CN117660369A - Triple inactivated vaccine of porcine epidemic diarrhea virus, porcine rotavirus and porcine delta coronavirus and preparation method thereof - Google Patents
Triple inactivated vaccine of porcine epidemic diarrhea virus, porcine rotavirus and porcine delta coronavirus and preparation method thereof Download PDFInfo
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Abstract
The invention provides a triple inactivated vaccine of porcine epidemic diarrhea virus, porcine rotavirus and porcine delta coronavirus and a preparation method thereof, and provides a safe and reliable triple inactivated vaccine of porcine epidemic diarrhea virus, porcine rotavirus and porcine delta coronavirus for emergency vaccination with high immune protection rate, good stability and high yield by adopting a cell serial passage method and a cell suspension culture technology, thereby providing an effective means for preventing and controlling PEDV, poRV and PDCoV.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a triple inactivated vaccine of porcine epidemic diarrhea virus, porcine rotavirus and porcine delta coronavirus and a preparation method thereof.
Background
Porcine epidemic diarrhea virus (Porcine epidemic diarrhea virus, PEDV) is a single-stranded positive strand RNA virus, belongs to coronavirus members of coronaviridae, has a virus diameter of about 95-190 nm, has a capsule membrane structure, and has spherical fiber at the tail end on the surface of the capsule membrane, wherein the fiber is regularly arranged in a crown shape. The full length of the viral genome is about 28kb, encoding 4 structural proteins (spike protein S, envelope protein E, membrane protein M and nucleocapsid protein N), 16 non-structural proteins (Nsp 1-Nsp 16) and one accessory protein ORF3. Porcine epidemic diarrhea (Porcine epidemic diarrhea, PED) caused by PEDV is a viral infectious disease, and the main clinical symptoms are acute watery diarrhea, vomiting, dehydration and the like. All day-old pigs can be infected and developed, with the incidence of newborn piglets being as high as 100%. Porcine epidemic diarrhea has become endemic and globally popular for many times since the discovery, and causes great economic loss to the pig raising industry, which severely restricts the development of the pig raising industry. Vaccination is still the most effective method of preventing and controlling PEDV.
Porcine rotavirus (Porcine Rotavirus, RV) is a member of the genus rotavirus of the family reoviridae, belongs to segmented double stranded RNA viruses, has a virion diameter of about 70nm, has no envelope structure, and is icosahedral symmetric. The genome encodes 6 structural proteins (VP 1-VP 4, VP6 and VP 7) and 6 non-structural proteins (NSP 1-NSP 6). The virion consists of three layers of capsid proteins, the outer capsid consists of VP7 and VP4 protein molecules, the inner capsid consists of VP6 protein molecule trimer, and the virion core consists of 11 dsRNA fragments and 3 structural proteins (VP 1-VP 3) and 6 non-structural proteins (NSP 1-NSP 6). Rotaviruses have different serotypes and serotypes, and are divided into 7 antigenically distinct serotypes (a-G) and two subgroups (i and ii) based on VP6 protein, and the podv can be divided into different P-types based on VP4 protein and into different G-types based on VP7 protein. Cross-reactivity between different serotypes is poor. Porcine rotavirus is a common pathogen causing gastroenteritis of piglets, and can cause the piglets to have clinical symptoms such as diarrhea, vomiting, emaciation, dehydration and the like. Rotavirus mainly propagates in small intestinal villus epithelial cells, cecum or colonic epithelial cells to cause intestinal villus atrophy, and intestinal tract absorption cells lose functions to cause malabsorption, so that a large amount of diarrhea is caused, and serious patients die. Porcine rotavirus is widely available worldwide, and its rapid prevalence and transmission causes a huge economic loss to the pig industry.
Porcine delta coronavirus (Porcine Deltacoronavirus, PDCoV) is a single-stranded positive strand RNA belonging to the order nimodiales, family coronaviridae, subfamily coronaviridae, genus delta coronaviridae. The virion is polymorphic, has a capsule and "imperial crown" like fibers, is about 60-180 nm in diameter, has a genome size of about 25.4kb, and encodes 4 structural proteins (spike protein S, envelope protein E, membrane protein M and nucleocapsid protein N), 15 non-structural proteins and 3 accessory proteins (NS 6, NS7 and 3' utr). PDCoV belongs to a novel porcine enteropathogenic coronavirus and can infect pig groups at different growth stages, wherein the highest infection rate of a suckling piglet can cause acute diarrhea, vomiting and dehydration of the suckling piglet.
PEDV, polv and PDCoV all cause severe diarrhea and dehydration in piglets, clinical symptoms and pathogenicity are highly similar, epidemiological investigation shows that mixed infection of three viruses often occurs, but if the viruses have similar antigenic structures, they may interfere with each other, resulting in poor immune response. Second, the immunogenicity of the three viruses also needs to be considered. In the preparation of triple vaccines, it is necessary to ensure that all three viruses stimulate an effective immune response in the body. There is no research on the triple inactivated vaccine of PEDV, poRV and PDCoV in the market at present. It is therefore very important and urgent to develop an effective and safe triple inactivated vaccine of PEDV, polv and PDCoV.
Disclosure of Invention
Aiming at the situation that no safe and effective triple inactivated vaccine for porcine epidemic diarrhea virus, porcine rotavirus and porcine delta coronavirus exists in the current market, the invention aims to provide the triple inactivated vaccine for porcine epidemic diarrhea virus, porcine rotavirus and porcine delta coronavirus and the preparation method thereof, and a safe and effective vaccine is developed to prevent and control the occurrence and transmission of three diseases.
To achieve the object of the invention, the invention provides a strain of porcine delta coronavirus which has been deposited in the China center for type culture collection, address: chinese university of Wuhan, post code 430072, preservation number CCTCCNO: V202379, preservation date 2023, 9 months and 15 days.
It is a further object of the invention to provide the use of said strain of porcine delta coronavirus in the preparation of a vaccine.
Further, a composition comprises a porcine epidemic diarrhea virus 2AKQ01 strain with a preservation number of CCTCC NO: V202005, a porcine rotavirus CH09 strain with a preservation number of CCTCC NO: V202191, a porcine delta coronavirus strain and a pharmaceutically acceptable carrier.
Further, the triple inactivated vaccine is prepared by respectively inoculating porcine epidemic diarrhea virus 2a KQ01 strain with the preservation number of CCTCC NO: V202005, porcine rotavirus PoRV CH09 strain with the preservation number of CCTCC NO: V202191 and porcine delta coronavirus strain with the preservation number of CCTCC NO: V202379 into susceptible cells for culture, inactivating the obtained virus liquid, mixing proportionally, and adding an adjuvant.
Porcine epidemic diarrhea virus 2a KQ01 strain is preserved in China Center for Type Culture Collection (CCTCC) in 12 months and 19 days of 2019, and the preservation number is CCTCC NO: v202005, deposit address: chinese, university of martial arts, martial arts.
Porcine rotavirus PoRV CH09 strain is preserved in China center for type culture collection (CCTCC NO) in 2021, 12 months and 15 days: v202191, deposit address: chinese, university of martial arts, martial arts.
Further, the virus titer of the harvested porcine epidemic diarrhea 2AKQ01 strain is not less than 10 7 TCID 50 The virus drop degree of the harvested porcine rotavirus CH09 strain is not less than 10 per ml 9 TCID 50 Per ml, the harvested porcine delta coronavirus has a droplet size of not less than 10 7 TCID 50 /ml。
Further, the adjuvant is water adjuvant IMS1313.
The third object of the invention is to provide a preparation method of triple inactivated vaccine of porcine epidemic diarrhea virus, porcine rotavirus and porcine delta coronavirus, comprising the following preparation steps,
step 1, suspending and culturing PK-15 cells and ST cells in a bioreactor, inoculating porcine rotavirus to the PK-15 cells, respectively inoculating porcine epidemic diarrhea virus and porcine delta coronavirus to the cultured ST cells, continuously culturing, harvesting virus liquid when cytopathy reaches more than 80%, taking naturally precipitated virus supernatant, filtering and concentrating to obtain virus liquid for preparing seedlings;
step 2, purifying virus liquid, filtering supernatant after virus liquid precipitation, and concentrating through a membrane bag;
step 3, inactivating the purified PEDV, poRV and PDCoV virus liquid stock solution or after dilution by formaldehyde, mixing the inactivated virus liquid with the same volume ratio, and mixing with an adjuvant;
the formaldehyde mass concentration is 0.25%, the adjuvant is water adjuvant IMS1313, and the volume ratio of the water phase of the virus mixed solution to the adjuvant is 3:1.
Further, in the step 1, the cell culture condition is that a serum-free cell culture medium with the total volume of 20% of the reactor is added, the culture condition is that the temperature is 37 ℃, the PH is 7.2-7.4, the Dissolved Oxygen (DO) is 40% -60%, and the rotating speed is 50rpm + -5 rpm.
The beneficial effects of the invention are as follows:
1. the current epidemic pig delta coronavirus PDCoV01262-9 strain is selected in the scheme, so that the seed toxicity advantage of the vaccine is ensured.
2. After the PEDV, poRV and PDCoV strains prepared by the scheme are cloned and purified, the purity and stability of the strains are better.
3. The scheme selects suspension cells to prepare the virus liquid for vaccine preparation, so that the vaccine production efficiency is higher.
4. The triple inactivated vaccine prepared by the research has the characteristics of high immune protection rate, good stability, high yield, safety and reliability, and can be used for preventive inoculation in epidemic outbreaks of PEDV, poRV and PDCoV.
The proposal provides a material basis and a technical support for preventing and controlling porcine epidemic diarrhea, porcine rotavirus and porcine delta coronavirus diseases and clearing in pig groups clinically, and has wide application prospect.
Drawings
FIG. 1 results of lesions of porcine epidemic diarrhea virus isolates inoculated onto Vero cells.
FIG. 2 shows the PCR detection results of porcine epidemic diarrhea virus isolates, M: DNA Marker DL2000;1: a sample; 2: negative control.
FIG. 3 shows the result of electron microscope identification of porcine epidemic diarrhea virus clone strain.
FIG. 4 results of lesions inoculated onto MA104 cells with porcine rotavirus isolates.
FIG. 5 shows the results of PCR detection of porcine rotavirus isolates, M: DNA Marker DL2000;1: a sample; 2: negative control.
FIG. 6 results of indirect immunofluorescence identification of porcine rotavirus isolates.
FIG. 7 depicts the results of electron microscope identification of porcine rotavirus clone strains.
FIG. 8 lesion results of porcine delta coronavirus isolates inoculated onto LLCPK cells.
FIG. 9 shows PCR detection results of porcine delta coronavirus isolates, M: DNA Marker DL2000;1: a sample; 2: negative control.
FIG. 10 shows the results of electron microscopy identification of porcine delta coronavirus cloned strains.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The instruments, reagents, etc. used in the examples are conventional instruments, reagents, etc. existing in the prior art, and are commercially available unless otherwise specified.
Example 1 isolation and identification of strains
1.1 isolation and identification of porcine epidemic diarrhea Virus
The porcine epidemic diarrhea virus seed virus is separated from a sick pig in a large-scale pig farm in Jiangsu of China, the filtrate of the swab carrying the virus in the sick pig is inoculated into cells for virus culture, the cytopathic effect is shown as figure 1, primers are designed by utilizing porcine epidemic diarrhea virus PEDV M protein genes as follows, and the upstream primer sequence is PEDV-F:5'-TATGGCTTGCATCATCACTCTTA-3', the downstream primer sequence is PEDV-R:5'-TTGACTGAACGACCAACACG-3', amplifying and identifying the target gene, wherein the PCR identification result is shown in figure 2, amplifying to obtain a specific fragment of 315bp, conforming to the expected size, and proving that the virus strain is obtained through separation.
PCR reaction system:
one step Enzyme Mix 1μL；
2X One-Step Reaction Solution(dye plus)12.5μL；
PEDV-F 1μL；
PEDV-R 1μL；
ddH2O 7.5μL；
template 2. Mu.L
Reaction conditions: reverse transcription is carried out at 50 ℃ for 30min, pre-denaturation is carried out at 94 ℃ for 2min, denaturation is carried out at 94 ℃ for 30sec, annealing is carried out at 55 ℃ for 30sec, extension is carried out at 72 ℃ for 20sec, and final extension is carried out at 72 ℃ for 5min
After separating and obtaining the porcine epidemic diarrhea virus, carrying out plaque purification for 3 times to successfully obtain the cloned virus of the porcine epidemic diarrhea virus, and carrying out passage on the attached Vero cells to transfer to F20 generation. The concentrated porcine epidemic diarrhea virus clone suspension is subjected to negative staining and then is subjected to electron microscopic observation, and the electron microscopic result is shown in figure 3. The virus is detected aseptically and exogenously according to the appendix of the current Chinese animal pharmacopoeia, and the bacteria, mycoplasma and exogenously virus are prevented from growing. And the virus titer is not less than 10 7 TCID 50 And (3) taking the virus as an antigen seed virus of the porcine epidemic diarrhea virus at the concentration of/mL, and storing the virus in a refrigerator at the temperature of-80 ℃ for later use.
1.2 isolation and identification of porcine rotavirus
The porcine rotavirus virus is separated in a large-scale pig farm in Jiangsu of China, the filtrate of a swab carrying the virus in a sick pig is inoculated in cells for virus culture, the cytopathic effect is shown as a graph in fig. 4, a specific primer is designed aiming at a PoRV VP6 protein gene as follows, and the upstream primer sequence is PoRV-F:5'-ATTTGATGGGAACTATGTGG-3', the downstream primer sequence is PoRV-R:5'-GGTGGACGAACTAATTGAAACG-3', the amplification and identification of target genes are carried out, the PCR identification result is shown in figure 5, the target fragment obtained by amplification is 350bp, the expected size is met, the indirect immunofluorescence identification result of the strain is shown in figure 6, and the virus strain is proved to be separated.
The PCR reaction system comprises:
one step Enzyme Mix 1μL；
2X One-Step Reaction Solution(dye plus)12.5μL；
PEDV-F 1μL；
PEDV-R 1μL；
ddH2O 7.5μL；
template 2. Mu.L
The reaction conditions are that reverse transcription is carried out for 30min at 50 ℃; after pre-denaturation at 94 ℃ for 2min, the mixture enters a cycle with the following cycle parameters: denaturation at 94℃for 30s, annealing at 55℃for 30s, and extension at 72℃for 20s; extension was performed at 72℃for 5min after 35 cycles.
After the porcine rotavirus is separated, the porcine rotavirus clone is successfully obtained through 3 times of plaque purification, and is passaged on the adherent MA-104 cells to be transferred to F20 generation. The concentrated porcine rotavirus clone suspension is subjected to negative staining and then is subjected to electron microscopic observation, and the electron microscopic result is shown in figure 7. The virus is detected aseptically and exogenously according to the appendix of the current Chinese animal pharmacopoeia, and the bacteria, mycoplasma and exogenously virus are prevented from growing. And the virus titer is not less than 10 9 TCID 50 And (3) at the time of/mL, taking the strain as porcine rotavirus antigen virus seed, and storing the strain in a refrigerator at the temperature of-80 ℃ for standby.
1.3 isolation and identification of porcine delta coronavirus
The pig delta coronavirus seed virus is separated from pig farm in Jiangsu Zhanghuan factory, the filtrate of the swab carrying the virus in the sick pig is inoculated into cells for virus culture, the cytopathic effect is shown in figure 8, a specific primer is designed aiming at the PDCoV M protein gene as follows, and the upstream primer sequence is PDCoV-F:5'-CATCTAAGAAGGACGCAGTT-3', the downstream primer sequence is PDCoV-R:5'-TGAAGTGGTTATGGTGTGAA-3', amplifying and identifying the target gene, wherein the PCR identification result is shown in figure 9, the target fragment is 437bp, which accords with the expected size, and the virus strain is obtained through separation. This strain was designated as porcine delta coronavirus PDCoV01262-9 (Porcine delta coronavirus PDCoV 01262-9) and the isolated porcine delta coronavirus strain was deposited at the chinese collection at 2023, 9 and 15, with the deposit number cctccc NO: v202379, deposit address: chinese, university of martial arts, martial arts.
The PCR reaction system comprises:
one step Enzyme Mix 1μL；
2X One-Step Reaction Solution(dye plus)12.5μL；
PEDV-F 1μL；
PEDV-R 1μL；
ddH2O 7.5μL；
template 2. Mu.L
The reaction conditions were reverse transcription at 50℃for 30min, pre-denaturation at 94℃for 2min, denaturation at 94℃for 30sec, annealing at 55℃for 30sec, extension at 72℃for 30sec, and final extension at 72℃for 5min.
After the porcine delta coronavirus is separated, the porcine delta coronavirus clone is successfully obtained through 3 times of plaque purification, and is passaged on the adherent LLCPK cells to be transferred to F20 generation. The concentrated porcine delta coronavirus clone suspension was subjected to negative staining and then subjected to electron microscopy, and the electron microscopy results are shown in fig. 10. The virus is detected aseptically and exogenously according to the appendix of the current Chinese animal pharmacopoeia, and the bacteria, mycoplasma and exogenously virus are prevented from growing. And the virus titer is not less than 10 7 TCID 50 And (3) at the time of/mL, taking the strain as the antigen strain of the porcine delta coronavirus, and storing the strain in a refrigerator at the temperature of-80 ℃ for standby.
The nucleotide sequence of the S1 protein expressed by the porcine delta coronavirus PDCoV01262-9 is shown as SEQ ID NO:01, the nucleotide sequence of the expressed S1 protein of the porcine delta coronavirus PDCoV01262-9 is shown as SEQ ID NO:02, the nucleotide sequence of the expressed N protein of the porcine delta coronavirus PDCoV01262-9 is shown as SEQ ID NO: shown at 03.
Example 2 preparation and testing of triple inactivated vaccine against porcine epidemic diarrhea Virus, porcine rotavirus and porcine Deltacoronavirus
2.1 preparation of virus liquid
2.1.1 expanded culture of cells: the bioreactor was autoclaved by calibrating Dissolved Oxygen (DO), PH and temperature electrodes of the bioreactor. When the density of suspension PK-15 cells or ST cells reaches 8×10 6 When the number of cells per mL and the viable cell rate reaches more than 95%, the suspended cells are 1 multiplied by 10 6 The density of each mL is transferred into a bioreactor, and serum-free cell culture medium with the total volume of 20% of the reactor is pumped into the bioreactor, the culture condition is 37 ℃, the PH is 7.2-7.4, the Dissolved Oxygen (DO) is 40% -60%, and the rpm is 50+/-5 rpm.
2.1.2 virus inoculation: when the PK-15 cell density in the bioreactor reached 8X 10 6 At each mL, it was diluted to 2X 10 with serum-free cell culture medium 6 Adding 6ug/mL pancreatin into each mL, and inoculating 0.5MOI porcine rotavirus into PK-15 cells; when the ST cell density in the bioreactor reached 8X10 6 At each mL, it was diluted to 2.5X10 with serum-free cell culture medium 6 A1 MOI porcine epidemic diarrhea virus and a porcine delta coronavirus were inoculated into ST cells, respectively, at a concentration of 10ug/mL pancreatin. The culture conditions are 37 ℃, the PH is 7.2-7.4, the DO (dissolved oxygen) is 40-60%, and the rpm is 50+/-5 rpm.
2.1.3 viral fluid harvesting: and after the virus is inoculated for 18 to 24 hours, the virus liquid is harvested when the cytopathy is more than 80 percent.
2.1.4 titer determination of virus: detecting the virus value of the virus liquid, wherein the virus titer of the porcine rotavirus is not lower than 10 9 TCID 50 The virus titer of the porcine epidemic diarrhea virus and the porcine delta coronavirus is not less than 10 per mL 7 TCID 50 /mL。
2.1.5 purity test: according to the appendices of the current Chinese animal pharmacopoeia, the virus liquid is subjected to aseptic and exogenous virus detection, and the virus liquid is subjected to aseptic growth, mycoplasma growth and exogenous virus pollution.
2.1.6 purification of virus liquid: the supernatant after natural precipitation of the virus solution was passed through a 0.65 μm filter and concentrated by a 300kDa membrane pack.
2.2 inactivation and semi-finished product detection
The purified PEDV, poRV and PDCoV virus liquid stock solution is diluted to obtain the titer of 10 9.25 TCID 50 Virus liquid of PoRV/ml with titer of 10 7.2 TCID 50 Virus solution of PEDV/ml with titer of 10 7.125 TCID 50 Viral fluid of PDCoV/ml. Respectively adding the components into an inactivation tank, adding formaldehyde with the final concentration of 0.25% for inactivation, uniformly mixing, and then inactivating at 37 ℃ for 48 hours, and stirring once every 2-4 hours. The sterility and exogenous virus inspection are carried out according to the annex of the current Chinese animal pharmacopoeia, and the sterility, mycoplasma growth and exogenous virus pollution are all required. Sampling the inactivated virus liquid, inoculating PEDV to the attached Vero cells, poRV to the attached MA104 cells, PDCoV to the attached LLCPK cells, harvesting the virus liquid after 36 hours, repeatedly freezing and thawing for 2-3 times, re-inoculating to the cells according to the method, and blindly transferring for 2 generations, wherein the cells do not generate any strainLesions.
2.3 preparation and testing of triple inactivated vaccine
The virus liquid of the inactivated PEDV, poRV and PDCoV is prepared according to the volume ratio of 1:1:1, mixing evenly, mixing the mixture with a water adjuvant IMS1313 according to a volume ratio of 3:1, mixing and emulsifying to obtain the PEDV, poRV and PDCoV triple inactivated vaccine. And (5) quantitatively split charging, capping and labeling after the detection is qualified. Performing character test on the vaccine sample, wherein the appearance of the vaccine is light yellow; according to the appendices of the current Chinese animal pharmacopoeia, the bacteria-free and exogenous virus detection is carried out, and the bacteria-free growth, mycoplasma-free growth and exogenous virus pollution are all required.
2.4 vaccine safety test
2.4.1 safety test on 3-5 day old piglets: the method comprises the steps of selecting 20 healthy piglets of 3-5 days old from sows with the PEDV and PDCoV neutralizing antibodies not higher than 1:4 and the PoRV neutralizing antibodies not higher than 1:8, dividing the healthy piglets into 4 groups, wherein 5 groups are respectively used for preparing 3 batches of three-combined inactivated vaccine of PEDV, poRV and PDCoV by intramuscular injection, 1.0 mL/head is respectively used for 1, 2 and 3 groups, the 4 groups are used as control groups, and continuously observing for 14 days.
2.4.2 safety test on 21-26 day old piglets: selecting 20 piglets of 21-26 days old, wherein the neutralizing antibodies of PEDV and PDCoV are not higher than 1:4, the neutralizing antibodies of PoRV are not higher than 1:8, and detecting the porcine pestivirus, the porcine circovirus type 2 and the porcine reproductive and respiratory syndrome virus as negative. Piglets were divided into 4 groups of 5 heads each. The 1 st and the 2 nd groups are respectively intramuscular injected with 1 head/head of triple inactivated vaccine, the 2 nd group is immunized for 14 days and then is intramuscular injected with 1 head/head, the 3 rd group is intramuscular injected with 2 head/head, and the 4 th group is a control group. The observation was continued for 4 weeks. The results show that compared with the control group, the piglets in the immune group are healthy and alive, and adverse reactions such as diarrhea, vomiting and dehydration symptoms do not occur.
The experimental results show that the PEDV, poRV and PDCoV triple inactivated vaccine provided by the invention has good safety.
2.5 test of efficacy
2.5.1 antibody detection method 10 piglets with 3-5 days old health susceptibility (3-5 days old health piglets produced by sow with PEDV and PDCoV neutralizing antibody not higher than 1:4 and PoRV neutralizing antibody not higher than 1:8) are divided into immune group and control group, each group has 5 piglets. Each immunization group was injected with 1.0mL of vaccine via neck muscle, the control group was injected with water adjuvant IMS1313, 21 days after immunization, along with the control group under the same conditions, blood was collected and serum was separated, and porcine epidemic diarrhea virus, porcine rotavirus, porcine delta coronavirus neutralizing antibodies in the serum were detected, respectively, and the results are shown in table 1, table 2, and table 3.
TABLE 1 detection results of neutralizing antibodies to porcine epidemic diarrhea virus
TABLE 2 detection results of porcine rotavirus neutralizing antibodies
TABLE 3 detection results of porcine delta coronavirus neutralizing antibodies
2.5.2 immunization and toxicity attack method 30 piglets are divided into 6 groups by 3-5 days old healthy susceptible piglets (3-5 days old healthy piglets produced by sows with a PEDV and PDCoV neutralizing antibody not higher than 1:4 and a PoRV neutralizing antibody not higher than 1:8), 5 groups of 5 piglets are immune groups, 1, 2 and 3 groups are immune groups, and 4, 5 and 6 groups are toxicity attack control groups. 1.0mL of the triple inactivated vaccine in the 2.3 is intramuscular injected into each immunization group through the neck, and 10.0mL of the oral porcine epidemic diarrhea virus virulent strain virus solution of the 1 st group and the 4 th group is 10 (the virus content is 10) 21 days after immunization 7.0 TCID 50 Per mL), 10.0mL (virus content 100 ID) of oral porcine rotavirus tissue venom of group 2 and group 5 50 Per mL), group 3 and group 6 oral porcine delta coronavirus virulent strain virus solution 10.0mL (virus content 10) 7.0 TCID 50 /mL), 14 days after challenge, the results showed that: all 5 piglets in the virus-counteracting control group showed diarrhea and vomitingClinical symptoms such as vomiting; none of the 5 piglets in the immunized group showed clinical symptoms of diarrhea and vomiting.
Claims (8)
1. The pig delta coronavirus strain is characterized by being preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of V202379.
2. Use of a strain of porcine delta coronavirus according to claim 1 in the preparation of a vaccine.
3. A composition comprising a porcine epidemic diarrhea virus 2AKQ01 strain having a accession number of CCTCC No. V202005, a porcine rotavirus CH09 strain having a accession number of CCTCC No. V202191, and a porcine delta coronavirus strain of claim 1, and a pharmaceutically acceptable carrier.
4. The triple inactivated vaccine is characterized in that porcine epidemic diarrhea virus 2AKQ01 strain with the preservation number of CCTCC NO: V202005 and porcine rotavirus CH09 strain with the preservation number of CCTCC NO: V202191 are respectively inoculated into susceptible cells for culture, and after the obtained virus liquid is inactivated, the mixed solution is mixed in proportion, and then an adjuvant is added to prepare the triple inactivated vaccine.
5. The triple inactivated vaccine according to claim 4, wherein the virus droplet size of the harvested porcine epidemic diarrhea 2AKQ01 strain is not less than 10 7 TCID 50 The virus drop degree of the harvested porcine rotavirus CH09 strain is not less than 10 per ml 9 TCID 50 Per ml, the harvested porcine delta coronavirus has a droplet size of not less than 10 7 TCID 50 /ml。
6. The triple inactivated vaccine according to claim 4, wherein the adjuvant is water adjuvant IMS1313.
7. A method for preparing the triple inactivated vaccine according to claims 4-6, comprising the steps of:
step 1, suspending and culturing PK-15 cells and ST cells in a bioreactor, inoculating porcine rotavirus to the PK-15 cells, respectively inoculating porcine epidemic diarrhea virus and porcine delta coronavirus to the cultured ST cells, continuously culturing, harvesting virus liquid when cytopathy reaches more than 80%, taking naturally precipitated virus supernatant, filtering and concentrating to obtain virus liquid for preparing seedlings;
step 2, purifying virus liquid, filtering supernatant after virus liquid precipitation, and concentrating through a membrane bag;
step 3, inactivating the purified PEDV, poRV and PDCoV virus liquid stock solution or after dilution by formaldehyde, mixing the inactivated virus liquid with the same volume ratio, and mixing with an adjuvant;
the formaldehyde mass concentration is 0.25%, the adjuvant is water adjuvant IMS1313, and the volume ratio of the water phase of the virus mixed solution to the adjuvant is 3:1.
8. The method according to claim 7, wherein the cell culture conditions in step 1 are that a serum-free cell culture medium is added in an amount of 20% of the total volume of the reactor, the culture conditions are 37 ℃, pH is 7.2-7.4, dissolved Oxygen (DO) is 40% -60%, and the rotation speed is 50rpm + -5 rpm.
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