Virus-like particle for expressing new coronavirus HR121 protein and RBD protein as well as preparation method and application of virus-like particle

Abstract

The invention relates to a virus-like particle for expressing new coronavirus HR121 protein and RBD protein as well as a preparation method and application of the virus-like particle, and belongs to the technical field of vaccines. The invention discloses a recombinant protein combination containing protein 1 and protein 2. The amino acid sequence of the protein 1 is as shown in SEQ ID NO: 5, and the amino acid sequence of the protein 2 is as shown in SEQ ID NO: 8. According to the invention, hepatitis B virus core protein (HBc) virus-like particles are utilized, and an HR121 sequence composed of new crown viruses HR1 and HR2 and RBD are displayed on the surfaces of the virus-like particles in a dimer form, so that the virus-like particles have immunogenicity of proteins of the new crown viruses HR1, HR2 and RBD at the same time, and a stronger protection effect is generated.

Description

Technical Field

[0001] The present invention relates to the field of vaccine technology, and in particular to virus-like particles expressing HR121 protein and RBD protein of the new coronavirus, and a preparation method and application thereof. Background Art

[0002] Virus-like particles (VLPs) are nanoparticles that are similar in structure to viruses but lack the viral genome and do not have the ability to replicate. They can be self-assembled from all or part of the viral structural proteins. VLP-based nanoparticle carriers are widely used in vaccine development and can be used to express and display exogenous antigen proteins. Their adjuvant effect can promote antigen-induced immune responses.

[0003] The neck S2 subunit of the novel coronavirus spike protein contains two connected heptad repeat regions HR1 and HR2, which are two highly conserved fragments. The HR121 dimer is a 6-helix structure composed of 4 HR1s and 2 HR2s formed by 2 HR121 molecules. Studies have found that the HR121 protein can effectively inhibit the fusion of the novel coronavirus with host cells, prevent the virus from invading host cells, and its antiserum also has a strong neutralizing effect, suggesting that HR121 is a good immunogen for the development of novel coronavirus vaccines. Patent CN113943375A discloses a recombinant fusion protein derived from the HR region of the novel coronavirus and its application, but it only connects HR1 and HR2 through a connecting peptide to obtain a recombinant fusion protein, and does not fuse the RBD protein, so HR121 and RBD protein cannot be stably displayed together.

[0004] Hepatitis B virus core protein (HBc) can spontaneously assemble into VLPs with an icosahedral structure after expression, which can be used as a nanocarrier for displaying exogenous antigens. The 72nd to 80th amino acids of HBc constitute the major immunodominant region (MIR), which can be used as an insertion site for exogenous antigens. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a virus-like particle that simultaneously expresses the HR121 protein and RBD protein of the new coronavirus, and a preparation method and application thereof.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] In a first aspect, the present invention provides a recombinant protein combination, comprising protein 1 and protein 2; the amino acid sequence of protein 1 is shown in SEQ ID NO: 5, and the amino acid sequence of protein 2 is shown in SEQ ID NO: 8.

[0008] The present invention inserts a recombinant protein containing HR121 and RBD proteins into the MIR region of the HBc protein. Since the two identical MIR insertion sites are inevitably closely connected in space, the two recombinant proteins form dimers under the action of disulfide bonds and are further assembled into virus-like particles, stably exposing the HR121 and RBD proteins on the surface of the virus-like particles.

[0009] In a second aspect, the present invention provides a gene encoding the recombinant protein combination, wherein the nucleotide sequence of the gene encoding protein 1 is shown in SEQ ID NO: 6, and the nucleotide sequence of the gene encoding protein 2 is shown in SEQ ID NO: 9.

[0010] In a third aspect, the present invention provides a recombinant expression vector comprising the encoding gene.

[0011] In a fourth aspect, the present invention provides a recombinant expression strain, which contains the encoding gene or the recombinant expression vector.

[0012] In a specific embodiment of the present invention, the nucleotide sequence of the recombinant vector is shown in SEQ ID NO:7.

[0013] In a fifth aspect, the present invention provides the use of the recombinant protein combination in the preparation of virus-like particles.

[0014] In a sixth aspect, the present invention provides a virus-like particle formed by self-assembly of the recombinant protein combination.

[0015] In a seventh aspect, the present invention provides a method for preparing the virus-like particles, culturing the recombinant engineered bacteria to express the virus-like particles.

[0016] Furthermore, the method for culturing the recombinant engineered bacteria comprises the following steps:

[0017] S1: mixing competent cells with the recombinant expression vector, heat-activating, and culturing to obtain competent cells into which the recombinant expression vector is transferred;

[0018] S2: culturing the competent cells into which the recombinant expression vector has been transferred in step S1 in a medium containing antibiotics for 10 to 12 hours to obtain monoclonal colonies;

[0019] S3: Cultivate the monoclonal colony described in step S2 using a culture medium containing antibiotics and isopropyl-β-D-thiogalactoside to induce the strain to express and obtain virus-like particles.

[0020] Furthermore, in step S1, the culture temperature is 37° C. and the culture time is 1 h.

[0021] Furthermore, in step S1, the volume ratio of the competent cells to the recombinant expression vector is 10:1.

[0022] In a specific embodiment of the present invention, the competent cells are BL21 Escherichia coli competent cells.

[0023] In a specific embodiment of the present invention, the culture medium used is lysing broth medium, namely LB medium.

[0024] In a specific embodiment of the present invention, the antibiotic is kanamycin.

[0025] Further, in step S3, 2 to 3 monoclonal colonies described in step S2 are picked to 10 to 15 mL of liquid LB culture medium containing 50 mg / mL kanamycin and cultured for 10 to 12 hours to obtain a cultured bacterial solution; 4 mL of the cultured bacterial solution is added to 400 mL of LB liquid culture medium containing 50 mg / mL kanamycin, and the culture is cultured until the OD600 value is 0.6 to 0.8, isopropyl-β-D-thiogalactoside (final concentration is 0.1 to 1 mM), and cultured at 20° C. for 10 to 12 hours to induce the expression of virus-like particles.

[0026] Furthermore, the cultured strain in step S3 is broken, the protein is extracted, and then purified by sucrose gradient centrifugation to obtain virus-like particles.

[0027] In an eighth aspect, the present invention provides the use of the virus-like particles in the preparation of products against the new coronavirus.

[0028] In a ninth aspect, the present invention provides the use of the virus-like particles in the preparation of products for preventing and / or treating diseases caused by the new coronavirus.

[0029] In a tenth aspect, the present invention provides a vaccine comprising a vaccine adjuvant and the virus-like particles.

[0030] In a specific embodiment of the present invention, the vaccine adjuvant may be QuickAntibody.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] The present invention utilizes hepatitis B virus core protein (HBc) virus-like particles, combined with the structural characteristics of HBc VLP, to display the recombinant proteins of HR121 protein and RBD protein composed of SARS-CoV-2 HR1 and HR2 on the surface of virus-like particles in the form of dimers, so that the resulting virus-like particle vaccine has the immunogenicity of SARS-CoV-2 HR1, HR2 and RBD proteins at the same time, producing a stronger protective effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Schematic diagram of the structure of HBc protein.

[0034] Figure 2 The figure is a schematic diagram of the structure of HR121-RBD VLP. A is the connection of the fragments of the HBc-HR121-RBD monomer; B is a schematic diagram of the HBc-HR121-RBD monomer structure; C is a schematic diagram of the HR121-RBD VLP structure.

[0035] Figure 3 The results of polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis of the components after sucrose gradient centrifugation of HR121-RBD VLP.

[0036] Figure 4 The results of non-denaturing gel electrophoresis (NAGE) and western blot analysis of the components after sucrose gradient centrifugation for HR121-RBD VLP.

[0037] Figure 5 This is a transmission electron microscopy image of HR121-RBD VLP.

[0038] Figure 6 Method for immunization of mice.

[0039] Figure 7 The double antibody sandwich enzyme-linked immunosorbent assay (ELISA) method was used to detect the RBD-specific antibody titer at each time point in mice immunized with HR121-RBD VLP. A is the time-course curve of the RBD-specific IgG antibody titer drawn every week; B is the specific RBD IgG antibody titer in the serum collected at 5 weeks. The adjusted p value was analyzed using one-way ANOVA and Tukey's multiple comparison test, *p<0.05, **p<0.01, ***p<0.001.

[0040] Figure 8The solid phase enzyme-linked immunospot technique (ELISPOT) was used to detect the secretion of IFN-γ by mouse spleen cells. A is a statistical graph of the number of spots after mouse spleen lymphocytes were incubated with RBD protein; B is a spot image after mouse spleen lymphocytes were incubated with RBD protein; C is a statistical graph of the number of spots after mouse spleen lymphocytes were incubated with HR121 protein; D is a spot image after mouse spleen lymphocytes were incubated with HR121 protein. DETAILED DESCRIPTION

[0041] To better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific examples. Other materials, reagents, etc. used in the examples, unless otherwise specified, can be obtained from commercial sources.

[0042] Example 1 Preparation of HR121-RBD VLPs

[0043] The HR121-RBD recombinant protein is inserted into the MIR region of HBc as an exogenous protein. Since the two identical MIR insertion sites are inevitably closely connected in space, the two HR121-RBD structures can naturally approach each other to form a dimer. The structural diagram of HBc is shown in Figure 1 As shown, the schematic diagram of the HBc-HR121-RBD structure is shown Figure 2 As shown. Two HBc-HR121-RBD monomers form dimers under the action of disulfide bonds and are further assembled into VLPs, and HBc-HR121-RBD is exposed on the surface of VLPs in the form of dimers.

[0044] 1. Experimental Methods

[0045] 1. Design of HR121-RBD VLP related fragments

[0046] (1) Design and synthesize the coreN-RBD-coreC fragment: coreN-linker-RBD-linker-coreC, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0047] The amino acid sequence of the linker is GGGGSGGGGSGGGGS; the amino acid sequence of the HBc protein is shown in SEQ ID NO: 2, the 1st to 79th amino acids of the HBc protein are coreN; the 81st to 183rd amino acids of the HBc protein are coreC.

[0048] Use a linker to connect the N-terminus of the RBD protein to the C-terminus of coreN, and connect the C-terminus of RBD to the N-terminus of coreC to obtain the coreN-RBD-coreC sequence.

[0049] (2) Design and synthesize the coreN-HR121 fragment, namely coreN-linker-HR121, whose amino acid sequence is shown in SEQ ID NO: 3. Use linker to connect the N-terminus of HR121 protein to the C-terminus of coreN to obtain the coreN-HR121 sequence.

[0050] The N-terminus of the HR2 protein was connected to the C-terminus of HR1 using linker1 (amino acid sequence: GGSG), and the C-terminus of the HR2 protein was connected to the N-terminus of another HR1 using linker2 (amino acid sequence: SGGRGG) to obtain the HR121 protein (HR1-linker1-HR2-linker2-HR1), the amino acid sequence of which is shown in SEQ ID NO: 4.

[0051] 2. Construction of expression vector

[0052] (1) A pET28a vector (pET28a-HBc-RBD) expressing the coreN-RBD-coreC fragment was constructed, and the coreN-RBD-coreC encoding gene was connected between the NdeI and XhoI restriction sites of the pET28a vector.

[0053] The pRSFDuet-1 vector (pRSFDuet-coreN-HR121) expressing the coreN-HR121 sequence was constructed, and the coreN-HR121 encoding gene was connected between the BamHI and AflII restriction sites of the pRSFDuet-1 vector.

[0054] Using pET28a-HBc-RBD as a template, primers F1 and R1 in Table 1 were designed according to the RBD fragment, and PCR amplification was performed to obtain PCR amplification product A. The PCR amplification system is shown in Table 2, and the PCR amplification conditions are shown in Table 3.

[0055] Using pRSFDuet-coreN-HR121 as a template, primers F2 and R2 in Table 1 were designed according to the coreN-HR121 fragment, and PCR amplification was performed to obtain PCR amplification product B. The PCR amplification system is shown in Table 4, and the PCR amplification conditions are shown in Table 5.

[0056] Table 1

[0057]

[0058]

[0059] Table 2

[0060] Element content 2×Super Pfx MasterMix 25μL Primer F1 (10 μM) 2.5μL Primer R1 (10 μM) 2.5μL pET28a-HBc-RBD 200ng <![CDATA[ddH2O]]> Make up the total reaction volume to 50 μL

[0061] Table 3

[0062]

[0063] Table 4

[0064] Element content 2×Super Pfx MasterMix 25μL Primer F2 (10 μM) 2.5μL Primer R2 (10 μM) 2.5μL pRSFDuet-coreN-HR121 200ng <![CDATA[ddH2O]]> Make up the total reaction volume to 50 μL

[0065] Table 5

[0066] program temperature time Pre-denaturation 94℃ 3min transsexual 94℃ 30s annealing 55℃ 30s Renaturation 72℃ 90s

[0067] Overlapping PCR was performed using PCR amplification product A and PCR amplification product B as templates and primers, and primers F1 and R2 were used for amplification to obtain overlapping PCR amplification products.

[0068] The overlapping PCR amplification system is shown in Table 6, and the overlapping PCR amplification conditions are shown in Table 7.

[0069] Table 6

[0070] Element content 2×Super Pfx MasterMix 25μL Primer F1 (10 μM) 2.5μL Primer R2 (10 μM) 2.5μL PCR amplification product A 100ng PCR amplification product B 100ng <![CDATA[ddH2O]]> Make up the total reaction volume to 50 μL

[0071] Table 7

[0072]

[0073]

[0074] (First, perform 15 cycles of pre-denaturation to extension without adding primers, then add primers and perform another 30 cycles)

[0075] (2) The overlapping PCR amplification products and the pRSFDuet-1 vector were double-digested with BamH I and Afl II at 37°C for 2 h to obtain the overlapping PCR amplification product digestion product and the pRSFDuet-1 vector digestion product, respectively, and then gel recovery and purification were performed.

[0076] (3) The overlapping PCR amplification product and the pRSFDuet-1 vector digestion product were ligated using a Quick Ligation Kit at 25°C for 30 minutes to obtain a ligation product.

[0077] Transform the ligation product into DH5α competent peptide cells: take 50 μL BL21 E. coli competent cells, thaw on ice for 15 minutes, add 5 μL ligation product, incubate on ice for 30 minutes, and gently flick to mix twice; heat activate in a 42°C water bath for 60 seconds, take out and immediately put in ice for 5 minutes, add 500 μL LB liquid culture medium (lysate broth culture medium), shake and culture at 37°C in a shaker at 220 rpm for 1 hour to obtain bacterial liquid.

[0078] Take 100 μL of bacterial solution and spread it on a LB solid culture plate containing 50 mg / mL kanamycin. After inverting and culturing in a 37°C incubator for 10 to 12 hours, a monoclonal colony was obtained. Pick a single clone and place it in 5 mL of LB liquid culture medium containing 50 mg / mL kanamycin. After culturing for 6 to 8 hours, sequence it. Keep the monoclonal strain with the correct sequencing sequence, extract the plasmid, and name it pRSFDuet-1-coreN-HR121-RBD intermediate vector. Connect the coreN-HR121-RBD encoding gene to the first T7 promoter of the pRSFDuet-1 vector.

[0079] coreN-HR121-RBD fragment, the C-terminus of coreN-HR121 is connected to the N-terminus of the RBD fragment, the amino acid sequence is shown in SEQ ID NO: 5, and the nucleotide sequence of the coreN-HR121-RBD encoding gene is shown in SEQ ID NO: 6.

[0080] (4) Using pET28a-HBc-RBD as a template, primers F3 and R3 in Table 1 were designed according to the coreC fragment, and PCR amplification was performed to obtain PCR amplification product C. The PCR amplification system is shown in Table 8, and the PCR amplification conditions are shown in Table 3.

[0081] Table 8

[0082] Element content 2×Super Pfx MasterMix 25μL Primer F3 (10 μM) 2.5μL Primer R3 (10 μM) 2.5μL pET28a-HBc-RBD 200ng <![CDATA[ddH2O]]> Make up the total reaction volume to 50 μL

[0083] According to the method of steps (2) and (3), double enzyme digestion and enzyme ligation were performed, and DH5α peptide-sensitive cells were transfected, and the coreC encoding gene was connected to the second T7 promoter of the pRSFDuet-1-coreN-HR121-RBD intermediate vector to prepare the pRSFDuet-coreN-HR121-RBD vector (nucleotide sequence is shown in SEQ ID NO: 7). The amino acid sequence of the coreC fragment is shown in SEQ ID NO: 8, and the nucleotide fragment of the coreC encoding gene is shown in SEQ ID NO: 9.

[0084] 3. Preparation of VLP particles

[0085] (1) Protein expression:

[0086] Take 50 μL of BL21 Escherichia coli competent cells, thaw on ice for 15 minutes, add 5 μL of pRSFDuet-coreN-HR121-RBD vector, incubate on ice for 30 minutes, and gently flick to mix twice; heat activate in a 42°C water bath for 60 seconds, take out and immediately put it in ice for 5 minutes, add 500 μL of LB liquid culture medium, and shake and culture at 220 rpm in a 37°C shaker for 1 hour to obtain BL21 competent cells transferred with the pRSFDuet-coreN-HR121-RBD vector.

[0087] Take 100 μL of BL21 competent cells transferred with the pRSFDuet-coreN-HR121-RBD vector, spread it onto an LB solid culture plate containing 50 mg / mL kanamycin, and culture it upside down in a 37°C incubator for 10 to 12 hours to obtain a monoclonal colony.

[0088] Pick 2 to 3 monoclonal colonies and culture them overnight (10 to 12 h) in 10 mL of LB liquid culture medium containing 50 mg / mL kanamycin to obtain a cultured bacterial solution; transfer 4 mL of the cultured bacterial solution to 400 mL of LB liquid culture medium containing 50 mg / mL kanamycin, culture until the OD600 value is 0.6 to 0.8, add isopropyl-β-D-thiogalactoside (IPTG) with a final concentration of 0.1 to 1 mM, and induce HR121-RBD VLP protein expression at 20°C overnight (10 to 12 h).

[0089] During protein expression, the coreN-HR121-RBD fragment interacts with coreC ( Figure 2 A) Combined to form a new HBc-HR121-RBD monomer ( Figure 2 B), two HBc-HR121-RBD monomers form dimers under the action of disulfide bonds and self-assemble into VLPs, exposing the HBc-HR121-RBD monomers on the surface of VLPs in the form of dimers ( Figure 2 C). Due to the use of pRSFDuet-1 vector, the N-terminus of the coreN-HR121-RBD fragment was connected to a 6×His tag, and the C-terminus of the coreC fragment was connected to an S-Tag tag, which facilitated protein purification and identification.

[0090] (2) The bacteria induced to express VLP protein in step (1) were ultrasonically disrupted and centrifuged at 4000 rpm and 4°C for 15 to 30 min. The bacteria were collected and resuspended in 3 to 6 mL of TN300 buffer (containing 25 mM Tris-HCl, pH = 7.5 and 300 mM NaCl) to obtain a resuspended bacterial solution. 25× protease inhibitor (1 / 25 of the volume of the resuspended bacterial solution), lysozyme with a final concentration of 0.1 mg / mL and 0.5% (v / v) TritonX-100 were added to the resuspended bacterial solution to obtain a mixed solution; the mixed solution was vortexed for 20 s and incubated on ice for 10 to 15 min until the mixed solution became viscous (DNA was released).

[0091] (3) Add nuclease to the mixed solution treated in step (2), vortex, and incubate on a shaker at room temperature for 10 to 45 minutes until the viscosity decreases (genomic DNA is fragmented).

[0092] (4) The mixed solution treated in step (3) was subjected to ultrasonic disruption at a power of 30%, with ultrasonication for 2 seconds and rest for 6 seconds, for a total time of 2 minutes, to obtain an ultrasonic sample. The ultrasonic sample was centrifuged at 8000 rpm and 4° C. for 30 minutes, and the supernatant was collected.

[0093] (5) Protein purification: The supernatant collected in step (4) was further purified by sucrose gradient centrifugation. A 10-60% (w / v) sucrose gradient was used and ultracentrifuged at 32,000 rpm for 3.5 h at 20°C in a SW32 rotor. After centrifugation, 14 fractions of equal size were collected from the top.

[0094] Example 2 Analysis of HR121-RBD VLPs

[0095] 1. Experimental Methods

[0096] 1. The 14 components collected in Example 1 were subjected to polyacrylamide gel electrophoresis (SDS-PAGE) and non-denaturing electrophoresis (NAGE), and analyzed by western blot. The expression of HR121, RBD, and HBc proteins in the 14 components were detected using HR antibody (manufacturer: SinoBiological, catalog number: 40590-T62), RBD antibody (manufacturer: SinoBiological, catalog number: 40592-T62) and HBc antibody (manufacturer: Guangzhou Miaobo Biological, catalog number: 2AHC21).

[0097] 2. Transmission electron microscopy observation of HR121-RBD VLP particles

[0098] 2. Experimental Results

[0099] 1. If Figure 3As shown, coreN-HR121-RBD (55 kDa) and coreC (15 kDa) in the expression vector of HR121-RBD VLP were successfully expressed and mainly distributed in layers 8 to 11, which is consistent with the typical distribution characteristics of HBc VLPs after sucrose gradient density centrifugation.

[0100] 2. If Figure 4 As shown, HR121-RBD VLP nanoparticles were successfully assembled and mainly distributed in layers 9 and 10, which was consistent with the results of SDS-PAGE and western blot.

[0101] 3. If Figure 5 As shown, transmission electron microscopy results showed that the diameter of HR121-RBD VLP was about 100 nm.

[0102] Example 3 HR121-RBD VLP induces immune response in mice

[0103] 1. Experimental Methods

[0104] (I) Mouse immunization method

[0105] Five BALB / c 6-8 week old female mice were prepared in each group. The experimental group was the HR121-RBD VLP group, and the control groups were the TN150 group and the HBc protein group. HR121-RBD VLP, TN150 and HBc protein were mixed with QuickAntibody adjuvant at a volume ratio of 1:1, and then injected into the hind leg muscle of mice. All mice were immunized in a booster immunization manner, once at 0 week and 3 weeks, respectively. Serum was collected once a week, and mice were killed in the fifth week. The immunization process is as follows: Figure 6 shown.

[0106] TN150 contains 25 mM Tris-HCl and 150 mM NaCl, the solvent is water, and the pH is 7.5.

[0107] (II) HR121-RBD VLP induces mice to produce RBD-specific antibodies

[0108] 1. The double antibody sandwich enzyme-linked immunosorbent assay (ELISA) method was used to detect the RBD-specific antibody titer at each time point after the mice were immunized in step (i) of this example, and the carrier was coated with RBD protein (prokaryotically expressed RBD protein purchased from Qingke Biotechnology Co., Ltd.).

[0109] The RDB IgG antibody titer in the sera of mice at the fifth week of immunization was detected and analyzed, expressed as the reciprocal of the endpoint dilution, and the RBD-specific IgG titer was calculated each week.

[0110] (1) Antigen coating: Add 50 μL of antigen (RBD protein 2 ng / μL, diluted with pH 7.4 PBS or pH 9.6 carbonate buffer) to each well of a 96-well plate and coat overnight at 4°C (10-12 h).

[0111] (2) Washing the plate: Pour off the antigen in step (1), spin dry, add 200 μL of phosphate-buffered saline (PBST) to each well to wash the plate, wash six times to obtain the washed plate.

[0112] (3) Blocking: After washing in step (2), PBST (blocking solution) containing 5% (w / v) skimmed milk powder was added to the 96-well plate, 250 μL per well, and the plate was blocked at 37° C. for 1 h to obtain a blocked plate.

[0113] (4) Washing the plate: Add 200 μL of PBST to each well of the plate blocked in step (3) to wash the plate. Wash the plate six times and keep it moist for later use.

[0114] (5) Adding the sample to be tested: Add 100 μL of the diluted mouse serum sample to be tested to each well after treatment in step (4), perform duplicate assays on each sample, and react at 37°C for 2 h.

[0115] Mouse serum was diluted in gradient concentrations, namely 1:625, 1:1250, 1:2500, 1:5k, 1:10k, 1:20k, 1:40k, 1:80k, 1:160k, 1:320k, 1:640k, 1:1280k, and negative serum was diluted in the same gradient as the control well. The diluent was a universal antibody diluent (manufacturer: Suzhou Xinsaimei, item number: WB500D)

[0116] (6) Washing: After the reaction in step (5), add 200 μL PBST to each well to wash the plate. Wash six times.

[0117] (7) Binding the secondary antibody: add 100 μL of diluted horseradish peroxidase (HRP)-labeled anti-mouse IgG (ELISA 1:2000) antibody to each well treated in step (6), incubate at 37° C. for 1 h; dilute with blocking solution.

[0118] (8) Washing: Add 200 μL PBST to each well after treatment in step (7) and wash the plate six times.

[0119] (9) Color development: Add 100 μL of color development solution (manufacturer: Huayun Biotechnology, catalog number: HYE960) to each well after treatment in step (8) and leave at room temperature for 5 to 10 min.

[0120] (10) After treatment in step (9), add 50 μL of stop solution (manufacturer: Huayun Biotechnology, catalog number: HYC1058) to each well to terminate the reaction.

[0121] (11) Result determination and analysis: OD450 value was measured at 450 nm using an ELISA plate spectrophotometer. If the value of the test was greater than 2.5 / negative control, and the OD of the test data was greater than 0.2, it was preliminarily determined to be positive.

[0122] 2. Detection of IFN-γ secretion by mouse spleen cells using solid phase enzyme-linked immunosorbent assay (ELISPOT)

[0123] ELISpot Plus: Mouse IFN-γ Kit (Manufacturer: MabTech, Catalog No.: 3321-4AST-2) was used for detection. Mice were killed 14 days after booster immunization, spleen mouse lymphocytes were taken and incubated with RBD protein and HR121 protein for 18 hours, biotin-labeled antibody for 1 hour, and the number of spots was recorded using an enzyme-linked spot analyzer after color development.

[0124] (1) On a sterile operating table, open the sealed INF-γ pre-coated ELISpot plate, place it in an additional plate frame, wash it four times with sterile PBS (200 μL / well), and place the remaining strips in a 50 mL centrifuge tube, wrap it with tin foil and store it at 4°C.

[0125] (2) Add 200 μL of R10 complete medium to each well after treatment in step (1) and block at 37°C for 2 to 4 hours.

[0126] (3) Set up positive control wells, negative control wells and experimental wells, and add 5×10 5 Resuspend spleen lymphocytes in R10 basal medium.

[0127] (4) Add 10 μL of positive stimulus to the positive control well, 10 μL of dimethyl sulfoxide (DMSO) to the negative control well, and 10 μL of 10 μg / mL RBD or HR121 protein to the experimental well. Cover the plate and incubate in a 37°C, 5% CO2 incubator for 18 h.

[0128] (5) Pour out the liquid in the wells after treatment in step (4), add ice-cold deionized water (200 μL / well), and place in a 4°C refrigerator for 10 min to hypotonic lyse the cells.

[0129] (6) Discard the liquid in the wells after treatment in step (5), add washing buffer (260 μL / well), let it stand for 1 min, then discard the liquid in the wells. Repeat 6 times, and each time dry on absorbent paper.

[0130] (7) Add the diluted biotin-labeled antibody working solution (100 μL / well) to each experimental well treated in step (6) and incubate at 37° C. for 1 h.

[0131] (8) Repeat the treatment of step (5) for each well treated in step (7), then add the diluted enzyme-labeled avidin working solution (100 μL / well) to each well and incubate at 37° C. for 1 h.

[0132] (9) Repeat step (5) for each well treated in step (8), then remove the base of the plate, wash the bottom surface of the membrane and the base with deionized water, carefully absorb the remaining water on the base and the bottom of the membrane with absorbent paper, close the base, add 260 μL of Washing buffer to each well, let it stand for 1 min, discard the liquid in the well, and thoroughly dry it.

[0133] (10) Add the prepared 3-amino-9-ethylcarbazole (AEC, 100 μL / well) colorimetric solution to each well treated in step (9), and incubate the wells in the dark at room temperature for 30 min. Then terminate the colorimetric development according to the spot formation.

[0134] (11) Pour out the liquid in the wells of step (10), uncover the base of the plate, wash the front and back surfaces and the base with deionized water 3 to 5 times to stop color development, place the plate in a cool place at room temperature, wait for it to dry naturally, and then close the base.

[0135] (12) Using an enzyme-linked spot analyzer, record various parameters of the spots after treatment in step (11).

[0136] 2. Experimental Results

[0137] like Figure 7 As shown in A, HR121-RBD VLP has the ability to induce RBD-specific immune response, and as Figure 7 As shown in B, the serum RBD-specific antibody titer was 10 3 There was a significant difference compared with the control group.

[0138] like Figure 8 A and Figure 8 As shown in B, when cells were stimulated with RBD protein, HR121-RBD VLP could induce IFN-γ secreting cells compared with the control group, indicating that HR121-RBD VLP could induce T cell immune response.

[0139] like Figure 8 C and Figure 8 As shown in D, when cells were stimulated with HR121 protein (, HR121-RBD VLPs also induced IFN-γ secreting cells compared with the control group.

[0140] In summary, the HR121-RBD VLP prepared by the present invention can induce T cell immune response.

[0141] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A recombinant protein combination, characterized in that: The recombinant protein combination contains protein 1 and protein 2; the amino acid sequence of protein 1 is shown in SEQ ID NO: 5, and the amino acid sequence of protein 2 is shown in SEQ ID NO:

8.

2. The coding gene of the recombinant protein combination according to claim 1, characterized in that: The nucleotide sequence of the gene encoding the protein 1 is shown in SEQ ID NO: 6, and the nucleotide sequence of the gene encoding the protein 2 is shown in SEQ ID NO:

9.

3. A recombinant expression vector, characterized in that: The recombinant expression vector contains the coding gene according to claim 2.

4. A recombinant expression strain, characterized in that: The recombinant expression strain contains the coding gene according to claim 2 or the recombinant expression vector according to claim 3.

5. Use of the recombinant protein combination according to claim 1 in preparing virus-like particles.

6. A virus-like particle, characterized in that It is formed by self-assembly of the recombinant protein combination according to claim 1.

7. The method for preparing the virus-like particle according to claim 5, characterized in that: Cultivate the recombinant engineered bacteria according to claim 4 to express virus-like particles.

8. Use of the virus-like particles according to claim 5 in the preparation of products against the new coronavirus.

9. Use of the virus-like particles according to claim 5 in the preparation of products for preventing and / or treating diseases caused by the new coronavirus.

10. A vaccine, characterized in that The vaccine contains a vaccine adjuvant and the virus-like particles according to claim 5.

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