A sars-cov-2 virus-like particle and preparation method and application thereof

Abstract

This invention discloses a SARS-CoV-2 virus-like particle, its preparation method, and its application. The genes encoding the structural proteins of the novel coronavirus include the S gene, M gene, or E gene. The nucleotide sequence of the S gene is shown in SEQ ID NO.1; the nucleotide sequence of the M gene is shown in SEQ ID NO.2; and the nucleotide sequence of the E gene is shown in SEQ ID NO.3. This invention provides, for the first time, a virus-like particle (VLP), which is self-assembled after expression of the three structural proteins S, M, and E of the SARS-CoV-2 virus via a baculovirus-insect cell expression system. This invention solves the problem of the current lack of a safe and effective VLP vaccine for the prevention and control of COVID-19. The VLP is suitable for mucosal immunization, improving the immunization effect while reducing vaccination and labor costs, and has potential economic benefits.

Description

Technical Field

[0001] This invention relates to a SARS-CoV-2 virus-like particle, its preparation method, and its application, belonging to the field of biotechnology. Background Technology

[0002] The novel coronavirus (SARS-CoV-2) is a single-stranded RNA virus belonging to the β-coronavirus family. It encodes 29 proteins, including 4 structural proteins and 25 non-structural and accessory proteins. The four main structural proteins of SARS-CoV-2 are the spike protein (S), nucleocapsid protein (N), membrane protein (M), and envelope protein (E). The S protein is composed of two functional subunits, S1 and S2. The receptor-binding domain (RBD) in the S1 subunit binds to angiotensin-converting enzyme 2 (ACE2) on the host cell membrane, mediating viral entry into the host cell (Jackson CB, Farzan M, Chen B, et al. Mechanisms of SARS-CoV-2 entry into cells[J]. Nat Rev Mol Cell Biol,2022,23(1):3-20.). Therefore, the S protein is a major target for the development of novel coronavirus vaccines.

[0003] As of March 2023, 183 COVID-19 vaccines had entered clinical trials. Subunit vaccines were the most numerous, accounting for 32%. Virus-like particles (VLPs) are highly structured protein particles composed of one or more viral structural proteins that assemble themselves. They do not contain viral genetic material and pose no risk of reverse mutations or pathogenic infection. VLPs can stimulate the immune system by recognizing repeating subunits, generating cellular and humoral immune responses. A, Melado MC, Castilho LR, et al. Virus-like particles in vaccine development[J]. Expert Rev Vaccines, 2010, 9(10): 1149-1176.). Currently, several VLP-based vaccines have been commercially produced, such as recombinant hepatitis B virus vaccine, human papillomavirus vaccine, and porcine circovirus type 2 vaccine, but there are still no reports of SARS-CoV-2 VLP vaccines being marketed.

[0004] Commonly used systems for expressing VLPs include bacteria, yeast, insect cells, and mammalian cells. Among them, bacteria and yeast are favored due to their rapid growth and high yield, but they cannot provide complex post-translational modifications. Mammalian cell production is costly and inefficient (Fuenmayor J, Gòdia F, Cervera L. Production of virus-like particles for vaccines[J].N Biotechnol,2017,39(Pt B):174-180.). For decades, baculovirus expression systems have been effectively used as a mature platform for expressing exogenous proteins in vaccine production and gene therapy. Its advantages are that the system can be mass-produced at low cost, and BEVS can insert large exogenous DNA fragments, express multiple exogenous proteins, and provide appropriate post-translational modifications (Hong Q, Liu J, Wei Y, et al. Application of baculovirus expression vector system (BEVS) in vaccine development[J].Vaccines(Basel),2023,11(7):1218.).

[0005] In summary, there is an urgent need to develop a safe, efficient, and economical method for preparing SARS-CoV-2 virus-like particles to provide technical and product support for the global prevention and control of the novel coronavirus and its variants. Summary of the Invention

[0006] Purpose of the invention: The technical problem to be solved by the present invention is to provide, for the first time, a SARS-CoV-2 virus-like particle, its preparation method and application.

[0007] Technical solution: To solve the above-mentioned technical problems, the present invention provides a gene encoding a structural protein of a novel coronavirus, wherein the structural protein gene includes an S gene, an M gene, or an E gene; the nucleotide sequence of the S gene is shown in SEQ ID NO.1; the nucleotide sequence of the M gene is shown in SEQ ID NO.2; and the nucleotide sequence of the E gene is shown in SEQ ID NO.3.

[0008] The present invention also provides a structural protein encoded by the said gene.

[0009] The present invention also provides an expression cassette, a recombinant vector, and a recombinant virus containing the gene of the structural protein.

[0010] The present invention also provides a recombinant rod-like particle containing the recombinant carrier.

[0011] The present invention also provides a recombinant baculovirus containing the recombinant baculovirus.

[0012] The present invention also provides a novel coronavirus virus-like particle, which is purified from Sf9 cells infected with the recombinant baculovirus.

[0013] The present invention also provides a method for constructing the novel coronavirus virus-like particles, comprising the following steps: transforming a recombinant vector containing the S gene, M gene or E gene into competent cells respectively, transfecting recombinant baculoviruses into Sf9 cells respectively, and co-infecting Sf9 cells with recombinant baculoviruses to obtain the novel coronavirus virus-like particles.

[0014] The present invention further provides a method for preparing VLPs, which includes the following steps:

[0015] I. Baculovirus P2-S and P2-ME were prepared using the Bac to Bac technique in the baculovirus expression system. The specific steps are as follows:

[0016] 1. Construction and identification of recombinant plasmids:

[0017] (1) Codon optimization of SARS-CoV-2 S, M and E genes;

[0018] (2) The S gene was synthesized into the pFastBac1 vector to obtain the recombinant plasmid pFastBac1-S;

[0019] (3) The M and E genes were synthesized into the pFast-Dual vector to obtain the recombinant plasmid pFast-Dual-ME;

[0020] (4) PCR identification of recombinant plasmids.

[0021] 2. Transformation of recombinant plasmids: The recombinant plasmids pFastBac1-S and pFast-Dual-ME were transformed into DH10Bac competent cells to obtain positive recombinant rod mids Bacmid-S and Bacmid-ME.

[0022] 3. Preparation of recombinant baculovirus:

[0023] (1) Extraction and PCR identification of recombinant rod-shaped particles;

[0024] (2) Recombinant baculoviruses Bacmid-S and Bacmid-ME were transfected into Sf9 insect cells, and the supernatant obtained was the P1 generation baculovirus.

[0025] (3) Infect Sf9 cells again with P1 generation baculovirus at low MOI. The supernatant obtained is P2 generation baculovirus, named P2-S and P2-ME.

[0026] (4) The titer of recombinant baculovirus was detected using the BacPAK Baculovirus Rapid Titer Kit.

[0027] 4. Identify the expression of S, M, and E proteins:

[0028] (1) Indirect immunofluorescence identification;

[0029] (2) Western blotting identification.

[0030] II. Preparation, purification, and identification of SARS-CoV-2 virus-like particle (VLP) vaccines

[0031] 1. Sf9 cells were co-transfected with P2-S and P2-ME.

[0032] 2. VLPs were purified using sucrose density gradient ultracentrifugation.

[0033] 3. Identification of SARS-CoV-2 VLPs:

[0034] (1) Western blotting identification;

[0035] (2) Observation by transmission electron microscopy;

[0036] (3) Immunoelectron microscopy observation;

[0037] (4) ACE2 binding activity.

[0038] III. Immunogenicity Study of SARS-CoV-2 VLPs

[0039] 1. Animal immunization and sample collection;

[0040] 2. Detection of IgG and IgG subtypes in mouse serum;

[0041] 3. Detection of neutralizing antibodies in mouse serum;

[0042] 4. Proliferative capacity of mouse spleen lymphocytes;

[0043] 5. ELISpot assay of spleen lymphocytes to detect the number of lymphocytes secreting IFN-γ and IL-4 cytokines.

[0044] The present invention also provides the application of the S gene, M gene or E gene, the structural protein, the expression cassette, the recombinant vector or recombinant virus, the recombinant baculosome, the recombinant baculovirus, and the novel coronavirus virus-like particles in the preparation of vaccines for the prevention of novel coronavirus infection.

[0045] The present invention also provides the use of the S gene, M gene or E gene, the structural protein, the expression cassette, the recombinant vector or recombinant virus, the recombinant baculosome, the recombinant baculovirus, and the novel coronavirus virus-like particle in the preparation of medicaments for the treatment and / or prevention of novel coronavirus infection.

[0046] In summary, the SARS-CoV-2 VLP formed by co-infecting Sf9 insect cells with P2-S and P2-ME recombinant viruses, followed by the expression of S, M, and E proteins and subsequent self-assembly, possesses a structure and biological characteristics similar to the natural product and exhibits good immunogenicity. When prepared as a vaccine for mice by mixing with flagellin protein (FliC) adjuvant, it further enhances the immune response level of VLP, inducing strong humoral and cellular immune responses. It has high safety and is of great significance for the development of COVID-19 vaccines, with broad application prospects.

[0047] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. The present invention provides a virus-like particle (VLP) for the first time, which is self-assembled by the expression of the three structural proteins S, M and E of SARS-CoV-2 virus through a baculovirus-insect cell expression system; 2. The present invention solves the problem that there is currently no safe and effective VLP vaccine for the prevention and control of COVID-19; 3. The VLP in the present invention is suitable for mucosal immunization, which can improve the immunization effect while reducing vaccination and labor costs, and has potential economic benefits. Attached Figure Description

[0048] Figure 1 The images show the PCR identification of recombinant plasmids pFastBac1-S and pFastBac-Dual-ME, where (A): pFastBac1-S, (B): pFastBac-Dual-ME, (C): pFastBac-Dual-ME, M: DL5000 DNA marker, 1: S gene amplification product, 2: M gene amplification product, and 3: E gene amplification product.

[0049] Figure 2 PCR identification of recombinant Bacmid-S and Bacmid-ME, where (A): Bacmid-S, (B): Bacmid-ME, (C): Bacmid-ME, M: DL5000 DNA marker, 1: primers SF / pUC / M13-R, 2: primers SF / R, 3: primers MR / pUC / M13-R, 4: primers MF / R, 5: primers EF / pUC / M13-R, 6: primers EF / R.

[0050] Figure 3 The results show the titer determination of recombinant baculoviruses P2-S and P2-ME, where (A): P2-S viral titer result, (B): P2-ME viral titer result, a: negative control, b: viral dilution to 10-1 -3 At the time of infection, c: virus diluted to 10 -4 At the time of infection focus, d: virus diluted to 10 -5 The site of infection at that time;

[0051] Figure 4 The results of indirect immunofluorescence experiments on the expression of S, M and E proteins are shown, where (A): S protein expression is identified, (B): M protein expression is identified, and (C): E protein expression is identified.

[0052] Figure 5 The results of Western blotting identification of S, M and E protein expression are shown. M: protein marker, 1, 3, 5: negative control, 2: identification of S protein expression, 4: identification of M protein expression, 6: identification of E protein expression.

[0053] Figure 6 To purify SARS-CoV-2 VLPs and identify them by Western blotting, (A): VLPs after ultracentrifugation with sucrose density gradient, (B): VLPs after Western blotting, M: protein marker, 1: samples with 20%-30% sucrose density, 2: samples with 30%-60% sucrose density.

[0054] Figure 7 Transmission electron micrographs of SARS-CoV-2 VLPs under different fields of view;

[0055] Figure 8 Immunoelectron microscopy images of SARS-CoV-2 VLPs under different fields of view;

[0056] Figure 9 This is a graph showing the ACE2 binding activity of VLPs.

[0057] Figure 10 This is a graph showing the detection of IgG antibodies in mouse serum 12 days after the second and third immunizations.

[0058] Figure 11 This is a graph showing the detection of IgG antibody subtypes in mouse serum 12 days after triple immunization;

[0059] Figure 12 The level of neutralizing antibodies against RBD protein in mouse serum two weeks after triple immunization;

[0060] Figure 13 The results of a mouse spleen lymphocyte proliferation experiment two weeks after triple immunization;

[0061] Figure 14 The results of ELISpot assay of spleen lymphocytes two weeks after the third immunization. Detailed Implementation

[0062] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0063] Example 1: Baculovirus P2-S and P2-ME prepared using Bac to Bac technology in a baculovirus expression system.

[0064] 1. Construction and identification of recombinant plasmids

[0065] (1) Gene sequences were optimized based on insect cell codon preferences. The optimized S gene (as shown in SEQ ID NO.1), M gene (as shown in SEQ ID NO.2), and E gene (as shown in SEQ ID NO.3) were synthesized by Genscript Biotech Co., Ltd. into the pUC57 vector. The synthesized vectors are as follows: pUC57-S sequence as shown in SEQ ID NO.4, pUC57-M sequence as shown in SEQ ID NO.5, and pUC57-E sequence as shown in SEQ ID NO.6. The optimized S, M, and E genes were amplified using SF / R, MF / R, and EF / R primers, respectively (primer sequences are detailed in Table 1). To facilitate subsequent identification, a 6×His tag was added after the M gene, and a Flag tag was added after the S and E genes. The PCR reaction system consisted of: 11 μL of 2×Taq Master Mix, 11 μL of ddH2O, 1 μL each of forward and reverse primers, 1 μL of plasmid, and a total volume of 25 μL. The PCR reaction program was as follows: 95℃, 3 min; 95℃, 30 s; 70℃, 30 s; 72℃, 4 min, 34 cycles; 72℃, 10 min. The PCR products were recovered separately. Simultaneously, the pFastBac1 and pFastBac-Dual plasmids were double-digested with restriction endonucleases (the PH promoter was digested with EcoR I and Hind III, and the p10 promoter with Xho I and Kpn I). The digestion system consisted of 2 μL of each restriction endonuclease, 2.5 μL of 10×M Buffer, 20 μL of plasmid, and dd H2O to a final volume of 50 μL. Digestion was carried out at 37°C for 3 h, and the digestion products were recovered. The S gene was ligated into the digested pFastBac1 plasmid using the ClonExpressUltraOneStepCloning Kit (Vazyme, C116-02), and the M and E genes were ligated into the digested pFastBac-Dual plasmid (the M gene was ligated after the p10 promoter, and the E gene was ligated after the PH promoter). The one-step ligation system was as follows: 1 μL of target fragment, 9 μL of vector, 10 μL of 2×ClonExpress Ultraone step cloning Mix, and ligation was carried out at 50℃ for 10 min. The ligation products were then transformed into DH5α competent cells, plated on 100 μg / mL ampicillin-resistant LB solid medium, and incubated overnight in an inverted incubator at 37℃.

[0066] Table 1 Primers for amplifying the S, M, and E genes

[0067]

[0068] (2) Identification of recombinant plasmids: Single colonies were picked, and pFastBac1-S and pFastBac-Dual-ME plasmids were extracted for PCR identification. SF / R primers were used to identify pFastBac1-S; MF / R primers were used to identify the M gene sequence in pFast-Dual-ME; and EF / R primers were used to identify the E gene sequence in pFast-Dual-ME (primer sequences are detailed in Table 1). The reaction system was: 11 μL of 2×Taq Master Mix, 11 μL of ddH2O, 1 μL each of forward and reverse primers, 1 μL of extracted recombinant plasmid, and a total volume of 25 μL. The PCR reaction program was: 95℃, 3 min; 95℃, 30 s, 70℃, 30 s; 72℃, 4 min, 34 cycles; 72℃, 10 min. The results showed that the corresponding target genes were amplified using different primers, and the band size was consistent with expectations. Figure 1 This indicates that the recombinant plasmids pFastBac1-S and pFast-Dual-ME were successfully constructed.

[0069] 2. Screening of recombinant rod particles

[0070] (1) Recombinant plasmids pFastBac1-S and pFast-Dual-ME were transformed into DH10Bac competent cells to obtain positive recombinant bacmids Bacmid-S and Bacmid-ME. The specific steps were as follows: DH10Bac competent cells were thawed on an ice-water mixture, and 1 μL of recombinant plasmids pFastBac1-S and pFast-Dual-ME were added respectively. The cells were incubated on ice for 30 min, then heat-shocked at 42℃ for 90 s. After that, the cells were allowed to stand in the ice-water mixture for 2 min, and 900 μL of LB medium preheated at 37℃ was added. The cells were then cultured in a shaker at 37℃ and 180 r / min for 4 h. The bacterial culture was then diluted to 10. -2 Then, the samples were spread on LB solid medium containing triple antibodies at concentrations of 50 μg / mL kanamycin, 7 μg / mL tetracycline, 10 μg / mL gentamicin, 40 μg / mL IPTG, and 200 μg / mL X-Gal, and incubated upside down in a 37°C incubator for 48 h. From the above plates, large white single colonies with good morphology were selected and purified by three-zone streak method until no more blue spots appeared.

[0071] (2) Extraction of recombinant rod particles Bacmid-S and Bacmid-ME.

[0072] (3) Identification of recombinant rod-like particles: Bacmid-S was identified using SF and pUC / M13-R primers and SF / R primers; the M gene sequence in Bacmid-ME was identified using MR and PUC / M13-R primers and MF / R primers; the E gene sequence in Bacmid-ME was identified using EF and pUC / M13-R primers and EF / R primers (Table 1). The reaction system was: 11 μL of 2×Taq Master Mix, 11 μL of ddH2O, 1 μL each of forward and reverse primers, 1 μL of extracted recombinant rod-like particles, and a total system volume of 25 μL. The PCR reaction conditions were: 95℃, 3 min; 95℃, 30 s, 70℃, 30 s; 72℃, 4 min, 34 cycles; 72℃, 10 min. The results showed that the target gene of the corresponding size was amplified using different primers, consistent with expectations. Figure 2 ).

[0073] 3. Preparation of recombinant baculovirus

[0074] (1) Insect cell culture: Sf9 cells (preserved by the Jiangsu Provincial Key Laboratory of Zoonoses) were seeded into 6-well cell culture plates to achieve a cell density of approximately 70% and cultured at 27°C for 1 hour; the cell culture medium was then replaced with 0.8 mL of plate culture medium (Sf-900 containing 1.5% FBS). TM IISFM); Preparation of transfection reagent: Take 4 clean, sterile centrifuge tubes and add 100 μL of serum- and antibiotic-free Sf-900 to each tube. TM II SFM medium was used to dilute the recombinant Bacmid and transfection reagent. 16 μg of Bacmid-S and Bacmid-ME were added to two centrifuge tubes, respectively, and gently mixed by pipetting. 8 μL of LipoInsect was added to the other two tubes. TM Mix the transfection reagent by gently pipetting. Let the diluted Bacmid (Bacmid-S and Bacmid-ME) and transfection reagent stand at room temperature for about 5 minutes. Gently add the diluted Bacmid to the diluted LipoInsect solution. TM Mix the transfection reagent by pipetting and let stand at room temperature for 30 minutes; add 200 μL of LipoInsect to each well. TM Add the transfection reagent-Bacmid mixture to the wells and mix well; incubate at 27°C for 4 hours, then replace with fresh, complete Sf-900. TMAfter incubating IISFM (containing 10% FBS and 1% penicillin and streptomycin) at 27°C for approximately 96 hours, collect the culture medium from each well and centrifuge at 1000 rpm for 10 minutes. The supernatant obtained is the P1 generation baculovirus (P1-S and P1-ME). Infect Sf9 cells again with the P1 generation baculovirus at MOI=0.1, collect the culture medium from each well, and centrifuge at 1000 rpm for 10 minutes. The supernatant obtained is the P2 generation baculovirus (P2-S and P2-ME).

[0075] (2) The titer of recombinant baculovirus was detected using the BacPAK Baculovirus Rapid Titer Kit. Detailed procedures are provided in the instruction manual. The calculated P2-S virus titer was 1.92 × 10⁻⁶. 8 IFU / mL, P2-ME viral titer is 1.8 × 10⁻⁶ 8 IFU / mL Figure 3 ).

[0076] 4. Identify the expression of S, M, and E proteins.

[0077] (1) Indirect immunofluorescence assay: Sf9 cells in 24-well cell culture plates were infected with P2-S virus and P2-ME virus, respectively. After 96 h, the cell culture supernatant was collected for Western blotting. Sf9 cells in the culture plate were fixed with pre-cooled methanol for 10 min, washed once with PBS for 5 min each time; permeabilized with 0.1% Triton X-10 for 5 min, washed twice with PBS for 5 min each time; blocked with PBS containing 5% BSA at room temperature for 2 h, washed three times with PBS; Anti-RBD polyclonal antibody (Sino Biological, 40592-T62; Anti-RBD polyclonal antibody: PBS containing 5% BSA = 1:1000, v / v) diluted with 5% BSA was added and incubated overnight at 4 °C; Goat anti-rabbit IgG-FITC diluted with BSA in PBS (Beyotime, A0562; Goat anti-rabbit IgG-FITC: PBS containing 5% BSA = 1:5000, v / v) was incubated at 37°C in the dark for 2 h, followed by 3 washes with PBS. Then, 4',6-diamidinyl-2-phenylindole (DAPI) was added and incubated at room temperature for 10 min, followed by 3 washes with PBS. The slides were mounted with a small amount of 20% glycerol and observed under a fluorescence inverted microscope. The results showed that Sf9 cells infected with recombinant baculovirus P2-S exhibited significant green fluorescence, while uninfected Sf9 cells showed no fluorescence. Figure 4A); Following the same method described above, the expression of M protein was identified using Anti-His monoclonal antibody (Beyotime, AF5060; Anti-His monoclonal antibody: PBS containing 5% BSA = 1:1000, v / v) as the primary antibody and goat anti-mouse IgG-FITC (Beyotime, A0568; goat anti-mouse IgG-FITC: PBS containing 5% BSA = 1:5000, v / v) as the secondary antibody; and Anti-Flag monoclonal antibody ( Beyotime (AF519); Anti-Flag monoclonal antibody: PBS containing 5% BSA = 1:1000, v / v) was used as the primary antibody, and goat anti-mouse IgG-FITC (Beyotime, A0568; goat anti-mouse IgG-FITC: PBS containing 5% BSA = 1:5000, v / v) was used as the secondary antibody to identify E protein expression; the results showed that Sf9 cells infected with recombinant baculovirus P2-ME showed green fluorescence, while uninfected Sf9 cells showed no fluorescence. Figure 4 B,C).

[0078] (2) Western Blotting Identification: Sf9 cell culture supernatant samples infected with P2-S virus or P2-ME virus were added to loading buffer at a ratio of 4:1 (Sf9 cell culture supernatant samples infected with P2-S virus or P2-ME virus: loading buffer = 4:1, v / v), and incubated at 95℃ for 10 min; a 12% polyacrylamide separating gel and a 5% stacking gel were prepared, and electrophoresis was performed at 22 mA constant current for 75 min; proteins were transferred to an NC membrane and blocked at room temperature for 2 h (PBST containing 5% skim milk powder), washed 3 times with PBST, 5 min each time; Anti-S monoclonal antibody (Sino) diluted in PBST containing 5% BSA was added. Biological, 40592-MM117; 1:1000), incubated overnight on a shaker at 4℃, washed 5 times with PBST, 5 min each time; goat anti-rabbit IgG-HRP (ABClone, AS003; 1:5000) diluted with PBST containing 5% BSA was added, incubated on a shaker at room temperature for 1 h, washed 7 times with PBST, 5 min each time; color development was performed using ECL substrate, and the results showed that the samples infected with P2-S virus and P2-ME virus could react with specific antibodies, and the band size was consistent with expectations. Figure 5 ).

[0079] Example 2: Preparation, purification, and identification of SARS-CoV-2 virus-like particle (VLP) vaccine

[0080] 1. Co-infection of Sf9 cells with P2-S and P2-ME

[0081] The day before infection, prepare a 250mL cell culture shake flask, and add 1×10⁻⁶ cells / mL. 6Sf9 cells / mL were seeded into culture flasks, with a total culture volume of 50 mL. The flasks were then placed in a dry, CO2-free shaker at 27°C and 120 rpm for 3-5 days, until the cell density reached 2 × 10⁻⁶ cells / mL. 6 Live cells / mL, viability ≥90%; P2-S and P2-ME viruses (MOI=5) were inoculated into shake flasks at a 1:1 ratio and cultured at 27°C and 120 r / min for 96 h.

[0082] 2. Purification of SARS-CoV-2 VLPs using sucrose density gradient ultracentrifugation.

[0083] Ninety-six hours after co-infection, cells were counted and their viability was observed. When viability reached 60%-80%, cells were transferred to 50 mL centrifuge tubes and centrifuged at 3000 rpm for 15 min at 4°C. The cell pellet was resuspended in 5 mL PBS. Cells were lysed using an ultrasonic homogenizer and centrifuged at 12000 rpm for 10 min at 4°C. The cell lysate supernatant was filtered through a 0.45 μm filter and placed on ice. Pre-chilled 60%, 30%, and 20% sucrose were added sequentially to the ultracentrifuge tubes, followed by slow addition of the cell lysate supernatant. The tubes were then ultracentrifuged at 24100 rpm for 4.5 h at 4°C. A milky white ring-shaped band appeared at the boundary between the 20%-30% and 30%-60% sucrose concentrations. Figure 6 A) After extracting the SARS-CoV-2 VLPs, resuspend them in an appropriate amount of PBS, centrifuge at 24100 r / min for 1.5 h at 4 °C to remove sucrose; discard the supernatant, add 1 mL of PBS to each ultracentrifuge tube to resuspend the SARS-CoV-2 VLPs, and store at 4 °C.

[0084] 3. Identification of SARS-CoV-2 VLPs

[0085] (1) Western Blotting Identification: The purified SARS-CoV-2 VLPs sample was added to the loading buffer at a ratio of 4:1 (purified VLPs sample: loading buffer = 4:1, v / v), and incubated at 95℃ for 10 min; a 12% polyacrylamide separating gel and a 5% stacking gel were prepared, and electrophoresis was performed at a constant current of 22 mA for 75 min; the protein was transferred to an NC membrane and blocked at room temperature for 2 h (PBST containing 5% skim milk powder), and washed 3 times with PBST for 5 min each time; mouse Anti-S monoclonal antibody (Sino) diluted with PBST containing 5% BSA was added. Biological, 40592-MM117; 1:1000), incubated overnight on a shaker at 4℃, washed 5 times with PBST, 5 min each time; goat anti-rabbit IgG-HRP (1:5000) diluted with PBST containing 5% BSA was added, incubated on a shaker at room temperature for 1 h, washed 7 times with PBST, 5 min each time; color development was performed using ECL substrate, and the results showed that samples from 30%-60% of the cell lysis supernatant simultaneously showed specific bands of S, M and E proteins ( ). Figure 6 B).

[0086] (2) Transmission electron microscopy (TEM) observation: 10 μL of purified VLPs sample was placed on a 200-mesh copper grid and adsorbed at room temperature for 6 min. The residue was then blotted dry with filter paper. 2% phosphotungstic acid negative staining solution was added to the copper grid and negatively stained for 1 min. The residue was then blotted away with filter paper and dried with a tungsten lamp. The morphology of the VLPs was observed under a TEM. A membrane with a diameter of 50-100 nm was observed, and the surface had a distinct crown-like structure. The morphological characteristics were similar to those of wild-type SARS-CoV-2 virus particles. Figure 7 ).

[0087] (3) Immunoelectron microscopy observation: 10 μL of purified VLPs was added to a 200-mesh copper grid and allowed to stand for 6 min. The sample was then aspirated off with filter paper. 10 μL of 1% BSA (prepared by filtering high-pressure water through a 0.22 μm filter) was added to the copper grid and blocked for 50 s. The block was then quickly aspirated off with filter paper. 10 μL of ultrapure water was added to wash away the blocking solution, and the mixture was allowed to stand for 30 s. The block was then aspirated off with filter paper. This process was repeated once. 10 μL of primary antibody, i.e., mouse-derived Anti-S monoclonal antibody (prepared by filtering PBS through a 0.22 μm filter and diluted 1:1000), was added and incubated at room temperature for 1 h. The primary antibody was then aspirated off with filter paper. 10 μL of ultrapure water was added and the mixture was allowed to stand for 30 min. s, blot with filter paper, repeat once; add 10 μL of secondary antibody, namely colloidal gold-labeled goat anti-mouse IgG (Solarbio, K1031G-Gold; prepared by filtering PBS through a 0.22 μm filter, diluted 1:20), incubate at room temperature for 1 h, blot with filter paper bristles to remove the secondary antibody; add 10 μL of ultrapure water, incubate for 30 s, blot with filter paper bristles, repeat once; add 10 μL of phosphotungstic acid negative staining solution, negative stain for 1 min, blot with filter paper bristles to remove residual staining solution, dry with tungsten lamp; observe VLPs under transmission electron microscopy, the results show that specific antibody colloidal gold-labeled VLPs can be observed, and the coronal structure on the surface is connected to colloidal gold particles ( Figure 8 ).

[0088] (4) ACE2 binding activity: VLPs or OVA proteins were diluted to 1 μg / mL with carbonate buffer (0.05 M, pH 9.6), 100 μL / well, and coated overnight at 4°C. The next day, the coating solution was discarded, and the cells were washed three times with PBST and patted dry. ELISA blocking buffer (PBS containing 1% BSA) was added, 200 μL / well, and the cells were blocked at 37°C for 2 h. The supernatant was discarded, and the cells were washed three times with PBST and patted dry. ACE2-Fc protein (Absin, abs04756; initial 100 μg / mL, 2-fold gradient method) diluted with blocking buffer was added. Dilute), 100 μL / well, incubate at 37℃ for 2 h, discard the liquid, wash 5 times with PBST, and blot dry; add 100 μL of mouse anti-human IgG (Fc)-HRP enzyme-labeled antibody (GenScript, A01854; 1:5000 dilution) per well, incubate at 37℃ for 1 h; discard the enzyme-labeled antibody, wash 7 times with PBST, and blot dry; add 100 μL of single-component TMB chromogenic solution, incubate at 37℃ in the dark for 10 min; add 50 μL of 2 mol / L H2SO4 stop solution per well, and read the OD using a microplate reader. 450 The results showed that, compared with the OVA protein group and the PBS group, the binding activity of VLPs and ACE2 increased with increasing ACE2 protein concentration, and saturable binding was achieved, indicating that VLPs have a high affinity for ACE2. Figure 9 ).

[0089] Example 3: Immunogenicity Study of SARS-CoV-2 VLPs

[0090] 1. Animal immunization and sample collection

[0091] Twenty-four 6-8 week old female BALB / c mice (purchased from Beijing Vital River Laboratory Animal Co., Ltd.) were randomly divided into four groups of six mice each: a PBS group, a 10 μg VLPs immunization group, a VLPs+FliC immunization group (10 μg VLPs and 20 μg FliC adjuvant mixture), and a VLPs+Alum immunization group (10 μg VLPs and Alum adjuvant mixture 1:1 (w / v). Immunization was performed via intraperitoneal injection at a dose of 100 μL per mouse, on day 0, with booster immunizations on days 14 and 28. Two weeks after the third immunization, blood was collected from the orbital sinus to detect serum levels of IgG, IgG subtypes, and neutralizing antibodies. Additionally, spleen lymphocytes were collected to assess their proliferative capacity and the number of lymphocytes secreting IFN-γ and IL-4 cytokines, in order to evaluate the levels of VLP-induced humoral and cellular immune responses.

[0092] 2. Detection of SARS-CoV-2 VLPs-specific IgG and its subtypes in serum

[0093] Dilute SARS-CoV-2 VLPs to 0.2 μg / mL with carbonate buffer, add 100 μL to each well, and coat overnight at 4°C. Discard the coating solution, wash the ELISA plate three times with PBST containing 0.05% Tween-20, 5 min each time, and blot dry. Add 200 μL / well of PBS containing 1% BSA and block at 37°C for 2 h. Discard the liquid, wash the plate three times with PBST, 5 min each time, and blot dry. Add 100 μL / well of serum from each group of mice after the second and third immunizations (initial ratio 1:100, serially diluted 2-fold) diluted with blocking buffer, and incubate at 37°C for 2 h. Wash the ELISA plate five times with PBST, 5 min each time, and blot dry. Add enzyme-labeled secondary antibodies (IgG-HRP (ABClone, AS003; 1:5000 dilution) diluted with blocking buffer and IgG1-HRP (Southern) diluted with blocking buffer. Biotech, 1071-05 (1:3000 dilution), IgG2a-HRP (Southern Biotech, 1081-05; 1:3000 dilution), 100 μL per well, incubate at 37℃ for 1 h; wash the enzyme label 7 times with PBST, 5 min each time, and pat dry; add 100 μL of TMB chromogenic solution to each well, incubate at 37℃ in the dark for 10 min; add 50 μL of 2 mol / L H2SO4 stop solution per well, and read the OD using a microplate reader. 450 Value. (P(OD of the sample to be tested)) 450Value) - N (negative control OD) 450 Value)) / (C(blank control OD) 450 Value) - N (negative control OD) 450 A value ≥ 2.1 was considered positive. Results showed that 12 days after the second and third immunizations, antibody levels in the VLPs immunization group were significantly higher than those in the PBS control group; antibody levels in the FliC group and the Alum adjuvant (ThermoFisher, 77161) group were significantly higher than those in the VLPs group. Figure 10 Twelve days after the third immunization, the levels of IgG1 and IgG2a antibodies in the VLPs immunization group were significantly higher than those in the PBS group; the levels of IgG1 and IgG2a antibodies in the FliC adjuvant group were significantly higher than those in the VLPs immunization group; and the level of IgG1 antibody in the Alum adjuvant immunization group was significantly higher than that in the VLPs group. Figure 11 ).

[0094] 3. Neutralizing antibody level detection

[0095] Two weeks after the third immunization, the serum and reagents of each group of mice were diluted according to the instructions of the SARS-CoV-2 Surrogate Virus Neutralization Test (sVNT) Kit (ELISA) - Multiplex VOC Kit (GenScript, L00871): The serum of each group of mice was diluted (Sample Dilution Buffer: Mouse Serum = 9:1, v / v); the Standard Stock standard was diluted to 600 U / mL using the Sample Dilution Buffer as a positive control, and the Sample Dilution Buffer was used as a negative control; the diluted serum, positive control or negative control were diluted with Probe RBD working solution (diluted serum, positive control or negative control: Probe RBD working solution = 2:1), added to a low-adsorption 96-well plate, vortexed for 30 s, and incubated at 37°C for 30 min; 100 μL of the mixture in the 96-well plate was transferred to the Capture plate. Cover each well with the corresponding plate membrane and incubate at 37°C for 15 min. Remove the cover membrane, discard the reaction solution, add 260 μL of 1×Wash Solution to each well, and discard the wash buffer after 30 s intervals. Repeat this process 4 times. Add 100 μL of enzyme-labeled antibody working solution to each well and incubate at 37°C for 15 min. Remove the cover membrane, discard the reaction solution, add 260 μL of 1×Wash Solution to each well, and discard the wash buffer after 30 s intervals. Repeat this process 4 times. Add 100 μL of TMB chromogenic reagent to each well and incubate at 25°C in the dark for 15 min. Add 50 μL of stop solution to each well to terminate the reaction. Immediately read the OD value using a microplate reader. 450Value; The inhibition rate is used to determine the level of neutralizing antibodies against SARS-CoV-2 in a sample, and the formula is as follows: Inhibition rate = (1 - (sample OD) / ... 450 Value / Negative Control OD 450 Value))×100%. The results showed that VLPs, FliC, and Alum adjuvant groups had high inhibition rates, indicating that they could all effectively induce the body to produce neutralizing antibodies ( Figure 12 ).

[0096] 4. Preparation of spleen lymphocyte suspension

[0097] Two weeks after the third immunization, mice in each group were euthanized, and their spleens were removed under sterile conditions and placed in blood-free and antibiotic-free RPMI 1640 medium. The spleens were ground using a glass slide with a frosted surface and filtered through a 70 μm cell sieve into centrifuge tubes. RPMI 1640 medium was added, and the cells were centrifuged at 1000 rpm for 5 min, discarding the supernatant. The cells were resuspended in 7 mL of complete RPMI 1640 medium (containing 1% penicillin-streptomycin solution and 10% FBS) and added to 7 mL of lymphocyte separation medium (Sigma, 10771). The cells were centrifuged at 1800 rpm for 20 min at room temperature, and the white, cloudy cells in the center were collected, which were the spleen lymphocytes. The spleen lymphocytes were washed with complete RPMI 1640 medium and centrifuged at 1000 rpm for 5 min. The spleen lymphocytes were resuspended in 1.5 mL of complete RPMI 1640 medium and counted.

[0098] 5. BrdU-based cell proliferation assay

[0099] The splenic lymphocyte proliferation level was detected according to the BrdU-based Cell Proliferation ELISA kit (Roche, 11647229001). The specific steps are as follows: Splenic lymphocyte resuspension was added to a 96-well cell culture plate at a density of 2 × 10⁶ cells / well. 5Cells were stimulated with 10 μg / mL SARS-CoV-2 VLPs per well, 100 μL per well. RPMI was set simultaneously. The control group was prepared using 1640 medium, with two replicates per group. Cells were cultured at 37°C in a 5% CO2 incubator for 48 hours. 20 μL / well of BrdU labeling was added to a final concentration of 10 mM, and the cells were cultured at 37°C in a 5% CO2 incubator for another 12 hours. Cells were centrifuged at 1000 rpm for 10 minutes in 96-well plates to allow cell adhesion, the medium was discarded, and the cells were dried in a 60°C oven for 1 hour. 200 μL / well of FixDenat fixation solution was added, and the cells were incubated for 30 minutes to fix the BrdU-labeled DNA within the cells. Cells were washed twice with PBS, and 100 μL of peroxidase-conjugated anti-BrdU antibody was added, followed by incubation for 90 minutes. Cells were washed three times with PBS, and 100 μL / well of TMB chromogenic solution was added. The reaction was terminated with 50 μL of H2SO4, and the OD values ​​were read using a microplate reader. 450 and OD 690 The stimulation index (SI) is calculated using the following formula: SI = (OD of antigen-treated group) / (OD of antigen-treated group) 450 -OD 690 ) / (OD of untreated group 450 -OD 690 The results showed that the lymphocyte stimulation index (SI) in the VLPs group was significantly higher than that in the PBS control group, indicating that VLPs could promote the proliferation and activation of splenic lymphocytes; compared with the VLPs immunization group, the splenic lymphocyte proliferation level in the FliC adjuvant group (VLPs+FliC) was significantly increased under the stimulation of the specific antigen VLPs. Figure 13 ).

[0100] 6. ELISApot detection of secreted IFN-γ and IL-4

[0101] The number of splenic lymphocytes secreting IFN-γ and IL-4 was detected using Mouse IFN-γ ELISPOT Set (BD, 551083) and Mouse IL-4 ELISPOT Set (BD, 551017), respectively. The specific steps were as follows: The capture antibodies IFN-γ and IL-4 were diluted to 2 μg / mL with Duchenne phosphate-buffered saline (DPBS) and added to 100 μL per well of a 96-well ELISpot plate. The plate was incubated overnight at 4°C. The next day, the plates were washed twice with RPMI 1640 medium, and 100 μL of RPMI 1640 medium was added to each well. The plates were then blocked at room temperature for 2 h. Finally, 100 μL of splenic lymphocyte suspension from each group of mice was added, for a total of 2 × 10⁻⁶ cells. 5Cells were stimulated with 5 μg / mL SARS-CoV-2 VLPs at 100 μL / well and incubated at 37°C, 5% CO2 for 24 h. The cell suspension was discarded, and the cells were washed twice with ddH2O and three times with PBST, 5 min each time. 100 μL / well of biotin-labeled detection antibodies (biotin-IFN-γ and biotin-IL-4) were added, and the cells were incubated at room temperature for 2 h. The 96-well ELISpot plates were washed five times with PBST, 5 min each time. 100 μL / well of streptavidin-HRP (Abcam, ab7403; 1:1000 dilution) was added, and the plates were incubated at room temperature for 1 h. The plates were washed five times with PBST, 5 min each time. 100 μL / well of BCIP / NBT liquid substrate (Sigma, B1911) was added for the reaction. The reaction was stopped with ddH2O when a blue color appeared. The plates were air-dried at room temperature until completely dry. The cells were then analyzed using a Bioreader. The number of spots was analyzed using an ELISpot readersystem in a 5000-Vβ instrument. Results showed that the number of splenic lymphocytes secreting IFN-γ and IL-4 in the VLPs group was significantly higher than that in the PBS group, indicating that VLPs can induce a mixed Th1 / Th2 immune response; the number of splenic lymphocytes secreting IFN-γ and IL-4 in the FliC adjuvant group was significantly higher than that in the VLP immunization group. Figure 14 A) The number of splenic lymphocytes secreting IFN-γ in the Alum adjuvant group was not significantly different from that in the VLPs group, while the number of splenic lymphocytes secreting IL-4 was significantly higher in the Alum adjuvant group than in the VLPs immunization group. Figure 14 B) indicates that FliC can enhance the ability of VLPs to induce a mixed Th1 / Th2 immune response.

Claims

1. A gene encoding a structural protein of a novel coronavirus, characterized in that, The structural protein genes include S gene, M gene and E gene; the nucleotide sequence of the S 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 E gene is shown in SEQ ID NO.

3.

2. A structural protein encoded by the gene of claim 1.

3. An expression cassette, recombinant vector, or recombinant virus, characterized in that, Contains the gene encoding the structural protein of the novel coronavirus as described in claim 1.

4. A recombinant rod-like particle, characterized in that, It contains the recombinant vector as described in claim 3.

5. A recombinant baculovirus, characterized in that, It contains the recombinant rod particles as described in claim 4.

6. A SARS-CoV-2 virus-like particle, characterized in that, It is prepared by purifying Sf9 cells infected with the recombinant baculovirus as described in claim 5.

7. A method for constructing the SARS-CoV-2 virus-like particles of claim 6, characterized in that, Includes the following steps: Sf9 cells were co-infected with a recombinant baculovirus containing the S gene as described in claim 1 and a recombinant baculovirus containing the M and E genes to obtain the SARS-CoV-2 virus-like particles.

8. The use of the gene encoding the structural protein of the novel coronavirus as described in claim 1, the structural protein as described in claim 2, the expression cassette, recombinant vector or recombinant virus as described in claim 3, the recombinant baculovirus as described in claim 4, the recombinant baculovirus as described in claim 5, and the SARS-CoV-2 virus-like particle as described in claim 6 in the preparation of a vaccine for the prevention of novel coronavirus infection.

9. The use of the gene encoding the structural protein of the novel coronavirus according to claim 1, the structural protein according to claim 2, the expression cassette, recombinant vector or recombinant virus according to claim 3, the recombinant baculosome according to claim 4, the recombinant baculovirus according to claim 5, and the SARS-CoV-2 virus-like particle according to claim 6 in the preparation of a medicament for treating and / or preventing novel coronavirus infection.

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