SARS-CoV-2 Delta mutant S protein variant and its application

Abstract

This invention discloses a SARS-CoV-2 delta mutant S protein variant and its applications. Compared to the wild-type S protein, the SARS-CoV-2 delta mutant B.1.617.2 S protein variant contains one or more mutations in its S2 subunit. The SARS-CoV-2 delta mutant B.1.617.2 S protein variant can be used to prepare a vaccine against SARS-CoV-2 delta mutant B.1.617.2, providing a possibility for the prevention and / or treatment of novel coronavirus pneumonia caused by SARS-CoV-2 delta mutant B.1.617.2. This invention has significant application value.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the SARS-CoV-2 delta mutant S protein variant and its application, particularly the SARS-CoV-2 delta mutant B.1.617.2 S protein variant and its application in the preparation of novel coronavirus vaccines. Background Technology

[0002] With the widespread transmission of SARS-CoV-2, many viral mutant strains have emerged. Among them, the second-generation mutant strain B.1.617.2 (also known as the SARS-CoV-2 delta mutant B.1.617.2) has been defined by the WHO as a novel coronavirus mutant strain of concern. B.1.617.2 exhibits higher transmission characteristics than Alpha (B.1.1.7) and the highest resistance to neutralizing antibodies, similar to B.1.351. Existing studies have shown that the SARS-CoV-2 delta mutant strain B.1.617.2 can significantly impact the efficacy of existing COVID-19 vaccines.

[0003] SARS-CoV-2 uses its surface membrane protein Spike (also known as the S protein) to enter susceptible cells. The S protein consists of three domains: the S1 domain at the N-terminus, the S2 domain at the apical membrane, and the transmembrane domain. The susceptibility of SARS-CoV-2 to host cells is determined by the receptor-binding domain (RBD) on the S1 domain. Summary of the Invention

[0004] The purpose of this invention is to prevent or treat infection with SARS-CoV-2 delta mutant strain B.1.617.2.

[0005] The present invention first protects a SARS-CoV-2 delta mutant strain B.1.617.2S protein variant, which, relative to its wild-type S protein, contains one or more mutations in its S2 subunit.

[0006] The SARS-CoV-2 delta mutant strain B.1.617.2S protein variant consists of 1208 amino acid residues, and its S2 subunit contains one or more mutations compared to its wild-type S protein.

[0007] In any of the aforementioned SARS-CoV-2 delta mutant strains B.1.617.2S protein variants, one or more mutations are included at their S1 and S2 subunit cleavage sites.

[0008] In some implementations, mutations in the S2 subunit stalk of the SARS-CoV-2 delta mutant strain B.1.617.2S protein variant include K986P and / or V987P.

[0009] In some embodiments, the mutation sites of the S1 and S2 subunit cleavage sites of the SARS-CoV-2 delta mutant strain B.1.617.2S protein variant include one or more of R682, R683, and R685.

[0010] In some embodiments, the mutation at the R682 site of the SARS-CoV-2 delta mutant strain B.1.617.2S protein variant is R682G. The mutation at the R683 site is R683S. The mutation at the R685 site is R685S.

[0011] In some embodiments, the amino acid sequence of the SARS-CoV-2 delta mutant strain B.1.617.2S protein variant is as shown in SEQ ID NO:2 from N-terminus 1 to 1208, or is a sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity with SEQ ID NO:2 from N-terminus 1 to 1208.

[0012] In some embodiments, the amino acid sequence of the SARS-CoV-2 delta mutant strain B.1.617.2S protein variant is as shown in positions 1-1208 from the N-terminus of SEQ ID NO:4 or positions 1-1208 from the N-terminus of SEQ ID NO:6, or is a sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity with the sequence shown in positions 1-1208 from the N-terminus of SEQ ID NO:4 or SEQ ID NO:6.

[0013] The fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of any of the aforementioned SARS-CoV-2 delta mutant strain B.1.617.2S protein variants is also within the scope of protection of this invention.

[0014] The label can stabilize the structure, aid in purification, or facilitate identification.

[0015] In some implementations, a Folden sequence (amino acid sequence: GYIPEAPRDGQAYVRKDGEWVLLSTFLG; nucleotide sequence: GGATACATCCCAGAGGCACCTAGGGACGGACAGGCCTACGTGCGCAAGGATGGCGAGTGGGTGCTGCTGTCCACCTTTCTGGGC) is added at the S2C end (after position 1208) to stabilize the structure. A tag is added for purification and identification (amino acid sequence: SAWSHPQFEKGGGSGGGGSGGSAWSHPQFEKGSDYKDDDDK; nucleotide sequence: TCTGCCTGGAGCCACCCACAGTTCGAGAAGGGCGGCGGCAGCGGCGGCGGCGGCTCCGGCGGCTCTGCATGGTCTCACCCCCAGTTTGAAAAGGGCAGCGACTACAAGGACGACGATGATAAATGA) is added after the Folden sequence.

[0016] The amino acid sequence of the fusion protein may be as shown in SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6.

[0017] The present invention also protects a DNA molecule encoding any of the above-described SARS-CoV-2 delta mutant strain B.1.617.2 S protein variants or any of the above-described fusion proteins.

[0018] The nucleotide sequence of the DNA molecule is as shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:1 from position 41 to 3658 from the 5' end, SEQ ID NO:3 from position 41 to 3658 from the 5' end, or SEQ ID NO:5 from position 41 to 3658 from the 5' end, or is a sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity with SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:1 from position 41 to 3658 from the 5' end, SEQ ID NO:3 from position 41 to 3658 from the 5' end, or SEQ ID NO:5 from position 41 to 3658 from the 5' end.

[0019] The present invention also protects a recombinant plasmid obtained by inserting any of the above-described DNA molecules into the multiple cloning site of an expression vector.

[0020] This invention also protects a recombinant protein, which can be obtained by transfecting mammalian cells with any of the recombinant plasmids described above and culturing the transfected cells. The cells are preferably 293F cells;

[0021] The present invention also protects a composition for preventing SARS-CoV-2 infection, for neutralizing SARS-CoV-2, or for treating diseases caused by SARS-CoV-2 infection, characterized in that: the composition comprises any of the recombinant proteins described above;

[0022] The preferred SARS-CoV-2 strain is the SARS-CoV-2 delta mutant strain B.1.617.2.

[0023] This invention also protects the use of any of the above-described recombinant proteins or any of the above-described compositions in the preparation of medicaments for preventing SARS-CoV-2 infection, for neutralizing SARS-CoV-2, or for treating diseases caused by SARS-CoV-2 infection;

[0024] The preferred SARS-CoV-2 strain is the SARS-CoV-2 delta mutant strain B.1.617.2.

[0025] The wild-type S protein mentioned above is specifically the S protein of SARS-CoV-2WA1 / 2020.

[0026] The present invention also protects a vaccine comprising an S protein variant of any of the SARS-CoV-2 delta mutant strain B.1.617.2S protein variants described above, any of the fusion proteins described above, any of the DNA molecules described above, any of the recombinant plasmids described above, any of the recombinant proteins described above, or any of the compositions described above.

[0027] Preferably, the vaccine may be an mRNA vaccine, an adenovirus vector vaccine, or a recombinant protein vaccine.

[0028] The use of any of the above-described SARS-CoV-2 delta mutant strain B.1.617.2S protein variants, any of the above-described fusion proteins, any of the above-described DNA molecules, any of the above-described recombinant plasmids, any of the above-described recombinant proteins, or any of the above-described compositions in the preparation of vaccines also falls within the scope of protection of this invention. The vaccine may be an mRNA vaccine, an adenovirus vector vaccine, or a recombinant protein vaccine.

[0029] Any of the vaccines mentioned above may be SARS-CoV-2 vaccines.

[0030] The recombinant protein or composition containing it provided by this invention incorporates multiple modified sites or peptides, stabilizing the structure of the S protein and enhancing its immunogenicity. This offers an effective strategy for the prevention and / or treatment of COVID-19 caused by mutant viruses, thus possessing broad application prospects. This invention has significant application value. Detailed Implementation

[0031] The object of this invention is to provide a composition comprising a recombinant protein containing a sequence encoding a protein that is a coding sequence for the extracellular region of a SARS-CoV-2 delta mutant strain B.1.617.2S protein containing a mutation and / or polypeptide. This invention also includes administering the composition comprising the recombinant protein of this invention to a desired subject to induce effector and memory T-cell and B-cell immune responses in the subject to treat and / or prevent SARS-CoV-2 infection, particularly SARS-CoV-2 delta mutant strain B.1.617.2.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While any methods and materials similar to or equivalent to those described herein may be used in the practice of testing the invention, preferred materials and methods are described herein. The following terminology will be used in describing and claiming protection for this invention.

[0033] As used herein, the terms “comparison” or “reference” are used interchangeably and refer to the value used as a standard for comparison.

[0034] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds. Polypeptides include any peptide or protein comprising two or more amino acids linked together by peptide bonds. The polypeptide sequences of the present invention can be produced by any suitable means, including recombinant production, chemical synthesis, or other synthetic methods. Suitable production techniques are well known to those skilled in the art. Alternatively, peptides can also be synthesized using well-known solid-phase peptide synthesis methods.

[0035] As used herein, a “fusion protein” refers to a protein comprising two or more proteins linked together by peptide bonds or other chemical bonds. Proteins may be directly linked together by peptide bonds or other chemical bonds, or may have one or more amino acids between two or more proteins, referred to herein as a spacer region.

[0036] As used herein, “mutation” refers to a change in the DNA sequence and the protein it encodes that results in an alteration of its natural state. In this invention, the S protein expressed by the DNA molecule inserted into the multiple cloning site of the pCAGGS vector is a variant of the SARS-CoV-2 delta mutant strain B.1.617.2 S protein, which, relative to its wild-type S protein, contains one or more mutations in the region approximately at sites 980 to 990 of its S2 subunit, such as one or more of the K986P and V987P mutations (the amino acid sites are numbered according to the amino acid numbering of the SARS-CoV-2WA1 / 2020 wild-type S protein), for example, the variant K986P+V987P shown in SEQ ID NO:2 (referring to a variant with only the two mutations K986P and V987P relative to the wild-type S protein, the same below), or variant sequences with at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity with these variants.

[0037] The SARS-CoV-2 delta mutant strain B.1.617.2S protein variant of the present invention may further include one or more mutations at its S1 and S2 subunit cleavage sites, for example, one or more mutations at sites R682, R683, and R685, wherein site R682 may be mutated to G or other amino acids of similar properties, site R683 may be mutated to S or other amino acids of similar properties, and site R685 may be mutated to S or other amino acids of similar properties (for information on the chemical properties of amino acids, see, for example, Stryer et al., Biochemistry, 5th edition, 2002, pp. 44-49). For example, the SARS-CoV-2 delta mutant strain B.1.617.2S protein variant may be a variant of SEQ ID NO:4 or SEQ ID NO:6, or a variant sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity with SEQ ID NO:4 or SEQ ID NO:6.

[0038] As used herein, the term "nucleic acid" refers to polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The term should also be understood to include RNA or DNA analogs prepared from nucleotide analogs as equivalents, and, as applicable to the described embodiments, to include single-stranded polynucleotides (sense or antisense) and double-stranded polynucleotides.

[0039] As used herein, a "vector" is a composition comprising isolated nucleic acids and a substance that can be used to deliver the isolated nucleic acids into the interior of a cell. Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. In this document, the term "vector" includes autonomously replicating viruses.

[0040] The term "transfected cell" as used in this article (sometimes referred to in the industry as "production cell," "complementary cell," or "host cell") can be any cell capable of proliferating and expressing the desired recombinant protein.

[0041] The recombinant protein of the present invention can be formulated into a pharmaceutical composition. Such a pharmaceutical composition may be in a form suitable for administration to a subject, or the pharmaceutical composition may further comprise one or more pharmaceutically acceptable carriers, one or more additional ingredients, or a combination thereof.

[0042] Vaccine compositions containing recombinant proteins disclosed herein can be used to induce immunity against encoded antigen proteins. Vaccines can be formulated using standard techniques and, in addition to encoding the desired protein, may include pharmaceutically acceptable media, such as phosphate-buffered saline (PBS) or other buffer solutions, as well as other components, such as antibacterial and antifungal agents, isotonic and absorption-delaying agents, adjuvants, etc. In some embodiments, the vaccine composition is administered in combination with one or more other vaccines.

[0043] As used herein, the term “prevention” means the prior provision of medication, which may be prior to exposure to a pathogen (pre-exposure prophylaxis) or before the development of disease symptoms (post-exposure prophylaxis). The term “treatment” means the administration of medication during illness.

[0044] As used herein, "subject" or "patient" can refer to a human or a non-human mammal. Non-human mammals include, for example, livestock and pets, such as sheep, bovine, swine, canine, feline, and rodent mammals. Preferably, the subject is a human. The vaccine or pharmaceutical composition of the present invention can be administered via intramuscular injection, intravenous injection, intraperitoneal injection, subcutaneous injection, epidermal application, intradermal application, nasal application, rectal application, or oral administration.

[0045] As used herein, the term "effective amount" or "therapeutic effective amount" refers to the amount of the recombinant protein or composition of the present invention that is necessary for the prevention of a specific condition, or for reducing the severity of the condition or at least one of its symptoms or related symptoms and / or improving the condition or at least one of its symptoms or related symptoms.

[0046] The recombinant proteins, compositions, and methods of the present invention may have one or more of the following superior features compared to the prior art, including but not limited to higher productivity, increased transgenic expression, improved immunogenicity and stability, improved antibody neutralizing activity, or a unique serological cross-reactivity profile. In particular, improved immunogenicity and improved antibody neutralizing activity are significant advantages.

[0047] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0048] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0049] In the quantitative experiments in the following examples, three replicate experiments were set up, and the average value of the results was taken.

[0050] The pCAGGS vector is a circular plasmid, and its nucleotide sequence is shown in SEQ ID NO: 7.

[0051] The amino acid sequence of the SARS-CoV-2 delta mutant B.1.617.2 S protein (i.e., the wild-type S protein of the SARS-CoV-2 delta mutant B.1.617.2) is shown in SEQ ID NO:12.

[0052] Example 1: Preparation and Identification of Recombinant Protein

[0053] I. Construction of Recombinant Plasmids

[0054] 1. The DNA fragment between the restriction endonucleases EcoRI and XhoI in the pCAGGS vector was replaced with the DNA molecule shown in SEQ ID NO:1 using homologous recombination to obtain the recombinant plasmid pCAGGS-DeltaS2P.

[0055] The recombinant plasmid pCAGGS-DeltaS2P expresses the recombinant protein pCAGGS-DeltaS2P shown in positions 1-1208 from the N-terminus of SEQ ID NO:2, which is the protein of the SARS-CoV-2 delta mutant strain B.1.617.2S containing K986P and V987P mutations.

[0056] 2. The DNA fragment between the restriction endonucleases EcoRI and XhoI in the pCAGGS vector was replaced with the DNA molecule shown in SEQ ID NO:3 using homologous recombination to obtain the recombinant plasmid pCAGGS-DeltaS-GSAS.

[0057] The recombinant plasmid pCAGGS-DeltaS-GSAS expresses the recombinant protein pCAGGS-DeltaS-GSAS shown in SEQ ID NO:4 from N-terminus 1 to 1208, which is the protein of the SARS-CoV-2 delta mutant strain B.1.617.2S containing R682G, R683S and R685S mutations.

[0058] 3. The DNA fragment between the restriction endonucleases EcoRI and XhoI in the pCAGGS vector was replaced with the DNA molecule shown in SEQ ID NO:5 using homologous recombination to obtain the recombinant plasmid pCAGGS-DeltaS-2PMT.

[0059] The recombinant plasmid pCAGGS-DeltaS-2PMT expresses the recombinant protein pCAGGS-DeltaS-2PMT, which is the protein of the SARS-CoV-2 delta mutant strain B.1.617.2S containing the R682G, R683S, R685S, K986P and V987P mutations, as shown in SEQ ID NO:6 from N-terminus 1-1208.

[0060] 4. The DNA fragment between the restriction endonucleases EcoRI and XhoI in the pCAGGS vector was replaced with the DNA molecule shown in SEQ ID NO:8 using homologous recombination to obtain the recombinant plasmid pCAGGS-WH-S-2PMT.

[0061] The recombinant plasmid pCAGGS-WH-S-2PMT expresses the control protein shown in positions 1-1208 from the N-terminus of SEQ ID NO:9.

[0062] II. Preparation of recombinant protein and control protein

[0063] Cell culture conditions: constant temperature culture at 125 rpm, 37°C, and 5% CO2.

[0064] 1. Use FreeStyle TM 293F cells were cultured in Expression Medium until the cell density reached 2-3 × 10⁻⁶ cells / year. 6 / ml. During passage, the cell ratio is 1:3-4, and the cell expansion rate is approximately 24 hours per generation.

[0065] 2. After completing step 1, take the recombinant plasmid pCAGGS-DeltaS2P, use the transfection reagent PEI to prepare the transfection system and transfect 293F cells, and culture for 72 hours.

[0066] 3. After completing step 2, centrifuge and collect the supernatant; use the Twin-Strep-tag carried by the protein and the binding filler StrepTactin-XT to purify the protein and collect the protein solution.

[0067] 4. After completing step 3, use a 30 kDa concentration tube to concentrate the volume to less than 1 ml.

[0068] 5. Further component separation, purification, and buffer replacement (PBS) were performed using molecular sieves to obtain the recombinant protein pCAGGS-DeltaS2P.

[0069] Following the steps described above, replace the recombinant plasmid pCAGGS-DeltaS2P with the recombinant plasmid pCAGGS-DeltaS-GSAS, keeping all other steps unchanged, to obtain the recombinant protein pCAGGS-DeltaS-GSAS.

[0070] Following the steps described above, replace the recombinant plasmid pCAGGS-DeltaS2P with the recombinant plasmid pCAGGS-DeltaS-2PMT, keeping all other steps unchanged, to obtain the recombinant protein pCAGGS-DeltaS-2PMT.

[0071] Following the steps described above, replace the recombinant plasmid pCAGGS-DeltaS2P with the recombinant plasmid pCAGGS-WH-S-2PMT, keeping all other steps unchanged, to obtain the control protein.

[0072] III. Identification of Recombinant Proteins

[0073] HumanACE2 protein is a product of Acrobiosystems, catalog number AC2-H52H8; it is the human ACE2 protein extracellular domain (18-740). AM122 is a product of Acrobiosystems, catalog number S1N-M122; it is an RBD-binding antibody. AM121 is a product of Acrobiosystems, catalog number SPD-M121; it is an NTD-binding antibody.

[0074] The recombinant protein is recombinant protein pCAGGS-DeltaS2P, recombinant protein pCAGGS-DeltaS-GSAS, or recombinant protein pCAGGS-DeltaS-2PMT.

[0075] 1. Identification of protein expression and purity

[0076] Proteins were collected by measuring the elution time and height of each recombinant protein component in the molecular sieve tubes. The OD value of the recombinant protein was detected by spectrophotometry for the peak with higher UV absorption, and the protein concentration was calculated using the theoretical absorption coefficient. At the same time, representative component tubes were selected for denatured protein gel electrophoresis analysis. Based on the comparison with protein markers, the band located at approximately 120 kDa was identified as the target band.

[0077] The high-purity fractions were combined into a single tube, and the concentration of all recombinant proteins was adjusted to 1 mg / ml using PBS buffer. The isoelectric point and purity of each purified protein were then determined using capillary electrophoresis.

[0078] 2. Identification of activity

[0079] (1) The binding affinity of the recombinant protein to the receptor humanACE2 protein was determined using the SPR method.

[0080] (2) The ACE2-his protein was coupled to the CM5 chip, and the Kon, Koff and affinity of the protein were detected by Biacore machine using different concentrations of recombinant protein.

[0081] (3) The antigenicity of recombinant proteins was identified by ELISA. Each recombinant protein was coated with 100 ng / well of ELISA plate, and the binding ability of the recombinant proteins was detected by AM121 and AM122, respectively.

[0082] The results showed that the recombinant plasmid pCAGGS-DeltaS2P could express the correct-sized Spike protein, and due to the retention of the Folin-Ciocalteu restriction site, detached S1 protein was also present. The recombinant plasmid pCAGGS-DeltaS-GSAS could express the correct-sized Spike protein, and due to the modification of the Folin-Ciocalteu restriction site, detached S1 protein was not produced simultaneously, resulting in larger protein bands. The recombinant plasmids pCAGGS-DeltaS-2PMT and pCAGGS-WH-S-2PMT could express the correct-sized Spike protein, and due to the modification of the Folin-Ciocalteu restriction site, detached S1 protein was not produced simultaneously, resulting in larger protein bands. They also contained the Folden tag, which stabilizes the protein structure, resulting in higher protein yield, higher purity, and more symmetrical peak shapes in the molecular sieve pattern. All Spike proteins could bind to the receptor ACE2 protein.

[0083] Recombinant proteins pCAGGS-DeltaS2P, pCAGGS-DeltaS-GSAS, and pCAGGS-DeltaS-2PMT all showed good binding to AM122, but poor binding to AM121. Control proteins could bind to both AM121 and AM122.

[0084] Example 2: Application of Recombinant Protein

[0085] I. Animal Immunization

[0086] Six-week-old female BALB / c mice were randomly divided into five groups: the first four groups had ten mice each, and the last group had five mice. The recombinant proteins pCAGGS-DeltaS2P, pCAGGS-DeltaS-GSAS, and pCAGGS-DeltaS-2PMT prepared in Example 1, along with the control protein, were mixed with CpG adjuvant and administered intramuscularly in 100 μl volumes. A total of three immunizations were performed: the initial immunization was 100 μg; the second immunization was 100 μg three weeks after the initial immunization; and the third immunization was 50 μg two months after the initial immunization. Blood was collected starting two weeks after the initial immunization, and every two weeks (from the cheek) until four weeks after the third immunization.

[0087] Group 1 (G1 group): The immunizing agent is recombinant protein pCAGGS-DeltaS2P.

[0088] Group 2 (G2 group): The immunizing agent is recombinant protein pCAGGS-DeltaS-GSAS.

[0089] Group 3 (G3 group): The immunizing agent was recombinant protein pCAGGS-DeltaS-2PMT.

[0090] Group 4 (G4): The immunizing agent was the control protein.

[0091] Group 5 (G5): Adjuvant injection control group.

[0092] II. Preparation of SARS-CoV-2 pseudoviruses

[0093] 1. Replace the small DNA fragment between the restriction endonucleases BamHII and EcoRI in the pcDNA3.1(+) vector with the double-stranded DNA molecule shown in SEQ ID NO:10 (the gene encoding the full-length S protein of the SARS-CoV-2 delta mutant strain B.1.617.2) to obtain the SARS-CoV-2 delta mutant strain B.1.617.2 S protein particle.

[0094] 2. Replace the small DNA fragment between the restriction endonucleases BamHII and EcoRI in the pcDNA3.1(+) vector with the double-stranded DNA molecule shown in SEQ ID NO:11 (the gene encoding the full-length S protein of SARS-CoV-2WA1 / 2020) to obtain the SARS-CoV-2WA1 / 2020S protein particle.

[0095] 3. The SARS-CoV-2 delta mutant strain B.1.617.2S protein grain and backbone plasmid pNL4-3R-E-luciferase (described in the following literature: He J, Choe S, Walker R, Di Marzio P, Morgan DO, Landau NR. Human immunodeficiency virus type 1 viral protein R (Vpr) arrests cells in the G2 phase of the cell cycle by inhibiting p34cdc2 activity. J Virol; 69:6705–6711, 1995) were co-transfected into 293T cells. After incubation, an infectious but non-replicating pseudovirus of the SARS-CoV-2 delta mutant strain B.1.617.2 was obtained, with infectivity similar to that of the live virus.

[0096] The specific steps are as follows: SARS-CoV-2 delta mutant strain B.1.617.2S protein particle and backbone plasmid pNL4-3R-E-luciferase were co-transfected into 293T cells, incubated at 37°C, and the cell culture supernatant was collected 48 hours after transfection. This was the viral fluid containing the SARS-CoV-2 delta mutant strain B.1.617.2 pseudovirus.

[0097] 4. Following the method in step 3, replace the SARS-CoV-2 delta mutant strain B.1.617.2S protein particle with the SARS-CoV-2WA1 / 2020S protein particle, keeping all other steps unchanged, to obtain the SARS-CoV-2WA1 / 2020 pseudovirus, whose infectivity is similar to that of the live virus.

[0098] 5. The viral titers of SARS-CoV-2 delta mutant strain B.1.617.2 and SARS-CoV-2 WA1 / 2020 viral fluid were detected using an ELISA kit for p24 quantitative detection (HIV P24 antigen quantitative detection kit, KEY-BIO, 96T).

[0099] The results showed that the OD of the viral fluid of the SARS-CoV-2 delta mutant strain B.1.617.2 was... 450nm The absorbance value was 1 (1021 TCID50 / ml), indicating the OD value of the SARS-CoV-2WA1 / 2020 viral fluid. 450nm The absorbance value is 1.7 (1735.7 TCID50 / ml). A higher absorbance value indicates a higher viral load.

[0100] III. Detection of total antibodies induced by vaccines

[0101] Take the blood samples obtained in step one, separate the serum, and use ELISA to detect total bound IgG. For total IgG detection, coat the ELISA plate with recombinant protein pCAGGS-DeltaS-2PMT or recombinant protein pCAGGS-WH-S-2PMT (100 ng / well). The serum was first diluted to 200-fold volume, and then serially diluted 3-fold (200, 600, 1800, 5400, 16200, 48600, 145800 and 437400, a total of 8 dilutions, using PBS buffer at pH 7.2 as the dilution solvent). The secondary antibody was Anti-mouse IgGHRP.

[0102] The results showed that serum immunized with recombinant proteins pCAGGS-DeltaS2P, pCAGGS-DeltaS-GSAS, or pCAGGS-DeltaS-2PMT bound to both pCAGGS-DeltaS-2PMT and pCAGGS-WH-S-2PMT very well. Serum from group G3 showed stronger binding to pCAGGS-DeltaS-2PMT than the other four groups. The control group (G5) showed virtually no binding.

[0103] IV. Detection of antibody neutralizing activity in animal serum after vaccination

[0104] The test solution is serum obtained from the blood sample obtained in step one.

[0105] 1. The test solution was diluted 48 times with DMEM medium containing 10% FBS, and then serially diluted 3 times to obtain different serum concentrations (6 dilutions in total: 48, 144, 432, 1296, 3888 and 11664).

[0106] 2. Mix 100 μl of the diluent obtained in step 1 with 50 μl of the SARS-CoV-2 delta mutant strain B.1.617.2 virus solution or SARS-CoV-2WA1 / 2020 virus solution (virus content of 100 TCID50) prepared in step 2, and incubate at 37°C for 1 h to form the experimental group.

[0107] Mix 100 μl of DMEM medium containing 10% FBS with 50 μl of SARS-CoV-2 delta mutant strain B.1.617.2 virus solution or SARS-CoV-2WA1 / 2020 virus solution (virus content of 100 TCID50) prepared in step two, and incubate at 37°C for 1 h as a blank control group.

[0108] 3. After completing step 2, add 50 μl of Huh7 cell culture medium (containing approximately 2 × 10⁻⁶ cells). 4 (1 Huh7 cells), incubated at 37°C for 48 hours (in practical applications, 48-72 hours is acceptable).

[0109] 4. After completing step 3, add 100 μl of PBS buffer and 50 μl of cell lysis buffer (Bright-Globe). TM The Luciferase Assay System (Promega, E2650) was used to allow the sample to stand for 2 minutes. Then, the luciferase activity was detected using a chemiluminescence analyzer, and the neutralization activity was further calculated.

[0110] Neutralization activity = (fluorescence intensity of blank control group - fluorescence intensity of experimental group) / fluorescence intensity of blank control group × 100%.

[0111] Three replicate wells were set for each treatment, and the results were averaged.

[0112] The serum dilution factor corresponding to a neutralizing activity of 50% is ID50.

[0113] The results showed that immune sera using recombinant protein pCAGGS-DeltaS2P, recombinant protein pCAGGS-DeltaS-GSAS, or recombinant protein pCAGGS-DeltaS-2PMT could effectively neutralize SARS-CoV-2 delta mutant strain B.1.617.2 pseudovirus and SARS-CoV-2WA1 / 2020 pseudovirus, and their neutralization of SARS-CoV-2 delta mutant strain B.1.617.2 pseudovirus was stronger than that of immune sera using control proteins (G4 group).

[0114] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A SARS-CoV-2 delta mutant strain B.1.617.2 S protein variant, which is A1), A2), or A3). A1) The amino acid sequence is shown as positions 1-1208 from the N-terminus of SEQ ID NO:2; A2) The amino acid sequence is shown as positions 1-1208 from the N-terminus of SEQ ID NO:4; A3) The amino acid sequence is shown as positions 1-1208 from the N-terminus of SEQ ID NO:

6.

2. A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the SARS-CoV-2 delta mutant strain B.1.617.2 S protein variant as described in claim 1.

3. A DNA molecule encoding the SARS-CoV-2 delta mutant strain B.1.617.2 S protein variant of claim 1 or the fusion protein of claim 2.

4. The DNA molecule according to claim 3, characterized in that: The nucleotide sequence of the DNA molecule is B1), B2), B3), B4), B5), or B6). B1) SEQ ID NO:1 is shown from position 41 to 3658 starting from the 5' end; B2) SEQ ID NO:3 is shown from position 41 to 3658 starting from the 5' end; B3) SEQ ID NO:5 is shown from position 41 to 3658 starting from the 5' end; B4) As shown in SEQ ID NO:1; B5) As shown in SEQ ID NO:3; B6) as shown in SEQ ID NO:

5.

5. A recombinant plasmid obtained by inserting the DNA molecule of claim 3 or 4 into the multiple cloning site of an expression vector.

6. A recombinant protein obtained by transfecting mammalian cells with the recombinant plasmid of claim 5 and culturing the transfected cells.

7. The recombinant protein according to claim 6, characterized in that: The cells in question are 293F cells.

8. A composition for preventing SARS-CoV-2 infection, for neutralizing SARS-CoV-2, or for treating diseases caused by SARS-CoV-2 infection, characterized in that: The composition comprises the recombinant protein of claim 6 or 7.

9. The composition according to claim 8, characterized in that: The SARS-CoV-2 mentioned is the SARS-CoV-2 delta mutant strain B.1.617.

2.

10. Use of the recombinant protein of claim 6 or 7 or the composition of claim 8 or 9 in the preparation of a medicament for preventing SARS-CoV-2 infection, for neutralizing SARS-CoV-2, or for treating diseases caused by SARS-CoV-2 infection.

11. The application according to claim 10, characterized in that: The SARS-CoV-2 mentioned is the SARS-CoV-2 delta mutant strain B.1.617.

2.

12. A vaccine comprising the SARS-CoV-2 delta mutant strain B.1.617.2 S protein variant of claim 1, the fusion protein of claim 2, the DNA molecule of claim 3 or 4, the recombinant plasmid of claim 5, the recombinant protein of claim 6 or 7, or the composition of claim 8 or 9.

13. The vaccine according to claim 12, characterized in that: The vaccine is an mRNA vaccine, an adenovirus vector vaccine, or a recombinant protein vaccine.

14. The use of the SARS-CoV-2 delta mutant strain B.1.617.2 S protein variant of claim 1, the fusion protein of claim 2, the DNA molecule of claim 3 or 4, the recombinant plasmid of claim 5, the recombinant protein of claim 6 or 7, or the composition of claim 8 or 9 in the preparation of a vaccine.

15. The application according to claim 14, characterized in that: The vaccine is an mRNA vaccine, an adenovirus vector vaccine, or a recombinant protein vaccine.