A Seneca Valley virus A empty capsid, its preparation method and uses

Abstract

The present invention discloses a Seneca Valley virus A empty capsid and its preparation method and use. In the present invention, 10 mM guanidine hydrochloride is added at 8 h after cell infection to make all the complete virus particles of SVA form empty capsids, and then 10%-50% cesium chloride is used to separate the complete virus particles and empty capsids of SVA under the conditions of centrifugation at 230,000 g for 2.5 h. To determine the immune effects of separating the complete virus particles and empty capsids of Seneca Valley virus A, BALB / c mice are immunized by intramuscular injection at a dose of 2 μg / mouse, and then the changes in specific antibodies and neutralizing antibodies are detected. The results show that the neutralizing antibodies and specific antibodies induced after immunization with the complete virus particles and empty capsids of SVA are comparable. The proposal of the present invention provides a new technical means for the preparation of the Seneca Valley virus A empty capsid, provides technical support for the research of a safer and more effective Seneca Valley virus A vaccine, and also lays a foundation for the isolation and purification of other picornaviruses.

Description

Technical Field

[0001] The present invention relates to a Seneca virus A empty capsid, a preparation method and uses thereof, and belongs to the field of biotechnology. Background Art

[0002] Seneca virus A (SVA), also known as Seneca Valley virus, is a single-stranded positive-sense RNA virus of the genus Seneca virus in the family Picornaviridae. The clinical symptoms caused by SVA are extremely similar to those of foot-and-mouth disease, swine vesicular disease and swine vesicular stomatitis, making it difficult to distinguish, which poses a great challenge to clinical diagnosis. Since SVA was confirmed in 2008, many pig-raising countries have successively reported the incidence of SVA. As the number of countries reporting the epidemic continues to increase and the contaminated area continues to expand, as a major global pig-raising country, SVA has quickly attracted the attention of the Chinese people.

[0003] Guanidine hydrochloride is a typical hydrogen bond disruptor, which can cause conformational and aggregated state structure changes of proteins. Proteins will spontaneously unfold in a high-concentration guanidine hydrochloride solution, and this unfolding is reversible. Once returned to a low-concentration denaturant or a solution environment without denaturant, the proteins will fold back to the native state. Guanidine hydrochloride can change the conformation and active center of enzymes, resulting in changes in enzyme activity. Huang Wen et al. found that low-concentration guanidine hydrochloride has a strong inhibitory effect on the activity of RNaseA when studying the effect of guanidine hydrochloride on the activity of reverse micelle RNaseA. D Bamford and L Mindich found that when exploring the destruction of lipid-containing bacteriophage PRD1, two proteins P3 and P5 were released from the phage after treatment with guanidine hydrochloride, and these two proteins can form polyhedral capsids. In addition, guanidine hydrochloride also has a certain impact on the replication and proliferation process of viruses. Brown et al. found that guanidine hydrochloride has an inhibitory effect on the replication of foot-and-mouth disease virus in the kinetic study of protein and RNA synthesis induced by foot-and-mouth disease virus infection. Jacobson et al. found that guanidine hydrochloride can inhibit newly synthesized RNA from entering the interior of poliovirus particles when studying the association between the morphological changes of poliovirus and viral RNA and capsid proteins.

[0004] During the isolation and purification process of SVA, the present inventors found that there was a sporadic phenomenon of empty capsids with black in the middle and white on the edge around the complete SVA virus particles. Therefore, the present inventors conducted relevant experiments on the effect of guanidine hydrochloride on the formation of empty capsids of complete SVA virus particles, and explored the optimal conditions for isolating complete SVA virus particles and empty capsids, as well as the immune effects of the isolated complete SVA virus particles and empty capsids, in order to provide technical guidance for the isolation and purification of picornaviruses, and at the same time provide data support for the quantification and quality control of SVA vaccines. Summary of the Invention

[0005] The object of the present invention is to provide a Seneca Valley virus A empty capsid and its preparation method and use.

[0006] To achieve the above object, the present invention adopts the following technical means:

[0007] A preparation method of a Seneca Valley virus A empty capsid of the present invention comprises the following steps:

[0008] (1) Preparation of Seneca Valley virus A empty capsid

[0009] 1) Add the cell suspension into a cell culture flask, culture it in a 37 °C, 5% CO2 cell culture incubator. When the cells grow to 80 - 90%, change to a 2% fetal bovine serum medium, inoculate the Seneca Valley virus A virus solution at a volume ratio of 1:100, continue to place it in the incubator for culture. When the SVA infects the cells for 8 h, add 10 mM guanidine hydrochloride and continue to culture. When the cell infection reaches 90%, harvest the virus to obtain a cell lysate containing SVA empty capsids, and store it in a -80 °C refrigerator for later use;

[0010] 2) Inactivation and concentration

[0011] The cell lysate is repeatedly frozen and thawed three times, add an inactivator, shake it on a shaker at 150 rpm / min for 28 h, add a blocker, and check whether the inactivation is complete; after inactivation, centrifuge the cell lysate at 6000 rpm / min for 30 min, take the supernatant and concentrate it according to the product manual of sartorius Vivaflow 200. The concentrated solution is then centrifuged at 10000 rpm / min for 30 min, take the supernatant and centrifuge it in an ultracentrifuge at 40000 rpm / min for 2 h. The precipitate is ground and dispersed with 1% Trition-100 PBS, left overnight at 4 °C, then centrifuged at 10000 rpm / min for 30 min, collect the supernatant, add an equal volume of trichloroethylene to degrease, centrifuge at 8000 rpm / min for 30 min, and collect the supernatant;

[0012] (2) Separation of SVA complete virus particles and empty capsids

[0013] Use Gradient Station TM Gradient preparation instrument (Bio Comp Instrument) to prepare a 10% - 50% w / w cesium chloride density gradient, add the supernatant obtained in step 2) and centrifuge at 230 000 g for 2.5 h to obtain purified Seneca Valley virus A empty capsids.

[0014] Among them, preferably, the cells in step 1) are porcine kidney cell line (IBRS-2), and the culture medium used is Dulbecco’s Modified Eagle’s Medium–high glucose containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin.

[0015] The Seneca virus A empty capsid prepared according to the described preparation method is also within the protection scope of the present invention.

[0016] Furthermore, the present invention also proposes the use of the described Seneca virus A empty capsid in the preparation of a Seneca virus A vaccine.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] During the cell infection process of SVA, complete virus particles and empty capsids can be formed, but the content of empty capsids is low and it is difficult to achieve separation and purification. The present invention uses guanidine hydrochloride to generate SVA empty capsids, and then performs ultracentrifugation under different density gradients, media, rotation speeds, and centrifugation times to screen the optimal conditions for separating SVA complete virus particles and empty capsids, and uses these conditions to separate SVA complete virus particles and empty capsids. To determine the immune effects of separated SVA complete virus particles and empty capsids, BALB / c mice were immunized by intramuscular injection at a dose of 2 μg / mouse, with PBS as the blank control. Blood was collected from the orbital venous plexus of the 4 groups of experimental mice at 1W, 2W, 3W, 4W, 5W, 6W, and 7W after immunization, and serum was separated, and then the changes in specific antibodies and neutralizing antibodies were detected. The results showed that adding 10 mM guanidine hydrochloride when SVA infected cells for 8 h could cause all SVA complete virus particles to form empty capsids; the best effect of separating SVA complete virus particles and empty capsids was achieved under the conditions of 10%-50% cesium chloride, 230,000 g, and centrifugation for 2.5 h; there was no significant difference in the neutralizing antibodies and specific antibodies induced after immunization with SVA complete virus particles and empty capsids, that is, their immunogenicities were equivalent. The acquisition of SVA empty capsids, the screening of the optimal separation conditions for SVA complete virus particles and empty capsids, and the immune effects provide new technical means for the preparation of Seneca virus A empty capsids, also provide technical support for the research of a safer and more effective Seneca virus A vaccine, and at the same time lay a foundation for the separation and purification of other small RNA viruses. Description of the Drawings

[0019] Figure 1For the acquisition of Seneca Valley virus A (SVA) complete virus particles. OD values measured after fractionation by 15%-45% sucrose density gradient centrifugation (a); SDS-PAGE corresponding to the peak fraction, Positive control is the unconcentrated SVA venom, Negative control is the IBRS cell lysate (b); Electron micrograph corresponding to the peak fraction (c);

[0020] Figure 2 For the cytotoxicity detection results of different concentrations of guanidine hydrochloride on cells;

[0021] Figure 3 For the treatment results of guanidine hydrochloride at different times and concentrations;

[0022] SVA infected with 10 mM guanidine hydrochloride for 2 h (a); SVA infected with 10 mM guanidine hydrochloride for 8 h (b); SVA infected with 5 mM guanidine hydrochloride for 8 h (c); SVA infected with 10 mM guanidine hydrochloride for 10 h (d); OD values measured after fractionation by 15%-45% w / w sucrose density gradient centrifugation (e); SDS-PAGE corresponding to the peak fraction, Positive control is the unconcentrated IBRS cell venom, Negative control is the IBRS cell lysate (f);

[0023] Figure 4 For the results of separating SVA complete virus particles and empty capsids under different sucrose density gradient conditions;

[0024] Figure 5 For the results of separating SVA complete virus particles and empty capsids with different media under 10%-50% w / w density gradient conditions;

[0025] Figure 6 For the results of separating SVA complete virus particles and empty capsids at different rotation speeds under 10%-50% w / w cesium chloride density gradient conditions;

[0026] Figure 7 For the results of separating SVA complete virus particles and empty capsids at different ultracentrifugation times under 10%-50% w / w cesium chloride at 230000 g;

[0027] Figure 8 For the serum antibody detection results after immunization of mice;

[0028] Collect serum at 1-7 weeks after immunization to detect specific antibodies (a) and neutralizing antibodies (b). Detailed implementation methods

[0029] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0030] Example 1 Preparation of Seneca Valley virus A empty capsid

[0031] 1 Materials and methods

[0032] 1.1 Test materials

[0033] 1.1.1 Main reagents and materials

[0034] Sucrose and skim milk powder were purchased from Amresco; phosphotungstic acid and cesium chloride were purchased from Solarbio; protein Marker was purchased from Fermentas; nitrocellulose membrane, ECL chromogenic solution, HRP-labeled rabbit anti-pig IgG, and cell culture medium Dulbecco’s Modified Eagle’s Medium-high glucose were purchased from Sigma; 300-mesh copper grids were purchased from Zhongke Jingyi; Fetal Bovine Serum (FBS) was purchased from Biological Industries; antibiotic G418 powder was purchased from Amresco.

[0035] 1.1.2 Viruses, cells and experimental animals

[0036] The SVA virus strain CH-HB-2017 (GenBank accession number: MN922286) and the porcine kidney cell line (IBRS-2) were stored in the laboratory. BALB / c mice were provided by the Animal Experiment Center of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0037] 1.2 Methods

[0038] 1.2.1 Cultivation of SVA complete virus particles

[0039] The porcine kidney cell line (IBRS-2) was cultured in Dulbecco’s Modified Eagle’s Medium–high glucose cell culture medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin in a 37 °C, 5% CO2 cell culture incubator. When the cells grew to 80-90%, they were changed to a 2% fetal bovine serum medium, and SVA was inoculated at a ratio of 1:100. When 90% of the cells were infected, the cell supernatant was frozen at -80 °C for later use.

[0040] 1.2.2 Virus concentration and purification

[0041] The cell lysate was repeatedly frozen and thawed three times, an inactivator was added, and it was shaken on a shaker at 150 rpm / min for 28 h. A blocker was added, and it was checked whether the inactivation was complete. The inactivated cell lysate was centrifuged at 6000 rpm / min for 30 min, and the supernatant was concentrated according to the product manual of sartorius Vivaflow 200, and the final volume was maintained at about 40 mL. The concentrated solution was centrifuged at 10 000 rpm / min for 30 min, and the supernatant was centrifuged at 40000 rpm / min for 2 h using an ultracentrifuge XPN-100 (Beakman). The precipitate was ground and dispersed with 1% Trition-100 PBS, and the final volume was less than 1 mL. After overnight incubation at 4 °C, it was centrifuged at 10000 rpm / min for 30 min, the supernatant was collected, an equal volume of trichloroethylene was added for defatting, and it was centrifuged at 8000 rpm / min for 30 min, and the supernatant was collected.

[0042] 1.2.3 Sucrose density gradient centrifugation

[0043] 15% w / w and 45% w / w sucrose solutions were prepared overnight, and a 15%-45% w / w continuous sucrose density gradient was made using a Gradient StationTM gradient preparation instrument (BioComp Instrument). The supernatant was added and centrifuged at 36000 rpm / min for 2.5 h. After centrifugation, 500 μL of the supernatant was aspirated from top to bottom each time for aliquoting, and its OD value was measured using a UV spectrophotometer.

[0044] 1.2.4 Transmission electron microscopy observation

[0045] The sample in the OD peak tube was added to the copper mesh and adsorbed for 3 min, then washed three times with PBS; stained with 2% phosphotungstic acid for 1 min and washed three times with PBS; the copper mesh was placed in a transmission electron microscope for observation.

[0046] 1.2.5 Polyacrylamide gel electrophoresis

[0047] The sample in the OD peak tube was mixed with 2×SDS loading buffer at a ratio of 1:1 and heated in a metal bath at 100 °C for 10 min; 10 μL was added to each well of the protein gel, at 60 V for 30 min and 90 V for 1 h; stained with Coomassie Brilliant Blue staining solution for 30 min; decolorized with Coomassie Brilliant Blue decolorizing solution for 2 h.

[0048] 1.2.6 Cytotoxicity determination of different concentrations of guanidine hydrochloride

[0049] According to 100 μL (10 6cell / mL) pig kidney cell (IBRS-2) suspension was added to a 96-well plate and cultured in a cell culture incubator at 37°C and 5% CO2 for 24 hours; guanidine hydrochloride was added to the plate at final concentrations of 100mM, 50mM, 20mM, 10mM, and 5mM, 8 replicates were made for each concentration, and the culture was continued for 24h, 48h, and 72h; 10μL / well of tetrazolyl blue salt compound (MTS) was added respectively, and the plate was placed in the incubator for another 3.5h, and the absorbance at 490nm was detected by an enzyme marker.

[0050] 1.2.7 Guanidine hydrochloride concentration and treatment time

[0051] The porcine kidney cell line (IBRS-2) was cultured in Dulbecco's Modified Eagle's Medium-high glucose containing 10% fetal bovine serum, 100U / mL penicillin, and 100μg / mL streptomycin in a cell culture medium at 37°C and 5% CO2. When the cells grew to 80-90%, the medium was replaced with 2% fetal bovine serum and inoculated with Seneca virus A virus solution at a volume ratio of 1:100. The cells were continued to be cultured in the incubator. At different times when SVA infected the cells, such as 8h and 10h, 100mM, 50mM, 20mM, 10mM, and 5mM guanidine hydrochloride were added respectively. When the cells were 90% infected, the virus was collected and placed in a -80°C refrigerator for use.

[0052] 1.2.8 Detection of viral nucleic acid content by fluorescent quantitative RT-PCR

[0053] The corresponding nucleic acid content was detected according to the SVA detection method established in the laboratory (MU S, ABDULLAH SW, ZHANG Y, HAN S, GUO H, LIM, DONG H, Xu J, Teng Z, Wen X and Sun S, 2020b. Development of novel SYBR green I-based quantitative RT-PCR assay for Senecavirus A detection in clinical samples of pigs. Mol Cell Probes, 53: 101643. DOI: 10.1016 / j.mcp.2020.101643).

[0054] 1.2.9 TCID50 determination after treatment with guanidine hydrochloride at different time and concentration

[0055] The SVA samples to be tested after treatment at different time and concentration were diluted 10 -1 -10 -9, add it to a 96-well plate at a volume of 100 μL / well, and make 8 replicates for each dilution; after the virus solution to be diluted is evenly dispersed in the 96-well plate, add porcine kidney cells IBRS-2 (about 1.5×10 6 cell / mL) at a volume of 100 μL / well; after mixing, place the 96-well plate in an incubator at 37 °C and 5% carbon dioxide for 72 h, determine the degree of cytopathic effect and the number of wells with cytopathic effect, and calculate the SVATCID50 by the Reed-Muench method.

[0056] 1.2.10 Optimal gradient for the separation of SVA intact virus particles and empty capsids

[0057] After obtaining SVA intact virus particles and empty capsids according to the methods in 1.2.1 - 1.2.9, perform large-scale culture according to the corresponding methods; prepare 10% w / w, 15% w / w, 45% w / w, 50% w / w sucrose overnight, and use a Gradient StationTM gradient preparation instrument (Bio Comp Instrument) to make 15% - 50% w / w, 10% - 50% w / w, 15% - 45% w / w continuous sucrose density gradients, and then follow the same steps as in 1.2.2 - 1.2.3.

[0058] 1.2.11 Optimal medium for the separation of SVA intact virus particles and empty capsids

[0059] After obtaining SVA intact virus particles and empty capsids according to the methods in 1.2.1 - 1.2.9, perform large-scale culture according to the corresponding methods; based on 1.2.10, select the gradient with the best separation effect for density gradient centrifugation with different media, such as potassium tartrate, cesium chloride, sucrose. Except for sucrose, the other media are manually prepared overnight before use. After centrifugation, collect the centrifugation products at 500 μL / tube, and detect the OD value of the collected samples.

[0060] 1.2.12 Optimal rotation speed for the separation of SVA intact virus particles and empty capsids

[0061] After obtaining SVA intact virus particles and empty capsids according to the methods in 1.2.1 - 1.2.9, perform large-scale culture according to the corresponding methods; based on 1.2.10 - 1.2.11, select the gradient and medium with the best separation effect for ultracentrifugation at different rotation speeds. According to the maximum centrifugal force of the horizontal rotor of the Beakman centrifuge, 4 rotation speeds are set in this experiment, which are: 150000g, 200000g, 230000g, 250000g. After centrifugation, collect the centrifugation products at 500 μL / tube, and detect the OD value of the collected samples.

[0062] 1.2.13 Optimal centrifugation time for the separation of SVA intact virus particles and empty capsids

[0063] Virus purification was carried out according to the previous method. Based on 1.2.10 - 1.2.12, the gradient, medium, and rotation speed with the best separation effect were selected for ultra - centrifugation with different centrifugation times. The times were selected as 2 h, 2.5 h, 3 h, and 3.5 h. After centrifugation, the centrifugation products were collected at 500 μL / tube, and the OD values of the collected samples were detected.

[0064] 1.2.14 Animal experiment design

[0065] Fifteen BALB / c mice were randomly divided into 3 groups, namely the SVA intact virus particle group, the SVA empty capsid group, and the PBS negative control group. Each group was immunized by intramuscular injection at a dose of 2 μg / mouse, and the negative control group was injected with the same volume of PBS.

[0066] 1.2.15 Antibody detection

[0067] Blood from the orbital venous plexus of mice was collected before immunization (0 d) and at 1W, 2W, 3W, 4W, 5W, 6W, and 7W after immunization, and the serum was separated. The indirect ELISA method was used to detect the changes in specific antibodies, and the neutralization antibody titer was detected for neutralizing antibodies.

[0068] 2 Results and analysis

[0069] 2.1 Characterization of SVA virus particles untreated with guanidine hydrochloride

[0070] After membrane - wrapping concentration and 15% - 45% w / w sucrose density gradient centrifugation, OD 280 nm peaks appeared at 1.0 mL and 6.5 mL ( Figure 1 a); SDS - PAGE was performed on the peak tubes. Three bands with sizes of approximately 36 kDa, 31 kDa, and 27 kDa could be clearly seen from the figure, which were consistent with the sizes of SVA VP2, VP3, and VP1 bands ( Figure 1 b); The peak tubes corresponding to the peaks were observed by electron microscopy. The first peak was the contaminating protein in the cell culture medium (results not shown), and the second peak was the SVA intact virus particles. It could be seen from the figure that SVA was arranged in a neat lattice pattern, was round, and had a size of approximately 25 nm ( Figure 1 c). This indicated that SVA intact virus particles were successfully obtained.

[0071] 2.2 Results of cytotoxicity determination of different concentrations of guanidine hydrochloride

[0072] The results of cytotoxicity detection of different concentrations of guanidine hydrochloride are as follows Figure 2As shown, PBS was set to 100%, and guanidine hydrochloride at different concentrations was compared with PBS. It can be seen from the figure that when IBRS-2 cells were treated with 5 mM, 10 mM, 20 mM, 50 mM, and 100 mM guanidine hydrochloride for 24 h, 48 h, and 72 h, the percentages were all around 100%, and there were no significant differences, that is, 5-100 mM guanidine hydrochloride had no obvious cytotoxicity to IBRS-2 cells and could be used for subsequent experiments.

[0073] 2.3 Viral nucleic acid levels after treatment with guanidine hydrochloride at different times and concentrations

[0074] Based on the successful acquisition of SVA intact virus particles, the virus was harvested, concentrated, and purified after SVA infected IBRS-2 cells for 2 h, 4 h, 6 h, and 8 h, and the OD values of each tube were measured. The results showed that no peak appeared within 0-6 h, and intact virus particles could only be obtained 8 h after infection. Therefore, when detecting the treatment with guanidine hydrochloride at different times and concentrations, it started from 8 h after infection. The specific nucleic acid detection results are shown in Table 1 below. According to the method established in the laboratory (MU S, ABDULLAH S W, ZHANG Y, HAN S, GUO H, LIM, DONG H, XU J, TENG Z, WEN X and SUN S, 2020b. Development of a novel SYBR green I-based quantitative RT-PCR assay for Senecavirus A detection in clinical samples of pigs. Mol Cell Probes, 53:101643. DOI:10.1016 / j.mcp.2020.101643), the detection range of Cq values was between 7.28 and 35.5, and values greater than 32 were negative. When 5 mM guanidine hydrochloride was added 8 h after SVA infection, the minimum Cq value was 25.42, and the Cq values were all around 30.44 when treated with 10 mM, 20 mM, 50 mM, and 100 mM guanidine hydrochloride; while when different concentrations of guanidine hydrochloride were added 10 h after SVA infection, the influence on Cq values was not significant, and the average value was around 19.40.

[0075] Table 1 Nucleic acid levels after treatment with guanidine hydrochloride at different times and concentrations

[0076]

[0077] 2.4 TCID of the virus treated with guanidine hydrochloride at different times and concentrations 50 Determination results

[0078] Perform TCID on the cell lysates treated with guanidine hydrochloride at different times and different concentrations 50Detection was carried out, and the results are shown in Table 2 below. It can be seen from the table that when 5 mM guanidine hydrochloride was added 8 h after SVA-infected cells, the TCID 50 measurement result was 10 -3.79 , and when treated with 10 mM guanidine hydrochloride, the TCID 50 was 10 -2.28 . As the concentration of guanidine hydrochloride increased, the virus titer was basically stable at around 10 -2.30 . When different concentrations of guanidine hydrochloride were added 10 h after SVA infection, the change in its TCID 50 was small, basically remaining around 10 -7.74 .

[0079] Table 2 TCID 50 measurement results after treatment with guanidine hydrochloride at different times and concentrations

[0080]

[0081] 2.5 Transmission electron microscopy results after treatment with guanidine hydrochloride at different times and concentrations

[0082] The samples treated with guanidine hydrochloride at different times and concentrations were concentrated, purified and observed by transmission electron microscopy. Only the key times and concentrations were selected for drawing, and the remaining results were not shown. The results are as Figure 3 shown. When 100 mM guanidine hydrochloride was added 8 h after SVA-infected cells, no complete SVA virus particles or empty capsids were observed ( Figure 3 a); when 10 mM guanidine hydrochloride was added 8 h after SVA-infected IBRS-2 cells, a large number of empty capsids appeared ( Figure 3 b); when 5 mM guanidine hydrochloride was added 8 h after SVA-infected IBRS-2 cells, a coexistence phenomenon of complete SVA virus particles and empty capsids was observed ( Figure 3 c); when 10 mM guanidine hydrochloride was added 10 h after SVA-infected IBRS-2 cells, only complete SVA virus particles appeared ( Figure 3 d); Figure 3 e is the OD value after Figure 3 b ultracentrifugation. It can be seen from the figure that SVA showed a peak at 6.5 mL; Figure 3 f is the SDS-PAGE corresponding to the peak tube in Figure 3 e. Three bands with sizes of approximately 36 kDa, 31 kDa, and 27 kDa can be seen from the figure, which are consistent with the band sizes of SVAVP2, VP3, and VP1. This shows that adding 10 mM guanidine hydrochloride 8 h after SVA infection can form all empty capsids.

[0083] 2.6 Results of establishing the best separation method for complete SVA virus particles and empty capsids

[0084] 2.6.1 Results of ultracentrifugation under different density gradient conditions

[0085] Using the obtained complete SVA virus particles and empty capsids, we found that when the complete SVA virus particles were mixed with the empty capsids, they could not be separated by sucrose density gradient ultracentrifugation at 15%-45% w / w; then the density gradient was changed to 10%-50% w / w, 15%-50% w / w. Considering that there was no sample in the range of 0-5 mL, only the OD values of 5-10 mL were selected for plotting when making the graph. 280nm The results showed that the separation of complete SVA virus particles and empty capsids by 10%-50% w / w and 15%-5% w / w was similar, with peaks appearing at 6.5 mL and 7 mL respectively ( Figure 4 ). Considering the experimental consumables and other aspects, a 10%-50% w / w density gradient was selected for the subsequent experiments.

[0086] 2.6.2 Results of ultracentrifugation under different medium conditions

[0087] There are many types of media for ultracentrifugation. The best medium was screened according to the existing media in this laboratory. As Figure 5 shown, the results showed that the separation of complete SVA virus particles and empty capsids by 10%-50% w / w cesium chloride was the greatest, with peaks appearing at 6.5 mL and 8 mL respectively; it can be seen from the figure that both the complete SVA virus particles and empty capsids separated by potassium tartrate were at 8.5 mL, so they could not be separated under the 10%-50% w / w potassium tartrate density gradient; similarly, the separation degree under the 10%-50% w / w sucrose density gradient was not very large, and the complete virus particles and empty capsids were at 6.5 mL and 7 mL respectively.

[0088] 2.6.3 Results of ultracentrifugation under different rotation speed conditions

[0089] According to the maximum rotation speed of the horizontal rotor of the Beckman ultracentrifuge XPN-100, four rotation speeds were set in this experiment, namely 150000g, 200000g, 230000g, and 250000g. It can be seen from the results that the separation of complete SVA virus particles and empty capsids was the best at a rotation speed of 230000g, with peaks appearing at 6.5 mL and 8 mL respectively; at rotation speeds of 150000g and 200000g, the separation of complete SVA virus particles and empty capsids was relatively small; at a rotation speed of 250000g, the peak of SVA empty capsids appeared at 9.5 mL, while the OD of complete SVA virus particles 280nm showed an upward trend all the time, and the specific positions of complete SVA virus particles and empty capsids could not be determined ( Figure 6 ).

[0090] 2.6.4 Results of ultracentrifugation under different centrifugation time conditions

[0091] According to the influence of centrifugation time on the centrifugation effect, in this experiment, centrifugation times of 2 h, 2.5 h, 3 h, and 3.5 h were selected to study the influence on the separation of SVA intact virus particles and empty capsids. The results showed that under the condition of 230,000 g of 10%-50% cesium chloride, the separation degree of SVA intact virus particles and empty capsids was the largest after centrifugation for 2.5 h, and peaks appeared at 6.5 mL and 8 mL respectively; followed by centrifugation for 2 h, and the peaks were at 7.5 mL and 8 mL respectively; when centrifuged for 3 h, the peaks of SVA intact virus particles and empty capsids appeared at the same place; when centrifuged for 3.5 h, the peak of SVA empty capsids appeared at 9 mL, but the OD value of intact virus particles kept increasing, that is, the peak of intact virus particles was uncertain( Figure 7 ).

[0092] In summary, the optimal conditions for separating SVA intact virus particles and empty capsids are centrifugation at 230,000 g of 10%-50% w / w cesium chloride for 2.5 h.

[0093] 2.7 Evaluation of the immunization effects of SVA intact virus particles and empty capsids

[0094] After immunization by intramuscular injection, blood was collected from the orbital venous plexus of mice, and specific antibodies and neutralizing antibodies were detected at 1-7 W respectively, as Figure 8 shown. There were no significant differences in the specific antibodies produced after immunization between the SVA intact virus particle group and the empty capsid group at 1-6 W, but the specific antibodies produced by the SVA empty capsid group were significantly higher than those of the SVA intact virus particle group at the 7th W after immunization; the neutralizing antibodies of SVA intact virus particles and empty capsids appeared at the 1st W after immunization and the difference between the two was extremely significant, with the SVA intact virus particle group being higher than the SVA empty capsid group; the neutralizing antibody titers of each group increased at 2-3 W after immunization, but the increase degrees were different; the neutralizing antibody titer of SVA intact virus particles was significantly higher than that of the empty capsid group at 4 W after immunization; then at 5-7 W, the neutralizing antibody titers of the SVA intact virus particle group and the empty capsid group began to decrease, but the decrease amplitude was small and there was no significant difference in the neutralizing antibody titers between the two groups; in summary, the immunization effects of SVA intact virus particles and empty capsids are equivalent.

Claims

1. A method for preparing the empty capsid of Seneca virus A (SVA), characterized in that, it comprises the following steps: (1) Preparation of the empty capsid of Seneca virus A 1) Add the cell suspension to a cell culture flask and culture it in a cell incubator at 37°C and 5% CO 2 2. When the cells grow to 80 - 90%, change to a 2% fetal bovine serum medium, inoculate the Seneca Valley virus A virus solution at a volume ratio of 1:100, continue to place it in the incubator for culture. When the SVA infects the cells for 8 h, add 10 mM guanidine hydrochloride and continue to culture. When the cell infection reaches 90%, harvest the virus to obtain a cell lysate containing SVA empty capsids and store it in a -80°C refrigerator for later use; (2) Inactivation and concentration The cell lysate is repeatedly frozen and thawed three times, an inactivator is added, and it is shaken on a shaker at 150 rpm / min for 28 h. A blocker is added, and it is checked whether the inactivation is complete; the inactivated cell lysate is centrifuged at 6000 rpm / min for 30 min, the supernatant is taken for concentration, the concentrated solution is centrifuged at 10000 rpm / min for 30 min, the supernatant is taken and centrifuged in an ultracentrifuge at 40000 rpm / min for 2 h. The precipitate is ground and dispersed with 1% Triton-100 PBS, left overnight at 4 °C, and then centrifuged at 10000 rpm / min for 30 min. The supernatant is collected, equal volume of trichloroethylene is added for defatting, and it is centrifuged at 8000 rpm / min for 30 min. The supernatant is collected; (2) Separation of SVA intact virus particles and empty capsids A 10%-50% w / w cesium chloride density gradient is prepared using a gradient maker, the supernatant obtained in step (2) is added, and it is centrifuged at 230000 g for 2.5 h to obtain purified empty capsids of Seneca virus A.

2. The preparation method according to claim 1, characterized in that, the cell described in step (1) is the porcine kidney cell line IBRS-2, and the culture medium used is a cell culture solution containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin.

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