Virus-like particle for displaying novel coronavirus spike protein receptor binding domain trimer as well as preparation method and application of virus-like particle

Abstract

The invention relates to a virus-like particle for displaying a novel coronavirus spike protein receptor binding domain trimer as well as a preparation method and application of the virus-like particle, and belongs to the technical field of biological medicines. The invention discloses a recombinant protein combination. The recombinant protein combination contains a fragment 1 and a fragment 2, the amino acid sequence of the fragment 1 is as shown in SEQ ID NO: 4, and the amino acid sequence of the fragment 2 is as shown in SEQ ID NO: 7. The structural characteristics of hepatitis B virus core protein virus-like particles are combined, and the RBD protein of SARS-CoV-2 is displayed on the surfaces of the virus-like particles in a trimer form, so that the natural conformation of the new crown virus RBD protein is better simulated, the immunogenicity of virus-like particle vaccines is enhanced, and a stronger protection effect is generated.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a virus-like particle displaying a trimer of the receptor binding domain of a novel coronavirus spike protein, and a preparation method and application thereof. Background Art

[0002] The spike protein (S) of the new coronavirus (SARS-CoV-2) is a homotrimeric structure on its virus particles, and the receptor binding domain (RBD) in it is a common target for vaccine development. However, the RBD antigens currently prepared are difficult to simulate its natural trimer structure and have low immunogenicity.

[0003] After expression, the hepatitis B virus core protein (HBc) can spontaneously assemble into an icosahedral structure consisting of 120 pairs (T=4) or 90 pairs (T=3) of HBc dimers, which can be used as a virus-like particle (VLP) nanocarrier for displaying exogenous antigens. The 72nd to 80th amino acid sequence of HBc is its major immunodominant region (MIR), which can be used as an insertion site for exogenous antigens.

[0004] Patent CN114752616A discloses a virus-like particle that displays the RBD protein of the new coronavirus on its surface, as well as its preparation and application. The RBD antigen is inserted into the immunodominant region (MIR) of HBc. The constructed fusion protein can self-assemble into virus-like particles in prokaryotic cells. At the same time, the RBD antigen is displayed on the outer surface in the form of a trimer, which can induce the body to produce a strong immune response. 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 displaying the trimer of the receptor binding domain of the novel coronavirus spike protein, 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, a recombinant protein combination is provided, wherein the recombinant protein combination comprises fragment 1 and fragment 2; the amino acid sequence of fragment 1 is shown in SEQ ID NO: 4, and the amino acid sequence of fragment 2 is shown in SEQ ID NO: 7.

[0008] The present invention inserts RBD into the MIR region of HBc, connects the C-terminus of RBD to the N-terminus of the carboxyl terminal part (coreC) of HBc to obtain RBD-coreC; connects the C-terminus of RBD to the N-terminus of coreC, and connects the N-terminus of RBD to the C-terminus of the amino terminal part (coreN) of HBc to obtain coreN-RBD-coreC, connects the N-terminus of coreN-RBD-coreC to the C-terminus of RBD-coreC with a flexible peptide (GGS) to obtain RBD-coreC-coreN-RBD-coreC; connects the N-terminus of RBD to the C-terminus of coreN to obtain coreN-RBD; coreN-RBD is combined with RBD-coreC-coreN-RBD-coreC to form an HBc dimer, which is further assembled into VLP, and three RBD monomers are displayed on each four-helix bundle formed by the assembly of the HBc dimer to form an RBD trimer to obtain RBD-Trimer VLP.

[0009] In a second aspect, the present invention provides the use of the recombinant protein combination in the preparation of virus-like particles.

[0010] In a third aspect, the present invention provides a virus-like particle formed by self-assembly of the recombinant protein combination.

[0011] In a fourth aspect, the present invention provides the use of the virus-like particles in the preparation of drugs for preventing and / or treating diseases caused by SARS-CoV-2.

[0012] In a fifth aspect, the present invention provides the use of the virus-like particles in the preparation of drugs against SARS-CoV-2.

[0013] In the sixth aspect, the present invention provides a nucleic acid molecule, which encodes the amino acid sequence of the recombinant protein combination, the nucleotide sequence of the nucleic acid molecule encoding the fragment 1 is shown in SEQ ID NO: 5, and the nucleotide sequence of the nucleic acid molecule encoding the fragment 2 is shown in SEQ ID NO: 8.

[0014] In a seventh aspect, the present invention provides a recombinant vector comprising the nucleic acid molecule.

[0015] In an eighth aspect, the present invention provides a recombinant engineered bacterium, wherein the recombinant engineered bacterium contains the recombinant vector.

[0016] In a specific embodiment of the present invention, the nucleotide sequence of the recombinant vector is shown as SEQ ID NO:6.

[0017] In a ninth aspect, the present invention provides a method for preparing the virus-like particles, culturing the recombinant engineered bacteria and expressing the virus-like particles.

[0018] Furthermore, the method for culturing cells comprises the following steps:

[0019] S1: culturing the recombinant engineered bacteria in a medium containing antibiotics for 10-12 hours to obtain a monoclonal colony;

[0020] S2: Cultivate the monoclonal colony described in step S1 with a culture medium containing isopropyl-β-D-thiogalactoside and antibiotics to induce the strain to express and obtain virus-like particles.

[0021] Furthermore, the recombinant engineered bacteria is BL21 Escherichia coli.

[0022] Furthermore, the antibiotic is kanamycin.

[0023] Furthermore, the culture medium used for the culture is LB culture medium.

[0024] Further, in step S2, 2-3 monoclonal colonies described in step S1 are picked to 10-15 mL of liquid LB culture medium containing 50 mg / mL kanamycin, and cultured for 10-12 hours to obtain a cultured bacterial solution; 4 mL of the cultured bacterial solution is transferred to 400 mL of LB culture medium containing 50 mg / mL kanamycin, cultured to an OD600 value of 0.6-0.8, and isopropyl-β-D-thiogalactoside is added at a final concentration of 0.1-1 mM, and cultured at 20°C for 10-12 hours to induce the expression of virus-like particles.

[0025] Furthermore, the cultured strain in step S2 is broken, the protein is extracted, and then purified by sucrose gradient centrifugation to obtain virus-like particles.

[0026] In a tenth aspect, the present invention provides a drug for fighting SARS-CoV-2, preventing and / or treating diseases caused by SARS-CoV-2, wherein the drug contains the virus-like particles and a pharmaceutically acceptable carrier.

[0027] In a specific embodiment of the present invention, the drug is a vaccine.

[0028] Furthermore, the vaccine contains a vaccine adjuvant.

[0029] In a specific embodiment of the present invention, the vaccine adjuvant may be QuickAntibody.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] The present invention is based on hepatitis B virus core protein virus-like particles and combines the structural characteristics of hepatitis B virus core protein virus-like particles to display the RBD protein of SARS-CoV-2 in the form of a trimer on the surface of the virus-like particles, thereby better simulating the natural conformation of the new coronavirus RBD protein, enhancing the immunogenicity of the virus-like particle vaccine, and producing a stronger protective effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Schematic diagram of the structure of HBc protein.

[0033] Figure 2 The figure is a schematic diagram of the structure of RBD-Trimer VLP. A is the connection of the fragments of the recombinant HBc dimer; B is a schematic diagram of the recombinant HBc dimer structure; C is a schematic diagram of the RBD-Trimer VLP structure.

[0034] Figure 3 This is a transmission electron microscopy image of RBD-Trimer VLP.

[0035] Figure 4 Polyacrylamide gel electrophoresis and western blotting analysis results of the components after sucrose gradient centrifugation of RBD-Trimer VLP.

[0036] Figure 5 The results of non-denaturing gel electrophoresis and protein immunoblot analysis of the components after sucrose gradient centrifugation of RBD-Trimer VLP.

[0037] Figure 6 The double antibody sandwich enzyme-linked immunosorbent assay was used to detect the RBD-specific antibody titer at each time point in mice immunized with RBD-Trimer 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.

[0038] Figure 7 The solid phase ELISA technique was used to detect the secretion of IFN-γ by mouse spleen cells. A is a statistical diagram 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. DETAILED DESCRIPTION

[0039] 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.

[0040] Example 1 Construction of a vector expressing RBD-Trimer VLP

[0041] RBD is inserted into the MIR region of HBc as an exogenous protein. The structure of HBc is shown in the figure below. Figure 1 As shown, the schematic diagram of the RBD-Trimer VLP structure is shown Figure 2 As shown. The C-terminus of RBD was connected to the N-terminus of coreC to obtain RBD-coreC; the C-terminus of RBD was connected to the N-terminus of coreC, and the N-terminus of RBD was connected to the C-terminus of coreN to obtain coreN-RBD-coreC. The N-terminus of coreN-RBD-coreC was connected to the C-terminus of RBD-coreC using GGS flexible peptide to obtain RBD-coreC-coreN-RBD-coreC; the N-terminus of RBD was connected to the C-terminus of coreN to obtain coreN-RBD; coreN-RBD was combined with RBD-coreC-coreN-RBD-coreC to form HBc dimers, which were further assembled into VLPs. Three RBD monomers were displayed on each four-helix bundle formed by the assembly of HBc dimers to form RBD trimers, and RBD-Trimer VLPs were obtained.

[0042] 1. Design RBD-Trimer VLP related fragments

[0043] (1) Design and synthesize the RBD-coreC fragment: the amino acid sequence is shown in SEQ ID NO: 1. The C-terminus of RBD is connected to the N-terminus of coreC via a linker.

[0044] The amino acid sequence of the linker is GGGGSGGGGSGGGGS.

[0045] (2) 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: 2.

[0046] The 1st to 79th amino acids of the HBc protein (amino acid sequence is shown in SEQ ID NO: 3) are coreN; the 81st to 183rd amino acids of the HBc protein are coreC.

[0047] 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 fragment.

[0048] 2. Construction of expression vector

[0049] (1) Construct a pET28a vector (pET28a-HBc-RBD) expressing coreN-RBD-coreC, and connect the coreN-RBD-coreC coding gene between the EcoRI and SalI restriction sites of the pET28a vector.

[0050] ① Using pET28a-HBc-RBD as a template, primers F1 and R1 were designed according to the RBD-coreC fragment, and PCR reaction was performed to obtain PCR reaction product 1, which was then recovered and purified by gel recovery.

[0051] The PCR reaction system was: 2×Super Pfx MasterMix 25 μL, primer F1 (10 μM) 2.5 μL, primer R1 (10 μM) 2.5 μL, pET28a-HBc-RBD 200 ng, and ddH2O added to make the total reaction system 50 μL.

[0052] The PCR reaction conditions were as follows: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, renaturation at 72°C for 90 s, 30 cycles; extension at 72°C for 5 min; and storage at 4°C.

[0053] F1: 5'-TCCGAATTCGCGTGTACAGCCG-3';

[0054] R1: 5'-CGCCGGAGCCTCCTGATCCACCACTTCCGCCTGAACCTCCAGAACTTCCACCTACTACGGTAGTTTCCGG-3'.

[0055] ② Using pET28a-HBc-RBD as a template, primers F2 and R2 were designed according to the coreN-RBD-coreC fragment, and PCR reaction was performed to obtain PCR reaction product 2, which was then recovered and purified by gel recovery.

[0056] The PCR reaction system was: 2×Super Pfx MasterMix 25 μL, primer F2 (10 μM) 2.5 μL, primer R2 (10 μM) 2.5 μL, pET28a-HBc-RBD 200 ng, and ddH2O added to make the total reaction system 50 μL.

[0057] The PCR reaction conditions were as follows: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s; annealing at 55°C for 30 s; and renaturation at 72°C for 90 s.

[0058] F2: 5'-GAAGTGGTGGATCAGGAGGCTCCGGCGGTAGCGGAGGCTCGACAATGGACATCGACCCATAC-3;'

[0059] R2: 5'-AGCTTGTCGACTTAACACTGCGGTTCACGAGACTGGCT-3'.

[0060] ③Overlap PCR

[0061] The PCR reaction system is: 2×Super Pfx MasterMix 25μL, PCR reaction product 1 of step ① 100ng, PCR reaction product 2 of step ② 100ng, primer F1 (10μM) 2.5μL, primer R2 (10μM) 2.5μL, and ddH2O to make up the total reaction system to 50μL.

[0062] PCR reaction conditions were as follows: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s; annealing at 55°C for 30 s; renaturation at 72°C for 90 s; extension at 72°C for 5 min; storage at 4°C (without adding primers, perform 15 cycles from pre-denaturation to extension, then add primers for another 30 cycles).

[0063] Overlapping PCR products were obtained, and the products were recovered and purified by gel to obtain overlapping PCR reaction products.

[0064] (2) The overlapping PCR reaction products and the pRSFDuet-1 vector were double-digested (EcoR I and Sal I enzymes) at 37°C for 2 h to obtain the overlapping PCR reaction product digestion product and the pRSFDuet-1 vector digestion product, respectively, and then the digestion products were recovered and purified by gel recovery.

[0065] (3) Ligate the overlapping PCR reaction product and the pRSFDuet-1 vector digestion product at 25°C with QuickLigation Kit for 30 min to obtain a ligation product; transform the ligation product into DH5α peptide-sensitive cells:

[0066] Take 50 μL of BL21 E. coli competent cells, thaw on ice for 15 min, add 5 μL of ligation product, incubate on ice for 30 min, flick to mix twice during the process, and obtain the vector strain incubation solution.

[0067] The vector strain incubation solution was heat activated in a water bath at 42°C for 60s, taken out and immediately placed in ice for 5min, 500μL LB liquid culture medium was added, and shaking culture was carried out at 220rpm in a shaker at 37°C for 1h to obtain the BL21 competent cell culture solution into which the ligation product was transferred.

[0068] Take 100 μL of bacterial solution and spread it onto a LB solid culture plate (lysate broth medium) containing 50 mg / mL kanamycin, and culture it upside down in a 37°C incubator for 10-12 hours to obtain a monoclonal colony.

[0069] Pick a single clone in 5 mL of resistant LB, culture it for 6-8 hours and then sequence it. Keep the single clone strain with the correct sequencing sequence, extract the plasmid, and name it as pRSFDuet-HBc-RBD trimer intermediate vector. Connect the RBD-coreC-coreN-RBD-coreC encoding gene to the first T7 promoter of the pRSFDuet-1 vector.

[0070] The RBD-coreC-linker-coreN-RBD-coreC fragment, i.e., RBD-coreC-coreN-RBD-coreC, is connected between the C-terminus of RBD-coreC and the N-terminus of coreN-RBD-coreC using linker' (amino acid sequence is GGS). The amino acid sequence of the RBD-coreC-coreN-RBD-coreC fragment is shown in SEQ ID NO: 4, and the nucleotide sequence of the RBD-coreC-coreN-RBD-coreC encoding gene is shown in SEQ ID NO: 5.

[0071] (4) Using pET28a-HBc-RBD as a template, primers F3 and R3 were designed according to the coreN-RBD fragment, and a PCR reaction was performed to obtain PCR reaction product 3.

[0072] The PCR reaction system was: 2×Super Pfx MasterMix 25 μL, primer F3 (10 μM) 2.5 μL, primer R3 (10 μM) 2.5 μL, pET28a-HBc-RBD 200 ng, and ddH2O added to make the total reaction system 50 μL.

[0073] The PCR reaction conditions were as follows: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, renaturation at 72°C for 90 s, 30 cycles; extension at 72°C for 5 min; and storage at 4°C.

[0074] F3: 5'-GGAGATATACATATGGACATCGACCCATACA-3';

[0075] R3: 5'-CCAGACTCGAGTTAGTTAACGCATTTGTTCTTCACCAGG-3'.

[0076] According to the method of steps (2) and (3), double enzyme digestion and enzyme ligation were performed, and DH5α peptide-sensitive cells were transfected, cultured, and plasmids were extracted. The coreN-RBD encoding gene was connected to the second T7 promoter of the pRSFDuet-HBc-RBD trimer intermediate vector to prepare the pRSFDuet-HBc-RBD trimer vector (nucleotide sequence is shown in SEQ ID NO: 6). The amino acid sequence of the coreN-RBD fragment is shown in SEQ ID NO: 7, and the nucleotide sequence of the coreN-RBD encoding gene is shown in SEQ ID NO: 8.

[0077] Example 2 Preparation of RBD-Trimer VLP

[0078] 1. Protein expression

[0079] (1) Take 50 μL of BL21 E. coli competent cells, thaw on ice for 15 min, add 5 μL of pRSFDuet-HBc-RBD trimer vector, and incubate on ice for 30 min. During this period, gently flick the cell tube to mix twice to obtain the vector strain incubation solution.

[0080] (2) The vector strain incubation liquid of step (1) was heat activated in a water bath at 42°C for 60 seconds, taken out and immediately placed in ice for 5 minutes, 500 μL of LB liquid culture medium was added, and shaking culture was carried out at 220 rpm in a shaker at 37°C for 1 hour to obtain a BL21 receptive cell culture liquid transferred with the pRSFDuet-HBc-RBD trimer vector.

[0081] (3) Take 100 μL of the BL21 competent cell culture medium transformed with the pRSFDuet-HBc-RBD trimer vector in step (2), spread it onto an LB solid culture plate containing 50 mg / mL kanamycin resistance, and culture it upside down in a 37°C incubator for 10-12 hours to obtain a monoclonal colony.

[0082] (4) Pick 2-3 monoclonal colonies from step (3) and culture them overnight 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 liquid LB culture medium containing antibiotics, culture until the OD600 value is 0.6-0.8, add isopropyl-β-D-thiogalactoside (IPTG) with a final concentration of 0.1-1 mM, and culture at 20°C for 10-12 h to induce RBD-Trimer VLP protein expression.

[0083] During protein expression, coreN-RBD is combined with RBD-coreC-coreN-RBD-coreC ( Figure 2 A), forming HBc dimer ( Figure 2 B), and further assembled into VLPs, each of the four-helix bundles formed by the assembly of HBc dimers displays three RBD monomers, namely RBD trimers, to obtain RBD-Trimer VLPs ( Figure 2 C). Due to the use of pRSFDuet-1 vector, the N-terminus of the RBD-coreC-coreN-RBD-coreC fragment was connected to a 6×His tag, and the C-terminus of the coreN-RBD fragment was connected to an S-Tag tag, which facilitated protein purification and identification.

[0084] 2. (1) The cells induced to express RBD-Trimer VLP protein in step 1 were ultrasonically disrupted, centrifuged at 4000 rpm for 15-30 min at 4°C, and the cells were collected and resuspended in 3-6 mL TN300 buffer to obtain a resuspended bacterial solution. The TN300 buffer contains 25 mM Tris-HCl, pH = 7.5 and 300 mM NaCl, and the solvent is water.

[0085] (2) Add TritonX-100 with a final concentration of 0.5% (v / v), 0.1 mg / mL lysozyme, and 1 / 25 volume of 25× protease inhibitor to the resuspended bacterial solution to obtain a mixed solution; vortex the mixed solution for 20 seconds and incubate on ice for 10-15 minutes until the mixed solution becomes viscous due to the release of DNA.

[0086] 3. Add nuclease to the mixed solution after treatment in step 2, vortex, and incubate on a shaker at room temperature for 10-45 minutes until the viscosity decreases due to genomic DNA fragmentation.

[0087] 4. Transfer the mixed solution after step 3 to a 5 mL centrifuge tube and perform ultrasonication at 30% power, ultrasonication for 2 seconds and stop for 6 seconds, for a total of 2 minutes to obtain the ultrasonic sample. Centrifuge the ultrasonic sample at 8000 rpm for 30 minutes at 4°C and collect the supernatant.

[0088] 5. Protein purification: The supernatant collected in step 4 was further purified by sucrose gradient centrifugation.

[0089] Using a 10-60% (w / v) sucrose gradient, ultracentrifugation was performed at 32,000 rpm in a SW32 rotor at 20° C. for 3.5 h. After centrifugation, 14 fractions of the same size were collected from the top for subsequent analysis.

[0090] Example 3 Electrophoresis, Western Blot Analysis and Microscopic Observation of RBD-Trimer VLP

[0091] 1. Experimental Methods

[0092] 1. Observe the morphology and size of RBD-Trimer VLP particles using a transmission electron microscope.

[0093] 2. The 14 components collected in Example 2 were subjected to polyacrylamide gel electrophoresis (SDS-PAGE), non-denaturing gel electrophoresis (NAGE), and western blot analysis. The expression of RBD and HBc proteins in the 14 components were detected using RBD antibody (manufacturer: SinoBiological, catalog number: 40592-T62) and HBc antibody (manufacturer: Guangzhou Miaobo Biological, catalog number: 2AHC21).

[0094] 2. Experimental Results

[0095] 1. If Figure 3 As shown, transmission electron microscopy revealed that layers 9 and 10 formed a complete RBD-Trimer VLP with a diameter of approximately 100 nm.

[0096] 2. If Figure 4 As shown, RBD-coreC-coreN-RBD-coreC (90 kDa) and coreN-RBD (34 kDa) in RBD-Trimer VLPs were successfully expressed and mainly distributed in layers 9-14.

[0097] 3. If Figure 5 As shown, RBD-Trimer VLPs were successfully assembled and mainly distributed in layers 8-10.

[0098] Example 4 RBD-Trimer VLP induces antibody production in mice

[0099] 1. Experimental Methods

[0100] 1. Immunize mice

[0101] The experimental group was the RBD-Trimer VLP group, and the control groups were the TN150 group (TN150 contained 25 mM Tris-HCl and 150 mM NaCl, the solvent was water, pH=7.5) and the HBc protein group.

[0102] RBD-Trimer VLP, TN150 and HBc proteins were mixed with QuickAntibody adjuvant at a volume ratio of 1:1 and then injected into the hind leg muscles of mice. Five BALB / c 6-8 week old female mice were prepared in each group. All mice were immunized once at 0 and 3 weeks, respectively, and serum was collected once a week. Mice were euthanized on the fifth week.

[0103] 2. Detection of RBD-specific antibody titer after mouse immunization RBD-specific IgG titer

[0104] 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 1, and the carrier was coated with RBD protein. The RBD protein was a prokaryotically expressed RBD protein purchased from Qingke Biotechnology Co., Ltd. The RDB IgG antibody titer in the mouse serum collected in the fifth week was detected, expressed as the reciprocal of the endpoint dilution, and the RBD-specific IgG titer was calculated each week.

[0105] (1) Dilute the RBD protein with PBS at pH 7.4 or carbonate buffer at pH 9.6 to obtain RBD protein dilution solution, add RBD protein (2 ng / μL, 50 μL / well) dropwise into a 96-well plate, and coat at 4°C for 10-12 h.

[0106] (2) Pour off the RBD protein dilution solution in step (1), spin dry, add phosphate-buffered saline (PBST) to wash the plate, 200 μL / well, wash 6 times, and obtain a washed plate.

[0107] (3) After washing in step (2), a blocking solution was added to each well of the 96-well plate, 250 μL, and the plate was blocked at 37° C. for 1 h to obtain a blocked plate; the blocking solution was PBST containing 5% (w / v) skimmed milk powder.

[0108] (4) After the wells were blocked in step (3), PBST was added to the wells for washing, 200 μL / well, and the wells were washed 6 times. The wells were kept moist for future use.

[0109] (5) Dilute the mouse serum in a gradient concentration to obtain the mouse serum dilution samples to be tested, which are 1:625, 1:1250, 1:2500, 1:5k, 1:10k, 1:20k, 1:40k, 1:80k, 1:160k, 1:320k, 1:640k, 1:1280k, respectively. The negative serum is diluted in the same gradient as the control well. The mouse serum diluent is a universal antibody diluent (manufacturer: Suzhou Xinsaimei, item number: WB500D)

[0110] After treatment in step (4), add the diluted mouse serum sample to be tested to each well, 100 μL / well, perform duplicate determination for each sample, and react at 37°C for 2 hours.

[0111] (6) After the reaction in step (5), add PBST to each well to wash the plate, 200 μL / well, and wash 6 times.

[0112] (7) Add secondary antibody (100 μL / well) to each well treated in step (6). The secondary antibody is horseradish peroxidase (HRP)-labeled anti-mouse IgG (ELISA 1:2000) antibody diluted in blocking solution. Incubate at 37°C for 1 h.

[0113] (8) Washing: Add PBST to each well after treatment in step (7) to wash the plate, 200 μL / well, and wash 6 times.

[0114] (9) Color development: Add color development solution (manufacturer: Huayun Biotechnology, catalog number: HYE960) to each well after treatment in step (8), 100 μL / well, and leave at room temperature for 5-10 min.

[0115] (10) Add stop solution (manufacturer: Huayun Biotechnology, catalog number: HYC1058) to each well after treatment in step (9), 50 μL / well, to terminate the reaction.

[0116] (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.

[0117] 2. Experimental Results

[0118] like Figure 6 A and Figure 6 As shown in B, RBD-Trimer VLP has the ability to induce RBD-specific immune response, and the serum RBD-specific antibody titer was 10 3 There was a significant difference compared with the control group.

[0119] Example 5 RBD-Trimer VLP induces mouse spleen cells to secrete IFN-γ

[0120] 1. Experimental Methods

[0121] According to the method of Example 4, mice were boosted with immunization. BALB / c mice were euthanized 14 days after 3 weeks of immunization, and spleen lymphocytes were collected and tested using ELISpot Plus: Mouse IFN-γ Kit (Manufacturer: MabTech, Catalog No.: 3321-4AST-2).

[0122] Splenic lymphocytes were incubated with RBD protein for 18 h, biotin-labeled antibody was incubated for 1 h, and the number of spots was recorded using an enzyme-linked spot analyzer after color development.

[0123] 1. On a sterile operating table, open the sealed INF-γ pre-coated ELISpot plate, put it into an additional plate frame, wash it 4 times with sterile PBS, 200 μL / well, put the remaining strips into a 50 mL centrifuge tube, wrap it with tin foil and store it at 4°C.

[0124] 2. Add 200 μL of R10 complete medium to each well after treatment in step 1 and block at 37°C for 2-4 hours.

[0125] 3. Resuspend spleen lymphocytes in R10 basal medium to obtain spleen lymphocyte resuspension solution. Set up negative control wells and positive control wells for the experimental wells. Add spleen lymphocyte resuspension solution to each well, 5×10 5 Splenic lymphocytes.

[0126] 4. Add RBD protein or HR121 protein (10 μg / mL, 10 μL) to the experimental wells, add dimethyl sulfoxide (DMSO, 10 μL) to the negative control wells, add positive stimuli (10 μL) to the positive control wells, cover the plate, and culture in a 37°C, 5% CO2 incubator for 18 h.

[0127] 5. Pour out the liquid in the wells after incubation in step 4, add 200 μL / well of ice-cold deionized water, and place in a 4°C refrigerator for 10 min to lyse the cells by hypotonic means.

[0128] 6. Discard the liquid in the wells after cell lysis in step 5, add 260 μL / well washing buffer, let stand for 1 min, discard the liquid in the wells, repeat 6 times, and dry on absorbent paper each time.

[0129] 7. Add the diluted biotin-labeled antibody working solution to each experimental well treated in step 6, 100 μL / well, and incubate at 37°C for 1 hour.

[0130] 8. Pour out the liquid in each well after incubation in step 7, add 200 μL / well of ice-cold deionized water, place in a 4°C refrigerator for 10 minutes to lyse the cells by hypotonicity, then add the diluted enzyme-labeled avidin working solution to each well, 100 μL / well, and incubate at 37°C for 1 hour.

[0131] 9. Pour out the liquid in each well after incubation in step 8, add 200 μL / well of ice-cold deionized water, and place in a 4°C refrigerator for 10 minutes to lyse the cells by hypotonic means; uncover 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 Washing buffer, 260 μL / well, let stand for 1 minute, discard the liquid in the well, and dry it thoroughly.

[0132] 10. Add the prepared 3-amino-9-ethylcarbazole (AEC) colorimetric solution to each well treated in step (9), 100 μL / well, and incubate at room temperature in the dark for 30 min. Perform subsequent color development according to the spot formation.

[0133] 11. Pour out the liquid in well 10, uncover the base of the plate, wash the front and back sides and the base with deionized water 3-5 times to stop the color development, place the plate in a cool place at room temperature, and close the base after it dries naturally.

[0134] 12. Use an enzyme-linked spot analyzer to record various parameters of the spots after treatment in step 11, and perform solid phase enzyme-linked immunospot technique (ELISPOT) analysis.

[0135] 2. Experimental Results

[0136] like Figure 7 A and Figure 7 As shown in B, compared with the control group, RBD-Trimer VLP can induce IFN-γ secreting cells, indicating that RBD-Trimer VLP can induce T cell immune response.

[0137] 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 fragment 1 and fragment 2; the amino acid sequence of fragment 1 is shown in SEQ ID NO: 4, and the amino acid sequence of fragment 2 is shown in SEQ ID NO:

7.

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

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

4. Use of the virus-like particles according to claim 3 in the preparation of drugs for preventing and / or treating diseases caused by SARS-CoV-2.

5. Use of the virus-like particles according to claim 4 in the preparation of drugs against SARS-CoV-2.

6. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the amino acid sequence of the recombinant protein combination of claim 1, the nucleotide sequence of the nucleic acid molecule encoding the fragment 1 is shown in SEQ ID NO: 5, and the nucleotide sequence of the nucleic acid molecule encoding the fragment 2 is shown in SEQ ID NO:

8.

7. A recombinant vector, characterized in that: The recombinant vector contains the nucleic acid molecule according to claim 6.

8. A recombinant engineered bacterium, characterized in that: The recombinant engineered bacteria contains the recombinant vector according to claim 7.

9. The method for preparing virus-like particles according to claim 3, characterized in that: Cultivate the recombinant engineered bacteria described in claim 8 to express virus-like particles.

10. A drug for resisting SARS-CoV-2, preventing and / or treating diseases caused by SARS-CoV-2, characterized in that: The drug contains the virus-like particle according to claim 3 and a pharmaceutically acceptable carrier.

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