WO2023151173A1 - 表达SARS-CoV-2奥密克戎突变株病毒抗原肽的核酸序列及其应用 - Google Patents

表达SARS-CoV-2奥密克戎突变株病毒抗原肽的核酸序列及其应用 Download PDF

Info

Publication number
WO2023151173A1
WO2023151173A1 PCT/CN2022/085802 CN2022085802W WO2023151173A1 WO 2023151173 A1 WO2023151173 A1 WO 2023151173A1 CN 2022085802 W CN2022085802 W CN 2022085802W WO 2023151173 A1 WO2023151173 A1 WO 2023151173A1
Authority
WO
WIPO (PCT)
Prior art keywords
nucleic acid
cov
sars
omicron
acid molecule
Prior art date
Application number
PCT/CN2022/085802
Other languages
English (en)
French (fr)
Inventor
陈凌
杨臣臣
汪乾
关素华
Original Assignee
广州恩宝生物医药科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 广州恩宝生物医药科技有限公司 filed Critical 广州恩宝生物医药科技有限公司
Publication of WO2023151173A1 publication Critical patent/WO2023151173A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0684—Cells of the urinary tract or kidneys
    • C12N5/0686—Kidney cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5256—Virus expressing foreign proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C12N2510/02—Cells for production
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host

Definitions

  • the present invention relates to the technical field of biomedicine, in particular, to the nucleic acid sequence for expressing the virus antigen peptide of SARS-CoV-2 Omecro mutant strain and its application.
  • Vaccines are the most cost-effective interventions to prevent and control 2019-nCoV infection.
  • the S-protein that makes up the "crown" is an obvious target and has become the focus of most research teams.
  • a research team has successfully revealed the relationship between the S protein and its receptor ACE2 in the process of invading cells through computer simulation of the three-dimensional structure of the S protein. Therefore, the S protein plays an important role in mediating the binding of virions to host cell receptors and in inducing neutralizing antibodies. According to research reports, the S protein has a pre-fusion conformation and a post-fusion conformation.
  • the S protein binds to the ACEII receptor of the host cell and is cleaved by the furin of the host cell to divide the protein into S1 and S2, which promotes the fusion of the viral envelope with the host.
  • Cell membrane fusion realizes the infection and invasion of viruses, and the antibodies produced after fusion are mostly binding antibodies, which have no neutralizing effect. Therefore, how to maintain the S protein in the pre-fusion conformation in vaccine development and design is the key to the success of vaccine development.
  • the Omicron strain According to the report of the World Health Organization, there is a latest mutant strain, the Omicron strain, whose pathogenicity and transmission ability are greatly enhanced. Moreover, the S gene of the Omicron mutant strain has at least 27 mutations.
  • the existing vaccines have very poor immune protection effect on the Omicron mutant strain, and it is very easy to escape the neutralizing effect of the existing vaccine neutralizing antibody. It greatly weakens the immune protection effect of the current vaccine and tends to replace the Delta mutant strain.
  • the vaccines currently on the market are inactivated vaccines, subunit protein vaccines, mRNA vaccines and adenovirus vector vaccines. These vaccines are mainly aimed at the original strain of SARS-CoV-2, and their protective effects against the mutant strain of Omicron are all reduced.
  • the expression level of the natural spike protein S gene of SARS-CoV-2 is very low in human kidney cell HEK293, so if the original S codon of the SARS-CoV-2 Omicron strain is used as an antigen, its SARS -The original S gene sequence protein of the CoV-2 Omicron strain cannot be effectively and efficiently expressed in cells, and its vaccine may be ineffective or the potency is very low, which is not enough to resist virus infection.
  • the object of the present invention is to provide a nucleic acid sequence capable of expressing a polypeptide that is immunogenic to SARS-CoV-2, especially an Omicron mutant strain, and its application.
  • a first aspect of the present invention provides a nucleic acid molecule comprising the following nucleic acid sequence:
  • nucleic acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence homology to SEQ ID NO:2.
  • the nucleic acid molecule is used to express a protein that is immunogenic to SARS-CoV-2. In some examples, the nucleic acid molecule is used to express a protein that is immunogenic to a mutant strain of SARS-CoV-2 omecroron. In some examples, the nucleic acid molecule is used to express the original strain of SARS-CoV-2, the mutant strain of SARS-CoV-2 Delta, the mutant strain of SARS-CoV-2 Alpha, the mutant strain of SARS-CoV-2 Beta, the mutant strain of SARS-CoV-2 One or more immunogenic proteins in a CoV-2 Gamma mutant strain.
  • the nucleic acid molecule or the protein expressed thereof is used to prevent or treat infection caused by SARS-CoV-2. In some examples, the nucleic acid molecule or the expressed protein thereof is used to prevent or treat infection caused by a mutant strain of SARS-CoV-2 omecroron. In some examples, the nucleic acid molecule or its expressed polypeptide is used to prevent or treat SARS-CoV-2 original strain, SARS-CoV-2 Delta mutant strain, SARS-CoV-2 Alpha mutant strain, SARS-CoV-2 Infection caused by one or more of Beta mutant strains, SARS-CoV-2 Gamma mutant strains.
  • the protein can be in the human body:
  • the induced immune response includes antibody and cell-mediated immune response.
  • the second aspect of the present invention provides: an expression vector containing the nucleic acid molecule of the first aspect of the present invention.
  • the vector is a DNA plasmid, RNA expression plasmid or viral vector.
  • the viral vector is an adenoviral vector.
  • the third aspect of the present invention provides: an expression cell that can express a protein based on the nucleic acid molecule of the first aspect of the present invention.
  • the expression cell is a host cell transformed or transfected with the expression vector of the second aspect of the present invention. In some embodiments, the expressing cells do not include propagation material.
  • the fourth aspect of the present invention provides: a nucleic acid composition comprising the nucleic acid molecule of the first aspect of the present invention, or the expression vector of the second aspect of the present invention.
  • the nucleic acid composition further includes at least one of a pharmaceutically acceptable adjuvant, carrier, diluent or excipient.
  • the fifth aspect of this aspect provides the application of the nucleic acid composition described in the fourth aspect of the present invention in a drug for preventing or treating infection caused by SARS-CoV-2.
  • the infection is an infection caused by a mutant strain of SARS-CoV-2 omecroron.
  • the infection is a SARS-CoV-2 original strain, a SARS-CoV-2 Delta mutant, a SARS-CoV-2 Alpha mutant, a SARS-CoV-2 Beta mutant, a SARS-CoV-2 Gamma Infection caused by one or more of the mutant strains.
  • the sixth aspect of the present invention provides a method for preventing or treating infection caused by SARS-CoV-2 strains, comprising administering a prophylactically effective amount or a therapeutically effective amount of the fourth aspect of the present invention to a subject in need. nucleic acid composition.
  • the infection is an infection caused by a mutant strain of SARS-CoV-2 omecroron.
  • the infection is a SARS-CoV-2 original strain, a SARS-CoV-2 Delta mutant, a SARS-CoV-2 Alpha mutant, a SARS-CoV-2 Beta mutant, a SARS-CoV-2 Gamma Infection caused by one or more of the mutant strains.
  • the amino acid sequence of the spike protein (Spike protein, S) of the original strain of SARS-CoV-2 of the present invention is shown in NCBI accession number YP_009724390.1.
  • the whole genome sequence of the original strain of SARS-CoV-2 in the present invention is shown in NCBI accession number NC_045512.2.
  • the optimized sequence After the optimized sequence is expressed in the human body or human-derived cells, it can induce a higher titer of neutralizing antibodies against the Omicron strain of SARS-CoV-2, which can effectively protect the body from Omicron It can also induce specific antibodies against the original strain of SARS-CoV-2 and other types of mutant strains, exerting multiple protective effects.
  • Figure 1 is a flowchart of the construction of pAd35-S50.
  • Figure 2 shows the expression results of S protein after transfection with equal amounts of pGA1-S-Ori, PGA1-S50, PGA261-S50 and PGA351-S50 respectively.
  • Fig. 3 is a virus purification diagram of pAd35-S50.
  • Figure 4 is the serum binding antibody level of Ad35-S50 immunized mice against the new coronavirus Omicron strain.
  • Figure 5 is the serum binding antibody level of Ad35-S50 immunized mice against the original strain of the new coronavirus.
  • Figure 6 is the serum binding antibody level of Ad35-S50 immunized mice against the new coronavirus Delta strain.
  • Fig. 7 is the serum neutralizing antibody level of Ad35-S50 immunized mice.
  • the nucleic acid sequence of the Spike protein (S) of SARS-CoV-2 is shown in GISAID: EPI_ISL_6640916, named as SEQ ID NO: 1.
  • the pre-mRNA transcribed by eukaryotic cells can produce different mRNA splicing isoforms through different splicing methods (selecting different combinations of splicing sites), which ultimately leads to different proteins produced by the same gene sequence. This is very detrimental to protein expression.
  • the inventor optimized the codon of the wild-type natural nucleic acid sequence and removed potential variable splicing sites based on his own technology, which ensured the uniqueness of protein expression and reduced the occurrence of erroneous splicing of protein expression.
  • the optimized nucleic acid sequence is recorded as SEQ ID NO: 2 (hereinafter the vector is named S50).
  • Example 1 Carrying the construction of the S antigen vector of the Omicron mutant strain
  • S50 is SEQ ID NO: 2
  • S50-F is SEQ ID NO: 2
  • S50-R is primers
  • TaKaRa Primer Star Mix
  • S50-F gtaccgagctcggatccgccaccatgttcgtgttcctggtcctactgcc (SEQ ID NO: 3);
  • S50-R agaatagggccctctagactagtttatcaggtgtagtgcagcttt (SEQ ID NO: 4).
  • PCR program 98°C for 3min; 98°C for 10s, 60°C for 5s, 72°C for 30s, 28 cycles; 72°C for 5min, store at 4°C.
  • the target fragment PGA1 was amplified by PCR with Primer Star Mix (TaKaRa).
  • CMV-R ggatccgagctcggtaccaagcttaagtttaaacgctagagtccgg (SEQ ID NO: 5);
  • BGH-F tctagagggccctattctatagtgtc (SEQ ID NO: 6).
  • PCR program 98°C for 3min; 98°C for 10s, 60°C for 5s, 72°C for 30s, 28 cycles; 72°C for 5min, store at 4°C.
  • the target fragment S50 and the vector backbone pGA1 were recombined using a homologous recombinase (Vazyme) to obtain the shuttle plasmid pGA1-S50 carrying the S gene of the omecro mutant strain.
  • Vazyme homologous recombinase
  • S50 is SEQ ID NO: 2
  • S50-F is SEQ ID NO: 2
  • S50-R is primers
  • TaKaRa Primer Star Mix
  • S50-F gtaccgagctcggatccgccaccatgttcgtgttcctggtcctactgcc (SEQ ID NO: 3);
  • S50-R agaatagggccctctagactagtttatcaggtgtagtgcagcttt (SEQ ID NO: 4).
  • PCR program 98°C for 3min; 98°C for 10s, 60°C for 5s, 72°C for 30s, 28 cycles; 72°C for 5min, store at 4°C.
  • the target fragment PGA261 was amplified by PCR with Primer Star Mix (TaKaRa).
  • CMV-R ggatccgagctcggtaccaagcttaagtttaaacgctagagtccgg (SEQ ID NO: 5);
  • BGH-F tctagagggccctattctatagtgtc (SEQ ID NO: 6).
  • PCR program 98°C for 3min; 98°C for 10s, 60°C for 5s, 72°C for 30s, 28 cycles; 72°C for 5min, store at 4°C.
  • the target fragment S50 and the vector backbone pGA261 were recombined using a homologous recombinase (Vazyme) to obtain a shuttle plasmid pGA261-S50 carrying the S gene of the Omicron mutant strain.
  • Vazyme homologous recombinase
  • S50 is SEQ ID NO: 2
  • S50-F is SEQ ID NO: 2
  • S50-R is primers
  • TaKaRa Primer Star Mix
  • S50-F gtaccgagctcggatccgccaccatgttcgtgttcctggtcctactgcc (SEQ ID NO: 3);
  • S50-R agaatagggccctctagactagtttatcaggtgtagtgcagcttt (SEQ ID NO: 4).
  • PCR program 98°C for 3min; 98°C for 10s, 60°C for 5s, 72°C for 30s, 28 cycles; 72°C for 5min, store at 4°C.
  • PGA351-EGFP plasmid (carrying the homologous recombination arm plasmid in the Ad35E1 region, preserved by Guangzhou Enbao Biomedical Technology Co., Ltd.) as a template, using CMV-R and BGH-F as primers, using Primer Star Mix (TaKaRa) PCR amplification
  • the target fragment PGA351 was obtained.
  • CMV-R ggatccgagctcggtaccaagcttaagtttaaacgctagagtccgg (SEQ ID NO: 5);
  • BGH-F tctagagggccctattctatagtgtc (SEQ ID NO: 6).
  • PCR program 98°C for 3min; 98°C for 10s, 60°C for 5s, 72°C for 30s, 28 cycles; 72°C for 5min, store at 4°C.
  • the target fragment S50 and the vector backbone pGA351 were recombined using a homologous recombinase (Vazyme) to obtain a shuttle plasmid pGA351-S50 carrying the S gene of the Omicron mutant strain.
  • Vazyme homologous recombinase
  • S-Ori is the original sequence of the S gene of Omicron strain, ie, SEQ ID NO: 1) plasmid as a template, SOri-F and SOri-R was used as a primer, and the target fragment S-Ori was amplified by PCR with Primer Star Mix (TaKaRa).
  • SOri-F gtaccgagctcggatccgccaccatgtttgtttttcttgtttttgccact (SEQ ID NO: 7);
  • SOri-R tagaatagggccctctagactagtttattatgtgtaatgtaatttgactcctt (SEQ ID NO: 8).
  • PCR program 98°C for 3min; 98°C for 10s, 60°C for 5s, 72°C for 30s, 28 cycles; 72°C for 5min, store at 4°C.
  • the target fragment PGA1 was amplified by PCR with Primer Star Mix (TaKaRa).
  • CMV-R ggatccgagctcggtaccaagcttaagtttaaacgctagagtccgg (SEQ ID NO: 5);
  • BGH-F tctagagggccctattctatagtgtc (SEQ ID NO: 6).
  • PCR program 98°C for 3min; 98°C for 10s, 60°C for 5s, 72°C for 30s, 28 cycles; 72°C for 5min, store at 4°C.
  • the target fragment S-Ori and the vector backbone pGA1 were recombined using a homologous recombinase (Vazyme) to obtain the shuttle plasmid pGA1-S-Ori carrying the original S gene of the Omicron mutant strain.
  • Vazyme homologous recombinase
  • the CMV-S50-BGH target fragment carrying the homologous recombination arm was amplified by PCR, and recovered by gel.
  • Ad35-SB-F agaattggatccgaattcgcggccgcgcgatcgccatcatcaataatatacctt (SEQ ID NO: 9);
  • Ad35-SB-R gcgtcgcagatccgaattcgtatacccatccaagctgcacgataa (SEQ ID NO: 10).
  • PCR program 98°C for 3min; 98°C for 10s, 60°C for 5s, 72°C for 50s, 28 cycles; 72°C for 5min, use the gel recovery kit to recover the target fragment, and store at 4°C.
  • pAd35 ⁇ E1 ⁇ E3 was linearized with PmeI and recovered by ethanol precipitation; the CMV-S50-BGH target fragment carrying the homologous recombination arm and the linearized PAd35 ⁇ E1 ⁇ E3 (5E4) were co-transformed into BJ5183, and the pAd35-S50 plasmid carrying the S50 gene was obtained by homologous recombination. The process is shown in Figure 1.
  • cationic liposomes 2.5 g of pGA1-S-Ori (carrying the original sequence of the S gene of Omicron strain, i.e. SEQ ID NO: 1), PGA1-S50, and PGA261 prepared in Example 1 were used respectively.
  • -S50 and PGA351-S50 transfection methods were used to transfect HEK293 cells. After 48 hours of transfection, the cells were collected, and the samples were processed according to the conventional Western Blot method, and protein detection was performed.
  • Ad35-S50 was linearized with AsisI, recovered by ethanol precipitation, and transfected into 293 cells by cationic liposome transfection; 2) 4 hours after transfection, 2 ml of DMEM containing 5% fetal bovine serum was added to culture 3) After detoxification, collect cells and culture supernatant, freeze and thaw 3 times in 37-degree water bath and liquid nitrogen, and centrifuge to remove cell debris, supernatant infects 10 cm dish ;4) After 2-3 days, collect cells and culture supernatant, freeze-thaw repeatedly 3 times and centrifuge to remove cell debris, supernatant infects 3-5 15 cm dishes; 5) After 2-3 days, collect cells, freeze-thaw repeatedly 3 times and centrifuged to remove cell debris; 6) After the supernatant infected 30 15 cm dishes for 2-3 days, collect the cells, freeze and thaw 3 times and centrifuge to remove cell debris; 7) Add the supernatant to a cesium chloride density gradient
  • Embodiment 4 animal immunogenicity evaluation
  • mice purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.
  • S50 dose 5 ⁇ 10 9 vp/monkey
  • Ad35-empty dose 5 ⁇ 10 9 vp/bird in the control group (ie negative group)
  • Enzyme-linked immunosorbent assay was used to detect the antibody level in serum using the RBD protein of the new coronavirus Omicron mutant strain, the original strain, and the Delta mutant strain (purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd.) as antigens.
  • the serum to be tested was inactivated in a water bath at 56°C for 30 minutes, and centrifuged at 6000g for 3 minutes; the serum was serially diluted 30 times;
  • inhibition rate [1-(mean value of luminous intensity of sample group - mean value of luminous intensity of blank control)/(mean value of luminous intensity of negative group - mean value of luminous intensity of blank control value)] ⁇ 100%;
  • blank The control refers to the background value of 96-well cells;
  • the negative group refers to the Ad35-empty immune group;
  • the vaccine can stimulate the mice to produce a higher titer of specific neutralizing antibodies against the new coronavirus of the Omicron strain.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Virology (AREA)
  • General Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Urology & Nephrology (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Oncology (AREA)
  • Cell Biology (AREA)
  • Communicable Diseases (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Immunology (AREA)
  • Mycology (AREA)
  • Epidemiology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

本发明提供表达SARS-CoV-2奥密克戎突变株病毒抗原肽的核酸序列及其应用。奥密克戎株原始的S基因序列蛋白不能有效在细胞内高效表达；本发明采用密码子偏好性进行优化得到新的S基因序列，使其能高效在人源细胞内高效表达，产生相应的多肽，诱导产生相应的免疫保护反应，为SARS-CoV-2奥密克戎株的疫苗的研发提供基础。

Description

表达SARS-CoV-2奥密克戎突变株病毒抗原肽的核酸序列及其应用 技术领域
本发明涉及生物医药技术领域，具体而言，涉及表达SARS-CoV-2奥密克戎突变株病毒抗原肽的核酸序列及其应用。
背景技术
面对新冠肺炎疫情，做好预防，阻断病毒的传播是控制疫情的关键。疫苗是预防和控制新型冠状病毒感染最经济有效的干预措施。SARS-CoV-2冠状病毒的病毒颗粒结构中，组成“皇冠”的S-蛋白是一个明显的靶点，成为大多数研究团队研究的重点。已有研究团队通过对S蛋白三维结构计算机模拟，成功地揭示了S蛋白与其侵入细胞过程中的受体ACE2的关系。因此，S蛋白在介导病毒粒子与宿主细胞受体的结合以及诱导中和抗体中起重要作用。根据研究报道，S蛋白存在融合前构象和融合后构象，S蛋白与宿主细胞的ACEII受体结合，通过宿主细胞的弗林蛋白酶进行切割，使蛋白分为S1和S2，促进病毒囊膜与宿主细胞膜融合实现病毒的感染入侵，融合后产生的抗体多为结合抗体，没有中和作用，所以在疫苗研发设计中如何维持S蛋白保持融合前构象是疫苗研发成功的关键。
根据世界卫生组织报告，有一最新突变株，奥密克戎株，其致病力和传播力均大大的增强。并且奥密克戎突变株其S基因发生至少27个突变之多，现有的疫苗对奥密克戎突变株免疫保护效果极差，极易逃逸现有疫苗中和抗体的中和作用，极大的削弱了目前疫苗的免疫保护作用，有取代Delta突变株的趋势。目前上市疫苗为灭活疫苗、亚单位蛋白疫苗、mRNA疫苗和腺病毒载体疫苗，这些疫苗主要针对的是原始株SARS-CoV-2，其对奥密克戎突变株的保护效果均有下降，都不能做到免疫后对奥密克戎株产生十分理想的免疫效果。此外，天然的SARS-CoV-2的刺突蛋白S基因在人肾细胞HEK293表达水平很低，因此如果以SARS-CoV-2奥密克戎株原始的S密码子来表达为抗原，其SARS-CoV-2奥密克戎株原始的S基因序列蛋白不能有效在细胞内高效表达，其疫苗可能无效或效价很低，不足以抵抗病毒的感染。
发明内容
本发明的目的在于提供一种能表达对SARS-CoV-2，特别是奥密克戎(Omicron)突变株，引起免疫原性的多肽的核酸序列及其应用。
本发明所采取的技术方案是：
本发明的第一个方面，提供一种核酸分子，所述核酸分子包含以下核酸序列：
a)SEQ ID NO：2所示核酸序列；或
b)与SEQ ID NO：2具有至少80％、85％、90％、95％、96％、97％、98％、或99％序列同源性的核酸序列。
在一些实例中，所述核酸分子用于表达对SARS-CoV-2引起免疫原性的蛋白。在一些实例中，所述核酸分子用于表达对SARS-CoV-2奥密克戎突变株引起免疫原性的蛋白。在一些实例中，所述核酸分子用于表达对SARS-CoV-2原始株、SARS-CoV-2 Delta突变株、SARS-CoV-2 Alpha突变株、SARS-CoV-2 Beta突变株、SARS-CoV-2 Gamma突变株中的一种或多种引起免疫原性的蛋白。
在一些实例中，所述核酸分子或其表达的蛋白用于预防或治疗SARS-CoV-2引发的感染。在一些实例中，所述核酸分子或其表达的蛋白用于预防或治疗SARS-CoV-2奥密克戎突变株引发的感染。在一些实例中，所述核酸分子或其表达的多肽用于预防或治疗SARS-CoV-2原始株、SARS-CoV-2 Delta突变株、SARS-CoV-2 Alpha突变株、SARS-CoV-2 Beta突变株、SARS-CoV-2 Gamma突变株中的一种或多种引发的感染。
在一些实例中，所述蛋白可在人体内：
诱导免疫应答；和/或
产生生物报告分子；和/或
产生用于检测的分子；和/或
调节基因功能；和/或
成为治疗性分子。
所述诱导免疫应答包括抗体与细胞介导的免疫应答。
本发明的第二个方面，提供：一种表达载体，所述的载体含有本发明第一方面的核酸分子。
在一些实例中，所述的载体为DNA质粒、RNA表达质粒或病毒载体。
在一些实例中，所述的病毒载体为腺病毒载体。
本发明的第三个方面，提供：一种表达细胞，所述表达细胞可基于本发明第一方面的核酸分子表达蛋白。
在一些实例中，所述表达细胞是采用本发明第二方面所述表达载体转化或转染的宿主细胞。在一些实施方式中，所述表达细胞不包括繁殖材料。
本发明的第四个方面，提供：一种核酸组合物，所述核酸组合物包括本发明第一方面的核酸分子、或本发明第二方面所述表达载体。
在一些实例中，所述核酸组合物还包括药学上可接受的佐剂、载体、稀释剂或赋形剂中的至少一种。
本方面的第五个方面，提供，本发明第四方面所述核酸组合物在预防或治疗SARS-CoV-2引发的感染的药物中的应用。
在一些实例中，所述感染是SARS-CoV-2奥密克戎突变株引发的感染。
在一些实例中，所述感染是SARS-CoV-2原始株、SARS-CoV-2 Delta突变株、SARS-CoV-2 Alpha突变株、SARS-CoV-2 Beta突变株、SARS-CoV-2 Gamma突变株中的一种或多种引发的感染。
本发明的第六个方面，提供，一种预防或治疗SARS-CoV-2株引发的感染的方法，包括给予有需要的受试者预防有效量或治疗有效量的本发明第四方面所述的核酸组合物。
在一些实例中，所述感染是SARS-CoV-2奥密克戎突变株引发的感染。
在一些实例中，所述感染是SARS-CoV-2原始株、SARS-CoV-2 Delta突变株、SARS-CoV-2 Alpha突变株、SARS-CoV-2 Beta突变株、SARS-CoV-2 Gamma突变株中的一种或多种引发的感染。
根据本发明的前述方面，在一些实例中，本发明所述SARS-CoV-2原始株的刺突蛋白(Spike protein，S)的氨基酸序列如NCBI登录号YP_009724390.1所示。在一些实例中，本发明所述SARS-CoV-2原始株的全基因组序列如NCBI登录号NC_045512.2所示。
本发明的有益效果是：
1)SARS-CoV-2奥密克戎株原始的S基因序列蛋白不能有效在细胞内高效表达，我们采用密码子偏好性进行优化得到新的S基因序列，其能高效在人源细胞内高效表达，产生相应的多肽，诱导产生相应的免疫保护反应。
2)优化的序列在人体或人源性细胞内表达后，可诱导产生较高滴度的针对奥密克戎株SARS-CoV-2的中和抗体，可以有效保护机体免受奥密克戎株的侵染；还可诱导针对SARS-CoV-2原始株以及其他类型突变株的特异性抗体，发挥多重保护作用。
附图说明
为了更清楚地说明本申请实施例中的技术方案，下面将对实施例描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本申请的一些实施例，对于本领域普通技术人员来讲，还可以根据这些附图获得其他的附图，而并不超出本申请要求保护的范围。
图1是pAd35-S50构建的流程图。
图2是分别转染等量的pGA1-S-Ori、PGA1-S50，PGA261-S50和PGA351-S50后S蛋白的表达结果。
图3是pAd35-S50的病毒纯化图。
图4是Ad35-S50免疫小鼠针对新冠病毒Omicron株的血清结合抗体水平。
图5是Ad35-S50免疫小鼠针对新冠病毒原始株的血清结合抗体水平。
图6是Ad35-S50免疫小鼠针对新冠病毒Delta株的血清结合抗体水平。
图7是Ad35-S50免疫小鼠的血清中和抗体水平。
具体实施方式
下面将结合本申请实施例中的附图，对本申请实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例是本申请一部分实施例，而不是全部的实施例。基于本申请中的实施例，本领域技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本申请保护的范围。
需要说明的是，在不冲突的情况下，本申请中的实施例及实施例中的特征可以相互组合。下面将结合实施例来详细说明本发明。
SARS-CoV-2的刺突蛋白(Spike protein，S)的核酸序列如GISAID：EPI_ISL_6640916所示,命名为SEQ ID NO：1。真核细胞转录的mRNA前体能够通过不同剪接方式(选择不同的剪接位点组合)产生不同的mRNA剪接异构体的过程，最终导致同一个基因序列产生的不同的蛋白质。这对蛋白的表达是非常不利的。发明人通过对野生型的天然核酸序列进行密码子优化，同时基于自有技术去除潜在的可变剪切位点，保证了蛋白表达的唯一性，减少了蛋白表达错误剪切的发生。优化得到的核酸序列记为SEQ ID NO：2(下文载体命名为S50)。
SEQ ID NO：1：
Figure PCTCN2022085802-appb-000001
Figure PCTCN2022085802-appb-000002
Figure PCTCN2022085802-appb-000003
SEQ ID NO：2：
Figure PCTCN2022085802-appb-000004
Figure PCTCN2022085802-appb-000005
以下结合具体实施例对本发明作进一步详细描述，这些实施例不能理解为限制本申请所要求保护的范围。
实施例1携带奥密克戎突变株的S抗原载体构建
1、构建奥密克戎突变株的S基因的穿梭质粒pGA1-S50
以pcDNA3.1-S50(由南京金斯瑞生物科技有限公司合成，S50即为SEQ ID NO：2)质粒为模板，以S50-F和S50-R为引物，采用Primer Star Mix(TaKaRa)PCR扩增得到目的片段S50。
S50扩增引物序列：
S50-F：gtaccgagctcggatccgccaccatgttcgtgttcctggtcctactgcc(SEQ ID NO：3)；
S50-R：agaatagggccctctagactagtttatcaggtgtagtgcagcttt(SEQ ID NO：4)。
PCR程序：98℃ 3min；98℃ 10s，60℃ 5s，72℃ 30s，28个循环；72℃ 5min，4℃保存。
以PGA1-EGFP质粒(由广州恩宝生物医药科技有限公司保存)为模板，以CMV-R和BGH-F为引物，采用Primer Star Mix(TaKaRa)PCR扩增得到目的片段PGA1。
pGA1骨架扩增引物序列：
CMV-R：ggatccgagctcggtaccaagcttaagtttaaacgctagagtccgg(SEQ ID NO：5)；
BGH-F：tctagagggccctattctatagtgtc(SEQ ID NO：6)。
PCR程序：98℃ 3min；98℃ 10s，60℃ 5s，72℃ 30s，28个循环；72℃ 5min，4℃保存。
将目的片段S50和载体骨架pGA1采用同源重组酶(Vazyme)进行重组，得到携带奥密克戎突变株S基因的穿梭质粒pGA1-S50。
2、构建奥密克戎突变株的S基因的穿梭质粒pGA261-S50
以pcDNA3.1-S50(由南京金斯瑞生物科技有限公司合成，S50即为SEQ ID NO：2)质粒为模板，以S50-F和S50-R为引物，采用Primer Star Mix(TaKaRa)PCR扩增得到目的片段S50。
S50扩增引物序列：
S50-F：gtaccgagctcggatccgccaccatgttcgtgttcctggtcctactgcc(SEQ ID NO：3)；
S50-R：agaatagggccctctagactagtttatcaggtgtagtgcagcttt(SEQ ID NO：4)。
PCR程序：98℃ 3min；98℃ 10s，60℃ 5s，72℃ 30s，28个循环；72℃ 5min，4℃保存。
以PGA261-EGFP质粒(由广州恩宝生物医药科技有限公司保存)为模板，以CMV-R和BGH-F为引物，采用Primer Star Mix(TaKaRa)PCR扩增得到目的片段PGA261。
pGA261骨架扩增引物序列：
CMV-R：ggatccgagctcggtaccaagcttaagtttaaacgctagagtccgg(SEQ ID NO：5)；
BGH-F：tctagagggccctattctatagtgtc(SEQ ID NO：6)。
PCR程序：98℃ 3min；98℃ 10s，60℃ 5s，72℃ 30s，28个循环；72℃ 5min，4℃保存。
将目的片段S50和载体骨架pGA261采用同源重组酶(Vazyme)进行重组，得到携带奥密克戎突变株S基因的穿梭质粒pGA261-S50。
3、构建奥密克戎突变株的S基因的穿梭质粒pGA351-S50
以pcDNA3.1-S50(由南京金斯瑞生物科技有限公司合成，S50即为SEQ ID NO：2)质粒为模板，以S50-F和S50-R为引物，采用Primer Star Mix(TaKaRa)PCR扩增得到目的片段S50。
S50扩增引物序列：
S50-F：gtaccgagctcggatccgccaccatgttcgtgttcctggtcctactgcc(SEQ ID NO：3)；
S50-R：agaatagggccctctagactagtttatcaggtgtagtgcagcttt(SEQ ID NO：4)。
PCR程序：98℃ 3min；98℃ 10s，60℃ 5s，72℃ 30s，28个循环；72℃ 5min，4℃保存。
以PGA351-EGFP质粒(携带Ad35E1区同源重组臂质粒，由广州恩宝生物医药科技有限公司保存)为模板，以CMV-R和BGH-F为引物，采用Primer Star Mix(TaKaRa)PCR扩增得到目的片段PGA351。
pGA351骨架扩增引物序列：
CMV-R：ggatccgagctcggtaccaagcttaagtttaaacgctagagtccgg(SEQ ID NO：5)；
BGH-F：tctagagggccctattctatagtgtc(SEQ ID NO：6)。
PCR程序：98℃ 3min；98℃ 10s，60℃ 5s，72℃ 30s，28个循环；72℃ 5min，4℃保存。
将目的片段S50和载体骨架pGA351采用同源重组酶(Vazyme)进行重组，得到携带奥密克戎突变株S基因的穿梭质粒pGA351-S50。
4、构建奥密克戎突变株的原始S基因的穿梭质粒pGA1-S-Ori
以pcDNA3.1-S-Ori(购自南京金斯瑞生物科技有限公司，S-Ori为奥密克戎株S基因原始序列，即SEQ ID NO：1)质粒为模板，以SOri-F和SOri-R为引物，采用Primer Star Mix(TaKaRa)PCR扩增得到目的片段S-Ori。
S-Ori扩增引物序列：
SOri-F：gtaccgagctcggatccgccaccatgtttgtttttcttgttttattgccact(SEQ ID NO：7)；
SOri-R：tagaatagggccctctagactagtttattatgtgtaatgtaatttgactcctt(SEQ ID NO：8)。
PCR程序：98℃ 3min；98℃ 10s，60℃ 5s，72℃ 30s，28个循环；72℃ 5min，4℃保存。
以PGA1-EGFP质粒(由广州恩宝生物医药科技有限公司保存)为模板，以CMV-R和BGH-F为引物，采用Primer Star Mix(TaKaRa)PCR扩增得到目的片段PGA1。
pGA1骨架扩增引物序列：
CMV-R：ggatccgagctcggtaccaagcttaagtttaaacgctagagtccgg(SEQ ID NO：5)；
BGH-F：tctagagggccctattctatagtgtc(SEQ ID NO：6)。
PCR程序：98℃ 3min；98℃ 10s，60℃ 5s，72℃ 30s，28个循环；72℃ 5min，4℃保存。
将目的片段S-Ori和载体骨架pGA1采用同源重组酶(Vazyme)进行重组，得到携带奥密克戎突变株原始S基因的穿梭质粒pGA1-S-Ori。
5、构建携带奥密克戎突变株的S基因的pAd35-S50
以pGA351-S50质粒为模板，PCR扩增得到携带同源重组臂的CMV-S50-BGH目的片段，胶回收。
CMV-S50-BGH目的片段扩增引物序列：
Ad35-SB-F：agaattggatccgaattcgcggccgcgcgatcgccatcatcaataatatacctt(SEQ ID NO：9)；
Ad35-SB-R：gcgtcgcagatccgaattcgtatacccatccaagctgcacgataa(SEQ ID NO：10)。
PCR程序：98℃ 3min；98℃ 10s，60℃ 5s，72℃ 50s，28个循环；72℃ 5min，采用胶回收试剂盒回收目的片段，4℃保存。
pAd35ΔE1ΔE3以PmeI线性化后乙醇沉淀回收；将携带同源重组臂的CMV-S50-BGH目的片段和线性化的PAd35ΔE1ΔE3(5E4)共转化BJ5183，同源重组得到携带S50基因的pAd35-S50质粒，技术流程如图1所示。
实施例2携带奥密克戎突变株的Spike基因表达检测
按照常规方法，利用阳离子脂质体，分别2.5g的实施例1制备得到的pGA1-S-Ori(携带奥密克戎株S基因原始序列，即SEQ ID NO：1)、PGA1-S50，PGA261-S50和PGA351-S50转染法转染HEK293细胞，转染48小时后，收集细胞，按照常规的WesternBlot方法处理样品，并进行蛋白检测。参见图2可以看出，pGA1-S-Ori样品中没有检测到S蛋白的表达，而经过密码子优化的PGA1-S50，PGA261-S50和PGA351-S50样品中均能够观察到高效的S蛋白的表达，说明我们优化的S50序列具有意料之外的效果。
实施例3 Ad35-S50载体的拯救与生产
1)按照常规方法，Ad35-S50以AsisI线性化，乙醇沉淀回收，阳离子脂质体转染法转染293细胞；2)转染后4小时，加入2毫升含5％胎牛血清的DMEM培养基，孵育7-10天，观察细胞病变；3)出毒后，收集细胞及培养上清，在37度水浴及液氮中反复冻融3次并离心去除细胞碎片，上清感染10厘米皿；4)2-3天后，收集细胞及培养上清，反复冻融3次并离心去除细胞碎片，上清感染3-5个15厘米皿；5)2-3天后，收集细胞，反复冻融3次并离心去除细胞碎片；6)上清感染30个15厘米皿2-3天后，收集细胞，反复冻融3次并离心去除细胞碎片；7)上清加至氯化铯密度梯度离心管；4℃，40000转，离心4小时；吸出病毒条带，脱盐，分装；8)以OD260吸光度测定病毒粒子滴度，计算公式为：病毒浓度＝OD260×稀释倍数×36/基因组长度(Kb)；病毒储存液于-80℃冻存。病毒纯化结果如图3所示。
实施例4动物免疫原性评价
6-8周龄Balb/c小鼠(购自北京维通利华实验动物技术有限公司)分为2组，每组5只；第0天，疫苗组(即样品组)肌注免疫Ad35-S50剂量：5×10 9vp/只，对照组(即阴性组)肌注免疫Ad35-empty剂量：5×10 9vp/只；第14天，眼眶取血并分离血清。
1、结合抗体
采用酶联免疫吸附测定(ELISA)，分别以新冠病毒奥密克戎突变株、原始株、Delta突变株的RBD蛋白(购自北京义翘神州科技有限公司)为抗原检测血清中的抗体水平。
ELISA结合抗体测定的具体操作为：
1)96孔板，每孔加50ng的RBD蛋白，4℃过夜；
2)吸掉上清，采用PBST洗3次，每孔加入200μl 5％BSA室温封闭2h；
3)PBST洗3次；每孔加入采用PBS以1:400，1:800，1:1600，1:3200，1:6400，1:12800，1:25600和，1:51200稀释的小鼠血清，37℃孵育2h；
4)加酶标抗体：加入100μl稀释后HRP标记IgG二抗，37℃孵育2h；
5)PBST洗6-8次；
6)加底物液显色：加入100μl TMB显色；
7)终止反应：加入50μl 1M硫酸终止反应；
8)结果判定：测OD值，OD值控制在0.1-4；
9)实验结果如图4、图5和图6所示，图4显示Ad35-S50能够诱异小鼠产生针对奥密克戎突变株RBD蛋白的特异性结合抗体。此外图5和图6显示Ad35-S50同样可以产生针对原始株新冠病毒和Delta突变株新冠病毒的的特异性结合抗体。
2、中和抗体
假病毒中和抗体的测定具体操作为：
1)将待检测的血清于56℃水浴灭活30min，6000g离心3min；将血清进行30倍梯度稀释；
2)用DMEM无血清培养基将奥密克戎假病毒(购买自南京诺唯赞生物科技股份有限公司，货号DD1768-02)稀释至1.3×10 4TCID50/ml与上述稀释的血清充分混匀，置于细胞培养箱中(37℃，5％CO 2)孵育1小时；
3)将孵育后的血清加入96孔板的ACEII细胞中，放入细胞培养箱中，37℃，5％CO 2培养72小时；
4)培养72小时后从细胞培养箱中取出96孔板，用多道移液器从每个上样孔中吸弃100μl上清，然后加入100μl荧光素酶检测试剂，室温避光反应2min；
5)计算中和抑制率：抑制率＝[1－(样品组的发光强度均值－空白对照发光强度均值)/(阴性组的发光强度均值－空白对照值发光强度均值)]×100％；空白对照指的是96孔细胞背景值；阴性组指的是Ad35-empty免疫组；
6)根据中和抑制率结果，利用Reed-Muench法计算IC50。
结果如图7所示：该疫苗可以刺激小鼠机体产生较高滴度的针对奥密克戎株新冠病毒的特异性中和抗体。
以上对本申请实施例进行了详细介绍，本文中应用了具体个例对本申请的原理及实施方式进行了阐述，以上实施例的说明仅用于帮助理解本申请的方法及其核心思想。同时，本领域技术人员依据本申请的思想，基于本申请的具体实施方式及应用范围上做出的改变或变形之处，都属于本申请保护的范围。综上所述，本说明书内容不应理解为对本申请的限制。

Claims (10)

  1. 一种核酸分子，所述的核酸分子包含：
    a)SEQ ID NO：2所示核酸序列；或
    b)与SEQ ID NO：2具有至少90％序列同源性的核酸序列。
  2. 根据权利要求1所述的核酸分子，其特征在于，所述的核酸分子可在人源细胞或人体内表达蛋白，所述的蛋白可在人体内：
    诱导免疫应答；和/或
    产生生物报告分子；和/或
    产生用于检测的分子；和/或
    调节基因功能；和/或
    成为治疗性分子。
  3. 一种表达载体，其特征在于，所述的表达载体含有权利要求1或2所述的核酸分子。
  4. 根据权利要求3所述的表达载体，其特征在于：所述的表达载体为DNA质粒、RNA表达质粒或病毒载体。
  5. 根据权利要求4所述的表达载体，其特征在于：所述的病毒载体为腺病毒载体。
  6. 一种表达细胞，其特征在于：所述的表达细胞可基于权利要求1或2所述的核酸分子表达蛋白。
  7. 一种核酸组合物，其特征在于：所述的核酸组合物包括权利要求1或2所述的核酸分子、或权利要求3-5任一项所述的表达载体。
  8. 根据权利要求7所述的核酸组合物，其特征在于：还包括药学上可接受的佐剂、载体、稀释剂或赋形剂中的至少一种。
  9. 权利要求7或8所述的核酸组合物在制备预防或治疗SARS-CoV-2引发的感染的药物中的应用。
  10. 根据权利要求9所述的应用，所述的感染为SARS-CoV-2奥密克戎突变株引发的感染。
PCT/CN2022/085802 2022-02-09 2022-04-08 表达SARS-CoV-2奥密克戎突变株病毒抗原肽的核酸序列及其应用 WO2023151173A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202210120115.9 2022-02-09
CN202210120115.9A CN114150004B (zh) 2022-02-09 2022-02-09 表达SARS-CoV-2奥密克戎突变株病毒抗原肽的核酸序列及其应用

Publications (1)

Publication Number Publication Date
WO2023151173A1 true WO2023151173A1 (zh) 2023-08-17

Family

ID=80450031

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/085802 WO2023151173A1 (zh) 2022-02-09 2022-04-08 表达SARS-CoV-2奥密克戎突变株病毒抗原肽的核酸序列及其应用

Country Status (2)

Country Link
CN (1) CN114150004B (zh)
WO (1) WO2023151173A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11878055B1 (en) 2022-06-26 2024-01-23 BioNTech SE Coronavirus vaccine

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114150004B (zh) * 2022-02-09 2022-04-22 广州恩宝生物医药科技有限公司 表达SARS-CoV-2奥密克戎突变株病毒抗原肽的核酸序列及其应用
CN114573667B (zh) * 2022-05-06 2022-08-02 艾棣维欣(苏州)生物制药有限公司 SARS-CoV-2病毒B.1.1.529突变株DNA疫苗及应用
CN114773463B (zh) * 2022-06-20 2022-08-26 北京市疾病预防控制中心 一种针对新冠病毒omicron株S蛋白的单域抗体VHH-1及编码序列和应用
CN115716866A (zh) * 2022-09-30 2023-02-28 珠海丽凡达生物技术有限公司 新型冠状病毒疫苗及其制备方法和应用

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111533812A (zh) * 2020-06-22 2020-08-14 艾立克(北京)生物科技有限公司 针对sars-cov-2病毒的dna疫苗及其用途
CN111778264A (zh) * 2020-07-14 2020-10-16 广州佰芮慷生物科技有限公司 基于新型腺病毒载体Sad23L和/或Ad49L的新型冠状病毒肺炎疫苗
CN112206318A (zh) * 2020-03-16 2021-01-12 广州恩宝生物医药科技有限公司 一种用于预防SARS-CoV-2感染的Ad7载体疫苗
CN112220918A (zh) * 2020-03-16 2021-01-15 广州恩宝生物医药科技有限公司 一种用于预防SARS-CoV-2感染的Ad35载体疫苗
CN113930452A (zh) * 2020-07-13 2022-01-14 中国科学院微生物研究所 基于黑猩猩腺病毒载体的重组载体、腺病毒、2019新型冠状病毒疫苗及其制备方法
CN114150004A (zh) * 2022-02-09 2022-03-08 广州恩宝生物医药科技有限公司 表达SARS-CoV-2奥密克戎突变株病毒抗原肽的核酸序列及其应用
CN114150005A (zh) * 2022-02-09 2022-03-08 广州恩宝生物医药科技有限公司 用于预防SARS-CoV-2奥密克戎株的腺病毒载体疫苗

Family Cites Families (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101098710A (zh) * 2004-06-02 2008-01-02 纽约血液中心 产生高度有效抗体的sars疫苗和方法
CN111821433B (zh) * 2020-02-06 2021-06-08 深圳市瑞吉生物科技有限公司 mRNA疫苗及其合成方法、试剂盒
CN110974950B (zh) * 2020-03-05 2020-08-07 广州恩宝生物医药科技有限公司 一种用于预防SARS-CoV-2感染的腺病毒载体疫苗
CN110951756B (zh) * 2020-02-23 2020-08-04 广州恩宝生物医药科技有限公司 表达SARS-CoV-2病毒抗原肽的核酸序列及其应用
CN111218458B (zh) * 2020-02-27 2020-11-20 珠海丽凡达生物技术有限公司 编码SARS-CoV-2病毒抗原的mRNA和疫苗及疫苗的制备方法
CN111088283B (zh) * 2020-03-20 2020-06-23 苏州奥特铭医药科技有限公司 mVSV病毒载体及其病毒载体疫苗、一种基于mVSV介导的新冠肺炎疫苗
WO2021222304A1 (en) * 2020-04-27 2021-11-04 Modernatx, Inc. Sars-cov-2 rna vaccines
CN111718951A (zh) * 2020-06-24 2020-09-29 宁波毓昌生物技术有限公司 重组新型冠状病毒covid-19 s蛋白、其制备方法及应用
CN112226445B (zh) * 2020-10-20 2021-05-11 成都欧林生物科技股份有限公司 编码SARS-CoV-2病毒刺突蛋白的核酸及其的应用
CN113024641B (zh) * 2021-03-11 2022-05-17 广东省农业科学院动物卫生研究所 新型冠状病毒的重组s蛋白及其制备方法和应用
CN113248581A (zh) * 2021-06-15 2021-08-13 江西浩然生物制药有限公司 用于产生新冠病毒中和抗体的新冠s抗原及其制备方法

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112206318A (zh) * 2020-03-16 2021-01-12 广州恩宝生物医药科技有限公司 一种用于预防SARS-CoV-2感染的Ad7载体疫苗
CN112220918A (zh) * 2020-03-16 2021-01-15 广州恩宝生物医药科技有限公司 一种用于预防SARS-CoV-2感染的Ad35载体疫苗
CN111533812A (zh) * 2020-06-22 2020-08-14 艾立克(北京)生物科技有限公司 针对sars-cov-2病毒的dna疫苗及其用途
CN113930452A (zh) * 2020-07-13 2022-01-14 中国科学院微生物研究所 基于黑猩猩腺病毒载体的重组载体、腺病毒、2019新型冠状病毒疫苗及其制备方法
CN111778264A (zh) * 2020-07-14 2020-10-16 广州佰芮慷生物科技有限公司 基于新型腺病毒载体Sad23L和/或Ad49L的新型冠状病毒肺炎疫苗
CN114150004A (zh) * 2022-02-09 2022-03-08 广州恩宝生物医药科技有限公司 表达SARS-CoV-2奥密克戎突变株病毒抗原肽的核酸序列及其应用
CN114150005A (zh) * 2022-02-09 2022-03-08 广州恩宝生物医药科技有限公司 用于预防SARS-CoV-2奥密克戎株的腺病毒载体疫苗

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
GARCIA-BELTRAN WILFREDO F.; ST. DENIS KERRI J.; HOELZEMER ANGELIQUE; LAM EVAN C.; NITIDO ADAM D.; SHEEHAN MAEGAN L.; BERRIOS CRIST: "mRNA-based COVID-19 vaccine boosters induce neutralizing immunity against SARS-CoV-2 Omicron variant", CELL, ELSEVIER, AMSTERDAM NL, vol. 185, no. 3, 6 January 2022 (2022-01-06), Amsterdam NL , pages 457, XP086949417, ISSN: 0092-8674, DOI: 10.1016/j.cell.2021.12.033 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11878055B1 (en) 2022-06-26 2024-01-23 BioNTech SE Coronavirus vaccine

Also Published As

Publication number Publication date
CN114150004B (zh) 2022-04-22
CN114150004A (zh) 2022-03-08

Similar Documents

Publication Publication Date Title
WO2023151173A1 (zh) 表达SARS-CoV-2奥密克戎突变株病毒抗原肽的核酸序列及其应用
CN110974950B (zh) 一种用于预防SARS-CoV-2感染的腺病毒载体疫苗
CN110951756B (zh) 表达SARS-CoV-2病毒抗原肽的核酸序列及其应用
WO2021184560A1 (zh) 一种以人复制缺陷腺病毒为载体的重组新型冠状病毒疫苗
WO2023151172A1 (zh) 用于预防SARS-CoV-2奥密克戎株的腺病毒载体疫苗
WO2021002776A1 (en) Immunobiological agent for inducing specific immunity against severe acute respiratory syndrome virus sars-cov-2
WO2021000968A2 (zh) 一种用于预防SARS-CoV-2感染的腺病毒载体疫苗
Liu et al. Polynucleotide viral vaccines: codon optimisation and ubiquitin conjugation enhances prophylactic and therapeutic efficacy
WO2022193552A1 (zh) 新型冠状病毒Ad26腺病毒载体疫苗及其制备方法与应用
CN111778264B (zh) 基于新型腺病毒载体Sad23L和/或Ad49L的新型冠状病毒肺炎疫苗
Kim et al. A single subcutaneous or intranasal immunization with adenovirus‐based SARS‐CoV‐2 vaccine induces robust humoral and cellular immune responses in mice
WO2021076009A1 (en) Expression vector against severe acute respiratory syndrome virus sars-cov-2
CN101591646A (zh) 一种治疗人宫颈癌的树突状细胞疫苗
Perez et al. A single dose of an MVA vaccine expressing a prefusion-stabilized SARS-CoV-2 spike protein neutralizes variants of concern and protects mice from a lethal SARS-CoV-2 infection
Cao et al. A single vaccine protects against SARS-CoV-2 and influenza virus in mice
Yun et al. TMPRSS12 is an activating protease for subtype B avian metapneumovirus
Ragonnaud et al. Therapeutic vaccine against primate papillomavirus infections of the cervix
US20220235376A1 (en) Expression vector against severe acute respiratory syndrome virus sars-cov-2
CN101280316A (zh) 人宫颈癌的一种治疗性疫苗
Khan et al. Adenovirus-vectored SARS-CoV-2 vaccine expressing S1-N fusion protein
WO2022007774A1 (zh) 一种新型黑猩猩腺病毒载体及其构建方法和应用
CN114717251B (zh) 一种用于预防SARS-CoV-2原始株和Beta株的腺病毒载体疫苗
KR20220012863A (ko) 아데노바이러스 유전자 요법 벡터 생산성 및 감염성을 증가시키는 아데노바이러스 폴리펩타이드 ix
WO2021184987A1 (zh) 一种用于预防SARS-CoV-2感染的Ad7载体疫苗
US20220331420A1 (en) Ad35-vectored vaccine for preventing sars-cov-2 infection

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22925534

Country of ref document: EP

Kind code of ref document: A1