Bovine coronavirus virus-like particles, methods of making and uses

Abstract

The application discloses a bovine coronavirus virus-like particle, a preparation method and application, and the bovine coronavirus virus-like particle is assembled after co-infection of five kinds of recombinant baculoviruses capable of stably secreting bovine coronavirus E, M, N, S and HE proteins, and contains complete bovine coronavirus E, M, N, S and HE proteins, and BCoV VLPs similar to natural BCoV virus particles in morphology and size are constructed; after bovine coronavirus virus-like particles obtained by the application are used for immunizing mice and calves, high-titer antibody levels can be generated, and cellular immunity of the body can be stimulated.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to bovine coronavirus virus-like particles, their preparation methods, and their applications. Background Technology

[0002] Bovine coronavirus (BCoV) is an enveloped virus, a non-segmented, single-stranded, positive-sense RNA virus. BCoV belongs to the order Nidovirales (…). Nidovirales Coronavirus family ( Coronaviridae Coronavirus ( ) Coronavirus Members of subgroup 2a. BCoV viral particles are pleomorphic, with most viral particles having a diameter between 65 and 210 nm. The genome is approximately 31 kb in length and encodes 10 open reading frames (ORFs). Among them, ORF3, ORF4, ORF8, ORF9, and ORF10 encode hemagglutinin protein (HE), spike protein (S), envelope protein (E), membrane protein (M), and nucleocapsid protein (N), respectively. These five proteins together constitute the major structural proteins of BCoV.

[0003] Bovine coronavirus disease (BCoV) is a viral disease caused by BCoV that infects the respiratory and intestinal tracts of cattle. BCoV is widely considered to be one of the important pathogens causing calf diarrhea (CD). In addition, it can also cause winter dysentery and respiratory-related symptoms in adult cattle. It is a virus that is ditropic to both the intestines and lungs.

[0004] The unique morphology of BCoV makes its isolation difficult. In recent years, BCoV has become widespread in my country, with a high overall positivity rate in cattle farms, severely hindering the development of the cattle industry. Studies have shown that BCoV can infect multiple hosts. While several BCoV vaccines exist abroad, no commercially available vaccine has yet been developed in China to prevent BCoV infection. Therefore, BCoV prevention and control in China remains a significant challenge. Summary of the Invention

[0005] In order to address the problems existing in the prior art, the present invention aims to provide bovine coronavirus virus-like particles, preparation methods, and applications.

[0006] In a first aspect, the present invention provides a bovine coronavirus structural protein gene, comprising at least one of the following proteins: E gene, M gene, N gene, S gene and HE gene;

[0007] the nucleotide sequence of the E gene is shown as SEQ ID NO. 1;

[0008] the nucleotide sequence of the M gene is shown as SEQ ID NO. 2;

[0009] the nucleotide sequence of the N gene is shown as SEQ ID NO. 3;

[0010] the nucleotide sequence of the S gene is shown as SEQ ID NO. 4;

[0011] the nucleotide sequence of the HE gene is shown as SEQ ID NO. 5.

[0012] In some embodiments of the present application, at least one of the E gene, M gene, N gene, HE gene is amplified, and the forward primer sequence of the nucleotide sequence of the E gene is shown as SEQ ID NO. 6, and the reverse primer sequence is shown as SEQ ID NO. 7;

[0013] the forward primer sequence of the nucleotide sequence of the M gene is shown as SEQ ID NO. 8; and the reverse primer sequence is shown as SEQ ID NO. 9;

[0014] the forward primer sequence of the nucleotide sequence of the N gene is shown as SEQ ID NO. 10; and the reverse primer sequence is shown as SEQ ID NO. 11;

[0015] the forward primer sequence of the nucleotide sequence of the HE gene is shown as SEQ ID NO. 12; and the reverse primer sequence is shown as SEQ ID NO. 13.

[0016] In a second aspect, the present application provides a recombinant plasmid comprising the aforementioned bovine coronavirus structural protein gene.

[0017] In a third aspect, the present application provides a recombinant bacmid comprising the aforementioned recombinant plasmid.

[0018] In a fourth aspect, the present application provides a recombinant baculovirus comprising the aforementioned recombinant bacmid.

[0019] In a fifth aspect, the present application provides a bovine coronavirus virus-like particle assembled by the aforementioned bovine coronavirus structural protein.

[0020] In a sixth aspect, the present application provides a preparation method of a bovine coronavirus virus-like particle, comprising:

[0021] (1) designing and amplifying bovine coronavirus structural protein genes;

[0022] (2) constructing a recombinant plasmid;

[0023] (3) Constructing a recombinant bacmid;

[0024] (4) Transfecting cells with the recombinant bacmid in (3) to obtain a recombinant baculovirus;

[0025] (5) Co-infecting cells with the recombinant baculovirus to obtain bovine coronavirus virus-like particles.

[0026] In a seventh aspect, the present application provides an immunogen comprising the aforementioned bovine coronavirus structural protein.

[0027] In an eighth aspect, the present application provides a pharmaceutical composition comprising the aforementioned bovine coronavirus structural protein.

[0028] The pharmaceutical composition can further comprise a pharmaceutically acceptable adjuvant and / or excipient.

[0029] The excipient refers to an excipient and an additional agent used in the production of a pharmaceutical composition and the dispensing of a prescription, which has the important functions of excipient, protection of active ingredients, improvement of stability, solubilization, solubilization aid, controlled release, etc., so as to make the pharmaceutical composition achieve a certain shelf life and bioavailability, thereby improving the safety and effectiveness of the pharmaceutical composition. The excipient that can be co-administered with the pharmaceutical composition of the present application includes but is not limited to sugars, proteins, amino acids, and high molecular weight polymers.

[0030] In a ninth aspect, the present application provides the use of the aforementioned bovine coronavirus structural protein gene in the preparation of a vaccine and / or a drug for treating and / or preventing bovine coronavirus infection.

[0031] The present application has the following beneficial effects:

[0032] (1) The present application expresses the complete E, M, N, S, HE five major structural proteins of bovine coronavirus, and enables the five major structural proteins to complete self-assembly, thereby constructing BCoV VLPs that are extremely similar in morphology and size to natural BCoV virions;

[0033] (2) In the present application, BALB / c mice, SD rats, and Holstein calves are used as experimental animals, an immunization scheme is designed, and immunogenicity evaluation is performed, and the results show that the highest ELISA specific IgG antibody titer of BALB / c mouse serum reaches 1:1.28x10 4 , and the neutralization titer reaches 1:128; the highest ELISA specific IgG antibody titer of Holstein calf serum reaches 1:1.024x10 5 , and the neutralization titer reaches 1:512;

[0034] (3) The BCoV VLPs are mixed with MF59 adjuvant, CpG 55.2 to prepare a vaccine, and after immunization, the BALB / c mice and Holstein calf in vivo can produce humoral and cellular immune responses, in addition, the vaccine can significantly reduce the lung and intestinal BCoV virus load of SD mice after BCoV attack, which shows that the BCoV VLPs are expected to become a new type of BCoV candidate vaccine. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 It is a Bovine coronavirus E, M, N, HE gene PCR amplification electrophoretogram;

[0036] Figure 2 It is a single enzyme digestion, double enzyme digestion verification of the recombinant plasmid;

[0037] Figure 3 It is the growth situation of colonies on three-antibiotic plates after transformation of the recombinant plasmid;

[0038] Figure 4 It is the PCR verification result of five kinds of recombinant bacmids;

[0039] Figure 5 It is the morphological change of Sf9 cells when blind-transferring to the fourth generation after transfection (A: normal Sf9 cell morphology; B: Sf9 cell morphology after inoculation of P4 rpFastBac-E; C: Sf9 cell morphology after inoculation of P4 rpFastBac-M; D: Sf9 cell morphology after inoculation of P4 rpFastBac-N; E: Sf9 cell morphology after inoculation of P4 rpFastBac-S; F: Sf9 cell morphology after inoculation of P4 rpFastBac-HE);

[0040] Figure 6 It is the genomic PCR verification result of P4 rpFastBac-E, rpFastBac-N, rpFastBac-S and rpFastBac-M;

[0041] Figure 7 It is the genomic PCR verification result of rpFastBac-HE;

[0042] Figure 8 It is the Western blot detection of BCoV S protein expression in Sf9 cells;

[0043] Figure 9 It is the Western blot detection of BCoV N protein expression in Sf9 cells;

[0044] Figure 10 It is the indirect immunofluorescence identification result of rpFastBac-N and rpFastBac-S.

[0045] Figure 11 Western blot detection and gray value analysis after infection with different MOI;

[0046] Figure 12 Western blot detection of BCoV VLPs;

[0047] Figure 13 Transmission electron microscopy observation of BCoV VLPs morphology (A: natural BCoV particle morphology; B-F: all are assembled BCoV VLPs);

[0048] Figure 14 IgG antibody titer growth in serum of BALB / c mice after immunization;

[0049] Figure 15 Neutralizing antibody titer growth in serum of BALB / c mice after immunization;

[0050] Figure 16 Flow cytometry detection of cells secreting IFN-γ and IL-4 in mouse spleen lymphocytes (A and B are the number of cells secreting IFN-γ in the spleen lymphocytes of immunized and Mock mice, respectively; C and D are the number of cells secreting IL-4 in the spleen lymphocytes of immunized and Mock mice, respectively);

[0051] Figure 17 Intracellular cytokine staining analysis results (A: IFN-γ analysis results of BALB / c mouse spleen lymphocytes; B: IL-4 analysis results of BALB / c mouse spleen lymphocytes);

[0052] Figure 18 IgG antibody titer growth in calf immune serum;

[0053] Figure 19 Neutralizing antibody titer growth in calf immune serum;

[0054] Figure 20 BCoV viral load detection after SD mouse challenge (A: viral load statistics of SD mouse lung tissue; B: viral load statistics of SD mouse intestinal tract front tissue; C: viral load statistics of SD mouse intestinal tract rear tissue);

[0055] Figure 21The results of HE staining of lung tissue of SD mice after challenge (A, B: lung tissue of normal mice (non-immunized) on the 4th day after challenge (400x and 100x); C, D: lung tissue of inactivated BCoV virus immunized mice on the 4th day after challenge (400x and 100x); E, F: lung tissue of BCoV VLPs mice on the 4th day after challenge (400x and 100x); G, H: lung tissue of negative mice (400x and 100x); the black square represents the 400x observation area; the black arrow represents the red cell exudation and other lesion areas). DETAILED DESCRIPTION

[0056] The application will be further described in detail by specific implementation examples and the accompanying drawings. It should be understood that these examples are only used to illustrate the application and are not used to limit the protection scope of the application. After reading the application, those skilled in the art can modify various equivalent forms of the application, which fall within the scope defined by the appended claims.

[0057] Unless otherwise specified, the culture medium, reagents and solutions used in the following examples are commercially available or can be prepared by known methods in the art.

[0058] Example 1

[0059] 1. Experimental materials

[0060] 1.1 Strains, cells, plasmids and antibodies

[0061] BCoV / SWUN / HXD-4 / 2021 strain and pFastBac-Dual carrier plasmid were obtained from the laboratory preservation; rabbit-derived anti-BCoV N protein polyclonal antibody and rabbit-derived anti-BCoV S2 protein polyclonal antibody were obtained from the laboratory preparation; Sf9 cells were obtained from Wuhan Pnuo Sai Co., Ltd.; pMD19-T carrier was obtained from Baosheng Bioengineering (Dalian) Co., Ltd.; goat anti-mouse IgG (H+L, HRP), goat anti-bovine IgG (H+L, HRP) and goat anti-rabbit IgG (FITC) were obtained from Beijing Doctorde Co., Ltd.

[0062] 1.2 Test reagents

[0063] T4 DNA Ligase, QuickCut Bam H I, QuickCut Eco R I, QuickCut Xho I and QuickCut KpnI, BacPAK™ baculovirus titer rapid assay kit was purchased from Baosheng Engineering (Dalian) Co., Ltd.; Cellfectin™ II Reagent was purchased from Thermo Fisher Scientific (China) Co., Ltd.; Sf9 cell complete / serum-free medium was purchased from Wuhan Punsense Co., Ltd.; Purification / gel recovery kit, E.Z.N.A. Plasmid Mini Kit I, E.Z.N.A. Endo-Free Plasmid Maxi Kit were purchased from OMEGA; Bovine serum albumin (BSA) was purchased from BioFroxx; BCA protein concentration determination kit was purchased from Beijing Comin Biotech Co., Ltd.; Hypersensitive ECL chemiluminescence substrate was purchased from Suzhou Sizuobai Biotechnology Co., Ltd.

[0064] 1.3 Test instrument

[0065] Optima L-100 XP ultracentrifuge was from Beckman; transmission electron microscope (JEM-1400FLASH) was from Japan Electron Co., Ltd. (JEOL); low-temperature centrifuge (centrifuge5427R), polymerase chain reaction instrument, pipettor were from Eppendorf Co., Ltd.; constant-temperature water bath was from Beijing Guangming Medical Instrument Co., Ltd.; protein electrophoresis instrument, membrane transfer instrument, multifunctional imaging system (ChemeiDoc MP Imageing System) were from Bio-Rad Co., Ltd.; ultrapure water instrument was from Thermo Fisher Scientific (China) Co., Ltd.; fluorescent inverted microscope (U-HGLGPS) was from OLYMPUS.

[0066] 2. Test method

[0067] 2.1 Amplification and identification of target protein gene

[0068] Five structural proteins of BCoV / SWUN / HXD-4 / 2021 strain (GenBank: OL456213.1) were selected as templates. Among them, the S gene was codon-optimized for Sf9 cells by Shengong Bioengineering (Shanghai) Co., Ltd. and synthesized, and connected to the pFastBac-Dual vector to obtain the pFastBac-Dual-S recombinant plasmid. The gene amplification primers carrying the enzyme digestion site were designed, and the primer information is shown in Table 1. The E, M, N and HE genes of the BCoV / SWUN / HXD-4 / 2021 strain of bovine coronavirus were amplified by PCR.

[0069]

[0070] 2.2 Construction and verification of recombinant plasmid

[0071] PCR products of each gene fragment of E, M, N and HE were sent to Shenguo Bioengineering (Shanghai) Co., Ltd. for sequencing. After the sequencing results were compared and corrected, the target gene fragments were recovered with gel, ligated to pMD-19T vector, transformed into DH5a competent cells, plated on plates, and single colonies were picked for enrichment, plasmid extraction, and PCR verification using universal primers of pMD-19T vector. After verification, the target gene fragments were obtained by double enzyme digestion, and were ligated to the two polyclonal sites of the final expression vector pFastBac-Dual, i.e. pH promoter and p10 promoter. Single enzyme digestion or double enzyme digestion was used to verify whether the vector was successfully constructed. After verification, the plasmid extraction kit was used to extract the plasmid, and the successfully constructed recombinant plasmids were named as pFastBac-Dual-2E, pFastBac-Dual-2M, pFastBac-Dual-2N, pFastBac-Dual-2HE, respectively, and were stored at -20°C for standby.

[0072] 2.3 Harvesting and verification of recombinant bacmids

[0073] The successfully constructed recombinant plasmids pFastBac-Dual-2E, pFastBac-Dual-2M, pFastBac-Dual-2N, pFastBac-Dual-2HE were transformed into DH10 Bac competent cells, respectively, and were plated on three-antibiotic LB agar plates containing gentamicin, kanamycin and tetracycline, and were placed in a 37°C carbon dioxide incubator for 24-48 hours. During the stationary culture, the morphology of the growing colonies on the plates was observed, and white round single colonies were picked for PCR verification using specific primers. The positive clones were further purified by streaking, at least twice, and false positives were removed by colony PCR verification. The endotoxin-free plasmid extraction kit was used to extract the five recombinant plasmids, which were named as Bacmid-E, Bacmid-M, Bacmid-N, Bacmid-S and Bacmid-HE. After the concentration was determined, each recombinant bacmid was aliquoted according to the mass units required in the subsequent transfection experiment, and was stored at -20°C for standby.

[0074] 2.4 Rescue of recombinant baculovirus

[0075] 5 μg of each of the recombinant bacmids Bacmid-E, Bacmid-M, Bacmid-N, Bacmid-S and Bacmid-HE was taken out, and Sf9 cell transfection was performed according to the instructions of Cellfectin™ II Reagent kit. The transfection process was as follows:

[0076] (1) Prepare the Sf9 cell culture medium for transfection with 1.5% fetal bovine serum, and do not add antibiotics.

[0077] (2) Passage the Sf9 cells, adjust the cell density to 4 x 107 cells / mL, and pass the cells into six-well plates, 2 mL per well, and incubate in a cell incubator for 6-8 h. 5

[0078] (3) Take out the six-well plates, stand at room temperature for 15 min, discard the liquid, and replace with 2.5 mL of the culture medium for transfection.

[0079] (4) Dilute the transfection reagent, add 0.1 mL of the insect cell culture medium into five 1.5 mL EP tubes, and then add 8 μL of the transfection reagent into the tubes, mix well, and stand at room temperature for 30 min.

[0080] (5) Take out 5 μg of the recombinant bacmids Bacmid-E, Bacmid-M, Bacmid-N, Bacmid-S, and Bacmid-HE, and add into five EP tubes, mix well, and stand at room temperature for 15-30 min.

[0081] (6) Add the five kinds of transfection mixtures dropwise into the six-well plates, and place into a 27°C constant-temperature cell incubator to incubate for 3-5 h.

[0082] (7) After the incubation, aspirate the liquid in the wells, add 2 mL of the complete Sf9 cell culture medium per well, and place into a cell incubator to incubate for 3-5 d.

[0083] (8) Harvest the virus liquid, which is used as the first generation virus (P1) and named as rpFastBac-E, rpFastBac-M, rpFastBac-N, rpFastBac-S, and rpFastBac-HE. After blind passage to P4, collect the virus liquid, store in aliquots in a -80°C refrigerator for standby use.

[0084] 2.5 Identification of the recombinant baculovirus

[0085] 2.5.1 Genomic PCR identification

[0086] Extract the genomic DNA of the recombinant baculovirus P4 virus liquid by phenol extraction. Use the extracted DNA of the recombinant baculovirus rpFastBac-E, rpFastBac-M, rpFastBac-N, rpFastBac-S, and rpFastBac-HE as a template to perform genomic PCR identification.

[0087] 2.5.2 Western blot identification ​

[0088] Western blot analysis was used to analyze the expression of BCoV N protein and S protein in Sf9 cells. Recombinant baculoviruses rpFastBac-N and rpFastBac-S were inoculated into monolayer Sf9 cells at a volume ratio of 3%, and after 48 h of static culture at 27°C, the culture solution was discarded, the cells were resuspended with PBS, centrifuged at 12000 rpm for 5 min, and the supernatant was discarded; 200 μL RIPA lysis buffer was added to lyse the cells, 2 μL protease inhibitor was added, and the sample was incubated in an ice bath for 15 min, then centrifuged again, and the supernatant was taken, 5% SDS-PAGE loading buffer was added, and the sample was heated at 95°C for 5 min to complete sample preparation. 5% skim milk was used as blocking solution, and the sample was incubated at 37°C for 2 h, and then washed with TBST for 3 times; Anti-BCoV-S2 protein polyclonal antibody or Anti-BCoV-N protein polyclonal antibody was used as the primary antibody, and the sample was incubated at 4°C for overnight, and then washed with TBST for 3 times; HRP-labeled Goat Anti-rabbit IgG was used as the secondary antibody, and the sample was incubated at 37°C for 2 h, and then washed with TBST for 3 times; the sample was developed using hypersensitive ECL chemiluminescence substrate, and the target band was observed.

[0089] 2.5.3 Indirect immunofluorescence identification

[0090] Sf9 cells with cell culture bottles were passaged, and the cell concentration was adjusted to 4 x 10 5 cells / mL using Sf9 special culture medium, and plated in a six-well plate with 2 mL per well. After 8-10 h, the recombinant baculovirus rpFastBac-S or rpFastBac-N was inoculated at a proportion of 3% of the cell culture solution. After 48 h of static culture at 27°C, the culture solution was discarded, and the cells were fixed with 80% acetone for 10 min and washed with PBS for 3 times; 5% skim milk was used for blocking for 2 h, and then washed with PBS for 3 times; Anti-BCoV-S2 protein polyclonal antibody or Anti-BCoV-N protein polyclonal antibody was used as the primary antibody, and the sample was incubated at 37°C for 2 h, and then washed with PBS for 3 times; FITC-labeled Goat Anti-rabbit IgG was used as the secondary antibody, and the sample was incubated at 37°C for 1 h, and then washed with PBS for 3 times; a few drops of DAPI staining solution were added, and the sample was incubated at room temperature for 10 min, and then washed with PBS for 3 times; the residual liquid was absorbed with filter paper, and then fluorescence microscopy imaging was performed.

[0091] 2.6 Determination of the titer of recombinant baculovirus

[0092] The titer of the P4 recombinant baculovirus rpFastBac-E, rpFastBac-M, rpFastBac-N, rpFastBac-S, and rpFastBac-HE was determined according to the BacPAK™ Baculovirus Titer Quick Assay Kit instructions. The specific steps are briefly described as follows:

[0093] (1) Passage Sf9 cells, 6.5 x 10 4 Sf9 cells were added to each well, and the 96-well plate was placed in a sealing bag matched with the kit, and was left in a cell incubator for 1 h.

[0094] (2) Dilute the recombinant baculovirus, and the dilution gradient was 10 -3 , 10 -4 , and 10 -5 three gradients.

[0095] (3) Discard the liquid in the well, and add 25 μL of the diluted recombinant baculovirus liquid, and 3 repeats were performed for each dilution gradient, and a negative control well was set up, and 25 μL of Sf9 cell culture medium was added to the negative control well.

[0096] (4) Place it in a matched sealing bag, and leave it at room temperature for 1 h.

[0097] (5) Discard the liquid in the well, and then add 50 μL of methyl cellulose to each well, and place it in a sealing bag, and leave it in a constant temperature cell incubator at 27°C for 43-47 h.

[0098] (6) Add 150 μL of pre-cooled acetone to each well, leave it for 10 min, then discard the liquid, and wash with 0.2 mL of PBST solution, 3 times, 5 min each time.

[0099] (7) Add 50 μL of normal goat serum to each well, and leave it at room temperature for 5 min, and discard the liquid.

[0100] (8) Add 50 μL of gp64 mouse monoclonal antibody to each well, and leave it at 37°C for 25 min, then discard the liquid, and wash with PBST 3 times, 5 min each time.

[0101] (9) Add HRP-labeled Goat anti-mouse antibody to each well, and leave it at 37°C for 25 min, then discard the liquid, and wash with PBST 3 times, 5 min each time.

[0102] (10) Add 50 μL of substrate to each well, and leave it at room temperature for 3 h to develop color, and after obvious blue spots appeared in the well, count the number of blue spots under a microscope, and calculate the baculovirus titer using the formula.

[0103] 2.7 Assembly of BCoV VLPs

[0104] 2.7.1 Optimization of assembly efficiency of BCoV VLPs

[0105] Infect Sf9 cells with rpFastBac-N or rpFastBac-S at MOI = 0.005, MOI = 0.01, MOI = 0.03, MOI = 0.05, MOI = 0.5, MOI = 1, respectively, and after 3 d, discard the supernatant, collect the cells, and prepare the samples according to the method in 1.9. Detect the expression of N protein or S protein after infecting Sf9 cells with baculovirus rpFastBac-N or rpFastBac-S by Western blot, and analyze the gray value of the N protein and S protein bands by Image J software to screen the best protein expression condition to maximize the protein expression and improve the assembly efficiency of BCoV VLPs.

[0106] 2.7.2 Assembly and harvesting of BCoV VLPs

[0107] Subculture the Sf9 cells that grow on the cell culture bottle, adjust the cell concentration to 4 x 10 5 cells / mL with Sf9 special medium, inoculate into T75 cm 2 cell bottles, add 15 mL of Sf9 cell suspension to each cell bottle, and after standing culture at 27°C for 5-6 h, co-infect the Sf9 cells according to the optimized conditions, continue to stand culture at 27°C for 120 h, freeze-thaw repeatedly for 3 times, centrifuge at 4°C at 3000 rpm for 20 min to collect the supernatant for standby, lyse the precipitate with RIPA lysis buffer, and at the same time, add a small amount of protease inhibitor to inhibit protein degradation during the lysis process, ice bath for 15 min, centrifuge at 4°C at 12000 rpm for 5 min, and collect the supernatant for standby. Mix the above supernatant uniformly, and then perform ultrasonic crushing, with crushing conditions of power 40%, 5 s on and 5 s off, for a total of 15 min. After crushing, centrifuge at 4°C at 10000 rpm for 20 min, and the supernatant is BCoV VLPs before purification. Concentrate BCoV VLPs by ultracentrifugation, centrifuge at 4°C at 30000 rpm for 2 h, resuspend the precipitate with PBS, and store in a -80°C refrigerator.

[0108] 2.8 Identification of BCoV VLPs

[0109] 2.8.1 Identification by Western blot

[0110] The purified BCoV VLPs were mixed with the loading buffer for SDS-PAGE at a ratio of 4:1 by volume, briefly vortexed, and boiled for 5-10 min. Western blot was performed using rabbit anti-BCoV-S2 protein polyclonal antibody and rabbit anti-BCoV-N protein polyclonal antibody as the primary antibody, and HRP-labeled goat anti-rabbit IgG as the secondary antibody, to observe the S and N protein bands.

[0111] 2.8.2 Electron microscope observation of BCoV VLPs morphology

[0112] The concentrated and purified BCoV VLPs solution was dropped onto a copper grid and left for 5 min. Excess liquid was absorbed with filter paper, and the copper grid was air-dried. Then 1% phosphotungstic acid was added, and the copper grid was stained at room temperature for 2-3 min. The liquid on the copper grid was absorbed with filter paper, and the copper grid was air-dried at room temperature. The morphology of the BCoV VLPs was observed and photographed using a JEM-1400 FLASH transmission electron microscope.

[0113] 3. RESULTS

[0114] 3.1 Amplification and identification of target protein genes

[0115] As shown in FIG. 1, the amplified E gene was about 250 bp in size, the amplified M gene was about 700 bp in size, the amplified N gene was about 1300 bp in size, and the amplified HE gene was about 1200 bp in size, indicating that the E, M, N, and HE genes were successfully amplified. Figure 1 3.2 Construction and verification of recombinant plasmids

[0116] As shown in FIG. 2, the recombinant plasmids pFastBac-Dual-2E, pFastBac-Dual-2M, pFastBac-Dual-2N, pFastBac-Dual-2HE, and pFastBac-Dual-S each produced two distinct band fragments after double digestion, which were the vector band fragment and the target band fragment, indicating that the five recombinant plasmids pFastBac-Dual-2E, pFastBac-Dual-2M, pFastBac-Dual-2N, pFastBac-Dual-2HE, and pFastBac-Dual-S were successfully constructed.

[0117] Figure 2 3.3 Harvesting and identification of recombinant bacmids

[0118] 3.3 Harvesting and identification of recombinant bacmids

[0119] ​The 3.2 identified successful plasmids were transformed into DH10Bac E. coli competent cells, respectively, and coated on LB agar plates containing gentamicin, kanamycin, and tetracycline. Round color or blue colonies were observed, and the blue colonies were untransformed or empty (Fig. 3.3). Figure 3 White colonies were selected by colony PCR to screen positive clones, as shown in Fig. 3.4, five recombinant bacmids each carrying the target gene, i.e., E gene, M gene, N gene, S gene, and HE gene, indicating that the five recombinant bacmids were successfully constructed. Figure 4

[0120] 3.4 Rescue of recombinant baculovirus

[0121] When the fourth generation (P4) was passed, typical lesions appeared in Sf9 cells under a microscope, and the specific lesions were large and round cells, slow cell growth, and cell shedding with time (Fig. 3.7). Figure 5

[0122] 3.5 Identification of recombinant baculovirus

[0123] 3.5.1 Genomic PCR detection results

[0124] As shown in Fig. 3.4, five recombinant bacmids each carrying the target gene, i.e., E gene, M gene, N gene, S gene, and HE gene, indicating that the five recombinant bacmids were successfully constructed. Figure 6~7

[0125] 3.5.2 Western blot identification results

[0126] As shown in Fig. 3.9, it showed specific binding with Anti-BCoV-S2 and Anti-BCoV-N protein polyclonal antibodies, indicating that the recombinant baculovirus infected Sf9 cells could effectively express the S target protein and N target protein of bovine coronavirus, and had good reactogenicity. Figure 8~9

[0127] 3.5.3 Indirect immunofluorescence results

[0128] Using an inverted fluorescence microscope, it was observed that the test group produced brighter green fluorescence compared to the normal Sf9 cell control well (Fig. 3.10), indicating that the constructed baculovirus effectively expressed the S target protein and N target protein. Figure 10

[0129] 3.6 Determination of recombinant baculovirus titer

[0130] The titers of recombinant baculoviruses rpFastBac-E, rpFastBac-M, rpFastBac-N, rpFastBac-S, and rpFastBac-HE were 1.16 x 10​​​​​8 IFU / mL, 4.45×10 8 IFU / mL, 2.1×10 8 IFU / mL, 1.016×10 7 IFU / mL, 1.44×10 8 IFU / mL; The recombinant baculovirus constructed in this invention has good quantity and density, and the viral titer meets the requirements, which can be applied to the inoculation of Sf9 cells to complete the expression of various target proteins.

[0131] 3.7 Assembly of BCoV VLPs

[0132] 3.7.1 Optimize the assembly efficiency of BCoV VLPs

[0133] like Figure 11 As shown in the figure, the analysis of protein band gray values ​​reveals that the expression level of S protein reached its highest after infecting Sf9 cells with rpFastBac-S at an MOI of 0.005; the expression level of N protein reached its highest after infecting Sf9 cells with rpFastBac-N at an MOI of 0.05. Therefore, co-infecting Sf9 cells with recombinant baculoviruses rpFastBac-S, rpFastBac-N, rpFastBac-E, rpFastBac-M, and rpFastBac-HE at MOIs of 0.005, 0.05, 0.05, 0.05, and 0.05, respectively, is the optimal infection condition for subsequent assembly of BCoV VLPs, increasing protein expression and thus improving the assembly efficiency of BCoV VLPs.

[0134] 3.7.2 Assembly and Identification of BCoV VLPs

[0135] like Figure 12 As shown, the S protein band is located at approximately 250 kDa, and the N protein band is located at approximately 50 kDa; Figure 13 As shown, the morphology of BCoV VLPs observed under a transmission electron microscope is similar to that of natural bovine coronavirus particles. The diameter of BCoV VLPs is approximately 70–120 nm, indicating that BCoV VLPs were successfully constructed.

[0136] Example 1: Immunogenicity study of bovine coronavirus-like particles

[0137] 1. Experimental Methods

[0138] 1.1 Immunization regimen for BALB / c mice

[0139] A total of 28 4-6 week old female BALB / c mice were purchased and randomly divided into 4 groups, namely BCoV VLPs group, BCoV inactivated virus control group, MF59 adjuvant control group and PBS control group, 7 mice in each group. Among them, the immunogen of BCoV VLPs group was 50 μg BCoV VLPs, 20 μg CpG 55.2, and the same volume of MF59 adjuvant, and was mixed and emulsified; the immunogen of BCoV inactivated virus group was 200 μL of inactivated virus liquid (dose of 2×10 4.67 TCID 50 ) and the same volume of MF59 adjuvant mixed and emulsified; the MF59 adjuvant control group was injected with 50 μL of MF59 adjuvant per mouse; the PBS group was injected with 50 μL of sterile PBS per mouse. All BALB / c mice were immunized by intramuscular injection. A total of two immunizations were performed, the first immunization and the second immunization were separated by 14 days, i.e. the second immunization was performed 14 days after the first immunization, and the mice were taken blood from the eye socket at the time points of before immunization, 7 days after the first immunization, 14 days after the first immunization, 7 days after the second immunization, and 14 days after the second immunization. When blood was collected, the blood was collected into a 1.5 mL EP tube, and the serum was separated after standing at room temperature for 3-6 h. After low-speed centrifugation at 5000 rpm for 10 min, the upper serum was aspirated and collected for subsequent evaluation of immunization effect.

[0140] 1.2 BALB / c mouse serum indirect ELISA test

[0141] The collected BALB / c mouse serum samples were detected for specific antibody levels, and the steps of indirect ELISA test were as follows: the BCoV S2 protein obtained by prokaryotic expression was diluted with 50 mM Tris-HCL solution to 0.1 μg / mL coating solution, 100 μL was added to each well of the enzyme-labeled plate, and after 1 h of coating at 37°C, the liquid in the well was discarded, and PBST was washed three times, each for 3 min; 100 μL of 5% skim milk was added to each well, and after 1 h of blocking at 37°C, the liquid was discarded, and PBST was washed three times, each for 3 min; the collected mouse serum was used as the primary antibody, and after gradient dilution with 2% cold water fish gelatin, 100 μL was added to each well, and after 1 h of incubation at 37°C, the liquid was discarded, and PBST was washed three times, each for 3 min; the HRP-labeled goat anti-mouse IgG was diluted with 2% cold water fish gelatin according to 1:10000, and 100 μL was added to each well, and after 1 h of incubation at 37°C, the liquid was discarded, and PBST was washed three times, each for 3 min; TMB color developing liquid was added, 100 μL was added to each well, and color development was performed at 37°C for 10 min in the dark, and finally 50 μL of stop solution was added to each well to terminate the reaction, and the OD450nm value was read using an enzyme-labeled instrument.

[0142] 1.3 BALB / c mice serum micro-neutralization test (VN)

[0143] HCT-8 cells in cell culture bottles were passaged into 96-well plates and incubated at 37°C for 8-10 h; mouse serum was placed in a 56°C water bath for inactivation for 30 min, and 2-fold dilutions were prepared with DMEM medium (without fetal bovine serum and antibiotics) and mixed with 200 TCID 50 of BCoV virus solution, and then placed in a 37°C cell incubator for 1 h; the 96-well plate containing HCT-8 cells was removed, the liquid was discarded, and the plate was washed twice with PBS. The mixed culture solution after the incubation was added to the 96-well plate and incubated for 96 h. The pathological changes in each well were observed and recorded, and the titer of the serum neutralizing antibody was calculated according to the Reed-muech method.

[0144] 1.4 Isolation of BALB / c mouse spleen lymphocytes

[0145] The eyes of the mice 14 days after the second immunization were bled, and the mice were then dislocated and sacrificed. The spleen lymphocytes were isolated by soaking the mice in 75% ethanol for 5-10 min, and the following steps were performed:

[0146] (1) The mouse spleen was removed and placed in a small dish with 4-5 mL complete medium, and was ground in a filter screen. The filter screen was washed with RPMI-1640 medium, and the cells were collected and centrifuged at 2000 rpm for 15 min.

[0147] (2) The supernatant was discarded, 20 mL of red blood cell lysis solution was added, and after lysis for 5 min, the cells were centrifuged at 2000 rpm for 15 min. The supernatant was discarded, and the operation was repeated once.

[0148] (3) The supernatant was discarded, 10 mL of RPMI-1640 medium was added to resuspend the cells, and the cells were centrifuged at 2000 rpm for 15 min. After centrifugation, the supernatant was discarded, and the washing was repeated once.

[0149] (4) The cell pellet was resuspended with 2 mL of RPMI-1640 medium, and the number of cells was counted.

[0150] (5) The cell density was adjusted to 1x106 cells / mL.

[0151] 1.5 Flow cytometry test

[0152] 1.5.1 Detection of spleen T lymphocyte subsets

[0153] The detection of BALB / c mouse spleen CD3 + , CD4 + , and CD8 + T cells was performed according to the following test steps:

[0154] (1) Take out the six-hole plate, add 2 mL of the above diluted cell suspension to each hole, add 20 μg of inactivated BCoV virus liquid and 2 μL of protein transport inhibitor, and set up a hole without adding stimulant as a control, and put it into the cell culture box for 6 h.

[0155] (2) Collect the cells in the six-hole plate, centrifuge at 2000 rpm for 15 min at room temperature. Discard the supernatant, and resuspend the cells with 1 mL of PBS solution containing 2% FBS.

[0156] (3) Take 100 μl of the cell suspension into a flow tube, add 1 μg of CD3, CD4, and CD8 fluorescent antibodies respectively, and incubate at 4°C for 30 min.

[0157] (4) Add 2 ml of PBS solution to resuspend the cells, centrifuge at 300 g, and discard the supernatant. Repeat this step once.

[0158] (5) Add 400 μl of PBS solution to resuspend the cells, and immediately detect on the machine.

[0159] (6) Analyze the results with Everest software.

[0160] 1.5.2 Detection of IL-4 and IFN-γ in Spleen Lymphocytes

[0161] The detection of IL-4 and IFN-γ in the spleen cells of BALB / c mice is carried out according to the following test steps:

[0162] (1) Add 2 mL of cell suspension to the six-hole plate, add 20 μg of inactivated BCoV virus liquid and 2 μL of protein transport inhibitor, and set up a control hole, and incubate in the cell culture box for 6 h.

[0163] (2) Collect the cells, centrifuge at 2000 rpm for 15 min. Discard the supernatant, and resuspend the cells with 1 mL of PBS.

[0164] (3) Take 100 μL of the cell suspension into a flow tube, add 1 mL of Foxp3 fixing / breaking membrane working solution to each tube and pulse vortex, and incubate at room temperature for 40 min.

[0165] (4) Add 2 mL of breaking membrane liquid to each tube, centrifuge the sample at 500 g for 5 minutes at room temperature. Discard the supernatant. Repeat the operation once.

[0166] (5) Add 1 μg of IL-4 and IFN-γ fluorescent antibodies, incubate at 4°C for 30 min, add 2 ml of PBS to resuspend the cells, centrifuge at 500 g, discard the supernatant, and add 400 μL of PBS to resuspend the cells.

[0167] (6) Immediately run the test and analyze the results using Everest software.

[0168] 1.6 Immunization Program for Holstein Calves

[0169] To evaluate the immunization efficacy of the BCoV VLPs vaccine in cattle, nine 1- to 2-month-old female calves were selected as experimental animals and randomly divided into three groups of three calves each: the VLP group, the BCoV inactivated virus control group, and the PBS control group. Two mg of BCoV VLPs were mixed with an equal volume of MF59 adjuvant, emulsified, and used to prepare the vaccine, which was then administered to the VLP group calves. Two × 10⁻⁶ sachets were used as the control group. 4.67 TCID 50 An equal volume of MF59 adjuvant was mixed with inactivated BCoV virus solution and used to immunize calves in the BCoV inactivated virus control group; 2 mL of PBS was used to immunize calves in the PBS control group. Two immunizations were administered, 21 days apart. Blood samples were collected before immunization and at 21, 38, and 55 days after immunization to collect serum.

[0170] 1.7 Indirect ELISA test of calf serum

[0171] The specific procedure for this experiment is the same as in 1.2. The antigen coated on the ELISA plate is BCoV S2 protein, the primary antibody is serum collected from Holstein calves immunized with BCoV VLPs vaccine, and the secondary antibody is Goat anti-Bovine IgG (HRP-labeled).

[0172] 1.8 Calf serum micro-neutralization test

[0173] The specific procedures for this experiment are the same as in 1.3. The strain used in the neutralization experiment was BCoV / SWUN / HXD-4 / 2021, and the cell line was HCT-8. The serum neutralizing antibody titer was calculated using the Reed-Muech method.

[0174] 2 Results

[0175] 2.1 Analysis of Specific IgG Antibody Titer in BALB / c Mice

[0176] like Figure 14 As shown, the serum antibody titer of mice in the inactivated BCoV virus group began to gradually increase one week after the first immunization, but the overall antibody level was not high. The highest titer reached one week after the booster immunization was only 1:1600, which was significantly lower than that in the BCoV VLPs group. p<0.01). The mice in the BCoV VLPs group were able to detect specific IgG antibodies against S protein in serum 1 week after the first immunization, and the specific IgG antibody titers of the serum of the mice in the BCoV VLPs group showed an upward trend after the first immunization, and the antibody titers reached 1:12800 2 weeks after the second immunization, which was significantly higher than that of the inactivated virus group and the PBS control group p <0.05). Compared with the inactivated BCoV virus control group, the S protein IgG antibody titer of the mice in the BCoV VLPs group was about 5 times higher. The above test results show that the BCoV VLPs vaccine can induce an effective humoral immune response in BALB / c mice.

[0177] 2.2 Analysis of neutralizing antibody titers in the immune serum of BALB / c mice

[0178] As shown in Figure 15 , 1 week after the first immunization, the serum of the mice in the BCoV VLPs group and the inactivated BCoV virus group could detect anti-BCoV neutralizing antibodies, and the neutralizing antibody titer of the BCoV VLPs group reached a peak 1 week after the second immunization, with the highest serum neutralization titer being 1:128. The neutralization titer of the inactivated BCoV virus group was only 1:32 at most, and the neutralization titer showed a gradual downward trend after the second immunization. Compared with the inactivated BCoV virus control group, the neutralization titer of the serum of the mice in the BCoV VLPs group was about 4.6 times higher p <0.01). The above results show that the neutralizing antibody titer produced by the BCoV VLPs group is higher than that of the inactivated BCoV virus group, and the BCoV VLPs vaccine has a stronger ability to induce BCoV neutralizing antibodies in mice than the inactivated BCoV.

[0179] 2.3 Intracellular factor staining analysis

[0180] As shown in Figure 16~17 , compared with the control group, the BCoV VLPs immunized mice induced higher levels of IFN-γ, showing a very significant difference p <0.01), but no significant difference in the secretion level of IL-4 was observed p >0.05).

[0181]

[0182] 2.4 Analysis of IgG antibody titers in calf immune serum

[0183] As shown in Figure 18As shown, the serum IgG antibody titers of the BCoV VLPs group and the inactivated BCoV virus group of the calves increased after the first immunization, and the titers were comparable before the second immunization. However, after the second immunization, the serum IgG antibody titers of the calves of the BCoV VLPs group increased rapidly, and reached the highest at 4 weeks after the second immunization, with the highest IgG antibody titer of 1:102400, while the highest serum IgG antibody titer of the calves of the inactivated BCoV group reached only 1:12800 during the period. The serum IgG antibody titer of the calves of the BCoV VLPs group was about 5 times higher than that of the inactivated BCoV control group. The above test results show that the constructed BCoV VLPs can induce a strong humoral immune response in the calves, and the immunization effect is better than that of the inactivated BCoV virus liquid.

[0184] 2.5 Analysis of neutralizing antibody titers in the immune serum of the calves

[0185] As shown, the serum IgG antibody titers of the BCoV VLPs group and the inactivated BCoV virus group of the calves increased after the first immunization, and the titers were comparable before the second immunization. However, after the second immunization, the serum IgG antibody titers of the calves of the BCoV VLPs group increased rapidly, and reached the highest at 4 weeks after the second immunization, with the highest IgG antibody titer of 1:102400, while the highest serum IgG antibody titer of the calves of the inactivated BCoV group reached only 1:12800 during the period. The serum IgG antibody titer of the calves of the BCoV VLPs group was about 5 times higher than that of the inactivated BCoV control group. The above test results show that the constructed BCoV VLPs can induce a strong humoral immune response in the calves, and the immunization effect is better than that of the inactivated BCoV virus liquid. Figure 19 p <0.05). It is shown that the BCoV VLPs immunized calves can effectively induce the production of neutralizing antibodies, and the effect of inducing the production of neutralizing antibodies is better than that of the inactivated BCoV virus liquid.

[0186] Test Example 2 Evaluation of the immunization effect of BCoV VLPs in SD rats and suckling mice

[0187] 1 Experimental method

[0188] 1.1 Immunization of SD pregnant mice and challenge of suckling mice

[0189] ​Four SD pregnant female mice were purchased as test animals and divided into four groups, namely BCoV VLPs group, BCoV S protein group, BCoV inactivated virus control group and normal control group, one in each group. Among them, the immunogen of BCoV VLPs group was 200 μg BCoV VLPs, 20 μg CpG 55.2 and the same volume of MF59 adjuvant mixed and emulsified, the immunogen of BCoV S protein group was 200 μg BCoV S protein mixed with the same volume of MF59 adjuvant, the immunogen of BCoV inactivated virus group was 150 μL of inactivated BCoV virus liquid (i.e. 1.5 × 10 4.67 TCID 50 ) mixed with the same volume of MF59 adjuvant, and the normal control group was not treated. The first immunization was performed on the first day of pregnancy of the SD female rats, and the second immunization was performed 14 days later. Blood was collected 4 days after the second immunization, and the serum was separated for use.

[0190] After the SD pregnant female mice gave birth, the female mice and the suckling mice were raised in the same cage for 7 days, and then challenged. The challenge method was gavage, and the dose was 0.1 mL of BCoV virus liquid (i.e. 10 4.67 TCID 50 ). One day, 3 days, 5 days, 7 days and 9 days after the challenge, 4 suckling mice were randomly killed from each group, marked, and the lung and intestinal tissue samples of the suckling mice were collected after the killing.

[0191] 1.2 Indirect ELISA test of SD pregnant mouse serum

[0192] The specific operation method of this test is the same as that of 1.2. The antigen coated on the enzyme-labeled plate is BCoV S2 protein, the first antibody is the serum collected from the SD pregnant mice immunized with BCoV VLPs vaccine, and the second antibody is Goat anti-Mouse IgG (HRP labeled).

[0193] 1.3 Micro-neutralization test of SD pregnant mouse serum

[0194] The specific operation method of this test is the same as that of 1.3. The strain used in the neutralization test is BCoV / SWUN / HXD-4 / 2021, and the cell line is HCT-8. The titer of the neutralizing antibody in the serum is calculated according to the Reed-muech method.

[0195] 1.4 Detection of BCoV virus load in the lungs and intestines of SD suckling mice

[0196] After the SD suckling mice were decapitated, the tissues were collected, the total RNA of the lung and intestinal tissues was extracted, and the cDNA was reverse transcribed. The BCoV virus load in the lung and intestinal tissues was detected by the absolute fluorescent quantitative PCR method based on N protein established in this experiment, and then the experimental data was calculated, statistically analyzed and analyzed.

[0197] 1.5 SD rat lung tissue sections HE staining

[0198] The lung tissue of SD rat was fixed in a 10 mL tube with 4% paraformaldehyde solution, and the paraffin embedding and sectioning of the lung tissue sample of SD rat were performed by Chengdu Life Technologies Co., Ltd., and HE staining was completed. The stained sections were observed and recorded under a microscope, and finally the test data were statistically arranged and analyzed.

[0199] 2 Results

[0200] 2.1 Analysis of serum titer of SD pregnant mice

[0201] The detection results are shown in Table 3. After two immunizations, specific IgG antibody levels were detected in all groups except the normal control group. The IgG antibody titers and neutralizing antibody titers of the BCoV VLPs group of pregnant mice were 1:3200 and 1:32, respectively, and the IgG antibody titer was higher than that of other groups, while the neutralizing antibody titer was consistent with that of the S protein group. The IgG antibody titers and neutralizing antibody titers of the inactivated BCoV virus group of pregnant mice were 1:1600 and 1:16, respectively. The IgG antibody titers and neutralizing antibody titers of the S protein group of pregnant mice were 1:1600 and 1:32, respectively. No antibody level was detected in the normal control group. The above results show that BCoV VLPs vaccine can induce the production of humoral immune response in pregnant mice.

[0202]

[0203] 2.2 Detection of tissue viral load of BCoV-infected SD rat

[0204] As shown in Table 4, the viral load in the lung tissue of the BCoV VLPs group was significantly lower than that of the normal control group (P<0.05) on the 5th, 6th, 7th and 8th day after infection, indicating that BCoV VLPs had a protective effect on the infection of BCoV in rats. Similar to the detection results of lung tissue, the viral load in the anterior and posterior intestinal tissues of the BCoV VLPs group was significantly lower than that of the normal control group (P<0.01) on the 5th, 6th, 7th and 8th day after infection, indicating that BCoV VLPs played a certain protective role in the infection process of rats. Figure 20 p <0.05), indicating that BCoV VLPs had a protective effect on the infection of BCoV in rats. Similar to the detection results of lung tissue, the viral load in the anterior and posterior intestinal tissues of the BCoV VLPs group was significantly lower than that of the normal control group (P<0.01) on the 5th, 6th, 7th and 8th day after infection, indicating that BCoV VLPs played a certain protective role in the infection process of rats. p

[0205] 2.3 HE staining results of lung tissue of BCoV-infected rats

[0206] As shown in Table 4, the viral load in the lung tissue of the BCoV VLPs group was significantly lower than that of the normal control group (P<0.05) on the 5th, 6th, 7th and 8th day after infection, indicating that BCoV VLPs had a protective effect on the infection of BCoV in rats. Similar to the detection results of lung tissue, the viral load in the anterior and posterior intestinal tissues of the BCoV VLPs group was significantly lower than that of the normal control group (P<0.01) on the 5th, 6th, 7th and 8th day after infection, indicating that BCoV VLPs played a certain protective role in the infection process of rats. Figure 21 ​​As shown, the normal control group of unimmunized neonatal rat lungs showed local alveolar septum thickness increased, and inflammatory cell infiltration in the lung interstitium. The same phenomenon was observed in the inactivated BCoV group of neonatal rat lungs. In the BCoV VLPs group, the lesions observed in the HE staining of the neonatal rat lungs were lighter compared with the control group. The above results show that the BCoV VLPs vaccine reduces the invasion of BCoV to the lungs, and provides a certain degree of protection for the infected neonatal rats.

[0207] The principles and implementation manners of the present application are described by using specific examples in the present text, and the above example descriptions are only used to help understand the technical solutions and core ideas of the present application; those skilled in the art should understand that the technical solutions recorded in the foregoing examples can still be modified, or some technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for preparing bovine coronavirus virus-like particles, characterized in that, include: (1) Design and amplify bovine coronavirus structural protein genes; wherein the bovine coronavirus structural protein genes consist of the following proteins: E gene, M gene, N gene, S gene and HE gene; the nucleotide sequence of the E gene is shown in SEQ ID NO.1; the nucleotide sequence of the M gene is shown in SEQ ID NO.2; the nucleotide sequence of the N gene is shown in SEQ ID NO.3; the nucleotide sequence of the S gene is shown in SEQ ID NO.4; and the nucleotide sequence of the HE gene is shown in SEQ ID NO.

5. (2) Construct recombinant plasmids; (3) Constructing recombinant rod particles; (4) Transfect cells with the recombinant baculovirus from (3); (5) Recombinant baculoviruses were co-infected with cells to obtain bovine coronavirus virus-like particles; wherein, recombinant baculoviruses rpFastBac-S, rpFastBac-N, rpFastBac-E, rpFastBac-M, and rpFastBac-HE were co-infected with Sf9 cells at MOIs of 0.005, 0.05, 0.05, 0.05, and 0.05, respectively.

2. The method for preparing bovine coronavirus virus-like particles according to claim 1, characterized in that, At least one of the E gene, M gene, N gene, and HE gene is amplified; during amplification, the forward primer for the nucleotide sequence of the E gene is shown in SEQ ID NO.6, and the reverse primer sequence is shown in SEQ ID NO.7; The forward primer sequence for the nucleotide sequence of the M gene is shown in SEQ ID NO. 8; the reverse primer sequence is shown in SEQ ID NO.

9. The forward primer sequence for the N gene nucleotide sequence is shown in SEQ ID NO.10; the reverse primer sequence is shown in SEQ ID NO.

11. The forward primer sequence for the HE gene is shown in SEQ ID NO. 12; the reverse primer sequence is shown in SEQ ID NO.

13.

3. A bovine coronavirus virus-like particle, characterized in that, It is obtained by the method for preparing bovine coronavirus virus-like particles as described in claim 1 or 2.

4. An immunogen, characterized in that, It includes bovine coronavirus virus-like particles obtained by the method for preparing bovine coronavirus virus-like particles as described in claim 1 or 2.

5. A pharmaceutical composition, characterized in that, include: Bovine coronavirus virus-like particles obtained by the method for preparing bovine coronavirus virus-like particles according to claim 1 or 2.

6. Use of the bovine coronavirus virus-like particles obtained by the method for preparing bovine coronavirus virus-like particles according to claim 1 or 2 in the preparation of vaccines and / or medicaments for the treatment and / or prevention of bovine coronavirus infection.

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