Novel coronavirus recombinant protein and preparation method and application thereof

Abstract

This invention discloses a recombinant protein of the novel coronavirus, its preparation method, and its applications. Referring to the mutation sites of the RBD protein in different SARS-CoV-2 variants, five high-frequency mutation sites (417, 452, 478, 484, and 501) were selected to design an RBD-RBD tandem expression recombinant protein to construct different novel coronavirus recombinant proteins. Based on different mutation sites, three recombinant proteins were constructed, named NNR, TTR, and NTR, respectively. These recombinant proteins exhibit better immunogenicity and are more likely to stimulate a specific immune response compared to expressing a single RBD protein. The recombinant protein of this invention has advantages such as high yield, low production cost, and high protein purity. The obtained novel coronavirus recombinant protein has good immunogenicity and cross-protection, and there are no biosafety risks. It can induce immune protection against the original and variant strains of the novel coronavirus and is suitable for large-scale preparation of COVID-19 vaccines.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, and in particular to recombinant proteins of the novel coronavirus, their preparation methods, and applications. Background Technology

[0002] After SARS-CoV-2 enters the cell by binding its spike protein to angiotensin-converting enzyme 2 (ACE2), the host transmembrane serine protease 2 (TMPRSS2) cleaves the spike protein, thereby achieving cell membrane fusion. SARS-CoV-2 possesses a large RNA genome containing approximately 30,000 nucleotides. Its replication is mediated by RNA-dependent RNA polymerase (RdRP) and the associated proofreading enzyme ribonuclease (ExoN). Combined with the discontinuous nature of coronavirus transcription, this results in high recombination, insertion and deletion rates, and point mutation rates. During the COVID-19 pandemic, new variants and mutants of SARS-CoV-2 have emerged from time to time. The recently emerged Omicron variant RBD contains 11 common mutations. These variants possess high immune evasion capabilities and partial vaccine evasion capabilities, allowing the SARS-CoV-2 coronavirus to continue circulating.

[0003] After infection with SARS-CoV-2, humans do not develop an antibody response that prevents reinfection. Vaccine protection remains the most effective measure to protect humans against COVID-19, providing protective immunity against SARS-CoV-2 infection. To date, more than ten vaccines have been approved for use by the World Health Organization.

[0004] Global vaccine strategies offer two main vaccine groups: the classic group includes subunit vaccines, inactivated vaccines, live attenuated vaccines, and virus-like particle vaccines; newer approaches include RNA-based vaccines, which deliver RNA encoding target viral proteins into human cells. Compared to other types of vaccines, recombinant protein vaccines offer several advantages, primarily high safety and the potential to serve as a useful supplement to two-phase (primary-boost) vaccination; the use of appropriate adjuvants in recombinant protein vaccines can address the issue of low immunogenicity; and unlike mRNA and viral vector vaccines, recombinant protein vaccines have lower requirements for production, storage, and transportation.

[0005] In response to the continuous mutation of the SARS-CoV-2 virus, the development of a safe and effective SARS-CoV-2 vaccine is the most pressing and urgent issue today. This invention utilizes an insect rod-like expression system, combined with high-frequency SARS-CoV-2 RBD mutation sites, to express a novel recombinant protein. Immunoprotection experiments were conducted to verify its protective effect against novel coronavirus attack and its cross-protective effect against different variants. Summary of the Invention

[0006] The purpose of this invention is to propose a recombinant protein of the novel coronavirus, its preparation method, and its application. By rationally selecting protein fragments, the immunogenicity and cross-protective effect of the recombinant protein can be improved, enabling it to be used in the development and production of vaccines to prevent the original strain and variant strains of the novel coronavirus.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] In a first aspect, the present invention provides a recombinant protein of the novel coronavirus, wherein the recombinant protein is any one of NNR, TTR, and NTR proteins, respectively denoted as NNR, TTR, and NTR, wherein the amino acid sequence of the NNR protein is shown in SEQ ID NO.4, the amino acid sequence of the TTR protein is shown in SEQ ID NO.5, and the amino acid sequence of the NTR protein is shown in SEQ ID NO.6.

[0009] Furthermore, the gene sequence of the NNR protein is shown in SEQ ID NO.1.

[0010] Furthermore, the gene sequence of the TTR protein is shown in SEQ ID NO.2.

[0011] Furthermore, the gene sequence of the NTR protein is shown in SEQ ID NO.3.

[0012] Secondly, the present invention also provides a method for preparing the above-mentioned recombinant protein of the novel coronavirus, comprising the following steps:

[0013] Step 1: Obtain the coding gene of the recombinant protein using gene synthesis technology. Perform multiple cloning site analysis on the coding gene and the gene of the pFastBac1 plasmid. Select two restriction enzyme sites provided by the pFastBac1 plasmid but not present on the target protein. After double digestion, recover the linearized vector fragment and the gene fragment of the target protein. Insert the target fragment into the multiple cloning site after the PH promoter of the pFastBac1 plasmid using enzyme ligation. Then transform the cells into E. coli DH5α competent cells to obtain the recombinant shuttle plasmid pFastBac1-target protein.

[0014] Step 2: Transform the recombinant shuttle plasmid pFastBac1-target protein into DH10Bac competent cells to obtain the recombinant rod plasmid Bacmid. Use the recombinant rod plasmid Bacmid to transfect adherent Sf9 insect cells to rescue the recombinant baculovirus. At the time of transfection, the confluence of Sf9 insect cells reached more than 80%.

[0015] Step 3: Inoculate adherent Sf9 insect cells with recombinant baculovirus at MOI=2. Three days later, harvest the supernatant and purify it to obtain the recombinant protein of the novel coronavirus.

[0016] Furthermore, in the above-mentioned method for preparing recombinant proteins of the novel coronavirus, the two restriction enzyme sites in step 1 are selected as EcoRI and NotI.

[0017] Further, in the above-mentioned method for preparing recombinant novel coronavirus protein, the specific steps for obtaining the recombinant baculovirus plasmid in step 2 are as follows: 10 ng of pFastBac1-target protein shuttle plasmid is added to DH10Bac competent cells, incubated on ice for 30 min, heat-shocked at 42℃ for 45 s, then incubated on ice for 3 min, 600 μl of SOC is added, and the cells are shaken at 37℃ and 200 rpm for 4 h, centrifuged at 5500 rpm for 2 min, the excess supernatant is discarded, and 100 μl of the bacterial cells is resuspended. 50 μl of the solution is evenly spread onto LB plates containing X-gal and IPTG triple antibodies, and cultured at 37℃ for 48 h-72 h. The white spots selected by blue-white screening are the recombinant baculovirus plasmid Bacmid.

[0018] Furthermore, in the above-mentioned method for preparing recombinant protein of the novel coronavirus, the concentration of adherent SF9 insect cells during inoculation with the recombinant rod plasmid in step 3 is 2 × 10⁻⁶. 6 / mL or more.

[0019] Furthermore, in the above-mentioned method for preparing recombinant protein of the novel coronavirus, the purification method for the recombinant protein of the novel coronavirus in step 3 is nickel column affinity chromatography purification method. The specific process is as follows: Collect the supernatant of the virus from the P3 generation of adherent sf9 insect cells, inoculate it into High Five suspension cells at MOI=2 for large-scale amplification and culture for 72h, collect the suspension cell culture medium and centrifuge at 4℃ and 5000rpm for 20min to obtain cell supernatant, filter the collected cell supernatant through a 0.45um filter membrane, and then bind and purify it with a nickel column gravity column. Collect the protein eluent, pass it through an ultrafiltration centrifuge tube, and dialyze it with PBS to obtain the recombinant protein.

[0020] Thirdly, this invention also provides the application of the aforementioned recombinant protein of the novel coronavirus in the preparation of vaccines against the original or variant strains of the novel coronavirus.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] The novel coronavirus recombinant protein provided by this invention, referencing the mutation sites of RBD proteins from different SARS-CoV-2 variants, selects five high-frequency mutation sites: 417, 452, 478, 484, and 501, and designs an RBD-RBD tandem expression recombinant protein to construct different novel coronavirus recombinant proteins. Based on different site mutations, three recombinant proteins are constructed, named NNR, TTR, and NTR, respectively. Compared with expressing a single RBD protein, this recombinant protein has better immunogenicity and can better stimulate the body to produce a specific immune response.

[0023] The recombinant protein production process of this invention utilizes an insect rod-shaped expression system as an expression vector and cells as a bioreactor to produce recombinant proteins of the novel coronavirus, which has the advantages of high yield, low production cost, and high protein purity.

[0024] Furthermore, since the recombinant protein of the novel coronavirus of the present invention has good immunogenicity, good cross-protection, and no biosafety risks, it can stimulate immune protection against the original strain and variant strain of the novel coronavirus, and is suitable for large-scale preparation of COVID-19 vaccines. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0026] Figure 1 This is a schematic diagram of the gene composition of NNR, TTR, and NTR provided in an embodiment of the present invention.

[0027] Figure 2 The images show immunofluorescence patterns of recombinant proteins provided in embodiments of the present invention. In the images, A is an immunofluorescence pattern of recombinant protein NNR obtained using SARS-CoV-2 Spike RBD rabbit polyclonal antibody as the primary antibody; B is an immunofluorescence pattern of recombinant protein TTR obtained using SARS-CoV-2 Spike RBD rabbit polyclonal antibody as the primary antibody; C is an immunofluorescence pattern of recombinant protein NTR obtained using SARS-CoV-2 Spike RBD rabbit polyclonal antibody as the primary antibody; and DF is a negative control image obtained from adherent sf9 cells uninfected with baculovirus using SARS-CoV-2 Spike RBD rabbit polyclonal antibody as the primary antibody.

[0028] Figure 3The figures show the recombinant protein expression identification results provided in the embodiments of the present invention. In the figures, A represents the identification of NNR-rBV protein expression using SARS-CoV-2 RBD monoclonal antibody, B represents the identification of TTR-rBV protein expression using SARS-CoV-2 RBD monoclonal antibody, C represents the identification of NTR-rBV protein expression using SARS-CoV-2 RBD monoclonal antibody, D represents the identification of NNR-rBV protein expression using his-tag monoclonal antibody, E represents the identification of TTR-rBV protein expression using his-tag monoclonal antibody, and F represents the identification of NTR-rBV protein expression using his-tag monoclonal antibody.

[0029] Figure 4 The SDS-PAGE identification results of the recombinant protein after purification provided in the embodiments of the present invention. In the figure, A is the SDS-PAGE identification of purified NNR recombinant protein: 1. Marker; 2. Cell stock solution; 3. Flow-through buffer; 4. Washing buffer; 5-10, 250mM elution buffer; B is the SDS-PAGE identification of purified TTR recombinant protein: 1. Marker; 2. Cell stock solution; 3. Flow-through buffer; 4. Washing buffer; 5-10, 250mM elution buffer; C is the SDS-PAGE identification of purified NTR recombinant protein: 1. Marker; 2. Cell stock solution; 3. Flow-through buffer; 4. Washing buffer; 5-10, 250mM elution buffer; D is the Western blot identification of purified NNR recombinant protein with SARS-CoV-2 RBD monoclonal antibody: 1. Marker; 2. Cell stock solution; 3. Flow-through buffer; 4. Washing buffer; 5-10, 250mM elution buffer; E is the Western blot identification of purified TTR recombinant protein with SARS-CoV-2 RBD monoclonal antibody. Blot identification: 1. Marker; 2. Cell stock solution; 3. Flow-through buffer; 4. Washing buffer; 5-10. 250mM elution buffer. F is the Western blot identification of NTR recombinant protein purified with SARS-CoV-2 RBD monoclonal antibody.

[0030] Figure 5A The detection immunoassay provided in the embodiments of the present invention

[0031] Specific antibody levels of SARS-CoV-2 (2019-nCoV) RBD protein.

[0032] Figure 5B This invention provides a method for detecting the level of specific antibodies against the SARS-CoV-2BA.2RBD protein.

[0033] Figure 5C The detection immunoassay provided in the embodiments of the present invention

[0034] Antibody levels of IgG1 and IgG2a in SARS-CoV-2 (2019-nCoV) RBD protein.

[0035] Figure 5D The present invention provides a method for detecting the antibody levels of IgG1 and IgG2a against the SARS-CoV-2BA.2RBD protein.

[0036] Figure 5E The present invention provides a method for detecting the ratio of IgG2a / IgG1 after stimulation with SARS-CoV-2 (2019-nCov) RBD protein or SARS-CoV-2 BA.2 RBD protein.

[0037] Figure 6 The present invention provides a method for detecting the titer of neutralizing antibodies against SARS-CoV-2.

[0038] Figure 7 The percentage change in body weight of mice after challenge with SARS-CoV-2BA.2 is provided in this embodiment of the invention.

[0039] Figure 8A The results of viral load detection in the nasal turbinate bone of mice after challenge are provided in the embodiments of the present invention.

[0040] Figure 8B The results of viral load detection in the trachea of ​​mice after challenge are provided in the embodiments of the present invention.

[0041] Figure 8C The results of viral load detection in the lungs of mice after challenge are provided in the embodiments of the present invention. Detailed Implementation

[0042] To better understand this technical solution, the method of the present invention will be described in detail below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0043] The novel coronavirus recombinant protein provided by this invention is based on the Spike RBD genome reference sequence (NC_045512.2) of the novel coronavirus. Site mutations were designed, selecting five high-frequency mutation sites: 417, 452, 478, 484, and 501. Two RBD proteins containing these site mutations were linked to construct a new RBD-RBD recombinant protein. Depending on the site mutation, the tandem combination of the two proteins varied, resulting in three different recombinant proteins, denoted as NNR, TTR, and NTR, respectively. The gene structures of the recombinant proteins NNR, TTR, and NTR are as follows: Figure 1 As shown.

[0044] The recombinant protein NNR has the following sequence from N-terminus to C-terminus: bee venom signal peptide + SARS-CoV-2RBD protein 1 + SARS-CoV-2RBD protein 1 + 10His tag protein. The SARS-CoV-2RBD protein 1 contains the following site mutations: K417N, L452R, T478K, E484K, and N501Y. The sequences are linked by linking peptides. The gene sequence of the NNR protein is shown in SEQ ID NO.1, and the amino acid sequence of the NNR protein is shown in SEQ ID NO.4.

[0045] The gene sequence of the NNR protein (as shown in SEQ ID NO.1) is as follows:

[0046] atgaagttcc tggttaacgt ggctctggtg ttcatggtgg tgtacatctc ctacatctacgcccgcgttcagcccacaga atccatcgtg cgcttcccta acatcaccaa cctgtgtccc ttcggtgaggtgttcaacgccacccgtttc gcttcagtgt acgcctggaa ccgtaagcgt atcaact gtgttcaggctcagcgtcctcaact ccgctagctt cagcaccttc aagtgctacg gcgttagccc caccaagctgaacgacctgtgtttcactaa cgtgtacgct gactccttcg tgatccgtgg tgacgaagtg cgccagatcgcccccaggccagactggcaac atcgccgact acaactacaa gctgcccgac gacttcaccg gttgcgtgatcgcttggaacagcaacaacc tggacagcaa ggtgggtggt aactacaact accgctaccg cctgttccgcaagagcaacctgaagccatt cgagcgcgac atcagcactg agatctacca ggccggcagc aagccttgtaacggagtgaagggattcaac tgctacttcc ccctgcagag ctacggtttc caaccaacat acggcgtgggctaccagccttaccgcgtcg tggtgctgag cttcgaactc ctgcacgctc ctgccaccgt ttgtggtcccaagaagagcaccaacctcgt taagaacaag ggcggcggcg gtagcggcgg tggcggctctggtggcggaggcagccgcgt gcaaccaacc gagtccatcg tgcgcttccc caacatcacc aacctgtgccccttcggtgaagtctttaac gccacccgtt tcgctagcgt gtacgcctgg aaccgtaagc gcatcagcaactgcgtcgccgactactcag tgctctacaa ctccgccagcttctccacat tcaagtgtta cggtgtttctcctactaagctgaacgacct ctgtttcacc aacgtctacg ctgactcctt cgtgatccgt ggcgacgaggtgcgccaaatcgctcctggc cagactggaa acatcgctga ctacaactac aagctgcctg acgacttcacaggctgcgtgatcgcctgga acagcaacaa cctcgacagc aaggtcggcg gcaactacaa ctaccgttacaggctgttccgtaagagcaa cctgaagcct ttcgaacgtg acatctccac tgaaatctac caggccggttccaagccctgtaacggcgtg aagggtttca actgctactt cccactgcag tcctacggct tccagccaacctacggcgttggttaccagc cataccgcgt tgtggtcctc tccttcgagc tgctccacgc tcctgccaccgtctgcggtcctaagaagtc cacaaacctc gtgaagaaca aggctcatca tcaccatcac catcaccaccaccat

[0047] The amino acid sequence of the NNR protein (as shown in SEQ ID NO. 4) is:

[0048] MKFLVNVALV FMVVYISYIY ARVQPTESIV RFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSV LYNSASFSTFKCYGVSPTKL NDLCFTNVYA DSFVIRGDEV

[0049] RQIAPGQTGNIADYNYKLPD DFTGCVIAWN SNNLDSKVGGNYNYRYRLFR KSNLKPFERDISTEIYQAGSKPCNGVKGFNCYFPLQSYGF QPTYGVGYQP YRVVVLSFELLHAPATVCGPKKSTNLVKNKGGGGSGGGGS GGGGSRVQPT ESIVRFPNITNLCPFGEVFN ATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVS PTKLNDLCFT NVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCV IAWNSNNLDS KVGGNYNYRYRLFRKSNLKP FERDISTEIYQAGSKPCNGVKGFNCYFPLQSYGFQPTYGV GYQPYRVVVL SFELLHAPATVCGPKKSTNLVKNKAHHHHHHHHHH

[0050] The recombinant protein TTR has the following sequence from N-terminus to C-terminus: bee venom signal peptide + SARS-CoV-2RBD protein 2 + SARS-CoV-2RBD protein 2 + 10His tag protein. The SARS-CoV-2RBD protein 2 contains the following site mutations: K417T, L452R, T478K, E484K, and N501Y. The sequences are linked by linking peptides. The gene sequence of the TTR protein is shown in SEQ ID NO.2, and the amino acid sequence of the TTR protein is shown in SEQ ID NO.5.

[0051] The gene sequence of the TTR protein (as shown in SEQ ID NO.2) is as follows:

[0052] atgaagttcc tggtgaacgt ggccctggtg ttcatggtgg tgtacatcagctacatctacgctcgcgttc agcctactga atccatcgtt cgcttcccta acatcaccaa cctgtgtccattcggcgaagtgttcaacgc tacccgcttc gctagcgtgt acgcttggaa ccgcaagagg atctcaaactgtgtggctgactactccgtg ctgtacaact cagctagctt ctccacattc aagtgctacg gtgtgagccctactaagctgaacgacctgt gtttcaccaa cgtctacgct gactctttcg tgatccgtgg tgacgaagtgcgccagatcgctcctggtca gacaggtacc atcgctgact acaactacaa gctgcctgac gacttcactggttgtgtgatcgcctggaac agcaacaact tggatagcaa ggtcggtggc aactacaact accgctaccgtctgttccgtaagtctaacc tgaagccctt cgagcgtgat atctcaaccg aaatctacca ggctggttctaagccctgtaacggtgtcaa gggtttcaac tgctacttcc cactgcaatc ctacggcttc cagccaacatacggtgtgggctaccagcct taccgcgttg tggttctgag cttcgagctg ctccatgccc ctgctactgtgtggtcctaagaagtcca ccaacctcgt taagaacaag ggtggtggtg gttccggtgg tggcggttctggtggtggtggatctcgtgt gcagcctact gaatcaatcg tgcgtttccc taacatcaca aacctgtgtcccttcggtgaagtgttcaac gccactcgtt tcgcttccgt gtacgcctgg aacaggaagc gcatcagcaactgcgttgccgactacagcg ttctgtacaa ctccgcttcattcagcacct tcaagtgtta cggcgtgagccctactaagctcaacgacct ctgtttcacc aacgtttacg ctgactcttt cgtgatccgt ggcgacgaagtccgccagatcgctcctggt cagactggaa caatcgccga ctacaactac aagctgccag acgacttcaccggctgcgtgatcgcttgga actcaaacaa cctggacagc aaggtgggcg gcaactacaa ctaccgttacaggctgttccgcaagagcaa cctgaagcct ttcgagcgtg acatcagcac cgagatctac caggccggtagcaagccctgcaacggagtg aagggcttca actgctactt ccctctccaa tcctacggct tccaacctacatacggtgtgggctaccagc cctaccgtgt ggtggttctg agcttcgagc tgctgcacgc cccagctactgtgtgtggtcctaagaagtc aactaacctg gttaagaaca aggctcatca tcaccatcac catcaccaccaccat

[0053] The amino acid sequence of the TTR protein (shown in SEQ ID NO. 5) is:

[0054] MKFLVNVALV FMVVYISYIY ARVQPTESIV RFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSV LYNSASFSTFKCYGVSPTKL NDLCFTNVYA DSFVIRGDEV

[0055] RQIAPGQTGTIADYNYKLPD DFTGCVIAWN SNNLDSKVGGNYNYRYRLFR KSNLKPFERDISTEIYQAGSKPCNGVKGFNCYFPLQSYGF QPTYGVGYQP YRVVVLSFELLHAPATVCGPKKSTNLVKNKGGGGSGGGGS GGGGSRVQPT ESIVRFPNITNLCPFGEVFN ATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVS PTKLNDLCFT NVYADSFVIRGDEVRQIAPGQTGTIADYNYKLPDDFTGCV IAWNSNNLDS KVGGNYNYRYRLFRKSNLKP FERDISTEIYQAGSKPCNGVKGFNCYFPLQSYGFQPTYGV GYQPYRVVVL SFELLHAPATVCGPKKSTNLVKNKAHHHHHHHHHH

[0056] The recombinant protein NTR sequence from N-terminus to C-terminus is: bee venom signal peptide + SARS-CoV-2RBD protein 1 + SARS-CoV-2RBD protein 2 + 10His tag protein. The mutation sites of SARS-CoV-2RBD protein 1 are: K417N, L452R, T478K, E484K, N501Y; the mutation sites of SARS-CoV-2RBD protein 2 are: K417T, L452R, T478K, E484K, N501Y. The sequences are linked by linker peptides. The gene sequence of the NTR protein is shown in SEQ ID NO.3, and the amino acid sequence of the NTR protein is shown in SEQ ID NO.6.

[0057] The gene sequence of the NTR protein (as shown in SEQ ID NO.3) is as follows:

[0058] atgaagttcc tggtgaacgt ggccctggtt ttcatggttg tgtacatcag ctacatctacgcccgcgttcagccaactga atcaatcgtg cgcttcccaa acatcactaa cctgtgtcct ttcggcgaagtgttcaacgccactcgcttc gcttctgtgt acgcttggaa caggaagcgc atctccaact gcgttgccgactacagcgttctgtacaact ccgcttcctt ctcaactttc aagtgttacg gagtctcccc aactaagctgaacgacctgtgtttcactaa cgtctacgct gacagcttcg tgatccgcgg cgacgaggtc cgtcagatcgctccaggtcagacaggaaac atcgctgact acaactacaa gctgccagac gacttcacag gctgcgtgatcgcttggaacagcaacaact tggactctaa ggttggtggc aactacaact accgttaccg tttgttccgtaagagcaacctgaagccttt cgagcgtgac atctccacag agatctacca ggctggttcc aagccttgtaacggagtcaagggcttcaac tgctacttcc ctctgcagag ctacggcttc cagcccacat acggtgtgggttaccagccttaccgtgtgg tcgtgctctc cttcgaactg ctgcacgctc ctgccacagt gtgcggtcctaagaagagcaccaacctggt taagaacaag ggtggtggcg gttctggtgg tggcggttct ggcggcggtggtagccgtgtgcagcctaca gagtccatcg tgcgcttccc taacatcacc aacctctgcc cattcggtgaggtgttcaacgctactcgct tcgcttccgt gtacgcctgg aaccgtaagc gtatctccaa ctgcctcgctgactactccgtgctgtacaa ctctgcttccttctctacct tcaagtgtta cggcgtgagc cctaccaagctgaacgacttgtgtttcaca aacgtctacg ccgactcttt cgtgatccgt ggtgacgaag tccgtcagatcgcccctggtcagacaggaa ccatcgctga ttacaactac aagctgcctg acgacttcac aggttgcgtgatcgcctggaactccaacaa cctcgactcc aaggtgggtg gcaactacaa ctaccgttac cgcctgttccgtaagtctaacctgaagcct ttcgaacgtg acatcagcac cgaaatctac caagccggtt caaagccttgtaacggcgtgaagggtttca actgctactt ccctctccag agctacggtt tccagcccac atacggtgttggctaccagccttaccgtgt ggtggtgctc tccttcgaac tgctgcacgc tcctgctaca gtgtgtggacctaagaagagcaccaacctg gtgaagaaca aggctcatca tcaccatcac catcaccacc accat

[0059] The amino acid sequence of the NTR protein (shown in SEQ ID NO.6) is:

[0060] MKFLVNVALVFMVVYISYIYARVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNI ADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGSKPCNGVKGFNCYFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKGG GGSGGGGSGGGGSRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGTIADYNYKLP DDFTGCVIAWNSNNLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGSKPCNGVKGFNCYFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKAHHHHHHHHHH

[0061] This invention references the mutation sites of RBD proteins from different SARS-CoV-2 variants, selecting five high-frequency mutation sites (417, 452, 478, 484, and 501) to design RBD-RBD tandem expression recombinant proteins to construct different novel coronavirus recombinant proteins. Based on different site mutations, three recombinant proteins were constructed, named NNR, TTR, and NTR, respectively. Compared with expressing a single RBD protein, these recombinant proteins have better immunogenicity and can more effectively stimulate the body to produce a specific immune response.

[0062] The method for preparing recombinant protein of the novel coronavirus includes the following steps:

[0063] Step 1: Obtain NNR, TTR, and NTR proteins using gene synthesis technology. Perform multiple cloning site analysis on the coding genes of the three proteins and the gene of the pFastBac1 plasmid. Select two restriction enzyme sites provided by the pFastBac1 plasmid that are not present on the target protein. After double digestion, recover the linearized vector fragment and the gene fragment of the target protein. Insert the target fragment into the multiple cloning site after the PH promoter of the pFastBac1 plasmid using enzyme ligation. Then transform E. coli DH5α competent cells to obtain the recombinant shuttle plasmid pFastBac1-target protein.

[0064] In step 1 of this invention, the encoding genes of recombinant proteins NNR, TTR, and NTR and the restriction enzyme sites of the pFastBac1 vector are EcoRI and NotI.

[0065] Step 2: The recombinant shuttle plasmid pFastBac1-target protein was transformed into DH10Bac competent cells to obtain the recombinant rod plasmid Bacmid. The recombinant rod plasmid Bacmid was used to transfect adherent Sf9 insect cells to rescue the recombinant baculovirus. At the time of transfection, the confluence of Sf9 insect cells reached more than 80%.

[0066] In step 2 of this invention, the molecular cloning of the recombinant shuttle plasmid pFastBac1-target protein was obtained by conventional transformation of DH5α competent cells. The specific steps for obtaining the recombinant baculovirus plasmid are as follows: 10 ng of pFastBac1-target protein shuttle plasmid was added to DH10Bac competent cells, incubated on ice for 30 min, heat-shocked at 42℃ for 45 s, then incubated on ice for 3 min, 600 μL of SOC was added, and the cells were shaken at 37℃ and 200 rpm for 4 h, centrifuged at 5500 rpm for 2 min, the excess supernatant was discarded, and 100 μL of the bacterial cells were resuspended. 50 μL of the solution was evenly spread on LB agar plates containing X-gal and IPTG triple antibodies, and cultured at 37℃ for 48 h-72 h. The white spots selected by blue-white screening are the recombinant baculovirus plasmid Bacmid. DNA homologous recombination between the blank baculovirus plasmid in DH10Bac competent cells and the pFastBac1-target protein shuttle plasmid was achieved through DNA transposition.

[0067] Step 3: The recombinant baculovirus was inoculated into adherent Sf9 insect cells at an MOI of 2. The supernatant was harvested three days later to obtain the recombinant novel coronavirus protein. The concentration of adherent Sf9 insect cells at the time of inoculation with the recombinant baculovirus plasmid was 2 × 10⁻⁶. 6 / mL or more.

[0068] In step 3 of this invention, the recombinant protein is purified using nickel column affinity chromatography. The specific process is as follows: The supernatant of the virus from the P3 generation of adherent SF9 insect cells is collected and inoculated into High Five suspension cells at an MOI of 2 for large-scale amplification and culture for 72 hours. The suspension cell culture medium is then collected and centrifuged at 4°C and 5000 rpm for 20 minutes to obtain the cell supernatant. The collected cell supernatant is filtered through a 0.45 μm filter membrane, then bound and purified using a nickel column gravity chromatography. The protein eluent is collected, and after ultrafiltration and centrifugation, dialysis with PBS yields high-purity recombinant proteins with a purity of approximately 93.93% for NNR, approximately 94.52% for TTR, and approximately 93.77% for NTR.

[0069] Example 1

[0070] The following materials were selected to verify the recombinant protein preparation method and application of the present invention.

[0071] The donor plasmid pFastBac1 was provided by the Virology Laboratory of the Institute of Veterinary Medicine, Academy of Military Medical Sciences; the main reagents, such as E. coli DH5α and E. coli DH10Bac antibiotics, were purchased from Solarbio; protein gene synthesis was provided by General Biotech; technical services such as recombinant plasmid gene sequencing and primer synthesis were provided by Shanghai Sangon Biotech Co., Ltd.; insect cell transfection kits were purchased from Invitrogen; endonucleases and Grace adherent culture medium were purchased from Thermo Fisher Scientific; genome extraction kits were purchased from Axygen; SIM SF and SIM HF were purchased from Beijing Sinocare Biotech Co., Ltd.; and SARS-CoV-2 Spike RBD rabbit polyclonal antibody was purchased from GeneTex.

[0072] The experimental method is as follows:

[0073] S1. Using gene fragments of recombinant proteins NNR, TTR, and NTR obtained through gene synthesis, the target genes were cloned into the pFastBac1 plasmid using EcoRI and NotI restriction enzyme sites. The plasmid was then transformed into *E. coli* DH5α competent cells, plated overnight, and positive bacteria were screened. Sequencing, PCR, and restriction enzyme digestion were performed to identify the correct pFastBac1-target protein plasmid. SEQ ID NO. 7 and SEQ ID NO. 8 are the upstream and downstream primers for PCR identification of the target gene NNR, TTR, and NTR fragments, respectively.

[0074] The upstream primer sequence is: 5'-GCTATAGTTC TAGTGGTTGG CTACGT-3'.

[0075] The downstream primer sequence is: 5'-ATGATCCTCT AGTACTTCTC GACAAGCT-3'.

[0076] S2, constructing recombinant rod-shaped plasmids

[0077] DH10Bac competent cells were transformed with pFastBac1-NNR plasmid. White spots were selected from the blue-white colonies produced by bacterial growth, and the plasmid was extracted and confirmed by PCR to be correct, thus obtaining the recombinant rod plasmid NNR-Bacmid. TTR-Bacmid and NTR-Bacmid were obtained using the same method.

[0078] S3, saving recombinant baculoviruses

[0079] Three positive Bacmid plasmids were transfected into adherent Sf9 cells using the Cellreagent transfection kit. After three days of culture at 27°C, cell culture supernatant P1 was collected and seeded into new Sf9 cells at a volume ratio of 2%. Culture was continued to amplify viral virulence. After three days of culture, P2 generation supernatant was collected and seeded into new Sf9 cells at a volume ratio of 2%. After three days of culture, P3 generation supernatant was collected. DNA was extracted from the P3 generation supernatant for PCR verification, and morphological changes in P3 generation cells were observed and recorded. Indirect immunofluorescence was used to identify the expression of the virus in P3 generation cells. Figure 2 A~ Figure 2 C represents the assay results of Sf9 cells infected with three different baculoviruses obtained using indirect immunofluorescence assay with SARS-CoV-2 Spike RBD rabbit polyclonal antibody as the primary antibody. Figure 2 A~ Figure 2 C found that all three types of baculovirus-infected P3 generation Sf9 cells showed strong green fluorescence expression, indicating that Sf9 cells successfully expressed the three recombinant proteins.

[0080] S4, Large-scale expression and purification of recombinant proteins

[0081] The titer of P3 generation baculovirus was determined using a rapid baculovirus titer assay kit from Takara. P3 generation baculovirus was inoculated into a large number of cultured suspension High Five cells at an MOI of 2. After 72 hours, the cell supernatant was collected and cultured. The recombinant protein was obtained after purification by nickel column affinity chromatography.

[0082] (1) Validation of the three recombinant proteins prepared in the example using Western blot.

[0083] Western blot analysis using SARS-CoV-2 Spike RBD rabbit polyclonal antibody as the primary antibody revealed bands in the 55-70 kDa range for all three recombinant proteins. Figure 3 As shown in AC, this indicates that the three recombinant proteins are expressed correctly.

[0084] Western blot analysis using his-tag mouse monoclonal antibody as the primary antibody revealed bands in the 55-70 kDa range for all three recombinant proteins. Figure 3 As shown in DF, this indicates that the his-tagged protein is expressed correctly.

[0085] (2) The three recombinant proteins purified by nickel column were verified using SDS-PAGE and Western blot.

[0086] SDS-PAGE was used to verify the protein composition of the cell stock solution, transfer medium, and elution buffer during the nickel column purification process. The results are as follows: Figure 4As shown in AC, recombinant protein can be obtained after elution with imidazole.

[0087] Using SARS-CoV-2 Spike RBD rabbit polyclonal antibody as the primary antibody, Western blot analysis was performed to verify the protein composition of the cell stock solution, transfer medium, and elution buffer during the white nickel column purification process. The results showed that all three recombinant proteins exhibited bands in the 55-70 kDa range. Figure 4 As shown in DF, the three recombinant proteins of this invention were successfully purified.

[0088] Example 2

[0089] The following materials were used to study the immunogenicity of the recombinant protein of the novel coronavirus: BCA protein quantitative detection kit was purchased from Beyotime Biotechnology Co., Ltd.; HPR-labeled goat anti-mouse IgG, IgG1, and IgG2a were purchased from Abcam; ELISApot detection kit for mouse IFN-γ and IL-4 was purchased from Mabtech; AddaVax adjuvant was purchased from Invivogen; and 6-8 week old and 9 month old female BALB / c mice were purchased from Beijing Vital River Pharmaceutical Co., Ltd.

[0090] 1. The specific experimental procedure is as follows:

[0091] S1, Immunity and Attack

[0092] Protein concentration was detected using a BCA assay kit. At weeks 0 and 3, 6-8 week old young mice were intramuscularly injected with 0.1 mL of a vaccine containing 1 μg / 10 μg recombinant protein and AddaVax adjuvant. An AddaVax adjuvant control group and a PBS control group were also included. At weeks 0 and 3, 9-month-old young mice were intramuscularly injected with 0.1 mL of a vaccine containing 10 μg recombinant protein and AddaVax adjuvant. An AddaVax adjuvant control group and a PBS control group were also included. Fourteen days after the second immunization, a challenge protection experiment was conducted in aged mice. In a biosafety level 3 laboratory, challenge was performed using 10⁴ PFU of SARS-CoV-2BA.2 via nasal instillation. Each mouse was inoculated with 50 μL of viral fluid. All experimental conditions and procedures complied with the ethical guidelines of the International Association for the Study of Pain.

[0093] S2, Specific IgG Antibody Detection

[0094] Blood was collected from the orbital sinus of mice at 3 and 5 weeks post-immunization. After being left at room temperature for 2 hours, the blood was centrifuged at 4000 rpm for 10 minutes, and the supernatant serum was separated and stored at -20°C. SARS-CoV-2-specific IgG, IgG1, and IgG2a in the serum were detected using an ELISA assay. 96-well plates were coated with SARS-CoV-2 (2019-nCoV) Spike RBD protein and SARS-CoV-2BA.2 (Omicron) Spike RBD protein at 2 μg / mL overnight at 4°C, blocked with 5% BSA at room temperature for 2 hours, and diluted serum samples were added to the 96-well plates. The plates were incubated at 37°C for 1.5 hours, washed with PBST, and then incubated with HRP-labeled goat anti-mouse IgG, IgG1, and IgG2a at 37°C for 1 hour. After washing with PBST, TMB was added and the plates were incubated at 25°C for 30 minutes. Finally, 50 μL of ELISA stop solution was added to terminate the reaction, and the results were detected at 450 nm using a spectrophotometer.

[0095] S3, Neutralizing antibody titer assay

[0096] Blood was collected from the orbital rims of mice at 3 and 5 weeks post-immunization. After being left at room temperature for 2 hours, the blood was centrifuged at 4000 rpm for 10 minutes, and the supernatant serum was separated and stored at -20°C. In biosafety level 3 experiments, [the following was applied].

[0097] Wild-type virus neutralization assay. The virus was heat-inactivated at 56°C for 30 min. Serum diluted 2-fold (1:10–1:20480) with 100 TCID50 SARS-CoV-2 cells was incubated at 37°C for 1 h, then transferred to pre-coated Vero E6 cells in 96-well plates and incubated at 37°C for 60 h. After incubation, the cytopathic effect of cells in each well was observed under an electron microscope, and the neutralizing titer of the mouse immunized group serum was calculated using the Reed-Menuch method.

[0098] S4, Challenge Protection Experiment: Body weight changes in each group of mice were observed within 15 days after challenge. The body weight changes in each group are shown below. Figure 7 As shown.

[0099] S5, lung viral load assay

[0100] The supernatant of the homogenized lungs of dissected mice was homogenized and viral RNA was extracted using magnetic beads. One-step real-time fluorescence PCR was used to design specific primers and probes targeting the ORF1ab gene of the novel coronavirus. The novel coronavirus nucleic acid in the sample was quantitatively detected by detecting changes in fluorescence signal.

[0101] 2. Experimental Results

[0102] (1) Detection results of specific IgG antibodies induced by recombinant protein of novel coronavirus in mice

[0103] The results of detecting the levels of SARS-CoV-2 (2019-nCoV) Spike RBD protein or SARS-CoV-2BA.2 (Omicron) Spike RBD protein in the serum of mice immunized at 3 and 5 weeks of gestation are as follows: Figure 5A As shown in Figure B, both proteins stimulated strong specific antibody responses, with significantly higher IgG antibody titers in both the single-immunization and double-immunization groups compared to the control group. The double-immunization group showed significantly higher IgG antibody titers than the single-immunization group, and antibody titers increased positively with the immunization dose. Even after booster immunization, the high-dose group serum remained positive at a maximum dilution of 1:1024000. In summary, immunization of mice with the three recombinant proteins NNR, TTR, and NTR elicited strong humoral immunity, and the low- and high-dose TTR groups showed higher antibody levels in a single immunization. The IgG1 and IgG2a antibody subtypes in the serum of all immunization groups at week 5 were detected. Figure 5C As shown in Figure -E, the titers of IgG1 against the Wuhan SARS-CoV-2 Spike RBD and Omicron SARS-CoV-2 Spike RBD antigens were higher than those of IgG2a in all immunized groups, indicating that IgG1 is the predominant antibody in evoking a specific immune response to the virus. In the assessment of immunization efficacy, the low IgG2a / IgG1 level suggests that, compared to cellular immunity, immunization of mice with recombinant proteins primarily induces humoral immunity to provide protection.

[0104] (2) Detection results of neutralizing antibodies induced by recombinant protein of novel coronavirus in mice

[0105] The titers of neutralizing antibodies against SARS-CoV-2 in mouse serum were measured at weeks 3 and 5 post-immunization. The results are as follows: Figure 6 As shown, the neutralizing antibody titer was not significantly different from the control group after the first immunization, but the neutralizing antibody titer was significantly higher after the second immunization than the control group. Furthermore, the overall titer after two immunizations was significantly higher than that after a single immunization. Specifically, in the TTR group with a high immunization dose, neutralizing antibodies were still detectable even after a maximum dilution of 1280 times, while in the TTR group with a low immunization dose, neutralizing antibodies were still detectable even after a maximum dilution of 320 times.

[0106] (3) Study on the protection against challenge with recombinant protein of novel coronavirus

[0107] The curve of body weight change rate in mice challenged with SARS-CoV-2BA.2 is shown below. Figure 7As shown in the figure, the body weight of mice in the challenge control group showed a slow decreasing trend, while the difference in the rate of change among the mice in each challenge experimental group was small, and their body weight remained basically stable. The body weight percentage test results indicate that the three recombinant proteins of this invention have good protective effects against the SARS-CoV-2 variant.

[0108] (4) The recombinant protein of the novel coronavirus can inhibit the replication of SARS-CoV-2 virus.

[0109] On the fourth day after challenge, the nasal turbinate bones, trachea, and lungs of mice were ground up. Using primers targeting the ORF1a / b gene, real-time quantitative PCR was used to detect changes in viral load in the mouse lungs. Results are as follows: Figure 8A As shown in Figure C, high levels of SARS-CoV-2 viral RNA copy numbers were detected in the organs of control group mice; the SARS-CoV-2 viral RNA copy numbers in the organs of immunized group mice were lower than those in the viral control group. The experimental results indicate that the three novel coronavirus recombinant proteins of this invention can effectively inhibit the replication of SARS-CoV-2 virus. Furthermore, among the three novel coronavirus recombinant proteins, the TTR group showed the best overall immunoprotective effect. The TTR group produced a high level of challenge protection against SARS-CoV-2 virus, specific IgG antibodies, and neutralizing antibodies, inhibiting the replication of the novel coronavirus. The immunoprotective effect of the recombinant proteins was mainly humoral immunity.

[0110] The various embodiments in this specification are described in a related manner. Similar or identical parts between embodiments can be referred to mutually. Each embodiment focuses on describing the differences from other embodiments. In particular, the device embodiments, electronic device embodiments, computer-readable storage medium embodiments, and computer program product embodiments are basically similar to the method embodiments, so the descriptions are relatively simple; relevant parts can be referred to the descriptions of the method embodiments.

[0111] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. However, these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A recombinant protein of the novel coronavirus, characterized in that, The recombinant protein is a TTR protein, and the amino acid sequence of the TTR protein is shown in SEQ ID NO.

5.

2. The method for preparing the recombinant protein of the novel coronavirus according to claim 1, characterized in that, Includes the following steps: Step 1: Obtain the coding gene of the recombinant protein using gene synthesis technology. Perform multiple cloning site analysis on the coding gene and the gene of the pFastBac1 plasmid. Select two restriction enzyme sites, EcoRI and NotI, on the pFastBac1 plasmid. After double digestion, recover the linearized vector and the coding gene of the target protein. Insert the coding gene into the multiple cloning site after the PH promoter of the pFastBac1 plasmid using enzyme ligation. Then transform the cells into E. coli DH5α competent cells to obtain the recombinant shuttle plasmid pFastBac1-target protein coding gene. Step 2: Transform the recombinant shuttle plasmid pFastBac1-target protein encoding gene into DH10Bac competent cells to obtain the recombinant rod plasmid Bacmid. Use the recombinant rod plasmid Bacmid to transfect adherent Sf9 insect cells to rescue the recombinant baculovirus. At the time of transfection, the confluence of Sf9 insect cells reached more than 80%. Step 3: Inoculate adherent Sf9 insect cells with recombinant baculovirus at MOI=2. Three days later, harvest the supernatant and purify it to obtain the recombinant protein of the novel coronavirus.

3. The method for preparing the recombinant protein of the novel coronavirus according to claim 2, characterized in that, The specific steps for obtaining the recombinant baculovirus plasmid in step 2 are as follows: Add 10 ng of pFastBac1-target protein shuttle plasmid to DH10Bac competent cells, incubate on ice for 30 min, heat shock at 42℃ for 45 s, then incubate on ice for 3 min, add 600 µl of SOC, shake on a shaker at 37℃ and 200 rpm for 4 h, centrifuge at 5500 rpm for 2 min, discard the excess supernatant, keep 100 µl to resuspend the bacterial cells, take 50 µl of the solution and spread it evenly on LB agar plates containing X-gal and IPTG triple antibodies, and incubate at 37℃ for 48 h-72 h. The white spots selected by blue-white screening are the recombinant baculovirus plasmid Bacmid.

4. The method for preparing recombinant protein of the novel coronavirus according to claim 2, characterized in that, In step 3, the concentration of adherent SF9 insect cells during inoculation with recombinant baculovirus was 2 × 10⁻⁶. 6 / mL or more.

5. The method for preparing the recombinant protein of the novel coronavirus according to claim 2, characterized in that, In step 3, the recombinant protein of the novel coronavirus was purified using nickel column affinity chromatography. The specific process is as follows: The supernatant of the virus from the P3 generation of adherent sf9 insect cells was collected and inoculated into High Five suspension cells at an MOI of 2 for large-scale amplification and culture for 72 h. The suspension cell culture medium was then collected and centrifuged at 4℃ and 5000 rpm for 20 min to obtain the cell supernatant. The collected cell supernatant was filtered through a 0.45 µm filter membrane and then bound and purified by a nickel column gravity column. The protein eluent was collected, and after passing through an ultrafiltration centrifuge tube and dialysis with PBS, the recombinant protein was obtained.

6. The use of the recombinant protein of the novel coronavirus according to claim 1 in the preparation of a vaccine for preventing the original strain or variant strain of the novel coronavirus SARS-CoV-2, wherein the variant strain is SARS-CoV-2 BA.2.

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