NUCLEOTIDE SEQUENCES ENCODING PEPTIDE ANTIGENS OF SARS-COV-2 VIRUS AND USE THEREOF

Abstract

The present disclosure discloses nucleotide sequences as shown by SEQ ID NO: 1, SEQ ID NO: 2 and the nucleotide sequences having at least 90% homology therewith, which encode peptide antigens of SARS-CoV-2 virus. According to some embodiments of the present disclosure, the nucleotide sequences can be effectively expressed in human cells and produce the corresponding peptides, thereby inducing an immune-protective response accordingly. Thus, the nucleotide sequences of the present disclosure are expected to be developed as a vaccine against SARS-CoV-2 infection.

Claims

1. A nucleotide sequence encoding a peptide triggering immunogenicity against Novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), comprising at least one selected from: a) a nucleotide sequence as shown by SEQ ID NO: 1; b) a nucleotide sequence as shown by SEQ ID NO: 2; or c) a nucleotide sequence having at least 90% sequence identity with the nucleotide sequence as shown by SEQ ID NO: 1.

2. The nucleotide sequence according to claim 1, wherein the peptide is capable of: inducing an immune response in a human body; generating a molecular biology reporter; generating a molecule for detection; regulating a gene function; or functioning as a therapeutic molecule.

3. An expression vector comprising the nucleotide sequence according to claim 1.

4. The expression vector according to claim 3, wherein the vector is a viral vector.

5. The expression vector according to claim 4, wherein the viral vector is an adenovirus vector.

6. (canceled)

7. A nucleotide-based composition comprising the nucleotide sequence according to claim 1 or the expression vector according to claim 3.

8. The nucleotide-based composition according to claim 7, further comprising a pharmaceutically-acceptable adjuvant, carrier, diluent or excipient.

9. (canceled)

10. A method for preventing or treating COVID-19, comprising administering a prophylactically or therapeutically effective amount of the nucleotide-based composition according to claim 7 to a human.

11. The method according to claim 10, wherein the vector is a viral vector.

12. The method according to claim 11, wherein the viral vector is an adenovirus vector.

13. The method according to claim 10, wherein the nucleotide-based composition further comprises a pharmaceutically-acceptable adjuvant, carrier, diluent or excipient.

15. The method according to claim 11, wherein the nucleotide-based composition further comprises a pharmaceutically-acceptable adjuvant, carrier, diluent or excipient.

16. The method according to claim 12, wherein the nucleotide-based composition further comprises a pharmaceutically-acceptable adjuvant, carrier, diluent or excipient.

17. The nucleotide-based composition according to claim 7, wherein the vector is a viral vector.

18. The nucleotide-based composition according to claim 17, wherein the viral vector is an adenovirus vector.

19. The nucleotide-based composition according to claim 17, further comprising a pharmaceutically-acceptable adjuvant, carrier, diluent or excipient.

20. The nucleotide-based composition according to claim 18, further comprising a pharmaceutically-acceptable adjuvant, carrier, diluent or excipient.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0030] FIG. 1 shows the expression of S protein after transfecting with a same dose of pVAX-NB1, pVAX-NB2 and pVAX-NB4.

[0031] FIG. 2 shows the antibody levels in serum of the mice immunized with pVAX-NB1- and pVAX-NB2-DNA vaccines.

DETAILED DESCRIPTION

[0032] The Spike protein (S) of SARS-CoV-2 has an amino acid sequence as shown by YP 009724390.1, and the natural nucleotide sequence encoding the S protein of SARS-CoV-2 is designated as NB1.

[0033] Precursor mRNA in eukaryotic cells can produce various mRNA splice variants through different splicing manners (by selecting different combination of splice sites), eventually leading to different proteins produced from the same gene sequence. This is very unfavorable for the expression of a protein. The inventor performed codon optimization to the wild-type natural nucleotide sequence and removed potential variable cleavage sites based on self-owned technology, thereby ensuring the uniqueness of expressed protein and reducing the difficulty in subsequent protein purification. The optimized nucleotide sequence is designated as NB2, and is as shown by SEQ ID NO: 1:

TABLE-US-00001 (SEQ ID NO.: 1) ATGTTCGTGTTTCTGGTGCTGCTGCCTCTGGTGAGCTCCCAGTGCGTGA ACCTGACCACAAGGACCCAGCTGCCACCTGCCTATACCAATAGCTTCAC ACGGGGCGTGTACTATCCCGACAAGGTGTTTAGATCTAGCGTGCTGCAC TCCACCCAGGATCTGTTTCTGCCTTTCTTTTCTAACGTGACATGGTTCC ACGCCATCCACGTGTCCGGCACCAATGGCACAAAGCGGTTCGACAATCC AGTGCTGCCCTTTAACGATGGCGTGTACTTCGCCTCCACCGAGAAGTCT AACATCATCAGAGGCTGGATCTTTGGCACCACACTGGACAGCAAGACCC AGTCCCTGCTGATCGTGAACAATGCCACAAACGTGGTCATCAAGGTGTG CGAGTTCCAGTTTTGTAATGATCCCTTCCTGGGCGTGTACTATCACAAG AACAATAAGTCTTGGATGGAGAGCGAGTTTAGGGTGTATTCCTCTGCCA ACAATTGCACCTTTGAGTACGTGAGCCAGCCTTTCCTGATGGACCTGGA GGGCAAGCAGGGCAATTTCAAGAACCTGAGGGAGTTCGTGTTTAAGAAT ATCGATGGCTACTTCAAGATCTACTCCAAGCACACACCAATCAACCTGG TGCGCGACCTGCCACAGGGCTTCTCTGCCCTGGAGCCACTGGTGGATCT GCCCATCGGCATCAACATCACCCGGTTTCAGACACTGCTGGCCCTGCAC AGAAGCTACCTGACCCCAGGCGACAGCTCCTCTGGATGGACAGCAGGAG CTGCCGCCTACTATGTGGGCTATCTGCAGCCCCGCACCTTCCTGCTGAA GTACAACGAGAATGGCACCATCACAGACGCAGTGGATTGCGCCCTGGAC CCCCTGTCTGAGACCAAGTGTACACTGAAGAGCTTTACAGTGGAGAAGG GCATCTACCAGACCAGCAACTTCAGGGTGCAGCCAACAGAGTCCATCGT GCGCTTTCCCAATATCACCAACCTGTGCCCTTTTGGCGAGGTGTTCAAT GCCACACGCTTCGCCAGCGTGTACGCCTGGAATAGGAAGCGCATCTCCA ACTGCGTGGCCGACTATTCTGTGCTGTACAACAGCGCCTCCTTCTCTAC CTTTAAGTGTTATGGCGTGAGCCCCACCAAGCTGAATGATCTGTGCTTT ACAAACGTGTACGCCGATTCCTTCGTGATCAGGGGCGACGAGGTGCGCC AGATCGCACCAGGACAGACCGGCAAGATCGCAGACTACAATTATAAGCT GCCTGACGATTTCACAGGCTGCGTGATCGCCTGGAACTCTAACAATCTG GATAGCAAAGTGGGCGGCAACTACAATTATCTGTACCGGCTGTTTAGAA AGTCTAATCTGAAGCCATTCGAGCGGGACATCTCCACCGAGATCTACCA GGCCGGCTCTACACCCTGCAATGGCGTGGAGGGCTTTAACTGTTATTTC CCTCTGCAGTCCTACGGCTTCCAGCCAACCAACGGCGTGGGCTATCAGC CCTACAGAGTGGTGGTGCTGTCTTTTGAGCTGCTGCACGCACCTGCAAC CGTGTGCGGCCCAAAGAAGAGCACAAATCTGGTGAAGAACAAGTGCGTG AACTTCAACTTCAACGGACTGACCGGCACAGGCGTGCTGACCGAGAGCA ACAAGAAGTTCCTGCCATTTCAGCAGTTCGGCAGGGACATCGCAGATAC CACAGACGCCGTGCGCGACCCTCAGACCCTGGAGATCCTGGACATCACA CCATGTTCCTTCGGCGGCGTGTCTGTGATCACCCCAGGCACCAATACAT CCAACCAGGTGGCCGTGCTGTATCAGGACGTGAATTGCACAGAGGTGCC CGTGGCAATCCACGCAGATCAGCTGACCCCTACATGGCGGGTGTACTCT ACCGGCAGCAACGTGTTCCAGACAAGAGCCGGATGCCTGATCGGAGCAG AGCACGTGAACAATAGCTATGAGTGCGACATCCCTATCGGCGCCGGCAT CTGTGCCTCCTACCAGACCCAGACAAACTCCCCAAGGAGAGCCCGGTCT GTGGCCAGCCAGTCCATCATCGCCTATACCATGAGCCTGGGCGCCGAGA ACAGCGTGGCCTACTCCAACAATTCTATCGCCATCCCTACCAACTTCAC AATCAGCGTGACCACAGAGATCCTGCCAGTGAGCATGACCAAGACATCC GTGGACTGCACCATGTATATCTGTGGCGATTCCACAGAGTGTTCTAACC TGCTGCTGCAGTACGGCTCCTTTTGCACCCAGCTGAATAGAGCCCTGAC AGGCATCGCCGTGGAGCAGGACAAGAACACCCAGGAGGTGTTCGCCCAG GTGAAGCAGATCTACAAGACACCACCCATCAAGGACTTTGGCGGCTTCA ACTTCAGCCAGATCCTGCCCGATCCTAGCAAGCCATCCAAGCGGTCTTT TATCGAGGACCTGCTGTTCAACAAGGTGACCCTGGCCGATGCCGGCTTC ATCAAGCAGTATGGCGATTGTCTGGGCGACATCGCCGCCAGAGACCTGA TCTGCGCCCAGAAGTTTAATGGCCTGACCGTGCTGCCTCCACTGCTGAC AGATGAGATGATCGCACAGTACACCTCTGCCCTGCTGGCCGGCACCATC ACAAGCGGATGGACATTCGGCGCAGGAGCCGCCCTGCAGATCCCCTTTG CCATGCAGATGGCCTATCGGTTCAACGGCATCGGCGTGACCCAGAATGT GCTGTACGAGAACCAGAAGCTGATCGCCAATCAGTTTAACAGCGCCATC GGCAAGATCCAGGACTCTCTGAGCTCCACCGCCAGCGCCCTGGGCAAGC TGCAGGATGTGGTGAATCAGAACGCCCAGGCCCTGAATACACTGGTGAA GCAGCTGTCTAGCAACTTCGGCGCCATCTCCTCTGTGCTGAATGACATC CTGAGCCGGCTGGACAAGGTGGAGGCAGAGGTGCAGATCGACCGGCTGA TCACCGGCAGACTGCAGTCCCTGCAGACCTACGTGACACAGCAGCTGAT CAGGGCAGCAGAGATCAGGGCCTCTGCCAATCTGGCCGCCACAAAGATG AGCGAGTGCGTGCTGGGACAGTCCAAGAGGGTGGACTTTTGCGGCAAGG GCTATCACCTGATGAGCTTCCCACAGTCCGCCCCTCACGGAGTGGTGTT TCTGCACGTGACCTACGTGCCAGCCCAGGAGAAGAACTTCACCACAGCC CCCGCCATCTGTCACGATGGCAAGGCCCACTTTCCTAGGGAGGGCGTGT TCGTGAGCAACGGCACCCACTGGTTTGTGACACAGCGCAATTTCTACGA GCCACAGATCATCACCACAGACAATACCTTCGTGTCCGGCAACTGCGAC GTGGTCATCGGCATCGTGAACAATACAGTGTATGATCCTCTGCAGCCAG AGCTGGACTCTTTTAAGGAGGAGCTGGATAAGTACTTCAAGAATCACAC CAGCCCCGACGTGGATCTGGGCGACATCTCTGGCATCAATGCCAGCGTG GTGAACATCCAGAAGGAGATCGACAGACTGAACGAGGTGGCCAAGAATC TGAACGAGAGCCTGATCGATCTGCAGGAGCTGGGCAAGTATGAGCAGTA CATCAAGTGGCCCTGGTATATCTGGCTGGGCTTCATCGCCGGCCTGATC GCCATCGTGATGGTGACCATCATGCTGTGCTGTATGACAAGCTGCTGTT CCTGCCTGAAGGGCTGCTGTTCTTGTGGCAGCTGCTGTAAGTTTGATGA GGACGATTCCGAGCCTGTGCTGAAGGGCGTGAAGCTGCACTACACCTA A.

[0034] In order to reduce the GC-content of the gene, the inventor further optimized the codons based on NB2, resulting in a sequence designated as NB4. NB4 has a sequence as shown by SEQ ID No.: 2:

TABLE-US-00002 (SEQ ID NO: 2) ATGTTTGTCTTCCTGGTGCTGCTGCCTCTGGTCTCTTCTCAGTGTGTGA ACCTGACCACCAGGACACAGCTGCCTCCTGCTTACACCAACTCCTTCAC ACGGGGCGTCTACTACCCTGACAAGGTCTTCAGGTCCTCTGTGCTGCAC TCCACACAAGACCTGTTCCTGCCATTCTTCTCCAACGTGACCTGGTTCC ATGCCATCCATGTCTCTGGCACCAATGGCACCAAGAGGTTCGACAACCC TGTGCTGCCATTCAACGACGGCGTCTACTTTGCCTCCACCGAGAAGTCC AACATCATCAGGGGCTGGATCTTTGGCACCACCCTGGACTCCAAGACCC AGTCCCTGCTGATTGTGAACAATGCCACCAACGTGGTGATCAAAGTCTG CGAGTTCCAGTTCTGCAATGACCCATTCCTGGGCGTCTACTACCACAAG AACAACAAGTCCTGGATGGAGTCTGAGTTCAGGGTCTACTCCTCTGCCA ACAACTGCACCTTTGAATATGTCTCCCAGCCATTCCTGATGGACCTGGA GGGCAAGCAGGGCAACTTCAAGAACCTGAGGGAGTTCGTCTTCAAGAAC ATCGACGGCTACTTCAAGATCTACTCCAAGCACACACCCATTAATCTCG TCCGGGATCTGCCTCAGGGCTTCTCTGCCCTGGAGCCCCTGGTGGACCT GCCCATTGGCATCAACATCACACGCTTTCAGACCCTGCTGGCTCTGCAC CGGTCTTACCTGACACCTGGCGACTCCTCCTCTGGCTGGACAGCTGGCG CTGCTGCCTACTATGTCGGCTACCTGCAGCCTAGGACCTTCCTGCTGAA GTACAATGAGAATGGCACCATCACAGATGCTGTGGACTGTGCCCTGGAC CCCCTGTCTGAGACCAAGTGCACCCTGAAGTCCTTCACAGTCGAGAAGG GCATCTACCAGACCTCCAACTTCCGGGTGCAGCCCACAGAGTCCATTGT CAGGTTCCCCAACATCACCAACCTGTGCCCATTTGGCGAGGTCTTCAAT GCCACACGCTTTGCCTCTGTCTATGCCTGGAACAGGAAGAGGATCTCCA ACTGTGTGGCTGACTACTCTGTGCTGTACAACTCTGCCTCCTTCTCCAC ATTTAAGTGCTATGGCGTCTCCCCCACCAAGCTGAATGACCTGTGCTTC ACCAATGTCTATGCTGACTCCTTTGTGATCCGGGGCGATGAAGTTAGGC AGATTGCCCCTGGCCAGACAGGCAAGATTGCTGACTACAACTACAAGCT GCCTGATGACTTCACCGGCTGTGTGATTGCCTGGAACTCCAACAACCTG GACTCTAAAGTTGGCGGCAACTACAACTACCTGTACAGGCTGTTCAGGA AGTCCAACCTGAAGCCATTTGAGAGGGACATCTCCACAGAGATCTACCA GGCTGGCTCCACCCCATGCAATGGCGTGGAGGGCTTCAACTGCTACTTC CCCCTGCAATCCTATGGCTTCCAACCCACCAATGGCGTGGGCTACCAGC CATACAGGGTGGTGGTGCTGTCCTTTGAGCTGCTGCATGCCCCTGCCAC CGTCTGTGGCCCCAAGAAGTCCACCAACCTCGTGAAGAACAAATGTGTG AACTTCAACTTCAACGGCCTGACAGGCACAGGCGTGCTGACAGAGTCCA ACAAGAAGTTCCTGCCATTCCAACAGTTTGGCAGGGACATTGCTGACAC CACCGATGCTGTCAGGGACCCCCAGACCCTGGAGATCCTGGACATCACC CCATGCTCCTTTGGCGGCGTCTCTGTGATCACCCCTGGCACCAACACCT CTAATCAAGTCGCTGTGCTGTACCAGGACGTGAACTGCACAGAGGTTCC TGTGGCCATCCATGCTGACCAGCTGACCCCCACCTGGAGGGTCTACTCC ACCGGCTCCAATGTCTTCCAAACACGGGCTGGCTGCCTGATTGGCGCTG AGCATGTGAACAACTCCTATGAGTGTGACATCCCCATTGGCGCTGGCAT CTGTGCCTCCTACCAAACCCAGACCAACTCCCCTCGGAGGGCTCGCTCT GTGGCTTCTCAGTCTATCATTGCCTACACCATGTCCCTGGGCGCTGAGA ACTCTGTGGCCTACTCCAACAACTCCATTGCCATCCCCACCAACTTCAC CATCTCTGTGACCACCGAGATCCTGCCTGTCTCCATGACCAAGACCTCT GTGGACTGCACCATGTACATCTGTGGCGACTCCACAGAGTGCTCCAACC TGCTGCTGCAGTATGGCTCCTTCTGCACCCAACTGAACAGGGCCCTGAC AGGCATTGCTGTGGAGCAGGACAAGAACACCCAGGAGGTCTTTGCCCAA GTCAAGCAGATCTACAAGACCCCCCCCATCAAGGACTTTGGCGGCTTCA ACTTCTCCCAAATCCTGCCTGACCCATCCAAGCCATCCAAGAGGTCCTT CATTGAGGACCTGCTGTTCAACAAGGTTACCCTGGCTGATGCTGGCTTC ATCAAGCAGTATGGCGACTGCCTGGGCGACATTGCTGCCAGGGACCTGA TCTGTGCCCAAAAGTTCAATGGCCTGACAGTGCTGCCCCCCCTGCTGAC CGATGAGATGATTGCCCAGTACACATCTGCTCTGCTGGCTGGCACAATC ACCTCTGGCTGGACCTTTGGCGCTGGCGCTGCCCTGCAAATCCCATTTG CCATGCAAATGGCCTACAGGTTCAATGGCATTGGCGTGACCCAGAACGT GCTGTATGAGAACCAGAAGCTGATTGCCAACCAGTTCAACTCTGCCATT AGGCAAGATCCAGGACTCCCTGTCCTCCACCGCCTCTGCCCTGGGCAAG CTGCAGGATGTGGTGAACCAGAATGCCCAGGCCCTGAACACCCTGGTGA AGCAGCTGTCCTCCAACTTTGGCGCCATCTCCTCTGTGCTGAATGACAT CCTGTCCCGGCTGGACAAGGTGGAGGCTGAGGTGCAGATTGACGGCTGA TCACAGGCAGGCTGCAGTCCCTGCAAACCTATGTGACCCAGCAGCTGAT CAGGGCTGCTGAGATCAGGGCCTCTGCCAACCTGGCTGCCACCAAGATG TCTGAATGTGTGCTGGGCCAGTCCAAGAGGGTGGACTTCTGTGGCAAGG GCTACCATCTGATGTCCTTCCCCCAATCTGCCCCCCATGGCGTGGTCTT CCTGCATGTGACCTATGTGCCTGCTCAGGAGAAGAACTTCACCACAGCC CCTGCCATCTGCCATGATGGCAAGGCCCACTTCCCTCGGGAGGGCGTCT TTGTCTCCAATGGCACCCACTGGTTTGTGACCCAGAGGAACTTCTATGA GCCTCAGATCATCACCACAGACAACACCTTTGTCTCTGGCAACTGTGAT GTGGTGATTGGCATTGTGAACAACACAGTCTATGACCCCCTGCAGCCTG AGCTGGACTCCTTCAAAGAGGAGCTGGACAAGTACTTCAAGAACCACAC CTCCCCTGATGTGGACCTGGGCGACATCTCTGGCATCAATGCCTCTGTG GTGAACATCCAGAAGGAGATTGACAGGCTGAATGAGGTGGCCAAGAACC TGAATGAGTCCCTGATTGACCTGCAAGAGCTGGGCAAGTATGAGCAGTA CATCAAGTGGCCATGGTACATCTGGCTGGGCTTCATTGCTGGCCTGATT GCCATTGTGATGGTGACCATCATGCTGTGCTGCATGACCTCCTGCTGCT CCTGCCTGAAAGGCTGCTGCTCCTGTGGCTCCTGCTGCAAGTTTGATGA GGATGACTCTGAGCCTGTGCTGAAGGGCGTGAAGCTGCACTACACCTAA TAA

[0035] Hereinafter, the technical solution of the present disclosure will be further illustrated in combination with the experiments.

[0036] Construction of Vectors, pVAX-NB1, pVAX-NB2 and pVAX-NB4, for the Expression of the Spike Gene:

[0037] Using NB1 and NB2 as templates, respectively, the NB1 fragment was obtained through PCR amplification with primers NB1-F and NB1-R, the NB2 fragment was obtained through PCR amplification with primers NB2-F and NB2-R, and the NB4 fragment was obtained through PCR amplification with primers NB4-F and NB4-R. The vector plasmid backbone pVAX was PCR amplified, by using CMV-R and BGH-F as primers, and using a pVAX plasmid, which was owned by our company, as the template. Then, two-fragment in vitro recombination was carried out by using a homologous recombination enzyme (Exnase) with the NB1, NB2 or NB4 fragment, respectively, to obtain pVAX-NB1, pVAX-NB2 and pVAX-NB4.

[0038] Detection of Spike Gene Expression

[0039] According to a conventional method, transfecting HEK293 cells with 2.5 μg of pVAX-NB1, pVAX-NB2 and pVAX-NB4, respectively, with cationic liposomes for 48 hours, and then collecting the cells. Such samples were processed according to a conventional Western Blot method, and were detected for the protein (FIG. 1). It could be seen that no S protein expression was detected in the pVAX-NB1 sample, while the expression of the S protein could be observed in the codon-optimized pVAX-NB2 and pVAX-NB4 samples. The expression level of S protein for pVAX-NB4 was about 2-3 times of that of S protein for pVAX-NB2. This indicates that the NB4 sequence produced unexpected effect.

[0040] Animal Immunogenicity Experiments for DNA Vaccines

[0041] Mice were intramuscularly injected with the S protein-expressing plasmids pVAX-NB1 and pVAX-NB2, 100 μg DNA/animal. In particular, 100 μg plasmids were added with 10% glucose to make it up to 200 μl in volume, and added with 10 μl Turbofect (Thermosfisher) according to the manufacturer's instructions. Orbital blood was collected 14 days after the immunization, and the antibody level in the serum was detected by adopting the S protein of the novel coronavirus as the antigen through enzyme-linked immunosorbent assay (ELISA). The specific process was as follows:

[0042] 1) adding 100 μl of PBS and 50 ng of the S protein per well in a 96-well plate, incubating overnight at 4° C., for 16-18 h;

[0043] 2) removing the supernatant, washing with PBST once, adding 200 μl of 5% skim milk for blocking at room temperature for 2 h;

[0044] 3) washing with PBST twice;

[0045] 4) adding the samples: adding 100 μl of diluted serum samples, incubating at 37° C. for 2 h, and then washing with PBST for 5 times;

[0046] 5) adding enzyme-labeled antibodies: adding 100 μl of diluted HRP-labeled IgM or IgG secondary antibody, and incubating at 37° C. for 2 h;

[0047] 6) washing with PBST for 6-8 times;

[0048] 7) adding substrate solution for development: adding 100 μl of TMB for development;

[0049] 8) terminating the reaction: adding with 50 μl of 1 M sulfuric acid to terminate the reaction; and

[0050] 9) determining the results: determining the OD values, and controlling the OD values between 0.1 and 4.

[0051] The experimental results showed that, NB2 could induce the generation of antibodies in the mice (FIG. 2).