Application of IOX1 in culture of porcine epidemic diarrhea virus and vaccine production and preparation
By using IOX1 to promote the amplification of swine epidemic diarrhea virus in host cells, the problem that existing vaccines cannot effectively prevent infection with new variants is solved, and efficient and stable viral materials are provided for vaccine production.
Patent Information
- Application Number
- CN202411767361.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-04
- Publication Date
- 2025-04-11
AI Technical Summary
The existing swine epidemic diarrhea virus vaccine cannot effectively prevent and control infection of new variants, and there is a lack of new vaccines that are effective against both new and classic strains.
Using IOX1 as a drug provides viral materials for culture and vaccine production by promoting the amplification of swine epidemic diarrhea virus in host cells. IOX1 has no effect on cell activity within the concentration range of 5 to 40 μM, but significantly promotes virus proliferation within the concentration range of 20 to 40 μM.
IOX1 significantly promotes the amplification of swine epidemic diarrhea virus, provides stable and efficient viral materials for the preparation of vaccines, and solves the problem that existing vaccines are ineffective against new variant strains.
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Figure CN119799651A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of medicines, and specifically relates to the application of IOX1 in the cultivation of porcine epidemic diarrhea virus and the production and preparation of vaccines. Background Art
[0002] Porcine epidemic diarrhea virus (PEDV) is a member of the Coronavirus genus of the Coronaviridae family. It is a single-stranded positive RNA enveloped virus with a genome (excluding polyA) of approximately 28 kb. The genome consists of a 5' end, a 5' end cap, a 5' UTR, seven open reading frames, a 3' UTR, and a polyadenylation (polyA) tail at the 3' end. The seven open reading frames encode four structural proteins: S protein (spike protein), M protein (membrane protein), E protein (envelope protein), and N protein (nucleocapsid protein), as well as pp1a, pp1ab, and ORF3 proteins.
[0003] Porcine epidemic diarrhea (PED) is an acute, contact and highly contagious intestinal disease caused by porcine epidemic diarrhea virus (PEDV). The main symptoms are vomiting, watery diarrhea, dehydration, loss of appetite and fever in infected pigs. PEDV infects the small intestine and intestinal villus cells of pigs and spreads rapidly, resulting in a high mortality rate of newborn piglets (up to 100%), causing huge economic losses to the pig industry. At present, the prevention and control strategy for porcine epidemic diarrhea is mainly prevention and treatment. Among them, vaccination is the most critical. However, with the passage of time, due to the occurrence of strain drift and the emergence of new subtypes, new variants make existing vaccines unable to effectively prevent and control viral infection. Therefore, it is urgent to develop new vaccines that are effective against both emerging strains and classical strains. Studying the viral proliferation characteristics of PEDV and finding methods and drugs to promote PEDV proliferation can provide a certain theoretical basis and basis for the development of new PEDV vaccines. Summary of the invention
[0004] Purpose of the invention: The purpose of the present invention is to provide the use of IOX1 in the cultivation of porcine epidemic diarrhea virus and the production and preparation of vaccines.
[0005] Technical solution: Application of IOX1 described in the present invention in the cultivation of porcine epidemic diarrhea virus.
[0006] The application of IOX1 described in the present invention in the production and preparation of a vaccine for porcine epidemic diarrhea virus.
[0007] The structure of IOX1 is as follows:
[0008]
[0009] The porcine epidemic diarrhea virus is PEDV CV777.
[0010] Preferably, the culturing is to promote the infection of cells with porcine epidemic diarrhea virus or to promote the infection rate of porcine epidemic diarrhea virus in cells.
[0011] Preferably, the cells are IPEC-J2 cells.
[0012] Preferably, the IOX1 is used at a concentration of 5 to 40 μM without being toxic to cells.
[0013] Preferably, the concentration of IOX1 used to promote the proliferation of porcine epidemic diarrhea virus is 20-40 μM.
[0014] The pharmaceutical composition for porcine epidemic diarrhea virus culture or vaccine production of the present invention comprises IOX1 and a pharmaceutically acceptable carrier thereof.
[0015] Preferably, the pharmaceutical composition comprises a stereoisomer of IOX1, a solvate of IOX1, a pharmaceutically acceptable salt of IOX1 or a solvate of a pharmaceutically acceptable salt of IOX1 of the active ingredient.
[0016] Preferably, the pharmaceutical composition is an oral preparation or an injectable preparation.
[0017] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) The present invention is the first to discover that IOX1 has the activity of promoting the proliferation of porcine epidemic diarrhea virus, and provides an application in the culture and vaccine production of porcine epidemic diarrhea virus. IOX1 shows a significant effect of promoting the proliferation of porcine epidemic diarrhea virus in host cells, and has a good and stable effect of promoting the proliferation of porcine epidemic diarrhea virus; (2) Using porcine intestinal epithelial cells (IPEC-J2) as a cell model, IOX1 has no effect on cell activity within the concentration range of 5 to 40 μM, and has a significant effect of promoting the proliferation of porcine circovirus within the concentration range of 20 to 40 μM, and can be used for the culture of porcine epidemic diarrhea virus and the application in the vaccine production. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Figure 1 This is a graph showing the effect of different concentrations of IOX1 on IPEC-J2 cell activity detected by CCK-8 method;
[0019] Figure 2 This is a cell morphology observation diagram of the morphological changes of IPEC-J2 cells at different time points after PEDV infection;
[0020] Figure 3This is a graph showing the changes in the load of PEDV CV777 at different time points detected by fluorescent quantitative PCR;
[0021] Figure 4 It is the change graph of the load of PEDV CV777 at different time points detected by immunoblotting;
[0022] Figure 5 This is a graph showing the changes in viral load before and after IOX1 treatment detected by fluorescent quantitative PCR;
[0023] Figure 6 This is a diagram showing the distribution of viral nucleocapsid protein before and after IOX1 treatment detected by indirect immunofluorescence. DETAILED DESCRIPTION
[0024] In order to make the purpose, technical solution and advantages of the present application more clear, the present application is further described in detail below in conjunction with the examples. The cells, viruses and main reagents used in the present invention are as follows:
[0025] Cell line: Porcine intestinal epithelial cells (IPEC-J2 cells) were cultured in DMEM (DMEM, Gibco, Waltham, MA) supplemented with 10% fetal bovine serum (FBS, Gibco, Waltham, MA).
[0026] Strain: PEDV CV777 is an existing strain. The strain used in this application was provided by Yangzhou University. The applicant has also used PEDV CV777 before. For details, see the article (Qin W, Qi X, Xie Y, Wang H, Wu S, Sun MA, Bao W. LncRNA446 Regulates Tight Junctions by Inhibiting the UbiquitinatedDegradation of Alix after Porcine Epidemic Diarrhea Virus Infection. J Virol. 2023 Mar 30; 97 (3): e0188422. doi: 10.1128 / jvi.01884-22).
[0027] Main reagents: PEDV-N antibody was purchased from Medgene Labs (Medgene Labs, USA), IOX1 solution was purchased from MCE (MCE, NJ, USA), CCK-8 cell proliferation and toxicity detection kit was purchased from Vazyme (Vazyme, Nanjing, China), and real-time fluorescence quantification kit was purchased from Vazyme (Vazyme, Nanjing, China).
[0028] The reagents not described in detail in the application are all conventional reagents and can be obtained from commercial channels; the methods not described in detail are all conventional experimental methods and can be obtained from the prior art.
[0029] Example 1: Detection of IPEC-J2 cell activity by different concentrations of IOX1
[0030] 2000 IPEC-J2 cells were plated in each well of a 96-well cell plate and mixed evenly with DMEM containing 10% FBS (total volume of 100 μL), and cultured overnight in a 37°C, 5% CO2 incubator. When the cell density reached 80%, the DMSO-dissolved IOX1 concentrated stock was diluted with water to 5 μM, 10 μM, 20 μM, and 40 μM and added to the well plate (three replicates were set for each concentration). A control group was also set up, and the treatment time was set to 24 h. The cell viability was then detected using a CCK-8 kit, and 10 μL of CCK-8 solution was added to the well plate per well. The absorbance was detected at 450 nm using a Tecan Infinit200 microplate reader (Sunrise, Tecan, Switzerland). The data were analyzed using GraphPad 9.0 software, and statistical analysis was performed using the Student t test. ns There is no significant difference, the results are as follows Figure 1 shown.
[0031] Depend on Figure 1 It can be seen that compared with the blank group, IOX1 had no significant effect on cell activity within the concentration range of 5 to 40 μM.
[0032] Example 2 Cell morphological observation of morphological changes of IPEC-J2 cells at different time points after PEDV infection
[0033] The cultured IPEC-J2 cells were inoculated into a 12-well cell culture plate. When the cell density reached about 80%, the culture medium was discarded, and the cells were washed three times with PBS. Then, the cells were infected with PEDV virus solution (MOI = 0.1). After 2 hours of infection, the culture medium containing 0.1% trypsin (without FBS) was replaced. After culturing for different time periods (0h, 2h, 12h, 24h, 48h), the cell morphology changes were observed and photographed. The results are shown in Figure 2. Figure 2 shown.
[0034] Depend on Figure 2 It can be seen that as the virus infection time increases, IPEC-J2 cells gradually shrink and deform, and even die and fall off, indicating that PEDV CV777 infection can cause adverse changes in the morphology of IPEC-J2.
[0035] Example 3: Fluorescence quantitative PCR detection of in vitro proliferation of PEDV CV777 in IPEC-J2 cells
[0036] In order to detect the in vitro proliferation of PEDV CV777 in IPEC-J2 cells, the virus was added to IPEC-J2 cells and the cells were collected 0 to 48 hours after infection. According to the instructions of the FastPure Cell / Tissue Total RNA Isolation Kit (Vazyme, Nanjing, China), the purity of viral RNA was determined using the ND-1000 nucleic acid / protein concentration detector. According to the gene sequence published in the GenBank database, GAPDH was used as the internal reference gene, and qPCR primers were designed using Primer Premier 6.0 software. The upstream primer of PEDV-N was 5'-CACCTCCTGCTTCACGTACA-3', and the downstream primer was 5'-AGCTCCACGACCCTGGTTAT-3'. The primers were synthesized by Sangon Biotech Co., Ltd. (Sangon Biotech, Shanghai, China). qPCR analysis was performed using a real-time fluorescence quantitative kit (Vazyme, Nanjing, China), and 3 independent replicates were set for each analyzed sample. The data were analyzed using GraphPad 9.0 software, and the results are shown in the figure. Figure 3 shown.
[0037] Depend on Figure 3 It can be seen that after PEDV CV777 infected IPEC-J2 cells, PEDV-N expression gradually increased from 0 to 24 hours, reached a maximum at 24 hours, and decreased at 48 hours compared with 24 hours.
[0038] Example 4: Immunoblotting to detect the changes in the loading of PEDV CV777 at different time points
[0039] IPEC-J2 cells were infected with PEDV CV777, and a mock group was set up. Cells were collected at different time points, and a mixture containing protein lysis buffer (RIPA) (Applygen, Beijing, China) was added with a protease promoter (TargetMOI, MA, USA) for lysis on ice for 30 min, and the supernatant was obtained by centrifugation at 14,000 rpm at 4°C for 15 min. The protein concentration was adjusted using a BCA protein assay kit (Beyotime, Shanghai, China), and denatured at 98°C for 10 min for later use.
[0040] The samples were subjected to polyacrylamide gel electrophoresis and the proteins were transferred to PVDF membranes (Millipore, USA). After blocking with 5% skim milk powder at room temperature for 2 h, PEDV-N and GAPDH primary antibodies were used and incubated overnight at 4°C. The PVDF membrane was rinsed three times with 1×TBST and incubated with secondary antibodies (Proteintech, Wuhan, China) at room temperature for 2 h. The samples were washed three times with 1×TBST and then soaked in ECL developer for development analysis. The results are shown in Figure 2. Figure 4 shown.
[0041] Depend on Figure 4 It was found that after PEDV CV777 infected IPEC-J2 cells, PEDV-N expression gradually increased from 0 to 24 hours, reached a maximum at 24 hours, and decreased at 48 hours compared with 24 hours.
[0042] Example 5: Fluorescence quantitative PCR detection of the promoting effect of IOX1 on the proliferation of PEDV CV777 in vitro
[0043] In order to detect the promoting effect of IOX1 on PEDV CV777, PEDV virus was added to IPEC-J2 cells treated with 0μM, 5μM, 10μM, 20μM, and 40μM IOX1, and the cells were collected after 24h of culture. Viral RNA (Vazyme, Nanjing, China) was prepared according to the instructions of the FastPure Cell / Tissue Total RNA Isolation Kit, and the purity was determined using an ND-1000 nucleic acid / protein concentration detector. According to the gene sequence published in the GenBank database, GAPDH was used as the internal reference gene, and qPCR primers were designed using Primer Premier 6.0 software. The upstream primer of PEDV-N was 5'-CACCTCCTGCTTCACGTACA-3', and the downstream primer was 5'-AGCTCCACGACCCTGGTTAT-3'. The primers were synthesized by Sangon Biotech, Shanghai, China. qPCR analysis was performed using a real-time fluorescence quantitative kit (Vazyme, Nanjing, China), and three independent replicates were set for each analysis sample. Data were analyzed using GraphPad 9.0 software and statistical analysis was performed using the Student t test. ns Indicates no significant difference, **p<0.01 indicates extremely significant difference. Figure 5 shown.
[0044] Depend on Figure 5It can be seen that compared with the toxin-added group, virus proliferation was promoted after the addition of IOX1, and 20 μM IOX1 significantly promoted the proliferation of PEDV CV777.
[0045] Example 6: Indirect immunofluorescence assay to detect the promoting effect of IOX1 on the proliferation of PEDV CV777 in vitro
[0046] After adding 0μM and 10μM IOX1 and culturing for 24h, PEDV CV777 was added to infect the cells for 24 and then the cells were collected. After washing the cells with pre-cooled PBS, the cells were fixed with 4% paraformaldehyde at 4°C for 30min, and bovine serum albumin (BSA) (Solarbio, Beijing, China) was added to block at 37°C for 2h. PEDV-N was used as the primary antibody and incubated overnight at 4°C. The cells were gently washed 3 times with 1×PBST, and then the fluorescently labeled secondary antibody (Solarbio, Beijing, China) was added and incubated at room temperature in the dark for 2h. The cells were gently washed 3 times with PBST, and DAPI was added and incubated at 37°C for 5min for nuclear staining. Then a fluorescence inverted microscope (Leica Microsystems, Wetzlar, Germany) was used to observe the fluorescence of the cells with different treatments and take pictures.
[0047] The results are as follows Figure 6 As shown: Compared with the poisoning group, the distribution of PEDV-N protein increased significantly after the addition of 20 μM IOX1, indicating that 20 μM IOX1 significantly promoted the proliferation of PEDV.
[0048] The above results indicate that IOX1 has a stable effect of promoting the activity of porcine epidemic diarrhea virus and promoting the replication of porcine epidemic diarrhea virus.
Claims
1. Application of IOX1 in the cultivation of porcine epidemic diarrhea virus.
2. Application of IOX1 in the production and preparation of porcine epidemic diarrhea virus vaccine.
3. The use according to claim 1 or 2, characterized in that: The porcine epidemic diarrhea virus is PEDV CV777.
4. The use according to claim 1, characterized in that: The culture is for promoting the infection of cells with porcine epidemic diarrhea virus or promoting the infection rate of porcine epidemic diarrhea virus in cells.
5. The use according to claim 4, characterized in that: The cells are IPEC-J2 cells.
6. The use according to claim 5, characterized in that: The IOX1 is not toxic to cells at a concentration of 5 to 40 μM.
7. The use according to claim 1, characterized in that: The IOX1 is used at a concentration of 20 to 40 μM to promote the proliferation of porcine epidemic diarrhea virus.
8. A pharmaceutical composition for the cultivation of porcine epidemic diarrhea virus or the production of a vaccine, characterized in that: Includes IOX1 and a pharmaceutically acceptable carrier thereof.
9. The pharmaceutical composition according to claim 8, characterized in that The pharmaceutical composition comprises a stereoisomer of 10X1, a solvate of 10X1, a pharmaceutically acceptable salt of 10X1 or a solvate of a pharmaceutically acceptable salt of 10X1 of the active ingredient.
10. The pharmaceutical composition according to claim 8, characterized in that The pharmaceutical composition is an oral preparation or an injection preparation.