Optimization of the SARS-COV-2 Spike Protein S1 Gene, mRNA Containing the Gene, and Applications

Abstract

The present invention relates to an optimized sequence of the SARS-CoV-2 spike protein S1 and its application, belonging to the field of biotechnology. In the present invention, the S1 gene of the SARS-CoV-2 spike protein is segmented and spliced by Overlap PCR to obtain a new gene fragment, and the new gene fragments are spliced pairwise in sequence, and finally an optimized S1 gene is obtained. The mRNA constructed by the obtained optimized gene can show a higher intracellular expression level. When the obtained mRNA molecule is used as the current SARS-CoV-2 mRNA S1 antigen vaccine, it has a stronger in vivo immune response. At the same time, it is detected that the antibody response can be maintained for a longer time and has a good cross-protection effect in mice.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology. Specifically, it relates to an optimized sequence of the SARS-COV-2 spike protein S1 and its application. Background Art

[0002] The SARA-COV-2 virus continues to evolve in the human host, resulting in the emergence of different variants. From the original strain to the Alpha strain, Beta strain, Gamma strain, Delta strain, and Omicron strain, the transmissibility, immune escape ability, and infectivity of the virus variants have gradually increased. Although COVID-19 vaccines have been developed and used for vaccination at a relatively fast pace, the emergence of COVID-19 mutants has posed a continuous threat to the protective efficacy of currently approved vaccines.

[0003] The spike protein is an important structure for the binding of the COVID-19 virus to host cells and is also an important target for current vaccine research and development. Gene mutations in the spike protein change the infectivity and pathogenicity of the virus, thereby affecting the changes in the body's immune response. The spike protein S is an important pathogenic target protein of the novel coronavirus, which contains two subunits, S1 and S2. S1 mainly contains the receptor binding domain (RBD), and the coronavirus infects cells by binding to the cell surface receptor through the RBD.

[0004] Currently, the mutations of the COVID-19 virus mainly occur in the spike protein. The research of vaccines uses the whole virus or partial components of the virus as immunogens. After the spike protein of the virus strain changes, the protective efficacy of existing vaccines is greatly reduced. The current prevalence of the Omicron strain and the fast mutation rate of the COVID-19 virus have brought great troubles to the prevention and control of the COVID-19 pandemic. Based on the research of COVID-19 mutants, mutations at some important sites of the spike protein, such as K417N, L452R, N460K, N501Y, and D614G, have caused immune escape of the body's immune system. Although vaccine companies have also carried out research and development of second-generation vaccines for variant strains, bivalent vaccines against the original strain and the Omicron strain, and booster immunization strategies against BA.5. However, due to the increasing concentration of immune escape ability caused by mutations in new strains and the accelerating evolution rate of the RBD, the booster shot against BA.5 is not a good choice. Summary of the Invention

[0005] To overcome the problems existing in the background art, the present invention provides an optimized sequence of the SARS-COV-2 spike protein S1 and its application. By optimizing the S1 sequence of SARS-CoV-2, the mRNA constructed from the optimized S1 gene can show a higher intracellular expression level. When the obtained mRNA molecule is used as the current SARS-CoV-2 mRNA S1 antigen vaccine, it has a stronger in vivo immune response. At the same time, it is detected that the antibody response can be maintained for a longer time.

[0006] To achieve the above object, the present invention is realized through the following technical solutions:

[0007] The optimized SARS-COV-2 spike protein S1 gene is composed of the amino acid sequence shown in SEQ ID NO.1.

[0008] The gene encoding the optimized SARS-CoV-2 spike protein S1.

[0009] The coding sequence of the optimized SARS-CoV-2 spike protein S1 gene is the nucleotide sequence shown in SEQ ID NO.2.

[0010] The construction method of the optimized SARS-CoV-2 spike protein S1 gene includes the following steps:

[0011] (1) Segmenting and the first round of Overlap PCR amplification

[0012] The SARS-CoV-2 spike protein S1 gene is divided into twelve segments, and Overlap PCR amplification is carried out separately.

[0013] The twelve segments are 70-251, 223-310, 291-479, 461-660, 642-1032, 993-1147, 1126-1260, 1237-1389, 1363-1497, 1478-1650, 1628-1855, 1831-2067 of the S1 sequence.

[0014] Design primers to introduce optimized gene sites, and at the same time design 20bp homologous arm sequences, and twelve individual sequences are cloned respectively. Table 1 Amplification primers for twelve fragments

[0015]

[0016]

[0017] (2) The second round of PCR amplification

[0018] Perform PCR splicing on the first and second DNA sequences: Using the amplification products of the 70-251 and 223-310 fragments in step (1) as templates, and 1-F and 2-R as primers for amplification to obtain Gene Sequence 1.

[0019] Perform PCR splicing on the third and fourth DNA sequences: Using the amplification products of the 291-479 and 461-660 fragments in step (1) as templates, and 3-F and 4-R as primers for amplification to obtain Gene Sequence 2.

[0020] Perform PCR splicing on the fifth and sixth DNA sequences: Using the amplification products of the 642-1032 and 993-1147 fragments in step (1) as templates, and 5-F and 6-R as primers for amplification to obtain Gene Sequence 3.

[0021] Perform PCR splicing on the seventh and eighth DNA sequences: Using the amplification products of the 1126-1260 and 1237-1389 fragments in step (1) as templates, and 7-F and 8-R as primers for amplification to obtain Gene Sequence 4.

[0022] Perform PCR splicing on the ninth and tenth DNA sequences: Using the amplification products of the 1363-1497 and 1478-1650 fragments in step (1) as templates, and 9-F and 10-R as primers for amplification to obtain Gene Sequence 5.

[0023] Perform PCR splicing on the eleventh and twelfth DNA sequences: Using the amplification products of the 1628-1855 and 1831-2067 fragments in step (1) as templates, and 11-F and 12-R as primers for amplification to obtain Gene Sequence 6.

[0024] (3) Third-round PCR amplification

[0025] Perform PCR splicing on Gene Sequence 1 and Gene Sequence 2: Using Gene Sequence 1 and Gene Sequence 2 in step (2) as templates, and 1-F and 4-R as primers for amplification to obtain Gene Sequence 7.

[0026] Perform PCR splicing on Gene Sequence 3 and Gene Sequence 4: Using Gene Sequence 3 and Gene Sequence 4 in step (2) as templates, and 5-F and 8-R as primers for amplification to obtain Gene Sequence 8.

[0027] Perform PCR splicing on Gene Sequence 5 and Gene Sequence 6: Using Gene Sequence 5 and Gene Sequence 6 in step (2) as templates, and 9-F and 12-R as primers for amplification to obtain Gene Sequence 9.

[0028] (4) Fourth-round PCR amplification

[0029] Perform PCR splicing on Gene Sequence 7 and Gene Sequence 8: Using Gene Sequence 7 and Gene Sequence 8 in step (3) as templates, and 1-F and 8-R as primers for amplification to obtain Gene Sequence 10.

[0030] Perform PCR splicing on gene sequences 8 and 9: Using the gene sequences 8 and 9 in step (3) as templates, and 5-F and 12-R as primers for amplification to obtain gene sequence 11.

[0031] (5) Using gene sequences 10 and 11 as templates, and 13-F and 12-R as primers, splice them by Overlap PCR to obtain the optimized S1 gene.

[0032] Nucleotide sequence of primer 13-F: 5'-gccaccatggagaccgataccctgctgctgtgggtgctgctgctgtgggtgcctggctctaccggcgacgtaaacctgagaaccagaacccagc-3'.

[0033] A SARS-COV-2 spike protein mRNA, and the S1 gene of this spike protein mRNA is the above-mentioned optimized S1 gene.

[0034] For the said spike protein mRNA, its construction method includes the following steps:

[0035] (1) Using 5’-UTR as the main mRNA translation regulatory element, add a Kozak sequence gccacc behind it; meanwhile, add an IgK signal peptide sequence with secretory signal function in front of the S1 gene of SARS-COV-2, and add 3’-UTR after the TAA terminator of the target gene S1.

[0036] (2) Design a segmented PolyA tail.

[0037] (3) Recombinantly insert and modify it into the constructed pVAX vector by HindIII and BamHI.

[0038] (4) After cloning screening and sequencing, obtain the plasmid pMu-6 that stably transcribes and expresses mRNA.

[0039] (5) Prepare pMu-6 plasmid DNA, linearize it by BamHI digestion, and perform in vitro mRNA transcription according to the procedure;

[0040] (6) After the transcription is completed, treat it with RNase, purify it by column to obtain mRNA.

[0041] Furthermore, the said 5’-UTR is the 5’-UTR of the yellow fever virus 17D strain gene; the said 3’-UTR is the 3’-UTR of the ribosomal gene in the human mitochondrial gene.

[0042] The sequence of the segmented PolyA tail is shown in SEQ ID NO.4, SEQ ID NO.4:

[0043] aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaagcttatgactaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaccgcgtgctgaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa。

[0044] The IgK signal peptide sequence is 5’-atggagaccgataccctgctgctgtgggtgctgctgctgtgggtgcctggctctaccggcgac-3’.

[0045] The application of the mRNA in the preparation of a vaccine for preventing or treating COVID-19.

[0046] A vaccine for preventing or treating COVID-19, comprising the above mRNA.

[0047] Advantages of the present invention:

[0048] By optimizing the S1 gene of the SARS-CoV-2 spike protein, compared with the unoptimized S1 gene: the mRNA constructed from the optimized S1 gene of the present invention can show a higher intracellular expression level. When the obtained mRNA molecule is used as the current SARS-CoV-2 mRNA S1 antigen vaccine, it has a stronger in vivo immune response. At the same time, it is detected that the antibody response can be maintained for a longer time. Description of the drawings

[0049] Figure 1 It is a schematic diagram of the mRNA construction method in Example 2 of the present invention;

[0050] Figure 2 It is a comparison of the plasmid transcription and expression effects of the mRNA in Example 2 of the present invention;

[0051] Figure 3 It is a comparison of the expression effects of the mRNA transfection of 293 cells in Example 2 of the present invention;

[0052] Figure 4 It is a comparison of the humoral and cellular immune responses after immunizing mice with the S1 mRNA constructed in Example 4 of the present invention. Detailed implementation manners

[0053] To make the objectives, technical solutions and beneficial effects of the present invention clearer, the technical solutions of the present invention will be described in detail below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative efforts all fall within the scope of protection of the present invention.

[0054] To illustrate the present invention more clearly, the following examples are used for detailed description.

[0055] Example 1

[0056] An optimized SARS-CoV-2 spike protein S1 gene, and its construction method includes the following steps:

[0057] (1) Segmenting and first-round Overlap PCR amplification

[0058] The SARS-CoV-2 spike protein S1 gene is divided into twelve segments for PCR amplification. The PCR system: 1 μL of template (30 ng), 1 μL each of upstream and downstream primers (10 pM), 2 μL of Taq polymerase, 2 μL of 10xPCR buffer, 2 μL of dNTP mixture (2 mM), 11 μL of RNase-free water; PCR amplification program: denaturation at 98 °C for 15 s, annealing at 56 °C for 30 s, extension at 72 °C (1 kb / min), 34 cycles.

[0059] The twelve segments are 70-251, 223-310, 291-479, 461-660, 642-1032, 993-1147, 1126-1260, 1237-1389, 1363-1497, 1478-1650, 1628-1855, 1831-2067 of the S1 sequence.

[0060] Design primers to introduce optimized gene sites, and at the same time design 20-bp homologous arm sequences, and separately clone to obtain twelve individual sequences;

[0061] Introduce optimized gene sites during primer design, and separately clone to obtain twelve individual sequences.

[0062] Table 2 Primers for twelve fragments

[0063]

[0064]

[0065] (2) Second-round PCR amplification

[0066] ① PCR splice the first and second sequences to obtain gene sequence 1.

[0067] PCR system: 2 μL of template (30 ng each of the amplification products of the first 70 - 251 and the second 223 - 310), 1 μL each of the forward and reverse primers 1 - F / 2 - R (10 pM), 2 μL of Taq polymerase, 2 μL of 10x PCR buffer, 2 μL of dNTP mixture (2 mM), and 10 μL of RNase - free water. PCR amplification program: denaturation at 98 °C for 15 s, annealing at 56 °C for 30 s, extension at 72 °C (1 kb / min), 34 cycles.

[0068] ② PCR splice the third and fourth sequences to obtain Gene Sequence 2.

[0069] PCR system: 2 μL of template (30 ng each of the amplification products of the third 291 - 479 and the fourth 461 - 660), 1 μL each of the forward and reverse primers 3 - F / 4 - R (10 pM), 2 μL of Taq polymerase, 2 μL of 10x PCR buffer, 2 μL of dNTP mixture (2 mM), and 10 μL of RNase - free water. PCR amplification program: denaturation at 98 °C for 15 s, annealing at 56 °C for 30 s, extension at 72 °C (1 kb / min), 34 cycles.

[0070] ③ PCR splice the fifth and sixth sequences to obtain Gene Sequence 3.

[0071] PCR system: 2 μL of template (30 ng each of the amplification products of the fifth 642 - 1032 and the sixth 993 - 1147), 1 μL each of the forward and reverse primers 5 - F / 6 - R (10 pM), 2 μL of Taq polymerase, 2 μL of 10x PCR buffer, 2 μL of dNTP mixture (2 mM), and 10 μL of RNase - free water. PCR amplification program: denaturation at 98 °C for 15 s, annealing at 56 °C for 30 s, extension at 72 °C (1 kb / min), 34 cycles.

[0072] ④ PCR splice the seventh and eighth sequences to obtain Gene Sequence 4.

[0073] PCR system: 2 μL of template (30 ng each of the amplification products of the seventh 1126 - 1260 and the eighth 1237 - 1389), 1 μL each of the forward and reverse primers 7 - F / 8 - R (10 pM), 2 μL of Taq polymerase, 2 μL of 10x PCR buffer, 2 μL of dNTP mixture (2 mM), and 10 μL of RNase - free water. PCR amplification program: denaturation at 98 °C for 15 s, annealing at 56 °C for 30 s, extension at 72 °C (1 kb / min), 34 cycles.

[0074] ⑤ PCR splice the ninth and tenth sequences to obtain Gene Sequence 5.

[0075] PCR system: Template (30 ng each of the amplification products of the 9th paragraph 1363 - 1497 and the 10th paragraph 1478 - 1650) 2 μL, forward and reverse primers 9-F / 10-R (10 pM) 1 μL each, Taq polymerase 2 μL, 10xPCR buffer 2 μL, dNTP mixture (2 mM) 2 μL, RNase-free water 10 μL. PCR amplification program: Denaturation at 98 °C for 15 s, annealing at 56 °C for 30 s, extension at 72 °C (1 kb / min), 34 cycles.

[0076] ⑥ Perform PCR splicing on the 11th and 12th sequences to obtain Gene Sequence 6.

[0077] PCR system: Template (30 ng each of the amplification products of the 11th paragraph 1628 - 1855 and the 12th paragraph 1831 - 2067) 2 μL, forward and reverse primers 11-F / 12-R (10 pM) 1 μL each, Taq polymerase 2 μL, 10xPCR buffer 2 μL, dNTP mixture (2 mM) 2 μL, RNase-free water 10 μL. PCR amplification program: Denaturation at 98 °C for 15 s, annealing at 56 °C for 30 s, extension at 72 °C (1 kb / min), 34 cycles.

[0078] (3) Third-round PCR amplification

[0079] ① Perform PCR splicing on Gene Sequence 1 and Gene Sequence 2 to obtain Gene Sequence 7.

[0080] PCR system: Template (30 ng each of Gene Sequence 1 and Gene Sequence 2) 2 μL, forward and reverse primers 1-F / 4-R (10 pM) 1 μL each, Taq polymerase 2 μL, 10xPCR buffer 2 μL, dNTP mixture (2 mM) 2 μL, RNase-free water 10 μL. PCR amplification program: Denaturation at 98 °C for 15 s, annealing at 56 °C for 30 s, extension at 72 °C (1 kb / min), 34 cycles.

[0081] ② Perform PCR splicing on Gene Sequence 3 and Gene Sequence 4 to obtain Gene Sequence 8.

[0082] PCR system: Template (30 ng each of Gene Sequence 3 and Gene Sequence 4) 2 μL, forward and reverse primers 5-F / 8-R (10 pM) 1 μL each, Taq polymerase 2 μL, 10xPCR buffer 2 μL, dNTP mixture (2 mM) 2 μL, RNase-free water 10 μL. PCR amplification program: Denaturation at 98 °C for 15 s, annealing at 56 °C for 30 s, extension at 72 °C (1 kb / min), 34 cycles.

[0083] ③ Perform PCR splicing on Gene Sequence 5 and Gene Sequence 6 to obtain Gene Sequence 9.

[0084] PCR system: 2 μL of template (30 ng each of gene sequence 5 and gene sequence 6), 1 μL each of upstream and downstream primers 9-F / 12-R (10 pM), 2 μL of Taq polymerase, 2 μL of 10x PCR buffer, 2 μL of dNTP mixture (2 mM), 10 μL of RNase-free water. PCR amplification program: denaturation at 98°C for 15 s, annealing at 56°C for 30 s, extension at 72°C (1 kb / min), 34 cycles.

[0085] (4) Fourth-round PCR amplification

[0086] ① PCR splice gene sequences 7 and 8 to obtain gene sequence 10.

[0087] PCR system: 2 μL of template (30 ng each of gene sequence 7 and gene sequence 8), 1 μL each of upstream and downstream primers 1-F / 8-R (10 pM), 2 μL of Taq polymerase, 2 μL of 10x PCR buffer, 2 μL of dNTP mixture (2 mM), 10 μL of RNase-free water. PCR amplification program: denaturation at 98°C for 15 s, annealing at 56°C for 30 s, extension at 72°C (1 kb / min), 34 cycles.

[0088] ② PCR splice gene sequences 8 and 9 to obtain gene sequence 11.

[0089] PCR system: 2 μL of template (30 ng each of gene sequence 8 and gene sequence 9), 1 μL each of upstream and downstream primers 5-F / 12-R (10 pM), 2 μL of Taq polymerase, 2 μL of 10x PCR buffer, 2 μL of dNTP mixture (2 mM), 10 μL of RNase-free water. PCR amplification program: denaturation at 98°C for 15 s, annealing at 56°C for 30 s, extension at 72°C (1 kb / min), 34 cycles.

[0090] (5) Using gene sequences 10 and 11 as templates and 13-F and 12-R as primers, splice them into the optimized S1 gene by Overlap PCR (its amino acid sequence is shown in SEQ ID NO.1 and nucleotide sequence is shown in SEQ ID NO.2).

[0091] PCR system: 2 μL of template (30 ng each of gene sequence 10 and gene sequence 11), 1 μL each of upstream and downstream primers 13-F / 12-R (10 pM), 2 μL of Taq polymerase, 2 μL of 10x PCR buffer, 2 μL of dNTP mixture (2 mM), 10 μL of RNase-free water. PCR amplification program: denaturation at 98°C for 15 s, annealing at 56°C for 30 s, extension at 72°C (1 kb / min), 34 cycles.

[0092] Example 2 (In this example, the SARS-CoV-2 spike protein S1 gene is the optimized S1 gene from Example 1)

[0093] The S1 gene obtained in Example 1 was constructed into mRNA, and the construction method included the following steps:

[0094] (1) Select the 5'-UTR of the yellow fever virus 17D strain gene as one of the translation regulatory elements of mRNA, and add a Kozak sequence gccacc by PCR amplification after YFV 17D 5'-UTR; the upstream and downstream primers are primer-1F: 5'-agtaaatcctgtgtgctaattga-3' and primer-1R: 5'-ggtggcgttctggtcagttctctgctaa-3' respectively; PCR program: denaturation at 98°C for 15 s, annealing at 56°C for 30 s, extension at 72°C (1 kb / min), 34 cycles; PCR system: 1 μL of the 5'-UTR of the yellow fever virus 17D strain gene as a template (30 ng), 1 μL of each of the upstream and downstream primers (10 pM), 2 μL of Taq polymerase, 2 μL of 10xPCR buffer, 2 μL of dNTP mixture (2 mM), and 11 μL of RNase-free water.

[0095] Meanwhile, between the S1 gene of SARS-COV-2 and the Kozark sequence, the IgK signal peptide sequence with secretory signal function was introduced by PCR amplification: 5’-atggagaccgataccctgctgctgtgggtgctgctgctgtgggtgcctggctctaccggcgac-3’. The upstream and downstream primers were Primer-2F: 5'-gaactgaccagaacgccaccATGGAGACCGATACCCTGCTGCTGTGGGTGCTGCTGCTGTGGGTGCCTGGCTCTACCGGCGACGTGAACCTGACAACCAGAACCCAGC-3' and Primer-2R: TTATCTAGCTCTTCTAGGGCTATTG respectively. The PCR amplification program was as follows: denaturation at 98°C for 15 s, annealing at 56°C for 30 s, extension at 72°C (1 kb / min), for 34 cycles. The PCR amplification system was as follows: 1 μL of the optimized S1 gene of SARS-COV-2 as the template (30 ng), 1 μL each of the upstream and downstream primers (10 pM), 2 μL of Taq polymerase, 2 μL of 10xPCR buffer, 2 μL of dNTP mixture (2 mM), and 11 μL of RNase-free water. The IgK signal peptide sequence was 5’-atggagaccgataccctgctgctgtgggtgctgctgctgtgggtgcctggctctaccggcgac-3’.

[0096] After the TAA terminator of the target gene S1, add the 3'-UTR of the ribosomal gene in the human mitochondrial gene: Primer-3F: 5'-GTGAACCTGACAACCAGAACCCAG-3', Primer-3R: 5'-TTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCTTTATCTAGCTCTTCTAGGGCTATTG-3', Primer-4R: 5'-CACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAG-3', Primer-5R: 5'-CAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCT-3'. First, perform the first-round PCR amplification with Primer-3F and 3R to obtain product sequence 3. Then, use product sequence 3 as the template and -3F and 4R as primers to perform the second-round PCR amplification to obtain product sequence 4. Finally, use product 4 as the template and -3F and 5R as primers to perform the third-round PCR amplification to obtain product 5. Product 5 is the S1 sequence with the 3'-UTR of the ribosome in the human mitochondrial gene added. The three-round PCR amplification procedures and amplification systems are the same. Amplification procedure: Denaturation at 98°C for 15 s, annealing at 56°C for 30 s, extension at 72°C (1 kb / min), 34 cycles; Amplification system: Template (30 ng) 1 μL, upstream and downstream primers (10 pM) 1 μL each, Taq polymerase 2 μL, 10xPCR buffer 2 μL, dNTP mixture (2 mM) 2 μL, RNase-free water 11 μL.

[0097] (2) Design a segmented PolyA tail; the PolyA tail sequence is as shown in SEQ ID NO.4. Add a segmented PolyA tail after the 3'-UTR sequence: Primer 6R: 5'-ttttttttttttttttttttttttttttttttttttcagcacgcggttttttttttttttttttttttttttttttttttttagtcataagcttttttttttttttttttttttttttttttttttttcaagcacgcagcaatgcagctc-3'. Using product 5 as a template and -3F and 6R as primers, perform the fourth round of PCR amplification to obtain the S1 sequence (product 6) with 3'-UTR and segmented PloyA tail. PCR amplification program: denaturation at 98°C for 15 s, annealing at 56°C for 30 s, extension at 72°C (1 kb / min), 34 cycles. PCR amplification system: template (product 5, 30 ng) 1 μL, upstream and downstream primers (10 pM) 1 μL each, Taq polymerase 2 μL, 10xPCR buffer 2 μL, dNTP mixture (2 mM) 2 μL, RNase-free water 11 μL.

[0098] Finally, using products 1, 2, and 6 as templates and 1F and 6R as primers, perform overlap extension PCR (PCR amplification program: denaturation at 98°C for 15 s, annealing at 56°C for 30 s, extension at 72°C (1 kb / min), 34 cycles). Amplification system: template (30 ng each of products 1, 2, and 6) 3 μL, upstream and downstream primers (10 pM) 1 μL each, Taq polymerase 2 μL, 10xPCR buffer 2 μL, dNTP mixture (2 mM) 2 μL, RNase-free water 9 μL.

[0099] Amplify to obtain the target sequence with 5'-UTR-Kozak sequence-Igκ signal peptide-S1-3'-UTR-Poly A.

[0100] The primer sequences used in this example are shown in Table 3.

[0101] Table 3 Primer sequences used

[0102]

[0103] (3) Digest the vector pVAX1 into linear fragments using restriction endonucleases HindIII and BamHI (37°C, digestion for 6 h, digestion system: template DNA 5 μg (1 μg / μL), 10x digestion buffer 5 μL, restriction endonucleases 3 μL each, sterile enzyme-free water 34 μL). After the vector fragment is recovered by gel column, insert the target fragment and the vector fragment into the pVAX1 vector by ligation overnight at 16°C according to a molar ratio of 3:1.

[0104] (4) After transformation, plating, screening for monoclonal clones, amplification, and sequencing, the plasmid pMu-6 that stably transcribes and expresses mRNA was obtained.

[0105] (5) Prepare pMu-6 plasmid DNA and linearize it with BamHI digestion (digestion system: 2 μg of template pMu-6 plasmid DNA, 2 μL of restriction endonuclease BamHI, 5 μL of 10x digestion buffer, 41 μL of sterile enzyme-free water, digest at 37°C for 5 h). After column purification and recovery, the linearized fragment reacts with 10x reaction buffer (2 μL), T7 RNA polymerase (2 μL), yeast inorganic pyrophosphatase (0.4 μL), RNA inhibitor (0.5 μL), linearized template DNA (1 μg), and substrate (4.5 μL of 25 mM NTP mixture) at 37°C for 16 h for in vitro mRNA transcription. The preparation system is shown in Table 4.

[0106] (6) After transcription is completed, treat with RNase, purify through a column, and obtain mRNA (the sequence is shown in SEQ ID NO.3).

[0107] Table 4. In vitro mRNA transcription system

[0108]

[0109]

[0110] Example 3

[0111] Construct mRNA with the common SARS-CoV-2 spike protein S1 gene in the same method as in Example 2.

[0112] Example 4

[0113] Utilize the mRNA translation and expression systems obtained in Example 3 and Example 4 to induce an immune response in animals

[0114] The immunization procedure is as follows:

[0115] A: Divide into two groups to immunize Balb / c mice, with 5 mice in each group. Immunize with the original S1 mRNA (mRNA obtained in Example 3) and our sequence-optimized S1 mRNA (mRNA obtained in Example 2) respectively. The immunization dose for each group is 10 μg / mouse, and administer by intramuscular injection.

[0116] B: Immunize on days 0 and 21 respectively. At 21 days after the first immunization, 14 days, 28 days, 90 days, 180 days, and 270 days after the second immunization, collect blood to detect anti-SARS-CoV-2 binding antibodies and neutralizing antibodies.

[0117] Combined antibodies: Parallel detection was performed using ELISA kits against the Wuhan strain, Beta strain, Delta strain, and Omicron strain.

[0118] Neutralizing antibodies: A pseudovirus neutralizing antibody detection system against the Wuhan strain, Beta strain, Delta strain, and Omicron strain was used, and live viruses, including the Wuhan strain, Delta strain, and Omicron strain, were used to detect neutralizing antibodies in the Vero cell system.

[0119] The above work showed that immunization of mice with the optimized SARS-CoV-2 S1 sequence elicited a stronger immune response (attached Figure 4 ), including good combined antibody responses, neutralizing antibody responses, and cellular immune responses against different variants, which were more significant than those elicited by the currently available SARS-CoV-2 S1 sequence. At the same time, it was detected that the antibody response could be maintained for a long time (attached Figure 4 ). These data suggest that the optimized SARS-CoV-2 S1 sequence we designed has good cross-protective effects in mice.

[0120] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not restrictive. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

[0121] Sequence Listing

[0122] SEQ ID NO.1

[0123] Amino acid sequence of the SARS-CoV-2 spike protein S1

[0124]

[0125] SEQ ID NO.2

[0126] Nucleotide sequence of the SARS-CoV-2 spike protein S1

[0127]

[0128]

[0129] SEQ ID NO.3

[0130] mRNA nucleotide sequence

[0131]

[0132]

[0133] SEQ ID NO.4

[0134] PolyA tail

[0135]

Claims

1. An optimized SARS-CoV-2 spike protein S1, characterized in that, The amino acid sequence of the SARS-CoV-2 spike protein S1 described above is shown in SEQ ID NO.

1.

2. A gene encoding the optimized SARS-CoV-2 spike protein S1 according to claim 1.

3. The gene according to claim 2, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

4. The construction method of the S1 gene according to claim 1 or 2, characterized in that Comprising the following steps: (1) Segmenting and first-round Overlap PCR amplification The SARS-CoV-2 spike protein S1 gene is divided into twelve segments, which are 70-251, 223-310, 291-479, 461-660, 642-1032, 993-1147, 1126-1260, 1237-1389, 1363-1497, 1478-1650, 1628-1855, 1831-2067 of the S1 sequence respectively, and Overlap PCR amplification is carried out separately. ； (2) Second-round PCR amplification The first and second segment sequences are PCR spliced. Using the amplification products of the 70-251 and 223-310 segments in step (1) as templates, and 1-F and 2-R as primers for amplification to obtain gene sequence 1; The third and fourth segment sequences are PCR spliced. Using the amplification products of the 291-479 and 461-660 segments in step (1) as templates, and 3-F and 4-R as primers for amplification to obtain gene sequence 2; The fifth and sixth segment sequences are PCR spliced. Using the amplification products of the 642-1032 and 993-1147 segments in step (1) as templates, and 5-F and 6-R as primers for amplification to obtain gene sequence 3; The seventh and eighth segment sequences are PCR spliced. Using the amplification products of the 1126-1260 and 1237-1389 segments in step (1) as templates, and 7-F and 8-R as primers for amplification to obtain gene sequence 4; The ninth and tenth segment sequences are PCR spliced. Using the amplification products of the 1363-1497 and 1478-1650 segments in step (1) as templates, and 9-F and 10-R as primers for amplification to obtain gene sequence 5; The eleventh and twelfth segment sequences are PCR spliced. Using the amplification products of the 1628-1855 and 1831-2067 segments in step (1) as templates, and 11-F and 12-R as primers for amplification to obtain gene sequence 6; (3) Third-round PCR amplification The gene sequence 1 and gene sequence 2 are PCR spliced. Using the gene sequence 1 and gene sequence 2 in step (2) as templates, and 1-F and 4-R as primers for amplification to obtain gene sequence 7; The gene sequence 3 and gene sequence 4 are PCR spliced. Using the gene sequence 3 and gene sequence 4 in step (2) as templates, and 5-F and 8-R as primers for amplification to obtain gene sequence 8; The gene sequence 5 and gene sequence 6 are PCR spliced. Using the gene sequence 5 and gene sequence 6 in step (2) as templates, and 9-F and 12-R as primers for amplification to obtain gene sequence 9; (4) Fourth-round PCR amplification Perform PCR splicing on gene sequence 7 and gene sequence 8. Using the gene sequence 7 and gene sequence 8 in step (3) as templates, amplify with 1-F and 8-R as primers to obtain gene sequence 10; Perform PCR splicing on gene sequence 8 and 9. Using the gene sequence 8 and gene sequence 9 in step (3) as templates, amplify with 5-F and 12-R as primers to obtain gene sequence 11; (5) Using gene sequence 10 and gene sequence 11 as templates, and 13-F and 12-R as primers, splice them by Overlap PCR to obtain the optimized S1 gene; Primer 13-F is: 5'-gccaccatggagaccgataccctgctgctgtgggtgctgctgctgtgggtgcctggctctaccggcgacgtaaacctgagaaccagaacccagc-3'.

5. The construction method of the S1 gene according to claim 3, characterized in that, The PCR amplification conditions in steps (1) to (5) are the same. The PCR system is: 1 μL of template; 1 μL each of upstream and downstream primers at 10 pM; 2 μL of Taq polymerase, 10xPCR; 2 μL of buffer; 2 μL of dNTP mixture at 2 mM; 11 μL of RNase-free water; The PCR program is: denaturation at 98°C for 15 s, annealing at 57°C for 30 s, extension at 72°C, extension speed 1 kb / min, 34 cycles.

6. A SARS-COV-2 spike protein mRNA, characterized in that, Its S1 protein is as shown in claim 1 and claim 2.

7. The spike protein mRNA according to claim 6, wherein, Its construction method includes the following steps: (1) Using 5'-UTR as the main mRNA translation regulatory element, add a Kozak sequence after it, and the Kozak sequence is gccacc; at the same time, add an IgK signal peptide sequence with secretory signal function before the S1 gene of SARS-COV-2, and add 3'-UTR after the TAA terminator of the target gene S1; (2) Design a segmented PolyA tail; (3) Recombinantly insert and modify it into the constructed pVAX vector by HindIII and BamHI; (4) After cloning screening and sequencing, obtain the plasmid pMu-6 that stably transcribes and expresses mRNA; (5) Prepare pMu-6 plasmid DNA, linearize it with BamHI enzyme digestion, and perform in vitro mRNA transcription; (6) After the transcription is completed, treat it with RNase and purify it by column to obtain mRNA.

8. The construction method according to claim 7, characterized in that, The said 5'-UTR is the 5'-UTR of the yellow fever virus 17D strain gene; the said 3'-UTR is the 3'-UTR of the ribosomal gene in the human mitochondrial gene; The sequence of the said segmented PolyA tail is as shown in EQ ID NO.4; The said IgK signal peptide sequence is 5'-atggagaccgataccctgctgctgtgggtgctgctgctgtgggtgcctggctctaccggcgac-3'.

9. The application of the mRNA according to any one of claims 6 to 8 in the preparation of a vaccine for preventing and / or treating COVID-19.

10. A vaccine for preventing or treating COVID-19 infection, comprising the mRNA as described in any one of claims 6 to 8.

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