Porcine rotavirus strain and application thereof

Abstract

The invention provides a porcine rotavirus strain and application thereof, the porcine rotavirus strain is a group A porcine rotavirus G11P13 type PORV / SC / 2022089 strain, and the preservation number of the porcine rotavirus strain is CGMCC (China General Microbiological Culture Collection Center) No.45990; compared with the prior art, the porcine rotavirus strain provided by the invention has the beneficial effects that the porcine rotavirus strain which is good in passage stability, strong in pathogenicity, good in immunogenicity, capable of inducing an organism to generate a high-titer neutralizing antibody and long in maintenance time is provided, and after the porcine rotavirus strain is prepared into a vaccine, diseases caused by porcine rotavirus can be specifically prevented or treated; important medical values are provided for prevention, control and treatment of porcine rotavirus infection.

Description

Technical Field

[0001] The invention relates to the technical field of veterinary biological products, and in particular to a porcine rotavirus strain and application thereof. The porcine rotavirus strain is a group A porcine rotavirus G11P13 strain. Background Art

[0002] Porcine rotavirus (PoRV) can infect pigs of all ages, with suckling piglets and weaned piglets being the most susceptible, with an infection rate of 90%-100%, causing diarrhea and gastroenteritis in piglets, with clinical manifestations of watery diarrhea, lethargy, loss of appetite, etc. Since piglets infected with PoRV have a sharp drop in resistance, they often suffer from mixed infections with bacteria and other diarrhea viruses, which directly or indirectly affect the productivity of the pig herd and cause serious economic losses to the pig industry.

[0003] Porcine rotavirus (PoRV) is currently divided into four serotypes (A, B, C, E), among which Group A rotavirus (RVA) is the most widespread and is one of the main causes of acute diarrhea in piglets. The genome of RVA is a segmented double-stranded RNA, which consists of 11 segments, encoding 6 structural proteins (VP1-VP4, VP6, VP7) and 6 non-structural proteins (NSP1-NSP6). VP7 and VP4 are two important structural proteins of RVA. They are viral proteins that bind to host cell receptors, determine the virulence of the virus, and are also the main proteins that induce the body to produce neutralizing antibodies. The VP7 protein consists of 326 amino acids and has a molecular weight of 37kDa. It determines the G type of RVA. The VP4 protein is encoded by rotavirus gene segment 4, encoding a total of 775 amino acids with a relative molecular mass of about 87.6kDa. It also determines the P type of the virus. Different combinations will occur between the G type and the P type. To date, 36 G types and 51 P types have been discovered. The cross-protection induced by different types of strains is low. Among PoRVs that infect pigs, G3, G4, G5, G9 and G11 are the most popular G genotypes, which are often combined with P[5], P[6], P[7], P

[13] , P

[23] and P

[28] in pig farms.

[0004] There is no effective treatment for porcine rotavirus, and vaccination is an effective means to prevent and control RVA infection. In the prior art, there are inactivated vaccines for different serotypes of porcine rotavirus, such as G3P23 (disclosed in CN117737005 A), G5 (Shi Yueming. Preparation and immunological test of porcine rotavirus oil emulsion inactivated vaccine [D]. Northeast Agricultural University, 2009.), G1P1A (Rotarix produced by GlaxoSmithKline), etc. However, due to the characteristics of porcine rotavirus, there are many serotypes and no cross-protection between them, and the cross-protection effect between different rotavirus serotypes is very small. Therefore, in order to effectively prevent and control porcine rotavirus, it is necessary to collect different pathogen hosts in different regions and different ecological environments in my country to isolate porcine rotavirus pathogens, and conduct analysis and research in terms of morphology, serology, biological characteristics, pathogenicity, and simulated natural infection experiments, so as to obtain the wild strains of porcine rotavirus prevalent in the country and prepare them into highly effective inactivated vaccines for the prevention and treatment of porcine rotavirus epidemics, which is of great significance for preventing and controlling the outbreak and spread of porcine rotavirus among livestock in my country. Summary of the invention

[0005] 1. Technical issues to be solved

[0006] The purpose of the present invention is to provide a porcine rotavirus strain, an inactivated vaccine and an application thereof. The strain is a porcine rotavirus epidemic strain obtained by optimized separation and culture, which can be stably propagated on MA104 cells, providing a basis for effective vaccine preparation and porcine rotavirus prevention and control.

[0007] (II) Technical solution

[0008] In the first aspect, the present invention provides a porcine rotavirus strain, which is the group A porcine rotavirus G11P13 type PORV / SC / 2022089 strain. The strain was deposited in the China General Microbiological Culture Collection Center on June 24, 2024. The address of the depository unit is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; Postal Code: 100101, Classification Name: Porcine Rotavirus, and the Collection Number is CGMCC No.45990.

[0009] In a second aspect, the present invention provides use of the above-mentioned inactivated porcine rotavirus strain in the preparation of a drug, wherein the drug is used to prevent diseases caused by porcine rotavirus.

[0010] In a third aspect, the present invention further provides an inactivated vaccine comprising inactivated porcine rotavirus or a culture thereof.

[0011] Preferably, the virus content of the porcine rotavirus or its culture used to prepare the inactivated vaccine is 10 7.0 TCID 50 / mL or above.

[0012] Furthermore, the inactivated vaccine also includes an adjuvant.

[0013] Preferably, the adjuvant is ISA 201VG adjuvant.

[0014] In a fourth aspect, the present invention also proposes a method for preparing an inactivated vaccine, which comprises: inoculating the porcine rotavirus strain into cells for cultivation to obtain a virus liquid culture; and then inactivating the virus liquid culture and mixing it with an adjuvant.

[0015] Furthermore, the virus liquid culture is mixed with β-propiolactone, and then inactivated at 80-100 r / min and 0-8°C for more than 24 hours; the inactivated virus liquid is mixed with an adjuvant at a weight ratio of 1:0.5-1.5, and then emulsified at 30-33°C for more than 30 minutes.

[0016] In a fifth aspect, the present invention further proposes the use of the above-mentioned inactivated vaccine or the method for preparing the inactivated vaccine in the preparation of drugs for preventing diseases caused by porcine rotavirus.

[0017] (III) Beneficial effects

[0018] Compared with the prior art, the beneficial effects of the present invention are: the present invention provides a porcine rotavirus strain with good passage stability, strong pathogenicity, good immunogenicity, and the ability to induce the body to produce high-titer neutralizing antibodies with a long maintenance time. After being prepared into a vaccine, it can specifically prevent or treat diseases caused by porcine rotavirus, providing important medical value for the prevention, control and treatment of porcine rotavirus infection in pastoral areas. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 These are optical microscope observation photos of MA104 cells infected with the PORV / SC / 2022089 virus strain; among them, A is a 24h negative control; B is MA104 cells inoculated for 24h; C is a 48h negative control; D is MA104 cells inoculated for 48h; E is a 72h negative control; and F is MA104 cells inoculated for 72h.

[0020] Figure 2 This is the growth curve of PORV / SC / 2022089F12 generation.

[0021] Figure 3 This is an electron microscopy photo of the PORV / SC / 2022089 virus strain.

[0022] Figure 4 Information table of porcine rotavirus strain VP7.

[0023] Figure 5Information table of swine rotavirus strain VP4.

[0024] Figure 6 This is the result of homology analysis of the VP7 gene sequencing system of the PORV / SC / 2022089 virus strain.

[0025] Figure 7 This is the result of systematic evolutionary tree analysis of VP7 gene sequencing of PORV / SC / 2022089 virus strain.

[0026] Figure 8 This is the result of homology analysis of the VP4 gene sequencing system of the PORV / SC / 2022089 virus strain.

[0027] Fig. 9 This is the result of systematic evolutionary tree analysis of VP4 gene sequencing of PORV / SC / 2022089 virus strain.

[0028] Among them, the provided group A porcine rotavirus G11P13 type PORV / SC / 2022089 strain has been deposited in the China General Microbiological Culture Collection on June 24, 2024. The depository address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; Postal Code: 100101, Classification Name: Porcine Rotavirus, Latin Name: Porcine rotavirus; Deposit Number is CGMCC No.45990. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] The methods used in the following examples are all conventional methods unless otherwise specified. For specific steps, please refer to: Molecular Cloning: A Laboratory Manual (Sambrook, J., Russell, David W., Molecular Cloning: A Laboratory Manual, 3rd edition, 2001, NY, Cold Spring Harbor).

[0031] The methods for obtaining various biological materials described in the examples are only provided as an experimental method to achieve the specific disclosed purpose, and should not be construed as limiting the sources of the biological materials of the present invention. In fact, the sources of the biological materials used are extensive, and any biological material that can be obtained without violating laws and moral ethics can be replaced and used according to the instructions in the examples.

[0032] Isolating the porcine rotavirus strains that have been prevalent in recent years and stably propagating them on cells is the basis for effective vaccine preparation and porcine rotavirus prevention and control. In 2022, the inventor isolated a porcine rotavirus strain from intestinal tissue collected from a pig farm with diarrhea in Sichuan Province, and named it porcine rotavirus PORV / SC / 2022089. The strain is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, with the deposit number CGMCC N0.45990. The strain can be stably propagated on MA104 cells, has strong pathogenicity, good immunogenicity, and can induce the production of high-titer neutralizing antibodies.

[0033] The methods used in the examples are all conventional methods unless otherwise specified. For specific steps, please refer to: "Molecular Cloning Laboratory Manual" (Sambrook, J., Russell, David W., MolecμLar Cloning: A Laboratory Manual, 3rd edition, 2001, NY, Cold Spring Harbor).

[0034] Example 1 Isolation and identification of virus strains

[0035] 1.1 Cells and Pathological Materials

[0036] MA104 cells are preserved by Jinyu Baoling Biopharmaceutical Co., Ltd., and the pathological materials were collected from feces in a pig farm with diarrhea in Sichuan Province in 2022.

[0037] 1.2 Treatment of diseased materials

[0038] Before inoculating cells, the intestinal tissue of diarrheal pigs that were positive for PORV by RT-PCR was made into a suspension with DMEM at a volume ratio of 1:5, ground with a grinder, frozen and thawed once at -80℃, centrifuged at 12000r / min for 30min at 4℃, and the supernatant was filtered with a 0.22μm filter to obtain a virus suspension, added with double antibodies (100μg streptomycin / ml, 100U penicillin / ml) and placed in a -4℃ refrigerator for storage. When isolating the virus, the intestinal tissue suspension was quickly frozen at -80℃, which accelerated the release of the virus from the intestinal tissue and reduced the loss of live virus during the freezing process, thereby increasing the probability of successful virus isolation.

[0039] 1.3 Virus isolation and screening

[0040] The virus solution prepared in 1.2 was added with trypsin at a final concentration of 20 μg / mL (0.25% concentration) and incubated in a water bath at 37°C for 1 hour for activation. 2 ), discard the original culture medium, wash the cells once with PBS, add 1 ml of the above-obtained virus suspension, place in a 37°C, 5% CO2 incubator for adsorption for 1 hour, discard the inoculum, add 10 ml of DMEM culture medium containing double antibodies (100 μg streptomycin / ml, 100 U penicillin / ml) and trypsin (final concentration of 5 μg / mL) for culture. Observe the cells daily to see if they produce lesions CPE. If there is no lesion in the first generation, collect the culture medium on the 5th day, freeze and thaw 3 times, and continue blind transmission. In the second generation, obvious lesions can be seen in the cells. Figure 1 The lesions showed typical cell shrinkage, stringing, and shedding into clumps. When the cell lesions reached more than 80%, the virus solution was harvested after 3 freeze-thaw cycles. The virus was subcultured on MA104 adherent cells for acclimation culture.

[0041] 1.4 Plaque purification of viruses

[0042] (1) The F3 virus solution of porcine rotavirus PORV / SC / 2022089 strain was added with pancreatin at a final concentration of 20 μg / mL and incubated in a 37°C water bath for activation for 1 hour. After 10-fold gradient dilution with DMEM to 10-1 to 10-5, the culture medium in the six-well plate of MA104 cells grown into a dense monolayer was discarded. After washing twice with maintenance solution, 400 ul of the virus solution of different dilutions after activation was added to each well, and the well was placed in an incubator for adsorption for 1 hour. The liquid was discarded, and a first layer of nutrient agar with a thickness of about 3 mm was added. The nutrient agar refers to 2% low melting point agarose and 2×DMEM in equal volumes, mixed, and allowed to stand at room temperature for 30 minutes. After inverted culture in a CO2 incubator at 37°C and 5% CO2 for 3 days, a second layer of nutrient agar was added when the cells showed lesions. After standing at room temperature for 30 minutes, the well was inverted and cultured in the dark at 37°C and 5% CO2. The growth of plaques was observed and recorded daily;

[0043] (2) After plaques are formed, small and isolated plaques in the cell wells are selected, aspirated with a 10 μl pipette tip, and placed together with agarose in 100 ul of serum-free DMEM. The cells are frozen and thawed three times to fully release the virus. Trypsin with a final concentration of 20 μg / mL is added and activated in a 37°C water bath for 1 h. New blank MA104 cells are then inoculated for virus proliferation. This cloning process is repeated three times. Five plaques of the fourth-generation clones are randomly selected for subculture, i.e., F9-generation viruses are cultured, the virus content is determined, and the strain with the highest virus content is selected and continued to be subcultured through MA104 cells, which is the purified porcine rotavirus PORV / SC / 2022089 strain.

[0044] 1.5 Electron microscopy observation

[0045] Take the porcine rotavirus PORV / SC / 2022089 virus solution, centrifuge at 10000r / min for 30 minutes, take the supernatant and centrifuge at 40000r / min for 5 hours, discard the supernatant, dissolve the precipitate with 0.5ml deionized water, negatively stain with 2% sodium phosphotungstate for 30 minutes, dry naturally at room temperature, and observe under a transmission electron microscope. Figure 3 As shown, the isolate is approximately round, with a size of 65-70nm, and wheel-shaped virus particles, which is consistent with the characteristics of porcine rotavirus.

[0046] 1.6 PCR identification and sequence analysis

[0047] This experiment used the porcine group A rotavirus VP7 gene sequence included in GenBank to design VP7 primers and the VP4 gene amplification primers in the article "Isolation and Identification of Porcine Group A Rotavirus BJ Strain" published by Yuan Lin in Chinese Animal Husbandry and Veterinary Medicine in 2016. The nucleotide sequences of the primers are shown in Table 1:

[0048] Table 1

[0049]

[0050] The isolated virus strain was amplified by PCR using the above specific detection primers. The product was sent to Shanghai Bioengineering Company for sequencing and the results were spliced ​​with the VP4 and VP7 gene sequences of 23 strains of porcine rotavirus from China, the United States and other countries published in the GenBank database (such as Figure 4 , Figure 5 ) to compare homology and draw a phylogenetic tree, such as Figure 6 , Figure 7 , Figure 8 , Fig. 9As shown. The results showed that the nucleotide homology of the strain VP7 gene fragment was as high as 85.5% with the Nebraska23 strain (accession number: MN862274) published in the United States in 2009. The homology of the VP4 gene fragment was as high as 81.9% with the Chinese LNCY strain (accession number: MF462326) published in 2016. The genotype of the porcine rotavirus strain PORV / SC / 2022089 is G11P13.

[0051] 1.7 Determination of virus titer

[0052] Take a 96-well cell culture plate of MA104 cells that have grown into a well-contaminated monolayer, discard the cell culture medium, and wash twice with PBS. Dilute the virus solution of the serial passages in serum-free DMEM medium 10 times in series, and dilute 10 -2 -10 -6 The diluted virus solution was inoculated into 96-well cell culture plates, and 4 wells were inoculated for each dilution. Positive and negative controls were set up at the same time. The culture plates were placed in a 37°C, 5% CO2 incubator for 3-5 days, and CPE was observed and recorded daily. The virus titer was calculated according to the Reed-Muench method. The experimental results showed that the virus titers of the 3rd, 4th, 5th, 7th, 10th, 12th, 15th, 17th, and 20th generations were 10 4.0 TCID 50 / mL, 10 5.5 TCID 50 / mL, 10 5.0 TCID 50 / mL, 10 5.5 TCID 50 / mL, 10 6.0 TCID 50 / mL, 10 6.0 TCID 50 / mL, 10 6.0 TCID 50 / mL, 10 5.5 TCID 50 / mL, 10 6.0 TCID 50 / mL. This indicates that the porcine rotavirus strain can be stably propagated on MA104 cells and a high-titer virus liquid can be obtained, and can be used as a candidate strain for porcine rotavirus vaccine.

[0053] 1.8 Determination of virus growth curve

[0054] In order to study the growth characteristics of the virus, the virus was inoculated into MA104 cells to draw a growth curve. The virus liquid of the isolated virus F12 was inoculated into MA104 cells at 1% of the virus amount. After adsorption for 1 h in a 37°C, 5% CO2 incubator, the inoculum was discarded, and the maintenance liquid was added to continue the culture. Samples were taken every 8 hours until 72 hours after inoculation. The TCID 50 , based on the test results, the growth curve of the virus on MA104 cells was drawn, such as Figure 2 .

[0055] Example 2 Preparation of inactivated virus vaccine

[0056] 2.1 Preparation of porcine rotavirus antigen solution

[0057] (1) Preparation of virus solution:

[0058] Take MA104 adherent cells grown to a good monolayer, discard the original culture medium, add porcine rotavirus liquid F10 at a ratio of 0.5%-2% (v / v), and culture at 37°C. When the cell pathological rate is ≧80%, harvest the virus liquid and freeze-thaw it 3 times to obtain the basic seed virus.

[0059] Preparation of antigen solution:

[0060] Take out the PK-15 suspension cell cryopreservation tube from the liquid nitrogen tank, immediately put it in a 37℃ water bath, and gently shake the cryopreservation tube to melt the liquid as soon as possible. Centrifuge the cell suspension at 1000rpm / min for 5 minutes. Discard the supernatant, suspend the cells in 20ml culture medium for each cryopreservation tube in a 125ml cell culture bottle, and culture it in a shaker at 37℃ with 5% CO2 and 130r / min. Count the cells every day. When the cell density reaches 5-7×10 6 When the inoculation density is 1×10 6 / ml for expansion culture.

[0061] When the cells grow to 5-7×10 6 When the cell density is 2-4×10 / ml, serum-free medium containing 20ug / ml trypsin is used to adjust the cell density to 2-4×10 6 The virus culture medium prepared in (1) was inoculated at 1% and cultured in a shaker at 37°C, 5% CO2, and 130 rpm. Cell counts and viability tests were performed daily and the virus was collected after 24-30 hours.

[0062] Virus content determination:

[0063] The antigen solution prepared in (2) was diluted 10-fold in DMEM culture medium containing 5ug / ml trypsin. 3 -10 7A total of 5 dilutions were used to inoculate MA104 cells grown into a monolayer in a 96-well plate. Each dilution was inoculated into 4 wells, and 100ul of dilution solution was added to each well. Then 100ul of DMEM containing 5ug / ml pancreatin was added. Negative and positive controls were set up at the same time. The cells were cultured in a 37°C, 5% CO2 incubator for 5 days. The cell lesions were observed under a microscope, and the TCID was calculated according to the Reed-Muench method. 50 . It is calculated that the virus content of this strain is not less than 10 7 TCID 50 / ml.

[0064] 2.2 Purity test of virus solution for seedling preparation

[0065] The test was carried out according to the appendix of the current "Chinese Veterinary Pharmacopoeia", and the results showed that the basic toxin species was not contaminated with bacteria, mycoplasma and exogenous viruses.

[0066] 2.3. Inactivation of antigen solution for vaccine preparation

[0067] Use 1:4000 (V / V) β-propiolactone to inactivate on a shaker with a speed of 80-100 / min at 4℃ for 24h, and hydrolyze the residual β-propiolactone in a water bath at 37℃ for 2h to obtain the inactivated antigen solution. After diluting the inactivated antigen solution with DMEM culture medium, inoculate it into well-growing MA104 adherent cells at a ratio of 10% (V / V) of the culture medium. At the same time, set up two controls in the same way, one bottle is a normal cell group, and the other bottle is a maintenance solution containing β-propiolactone (the content of β-propiolactone is consistent with the inactivated antigen solution test sample). Place it in a 37℃, 5% CO2 incubator for 5 days, observe it every day, freeze and thaw the culture at -80℃ for 3 times, and blindly pass it for 3 generations according to the above method to observe the CPE of each generation of culture. The results show that no CPE is produced, and it is determined that the inactivation is complete.

[0068] 2.4 Vaccine preparation

[0069] (1) Preparation of aqueous phase: Heat the inactivated antigen solution to 31±1°C for later use.

[0070] (2) Preparation of oil phase: Use ISA 201VG adjuvant and preheat to 31±1°C for use.

[0071] (3) Emulsification: The oil phase and the water phase are mixed in a mass ratio of adjuvant: water phase = 1:1. The specific operation is: feed the adjuvant into the emulsification tank, and under stirring conditions, slowly feed the water phase into the emulsification tank under positive pressure, stir at 30-33°C for 30 minutes until the water phase and the adjuvant are fully mixed and emulsified into a two-phase oil emulsion vaccine. Then, quantitatively package, seal with a cap, label, and store at 2-8°C.

[0072] 2.5. Vaccine product testing

[0073] (1) Appearance: Milky white slightly viscous emulsion.

[0074] (2) Dosage form: water-in-oil-in-water type.

[0075] (3) Stability: Pipette 10 ml of the vaccine into a centrifuge tube and centrifuge at 3000 r / min for 15 minutes. No more than 0.5 ml of water phase should be precipitated at the bottom of the tube.

[0076] (4) Viscosity: Tested in accordance with the current Appendix of the Chinese Pharmacopoeia of Veterinary Medicine and in compliance with the regulations.

[0077] (5) Filling quantity inspection: Inspection shall be carried out in accordance with the appendix of the current Chinese Pharmacopoeia of Veterinary Medicine and shall comply with the regulations.

[0078] (6) Sterility test: Test according to the appendix of the current Chinese Pharmacopoeia of Veterinary Medicine, sterile growth.

[0079] (7) Safety inspection

[0080] Two guinea pigs weighing 350-450g were injected subcutaneously with 2ml of vaccine each; five mice weighing 18-22g were injected subcutaneously with 0.5ml of vaccine each. The animals were observed daily for 7 days, and no deaths or obvious local or systemic adverse reactions due to the injection of the vaccine occurred.

[0081] 2.6 Neutralizing antibody titer detection

[0082] Eight healthy rabbits weighing 2.0-3.0 kg (the titer of swine rotavirus neutralizing antibody was not higher than 1:4) were selected and divided into two groups. The first group of four rabbits were injected with 2.0 ml of vaccine into the leg muscle, as the immunization group; the second group of two rabbits were injected with 2.0 ml of DMEM into each leg, as the control group; after 21 days, the immunization was strengthened in the same way.

[0083] Blood was collected 21 days after the first immunization, 21 days after the second immunization, 60 days, and 90 days respectively, and the serum was separated to determine the titer of neutralizing antibodies against porcine rotavirus. The maintenance of antibodies in the body after immunization of rabbits was observed. The results are shown in Table 2.

[0084] Table 2

[0085]

[0086] It can be seen that the anti-porcine rotavirus neutralizing antibody titer levels in the serum of rabbits in the immunized group 21 days after the second vaccination were relatively high (not less than 1:256). As time went on, the immunized group was able to maintain a high titer level for a longer period of time, while the neutralizing antibody titer of the control group was lower than 1:4.

[0087] The above results show that the strain provided by the present invention can be stably propagated on cells, which can provide a basis for effective vaccine preparation and porcine rotavirus prevention and control. After immunizing the body with the inactivated vaccine prepared by the strain of the present invention, it can induce the production of high titer neutralizing antibodies and maintain them for a long time.

[0088] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A porcine rotavirus strain, characterized in that: It is group A porcine rotavirus G11P13 type PORV / SC / 2022089 strain, and its preservation number is CGMCC No.45990.

2. Use of the inactivated porcine rotavirus strain according to claim 1 in the preparation of a drug for preventing diseases caused by porcine rotavirus.

3. An inactivated vaccine, characterized in that: It contains the inactivated porcine rotavirus according to claim 1 or a culture thereof.

4. The inactivated vaccine according to claim 3, characterized in that The virus content of the porcine rotavirus or its culture for preparing the inactivated vaccine is 10 7.0 TCID 50 / mL or above.

5. The inactivated vaccine according to claim 3, characterized in that The inactivated vaccine also includes an adjuvant.

6. The inactivated vaccine according to claim 5, characterized in that The adjuvant was ISA 201VG adjuvant.

7. The method for preparing the inactivated vaccine according to claim 5, characterized in that: include: The porcine rotavirus strain of claim 1 is inoculated into cells for culture to obtain a virus culture fluid; the virus culture fluid is then inactivated and mixed with an adjuvant.

8. The method for preparing an inactivated vaccine according to claim 7, characterized in that: The virus liquid culture is mixed with β-propiolactone, and then inactivated at 80-100 r / min and 0-8° C. for more than 24 hours; the inactivated virus liquid is mixed with an adjuvant at a weight ratio of 1:0.5-1.5, and the mixture is emulsified at 30-33° C. for more than 30 minutes.

9. Use of the inactivated vaccine according to any one of claims 3 to 6, or the method for preparing the inactivated vaccine according to claim 7 or 8, in the preparation of a drug for preventing diseases caused by porcine rotavirus.

Images