detected in the vaccine-immunized protection group.

[0047] The above results show that the bivalent attenuated live vaccine against porcine epidemic diarrhea virus (PEDV) and porcine delta coronavirus (PDV) described in this invention can induce piglets to produce highly effective neutralizing antibodies against two variant strains of PEDV and against PPDV, effectively protecting piglets from the corresponding viral infections. This bivalent attenuated live vaccine is prepared using a serum-free, ST-cell fully suspended suspension process, combining high titer with low side effects, providing a reliable technical means for disease prevention and control.

[0048] The invention has been described in detail above with general descriptions and specific embodiments. Any modifications or improvements made to the invention without departing from its spirit are within the scope of protection of the invention.

Claims

1. A bivalent attenuated live vaccine containing porcine epidemic diarrhea virus and porcine delta coronavirus, characterized in that, The bivalent live attenuated vaccine contains viral fluid of a weakened strain of porcine epidemic diarrhea virus and a weakened strain of porcine delta coronavirus. The attenuated porcine epidemic diarrhea virus strain included the G2a genotype strain PEDV CH / HBXT-P240 / 2018, with a viral titer ≥10. 7.0 TCID 50 / mL, biological accession number CGMCC NO.45213; and G2b genotype strain PEDV CH / HNPJ-P240 / 2017, viral titer ≥10 7.0 TCID 50 / mL, with accession number CGMCC NO.5212; The attenuated porcine delta coronavirus strain was PDCoV CH / XJYN-P240 / 2016, with a viral titer ≥10. 9.0 TCID 50 / mL, with accession number CGMCC NO.45211.

2. The live attenuated bivalent vaccine of porcine epidemic diarrhea virus and porcine delta coronavirus according to claim 1, characterized in that, The attenuated strains PEDV CH / HBXT-P240 / 2018, PEDV CH / HNPJ-P240 / 2017, and PDCoV CH / XJYN-P240 / 2016 were all obtained by continuous passage of cells to attenuate them up to the 240th generation.

3. The live attenuated bivalent vaccine of porcine epidemic diarrhea virus and porcine delta coronavirus according to claim 1, characterized in that, The bivalent attenuated live vaccine also contains a pharmaceutically acceptable lyophilized protectant.

4. The live attenuated bivalent vaccine of porcine epidemic diarrhea virus and porcine delta coronavirus according to claim 3, characterized in that, The freeze-drying protectant is an aqueous solution, comprising, by 100% by mass: 3% gelatin, 5% sucrose, 2% trehalose, 0.5% L-arginine, 0.5% mannitol, 2% monosodium glutamate, 5% polyvinylpyrrolidone, with the remainder being pharmaceutical water.

5. The live attenuated bivalent vaccine of porcine epidemic diarrhea virus and porcine delta coronavirus according to claim 4, characterized in that, The vaccine is a freeze-dried preparation, which, after reconstitution, forms a homogeneous suspension.

6. The method for preparing the live attenuated bivalent vaccine of porcine epidemic diarrhea virus and porcine delta coronavirus as described in any one of claims 1-5, characterized in that, Includes the following steps: (1) Seed virus propagation: The attenuated strains PEDV CH / HBXT-P240 / 2018, PEDV CH / HNPJ-P240 / 2017 and PDCoV CH / XJYN-P240 / 2016 were inoculated into sensitive cells for seed virus propagation; (2) Preparation of virus solution: Three attenuated strains were cultured using cell suspension culture technology, and the virus solution was harvested when the cytopathic effect reached more than 80%. (3) Virus purification and concentration: The virus supernatant after natural precipitation was taken, filtered and concentrated, the virus titer was determined, and the purity test was passed before it was used as the virus solution for vaccine preparation. (4) Vaccine preparation: Mix the three virus solutions, add freeze-drying protectant, and mix well; (5) Packaging and freeze-drying: After quantitative packaging, freeze-drying is carried out to obtain the bivalent live attenuated vaccine.

7. The method for preparing the bivalent attenuated live vaccine of porcine epidemic diarrhea virus and porcine delta coronavirus according to claim 6, characterized in that, In step (1), the attenuated strains PEDV CH / HBXT-P240 / 2018 and PEDV CH / HNPJ-P240 / 2017 were inoculated into Vero cells, and the attenuated strain PDCoV CH / XJYN-P240 / 2016 was inoculated into LLC-PK1 cells.

8. The method for preparing the bivalent attenuated live vaccine of porcine epidemic diarrhea virus and porcine delta coronavirus according to claim 6, characterized in that, In step (2), the conditions for cell suspension culture are: temperature 37℃, pH 7.2, dissolved oxygen 50%, and stirring speed 60 r / min.

9. The method for preparing a bivalent attenuated live vaccine of porcine epidemic diarrhea virus and porcine delta coronavirus according to claim 6 or 8, characterized in that, The suspended cells used were ST cells, and the culture medium used for culturing ST cells was CD ST323 medium.

10. The method for preparing the bivalent attenuated live vaccine of porcine epidemic diarrhea virus and porcine delta coronavirus according to claim 6, characterized in that, Step (4) involves mixing the three types of viral solutions for vaccine preparation, namely PEDV G2a CH / HBXT / 2018, PEDV G2b CH / HNPJ / 2017 and PDCoV CH / XJYN / 2016, at a volume ratio of 1:1:2, and then mixing them with a freeze-drying protectant at a volume ratio of 1:1.

Images